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WO2021015218A1 - Prevention or treatment of fibrotic disease which targets transcription-associated factor - Google Patents

Prevention or treatment of fibrotic disease which targets transcription-associated factor Download PDF

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WO2021015218A1
WO2021015218A1 PCT/JP2020/028361 JP2020028361W WO2021015218A1 WO 2021015218 A1 WO2021015218 A1 WO 2021015218A1 JP 2020028361 W JP2020028361 W JP 2020028361W WO 2021015218 A1 WO2021015218 A1 WO 2021015218A1
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vgll3
myofibroblasts
expression
seq
fibrosis
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道雄 仲矢
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国立大学法人九州大学
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Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating fibrotic diseases.
  • the present invention relates to substances and nucleic acid molecules that prevent or treat fibrotic diseases, methods for screening them, and kits for them.
  • Fibrosis is a condition in which extracellular matrix proteins such as collagen are excessively accumulated in the interstitium of tissues, and is induced in almost all organs such as the heart, liver, lungs, intestines, and kidneys due to inflammation and aging.
  • parenchymal cells such as myocardial cells and hepatocytes undergo cell death, and extracellular matrix such as collagen accumulates to compensate for the loss of the parenchymal cells.
  • a characteristic of fibrotic organs is the accumulation of non-stretchable extracellular matrix, which makes the tissue extremely stiff.
  • Non-Patent Document 3 Excessive fibrosis causes organ dysfunction and can lead to death if left untreated. It has also been reported that fibrosis is involved in about 45% of all causes of death.
  • Organ fibrosis is carried out by a group of cells called myofibroblasts that produce collagen and the like. Myofibroblasts are rarely present when the tissue is normal. However, it is known that when inflammation occurs, humoral factors such as TGF- ⁇ secreted from immune cells are stimulated to differentiate resident fibroblasts into myofibroblasts ( Non-Patent Document 4). On the other hand, in recent years, it has been reported that not only cytokines such as TGF- ⁇ , but also pericellular hardness and mechanical stress play important roles in differentiation into myofibroblasts. (Non-Patent Document 5-8).
  • MRTF Myocardin-related transcription factor
  • YAP Yes-associated protein
  • Myofibroblasts trust your heart and let fate decide. Journal of molecular and cellular cardiology 70, 9-18, doi: 10.1016 / j.yjmcc.2013.10.019 (2014). Schroer, A. K. & Merryman, W. D. Mechanobiology of myofibroblast adhesion in fibrotic cardiac disease. Journal of cell science 128, 1865-1875, doi: 10.1242 / jcs.162891 (2015). Hinz, B. Mechanical aspects of lung fibrosis: a spotlight on the myofibroblast. Proceedings of the American Thoracic Society 9, 137-147, doi: 10.1513 / pats.201202-017AW (2012). Tomasek, J.
  • Pirfenidone has been clinically shown to suppress the decline in vital capacity by its antifibrotic effect in patients with idiopathic pulmonary fibrosis (Margaritopoulos, G.A., Vasarmidi, E. & Antoniou, K. M. Pirfenidone in the treatment of idiopathic pulmonary fibrosis: an evidence-based review of its place in therapy.
  • Nintedanib inhibits platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (EGFR), and is involved in the pathogenesis of idiopathic pulmonary fibrosis. It is thought to be a mechanism of action that suppresses cell proliferation and migration (Wollin, L. et al. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis.
  • PDGFR platelet-derived growth factor receptor
  • FGFR fibroblast growth factor receptor
  • EGFR vascular endothelial growth factor receptor
  • An object of the present invention is to provide an epoch-making and effective pharmaceutical composition for fibrosis targeting VGLL3. Another object of the present invention is to provide a method for screening candidate substances effective for the prevention or treatment of fibrotic diseases targeting VGLL3.
  • the present inventor seeks transcription-related factors involved in fibrosis expressed in myofibroblasts, aiming at the development of innovative therapeutic methods and agents for fibrotic pathologies for which sufficient therapeutic methods have not yet been established. I found VGLL3, whose function is almost unknown. Further research revealed for the first time that VGLL3 translocates into the nucleus in a mechanical stimulus-dependent manner and regulates the expression of fibrosis-related factors, and the present invention was completed based on this finding. is there.
  • the present invention includes the following aspects.
  • the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5.
  • nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing A nucleic acid that hybridizes with a base sequence complementary to the VGLL3 gene under stringent conditions.
  • the pharmaceutical composition according to [3], wherein the substance that inhibits the expression of a gene or nucleic acid is at least one selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme.
  • ⁇ Screening method> [9] The step of measuring the expression level of VGLL3 in cells in the presence of the test substance, When the expression level is lower than the expression level in the absence of the test substance, the step of determining that the test substance is an effective candidate substance for the prevention or treatment of fibrotic disease. Prepare, prepare A screening method for candidate substances that are effective in the prevention or treatment of fibrotic diseases.
  • a method of determining the degree of fibrosis (a10) Step of measuring the amount of VGLL3 (test biomarker amount) in myofibroblasts of a subject, (b10) A step of comparing the amount of the test biomarker with the amount of VGLL3 in the reference myofibroblast (control biomarker amount), and (c10) when the test biomarker amount is larger than the control biomarker amount.
  • a method for determining a subject to have a high degree of fibrosis [11] The method according to [10], wherein the reference amount of VGLL3 in myofibroblasts is the amount of VGLL3 in myofibroblasts of the same subject measured prior to step (a10).
  • VGLL3 in myofibroblasts is the amount of VGLL3 in healthy subjects.
  • a kit for detecting myofibroblasts which comprises a primer set for amplifying VGLL3 cDNA, a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding agent for VGLL3 protein.
  • VGLL3 is a factor that promotes fibrosis because the expression of extracellular matrix proteins such as collagen is suppressed when VGLL3 is knocked down. According to the present invention, it is possible to provide various techniques for preventing or treating fibrotic diseases related to inhibitors of VGLL3.
  • FIG. 1 is a schematic diagram showing that the expression of VGLL3 is related to fibrosis as a whole.
  • FIG. 1A is a schematic diagram of an MI (myocardial infarction) surgical heart. The procedure for MI surgery is occlusion of the left anterior descending coronary artery. In an infarcted heart, the ischemic necrotic region is recognized as the "infarcted” region and the rest of the infarcted region is recognized as the "non-infarcted” region.
  • FIG. 1B shows the procedure for identifying VGLL3 that may be associated with fibrosis. Gene expression in the hearts of sham and MI operated mice was comprehensively detected using microarray analysis.
  • FIG. 1C shows VGLL3 and fibrosis-related genes ( ⁇ SMA ( ⁇ -Smooth Muscle)) in the non-infarcted region (rem) and infarcted region (inf) of the sham-operated mouse heart (sham) and the MI-operated mouse heart.
  • ⁇ SMA ⁇ -Smooth Muscle
  • the mRNA expression is normalized to the expression of GAPDH and is intact. It is shown as a multiple change with respect to the mouse (day 0).
  • N 3 (0 days), 3 (sham 3 days), 5 (inf 3 days), 5 (rem 3 days), 4 (sham 7 days), 5 (inf 7 days), 5 (rem 7 days), 3-4 (sham 28 days), 6 (inf 28 days), and 6 (rem 28 days).
  • Group comparisons are on both sides without correspondence at each time point. Performed by Student's t-test. #P ⁇ 0.05, ### P ⁇ 0.001 (inf vs. sham); * P ⁇ 0.05, *** P ⁇ 0.001 (inf vs. rem).
  • FIG. 1D outlines a protocol for the regulation of myofibroblast differentiation by mechanical stimulation.
  • Myocardial fibroblasts were cultured on an adhesive dish for 7 days, some of them were separated and plated on a non-adhesive dish for an additional 7 days (dedifferentiation), and then some cells were recultured on an adhesive dish for 7 days. (Redifferentiation). Protein and RNA were collected from myofibroblasts at each time point.
  • FIG. 1F shows the collection of high-purity myocardial fibroblasts and the expression of fibrosis-related genes in myocardial fibroblasts cultured on a plate or suspension, and CD45 ⁇ +) cells by MACS (Magnetic cell sorting).
  • MACS Magnetic cell sorting
  • Histograms labeled with blue or red arrows represent heart cells stained with PE-conjugated anti-CD45 antibody or FITC-conjugated anti- ⁇ SMA antibody, respectively. Histograms without arrows represent unstained controls.
  • FIG. 1H shows fibrosis-related genes (ACTA2, COL1A1, COL1A2, COL3A1, FN1, LOX and CTGF) in cardiac fibroblasts of “adhesive”, “non-adherent” and “re-adherent” (Re). )
  • Is a graph showing mRNA expression. mRNA expression was normalized to that of GAPDH. Each n 5.
  • FIG. 1J shows differentiated or redifferentiated myofibroblasts up-regulated (16-fold or more) compared to undifferentiated myofibroblasts or infarcted mouse hearts compared to non-infarcted mouse hearts. It is a Ben diagram showing eight duplicate transcripts up-regulated (2 to the 1.5th power or higher) in.
  • FIG. 1K shows the protein expression distribution of VGLL3 in each human organ.
  • FIG. 1M shows the results of electrophoresis showing the expression levels of the VGLL families VGLL1, VGLL2, VGLL3 and VGLL4 in muscle fibroblasts isolated from the heart as PCR amplification.
  • FIG. 1N is a graph showing the results of absolute quantification of the expression levels of VGLL family VGLL1, VGLL2, VGLL3 and VGLL4 in muscle fibroblasts isolated from the heart.
  • FIG. 2A-G are photographs that replace the drawings, showing that VGLL3 is generally specifically expressed in fibrotic cardiac myofibroblasts.
  • the square in the leftmost photo shows the enlarged area in the right panel.
  • Arrows ( ⁇ ) indicate where the signals overlap in the overlay.
  • the scale bar indicates 30 ⁇ m.
  • FIG. 2A shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the myofibroblast marker ⁇ SMA (A).
  • FIG. 2B shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the macrophage marker CD68 (B).
  • FIG. 2C shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the cardiomyocyte marker TNNI3 (C).
  • FIG. 2D is a representative image of an in situ hybridization (RNA scope) experiment evaluating VGLL3 mRNA expression in LV of mouse heart on day 7 MI. Heart sections were co-stained with anti- ⁇ SMA antibody (D). Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows ( ⁇ ) indicate where the signals overlap in the overlay. The scale bar indicates 30 ⁇ m.
  • FIG. 2E is a representative image of an in situ hybridization (RNA scope) experiment evaluating VGLL3 mRNA expression in LV of mouse heart on day 7 of MI. Cardiac sections were co-hybridized with RNA scope probes for periostin mRNA (E). Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows ( ⁇ ) indicate where the signals overlap in the overlay. The scale bar indicates 30 ⁇ m.
  • FIG. 2F is a representative image of an in situ hybridization experiment in which VGLL3 mRNA and periostin mRNA expression in LV of day 7 sham mice was evaluated. Cardiac sections were co-stained with anti- ⁇ SMA antibody.
  • FIG. 2G is a representative image of an in situ hybridization experiment evaluating VGLL3 mRNA expression in an infarcted heart of a human patient. Cardiac sections were co-stained with anti- ⁇ SMA antibody. Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows ( ⁇ ) indicate where the signals overlap in the overlay. The scale bar indicates 30 ⁇ m.
  • FIG. 3A-D are photographs that replace the drawings, showing that the intranuclear translocation of VGLL3 is generally induced by mechanical stimulation. It is a representative confocal immunocytochemical image of VGLL3 and nucleus (DAPI) in myocardial fibroblasts isolated from the heart on the third day of MI. The cells were stained with Cell Trace to observe the overall picture of the cells.
  • FIG. 3A is an immunocytochemical image of VGLL3 (A). The upper panel shows suspended cultured myofibroblasts and the lower panel shows adherent cultured myofibroblasts.
  • FIG. 3B is an immunocytochemical image of YAP1 (B). The upper panel shows suspended cultured myofibroblasts and the lower panel shows adherent cultured myofibroblasts. The graph shows the ratio of nuclei to cytoplasmic VGLL3 or YAP1 strength in myofibroblasts cultured in the floating state (Float) and the adhesive state (Adherent) (n> 100 cells in each group). Scale bar: 20 ⁇ m.
  • FIG. 3C is an image of myofibroblasts overexpressing FLAG-tagged VGLL3.
  • FLAG-tagged VGLL3 was stained with anti-FLAG antibody.
  • the graph shows the ratio of nuclei to cytoplasmic FLAG-tagged VGLL3 strength in myofibroblasts cultured in the floating state (Float) and the adhesive state (Adherent) (n> 100 cells in each group). Scale bar: 20 ⁇ m.
  • FIG. 3D is an image of myofibroblasts adhering to a poly-L-lysine coated glass bottom dish at each time shown in the bar graph.
  • FIG. 4 is an alternative photograph showing that the intranuclear translocation of VGLL3 is regulated by the hardness of the extracellular matrix.
  • FIG. 4A is a schematic diagram showing the hardness of normal tissue and fibrous tissue. Elastic modulus is the hardness of an organ or cell.
  • FIG. 4B is a representative confocal immunocytochemical image of VGLL3 and nucleus (DAPI) in myocardial fibroblasts seeded on hydrogel plates with different hardnesses (moduli of 1, 8, 25, and 50 kPa). Is. The cells were stained with Cell Trace to observe the overall picture of the cells.
  • DAPI nucleus
  • the graph shows the ratio of nuclei to VGLL3 intensity of cytoplasm in myofibroblasts seeded on hydrogel plates with different hardness (n> 100 cells in each group). Intergroup comparisons were calculated by 1-way ANOVA followed by Newman-Keuls analysis; *** P ⁇ 0.001. Scale bar: 20 ⁇ m.
  • FIG. 5 is an alternative photograph showing that the nuclear translocation of VGLL3 is regulated by actin polymerization.
  • FIG. 5A is a schematic diagram of cytoskeleton formation in response to mechanical stimuli such as the hardness of the extracellular environment. Mechanical stimulation induced G-actin polymerization and F-actin formation.
  • FIG. 5B is a representative confocal image of myofibroblasts plated on a soft hydrogel (modulus of elasticity 1 kPa) and a hard hydrogel (modulus of elasticity 50 kPa). The nuclei were stained with DAPI and the cells were stained with Cell Trace to observe the whole picture of the cells. Scale bar: 20 ⁇ m.
  • FIG. 5C shows DMSO (control, 0.5%), F-actin inhibitor latrunculin A (Lat.A, 2 ⁇ M), non-muscle myosin inhibitor blebbistatin (Blebbist., 50 ⁇ M), ROCK inhibitor.
  • Rho is a member of the Ras homologue family of low molecular weight G proteins.
  • the cells were stained with Cell Trace to observe the overall picture of the cells. Scale bar: 20 ⁇ m.
  • the graph shows the ratio of nuclei to VGLL3 intensity in the cytoplasm of myofibroblasts (each group: n> 100 cells). Intergroup comparisons were calculated by 1-way ANOVA followed by Newman-Keuls analysis; *** P ⁇ 0.001.
  • FIG. 6 is an alternative photograph showing that intranuclear translocation of VGLL3 is regulated by activation of focal adhesions.
  • Figures 6A-B show VGLL3 in myocardial fibroblasts treated with DMSO (control, 0.5%), integrin ⁇ 1 inhibitor BTT-3033 (30 ⁇ M) (A), and FAK inhibitor VS-4718 (50 ⁇ M) (B). And is a typical confocal immunocytochemical image of the nucleus (DAPI). The cells were stained with Cell Trace to observe the overall picture of the cells. Scale bar: 20 ⁇ m. The graph shows the ratio of nuclei to VGLL3 intensity in the cytoplasm of myofibroblasts (each group: n> 100 cells). Intergroup comparisons were calculated by unpaired two-sided student's t-test; *** P ⁇ 0.001.
  • FIG. 7 is a graph showing that VGLL3 regulates the expression of fibrosis-related factors in myofibroblasts.
  • FIG. 7A shows the mRNA expression of VGLL3 and fibrosis-related genes (COL1A1, COL1A2, COL3A1) in myocardial fibroblasts transfected with control siRNA (siCtrl) or siRNA targeting VGLL
  • FIG. 8A shows VGLL3 binding protein candidates identified using a mass spectrometer.
  • the horizontal axis of the graph shows the amount of sediment of various proteins, and the vertical axis shows the reliability.
  • FIG. 8B is an alternative photograph showing the interaction of FLAG-VGLL3 and DDX5 in mouse myofibroblasts. FLAG-VGLL3 was immunoprecipitated from cell lysates from mouse myofibroblasts expressed using a retrovirus. DDX5 was detected by anti-DDX5 antibody.
  • FIG. 8C is an alternative photograph showing the in vitro binding assay between recombinant GST-VGLL3 and recombinant His-DDX5. GST-VGLL3 was precipitated with glutathione sepharose beads.
  • FIG. 8D is a schematic representation of wild-type DDX5 and deletion mutants of DDX5.
  • FIG. 8E lysed NIH3T3 cells expressed using the FLAG-VGLL3 and HA-tagged DDX5 wild-type or DDX5 deletion mutant retroviruses shown in FIG. 8D and subjected to immunoprecipitation with anti-FLAG antibody. It is a photograph that replaces the drawing showing the result. Immunoprecipitates and inputs were immunoblotted with anti-HA and anti-FLAG antibodies.
  • FIG. 8E is a schematic representation of wild-type DDX5 and deletion mutants of DDX5.
  • FIG. 8E lysed NIH3T3 cells expressed using the FLAG-VGLL3 and HA-tagged DDX5 wild-type or DDX5 deletion mutant retroviruses shown in FIG. 8D and subjected to immunoprecipitation with anti-FLAG antibody. It is a photograph that replaces the drawing showing the result. Immunoprecipitate
  • FIG. 8F is a schematic diagram of a mutant having only the N-terminal or a mutant having only the C-terminal of VGLL3.
  • FIG. 8G shows the results of overexpressing the N-terminal only mutant or C-terminal only mutant of VGLL3 and HA-DDX5 shown in FIG. 8F in NIH3T3 cells using a retrovirus and subjecting them to immunoprecipitation with an anti-FLAG antibody. It is a photograph that replaces the drawing showing. Immunoprecipitates and inputs were immunoblotted with anti-HA and anti-FLAG antibodies.
  • FIG. 10B is a graph showing the expression level of miR-29b (SEQ ID NO: 66: uagcaccauuugaaaucaguguu) in myocardial fibroblasts isolated from fibrotic hearts transfected with siRNA targeting DDX5.
  • FIG. 10D RNA immunoprecipitation using a DDX5 antibody was performed on myofibroblasts isolated from a fibrotic heart, and a primer set specific for Pri-miR29b-1 or Pri-miR29b-2 was used. It is a photograph instead of a drawing which shows the result of performing PCR. RT is reverse transcriptase, and (-) and (+) indicate its presence or absence.
  • FIG. 12B is a representative image of a unilateral ureteral obstruction (UUO) model-treated mouse kidney in which VGLL3 mRNA was in situ hybridized, myofibroblast marker ⁇ SMA was stained with an antibody against it, and further stained with DAPI. Arrows ( ⁇ ) indicate the respective signals in myofibroblasts expressing VGLL3. The scale bar is 30 ⁇ m.
  • FIG. 12A shows the results of unilateral ureteral obstruction (UUO) model treatment of wild-type (WT) mice, recovery of kidneys 10 days after treatment, and evaluation of VGLL3 mRNA expression level by real-time RT-PCR. It is a graph
  • FIG. 12C is a graph showing the results of unilateral ureteral obstruction (UUO) model treatment of WT and VGLL3KO mice, recovery of the kidney 10 days after the treatment, and evaluation of collagen expression level by real-time RT-PCR.
  • UUO unilateral ureteral obstruction
  • liver damage was caused by administration of CCl4 (carbon tetrachloride) for 4 weeks, and fibrosis was induced by administration of fibrotic liver (CCl4) and CCl4 for 4 weeks, and then 4 weeks had passed since the administration was stopped.
  • CCl4 carbon tetrachloride
  • fibrosis was induced by administration of fibrotic liver (CCl4) and CCl4 for 4 weeks, and then 4 weeks had passed since the administration was stopped.
  • CCl4 fibrotic liver
  • CCl4 fibrotic liver
  • FIG. 13C shows centrifuge hepatocytes (HC) and F4 / 80-positive Kupffer cells (KC) and F4 / 80-negative stars in non-precipitated cells after constant treatment of fibrotic-induced mouse liver by administration of CCL4. It is a graph which shows the result of having measured the mRNA of Cyp7a1, Cd68, and Acta2 which are marker molecules of hepatocyte, Kupffer cell, and activated stellate cell in each cell fraction of a cell (HSC).
  • FIG. 13D is a graph showing the results of measuring the expression level of VGLL3 mRNA in each cell fraction collected in FIG. 13C by real-time RT-PCR.
  • the present invention provides, in one embodiment, a composition comprising an inhibitor of VGLL3, specifically an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5. .
  • the composition includes a pharmaceutical composition or a food composition.
  • the pharmaceutical composition contains an inhibitor of VGLL3, specifically, an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5 as an active ingredient. It is a pharmaceutical composition for preventing or treating.
  • the food composition contains an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5, and assists in the treatment of fibrotic diseases, alleviates or prevents symptoms.
  • Food composition for use is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5, and assists in the treatment of fibrotic diseases, alleviates or prevents symptoms.
  • Food composition for use.
  • VGLL3 whose expression level is increased in the heart at the time of myocardial infarction, is a factor that changes intracellular localization depending on extracellular hardness and regulates fibrosis.
  • the infarction causes cardiomyocyte necrosis.
  • cardiomyocytes are necrotic, myofibroblasts rapidly produce extracellular matrix proteins such as collagen to prevent cardiac rupture. That is, the infarcted region is excessively replaced with extracellular matrix protein, and as a result, fibrosis is strongly induced (Fig. 1A).
  • VGLL3 is cytoplasmic by allowing myofibroblasts to receive mechanical stimuli such as the hardness of the extracellular environment by aggregates called focal adhesion spots and promote cytoskeleton formation by activating Rho / ROCK. It was clarified that it shifts from to into the nucleus. We also found that knockdown of VGLL3 reduced the expression of extracellular matrix proteins such as collagen, indicating that VGLL3 is a factor that promotes fibrosis. According to the present invention, it is possible to provide various techniques for preventing or treating fibrotic diseases related to inhibitors of VGLL3.
  • VGLL3 is a molecule similar to the gene Vgl involved in the formation of Drosophila wings, and is one of the four VGLL (Vestigial-like) family proteins in mammals (Williams et al., 1991, Genes Dev). . 5: 2481-95). VGLL family proteins are known to bind to the transcription factor TEAD (TEA domain family member). As for the function of VGLL3, it has been reported that the difference in the expression of VGLL3 between men and women may determine the difference in the incidence of autoimmune diseases (JCI Insight.2019 Apr 18; 4 (8). Pii: 127291.
  • TEAD and YAP / TAZ are known to promote fibrosis in myofibroblasts.
  • TEAD and YAP / TAZ are expressed not only in myofibroblasts but also in cells of various tissues during normal times, and are closely involved in cell functions during normal times. This is considered to be an obstacle to targeting TEAD and YAP / TAZ for drug discovery.
  • the protein expression distribution of VGLL3 found by the present invention in each human organ was investigated from The Human Protein ATLAS (http://www.proteinatlas.org/).
  • VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in vivo (Fig. 1K). This suggests that VGLL3 may be a target molecule for the development of innovative fibrotic therapies.
  • VGLL3 is described as an example of a classification marker for prostate cancer in Special Table 2015-501151, there is no specific description regarding myofibroblasts and fibrotic diseases.
  • VGLL is described as a transcription factor in Japanese Patent Application Laid-Open No. 2011-019520, there is no specific description regarding myofibroblasts and fibrotic diseases.
  • VGLL3 includes human VGLL3 and mouse VGLL3. It also includes those variants.
  • the amino acid and nucleic acid sequences of variants 1, variant 2 and variant 3 of human VGLL3 are shown in SEQ ID NOs: 1 and 2 of the Sequence Listing for variant 1, SEQ ID NOs: 3 and 4 of the sequence listing for variant 2, and for variant 3, respectively. It is shown in SEQ ID NOs: 5 and 6 of the sequence listing.
  • the amino acid and nucleic acid sequences of Variants 1, Variant 2 and Variant 3 of mouse VGLL3 are shown in SEQ ID NOs: 7 and 8 of the Sequence Listing for Variant 1, SEQ ID NOs: 9 and 10 of the Sequence Listing for Variant 2, and for Variant 3, respectively.
  • VGLL3 is specifically expressed in myofibroblasts and is associated with fibrosis-related factors such as ⁇ SMA, periostin, COL1A1 (collagen 1 ⁇ 1), COL1A2 (collagen 1 ⁇ 2) and COL3A1 (collagen 3 ⁇ 1). It promotes expression and has the property of translocating to the nucleus when the surroundings become hard. Therefore, inhibitors of VGLL3 can be used for the prevention or treatment of fibrotic diseases.
  • fibrosis refers to a phenomenon in which substances called extracellular matrix such as collagen fibers increase in the skin and internal organs, and as a result, the skin and internal organs become hard, and is also called "hardening".
  • the fibrotic disease in the present invention is due to fibrosis such as heart fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis, systemic scleroderma, skin sclerosis, and intractable cancer such as pancreatic cancer. Means disease.
  • inhibitortion of an “inhibitor” means inhibiting or suppressing VGLL3 expression, VGLL3 activity, nuclear translocation of VGLL3 or binding of VGLL3 to DDX5.
  • examples of the VGLL3 expression inhibitor include substances that inhibit the expression of the gene or nucleic acid shown in any of the following (a) to (d): (a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing, (b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
  • the gene or nucleic acid in the embodiment of inhibiting the expression of a gene or nucleic acid, is a myofibroblast-specific marker molecule and a molecule that promotes fibrosis.
  • the VGLL3 gene represented by the nucleotide sequence of SEQ ID NO: 2, 4 or 6 discovered by the present inventors, or the nucleotide sequence shown in SEQ ID NO: 2, 4 or 6 of the sequence listing. Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
  • nucleic acid that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid containing the base sequence shown in SEQ ID NO: 2, 4 or 6 of the sequence listing, according to SEQ ID NO: 1, 3 or 5.
  • Any nucleic acid may be used as long as it has a protein represented by an amino acid sequence or a nucleic acid having a base sequence encoding a protein having the same function as the protein.
  • it is the VGLL3 gene represented by SEQ ID NO: 2, 4 or 6 in the sequence listing.
  • one or several may be, for example, a range in which the gene of the above (b) encodes a substance having a VGLL3 function.
  • “1 or several” is, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 1 or 2 in the VGLL3 gene. is there.
  • identity is synonymous with homology or similarity.
  • “90% or more” is preferably 93% or more, more preferably 95% or more, still more preferably 98% or more.
  • the "stringent condition” may be, for example, any of a low stringent condition, a medium stringent condition, and a high stringent condition.
  • “Low stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C.
  • “Medium stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C.
  • “High stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C.
  • the degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time.
  • “Stringent conditions” are, for example, "Molecular Cloning: A Laboratory Manual 2nd Ed.” Edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989). )] Etc. can also be adopted.
  • the mRNA may be any mRNA encoded by the target nucleic acid or any one encoding a protein encoded by the target nucleic acid. It is preferably an mRNA encoded by the VGLL3 gene.
  • examples of the inhibitor of VGLL3 expression include siRNA, shRNA, miRNA, antisense, ribozyme, and compounds.
  • SiRNA small interfering RNA
  • RISC RNA-induced silencing complex
  • sense strand and antisense strand oligonucleotides are synthesized by a DNA / RNA automatic synthesizer, respectively, and denatured in an appropriate annealing buffer at 90 to 95 ° C. for about 1 minute, and then 30 to 70 ° C. It can be prepared by annealing in 1 to 8 hours.
  • ShRNA short hairpin RNA
  • the shRNA may be introduced into cells by a vector and expressed by the U6 promoter or the H1 promoter, or an oligonucleotide having the shRNA sequence may be synthesized by a DNA / RNA automatic synthesizer and self-annealed by the same method as siRNA. May be prepared by.
  • the hairpin structure of the shRNA introduced into the cell is cleaved into siRNA and binds to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that has a sequence complementary to siRNA. As a result, gene expression is suppressed in a sequence-specific manner.
  • RISC RNA-induced silencing complex
  • MiRNA is a functional nucleic acid that is encoded on the genome and finally becomes a microRNA of about 20 bases through a multi-step production process. miRNAs are classified as functional ncRNAs (non-coding RNAs, a general term for RNAs that are not translated into proteins), and play an important role in life phenomena by regulating the expression of other genes. There is. The expression of VGLL3 can be inhibited by administering a miRNA having a specific base sequence to a living body.
  • the ribozyme of the present invention means an RNA molecule that specifically cleaves another single-stranded RNA molecule by a mechanism similar to that of a DNA-restricted endonuclease.
  • Antisense nucleic acid is a nucleic acid complementary to the target sequence. Antisense nucleic acids inhibit transcription initiation by triple-strand formation, transcription inhibition by hybrid formation with a site where an open loop structure is locally formed by RNA polymerase, and transcription inhibition by hybrid formation with RNA whose synthesis is progressing.
  • the siRNA of the present invention refers to a double-stranded RNA that suppresses the expression of a target nucleic acid, and means "RNAi agent”, “short-chain interfering RNA”, “short-chain interfering nucleic acid”, “siRNA”, and is sequence-specific.
  • RNAi agent means "short-chain interfering RNA”, “short-chain interfering nucleic acid”, “siRNA”, and is sequence-specific.
  • a nucleic acid molecule capable of inhibiting or downwardly regulating gene expression or viral replication through RNA interference (RNAi) or gene silencing. It may consist only of RNA or it may be a fusion of DNA and RNA.
  • SiRNAs, shRNAs, miRNAs, ribozymes and antisense nucleic acids may contain various chemical modifications to improve stability and activity.
  • the phosphate residue may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate phosphorodithionate
  • at least a part thereof may be composed of nucleic acid analogs such as peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • siRNA, shRNA, miRNA, ribozyme and antisense nucleic acid of the present invention inhibit the expression of the target nucleic acid (VGLL3 gene), they prevent fibrosis inhibitors and thus fibrotic diseases as substances that regulate the expression of the target nucleic acid.
  • VGLL3 gene target nucleic acid
  • examples of such siRNA include siRNA having an RNA sequence shown in any of SEQ ID NOs: 40 to 51 in the sequence listing.
  • the nucleic acid having the antisense sequence of the present invention inhibits the expression of the target nucleic acid (VGLL3 gene), as a substance that regulates the expression of the target nucleic acid, a fibrosis inhibitor, and thus a pharmaceutical composition for preventing or treating a fibrotic disease. It can be used as a thing.
  • the compound or the like as an inhibitor of VGLL3 expression may be a compound or the like that inhibits or suppresses the expression of the VGLL3 gene, or may be a compound or the like that inhibits or suppresses the expression of the VGLL3 protein. However, it may be a compound or the like that inhibits or suppresses the activity of VGLL3.
  • the compounds include VGLL3 transcription factor inhibitors, VGLL3 protein translation factor inhibitors, VGLL3 proteolysis promoters, and specific binding substances for VGLL3 proteins (antibodies, antibody fragments, peptides, aptamers, low molecular weight compounds, etc.). ..
  • an antibody can be produced by immunizing an animal such as a mouse with the VGLL3 protein or a fragment thereof as an antigen. It may be a commercially available product. The antibody is preferably a monoclonal antibody.
  • the inhibitor of VGLL3 may be, for example, a substance that suppresses the expression of VGLL3 at the mRNA level or the protein level, an antagonist against VGLL3, or the like, or a substance that inhibits the activity of VGLL3. It may be.
  • antagonists to VGLL3 include substances that do not activate VGLL3 and substances that inhibit the binding of ligands to VGLL3 among specific binding substances to VGLL3.
  • the VGLL3 inhibitor in the present invention can broadly include a substance that inhibits the VGLL3-mediated fibrosis pathway and, as a result, suppresses fibrosis, and in that sense, as an inhibitor of VGLL3, G-actin Latrinclin A, which inhibits polymerization, blevisstatin, which inhibits the association of non-muscle myosin II, which is a constituent of the cytoskeleton, and actin, and kinase, which controls the phosphorylation of the myosin light chain required for cytoskeletal movement.
  • Y27632 an inhibitor of ROCK, C3 transferase (39), which inhibits the low molecular weight G protein Rho that activates ROCK, BTT-303343, an inhibitor of integrin ⁇ 1, and FAK (focal adhesion group kinase).
  • C3 transferase 39
  • FAK finacal adhesion group kinase
  • the pharmaceutical composition of the present invention contains an inhibitor of VGLL3 translocation into the nucleus.
  • VGLL3 like the transcriptional conjugation factor YAP / TAZ, migrates from extranuclear to intranuclear by mechanical stimulation, which is dependent on actin polymerization and contributes to excessive fibrosis. doing. Therefore, it is expected that inhibition of nuclear translocation of VGLL3 can inhibit, suppress, or reduce fibrosis.
  • the pharmaceutical composition of the present invention contains an inhibitor of the binding of VGLL3 and DDX5.
  • VGLL3 translocates via Rho / ROCK activity and contributes to excessive fibrosis. Therefore, it is expected that inhibition of the binding between VGLL3 and DDX5 can inhibit, suppress, and reduce fibrosis.
  • Specific examples of the inhibitor of the binding between VGLL3 and DDX5 include a peptide corresponding to the binding domain of VGLL3 with DDX5 and an inhibitory peptide corresponding to the binding domain of DDX5 with VGLL3.
  • treatment refers to (1) delaying the onset of fibrotic disease or fibrotic state; (2) slowing or stopping the progression, exacerbation or exacerbation of symptoms of fibrotic disease or fibrotic state. (3) Brings remission of the symptoms of fibrotic disease or fibrotic state; or (4) means a method or process aimed at curing fibrotic disease or fibrotic state. Treatment may be given as a precautionary measure before the onset of the disease or condition, or treatment may be given after the onset of the disease.
  • prevention means preventing the onset of fibrotic disease or fibrotic state.
  • the pharmaceutical composition usually means a drug for treating or preventing a disease, or for testing / diagnosis.
  • the pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art.
  • it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injectable suspension.
  • pharmacologically acceptable carriers or vehicles specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavors, excipients, vehicles, preservatives.
  • the sterile composition for injection can be formulated according to the usual preparation using a vehicle such as distilled water for injection.
  • aqueous solution for injection examples include isotonic solutions containing physiological saline, glucose and other auxiliary agents (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride).
  • auxiliary agents eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride.
  • Appropriate solubilizing agents such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) may be used in combination.
  • oily liquid examples include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent. It may also be blended with a buffer (eg, phosphate buffer and sodium acetate buffer), a soothing agent (eg, procaine hydrochloride), a stabilizer (eg, benzyl alcohol and phenol), and an antioxidant.
  • a buffer eg, phosphate buffer and sodium acetate buffer
  • a soothing agent eg, procaine hydrochloride
  • a stabilizer eg, benzyl alcohol and phenol
  • antioxidant e.g, benzyl alcohol and phenol
  • the pharmaceutical composition of the present invention is preferably administered by parenteral administration.
  • parenteral administration can be an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type composition.
  • it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
  • the administration method can be appropriately selected depending on the patient's age and symptoms.
  • the dose of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose may be 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values.
  • the dose and administration method vary depending on the weight, age, symptom and the like of the patient, but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.
  • the present invention in another aspect, relates to a method for preventing or treating a fibrotic disease, comprising administering an inhibitor of VGLL3 to a patient in need of such treatment or prevention.
  • the inhibitor of VGLL3 is a method for preventing or treating fibrotic disease, which is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5. ..
  • the present invention relates to, in another aspect, an inhibitor of VGLL3 for preventing or treating fibrotic diseases.
  • the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of the binding of VGLL3 to DDX5.
  • the invention relates to the use of an inhibitor of VGLL3 to produce a medicament for preventing or treating fibrotic disease.
  • the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5.
  • the VGLL3 inhibitor includes an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3 and an inhibitor of binding between VGLL3 and DDX5, and specific compounds, genes or nucleic acids thereof. Is included.
  • the present invention as another embodiment, (a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing, (b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
  • At least one nucleic acid molecule selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme of the present invention inhibits the expression of the target nucleic acid (VGLL3 gene), thus regulating the expression of the target nucleic acid.
  • VGLL3 gene target nucleic acid
  • it can be used as a fibrosis inhibitor, and thus as a pharmaceutical composition for preventing or treating a fibrosis disease.
  • the nucleic acid molecule of the present invention is expressed in a vector.
  • gene therapy often refers to treatment for a genetic disorder, but in the present invention, in order to prevent or treat a fibrotic disease or suppress the progression of the fibrotic disease. Means the treatment of.
  • gene therapy may include in vivo copy insertion of a gene into the cell of a patient with a fibrotic disease. Gene therapy may also include stopping the gene. Genetic recombination may also be used in exobibo gene therapy.
  • human stem cells, immune cells or cancer cells may be genetically modified for a variety of uses. Cells are modified to induce differentiation, transdifferentiation or reprogramming. The cells may also be modified to serve as vehicles that deliver therapeutic proteins.
  • the present invention further provides a cell containing the vector expressing the nucleic acid molecule of the present invention.
  • the cells of the present invention can be used for cell therapy for preventing or treating fibrotic diseases or suppressing the progression of fibrotic diseases.
  • the cells of the invention are myofibroblasts from the heart, lung, kidney, liver or intestine containing the vectors of the invention.
  • Example 10 of the present specification demonstrates the attenuation of cardiac fibrosis after MI in VGLL3 knockout mice, and
  • Example 11 demonstrates the attenuation of renal fibrosis after unilateral ureteral obstruction in VGLL3 knockout mice. The effect of inhibiting the expression of is confirmed.
  • the present invention provides, in another embodiment, a method of screening for candidate substances for inhibitors of VGLL3. More specifically, the step of measuring the expression level of VGLL3 in cells in the presence of the test substance, and the case where the expression level is lower than the expression level in the absence of the test substance.
  • the present invention provides a method for screening a candidate substance for a VGLL3 inhibitor, which comprises a step of determining that the test substance is a candidate substance having a VGLL3 inhibitory action. Further, as described above or described later in Examples, since an inhibitor of VGLL3 is effective in preventing or treating fibrotic diseases, a screening method for candidate substances effective in preventing or treating fibrotic diseases is provided.
  • a method for screening a candidate substance effective for prevention or treatment of fibrotic disease which comprises a step of determining that the test substance is an effective candidate substance for prevention or treatment of fibrotic disease.
  • the cells are not limited as long as they are cultured cells.
  • the cells are preferably myofibroblasts, human myofibroblasts, and more preferably heart, lung, kidney, liver or intestinal myofibroblasts.
  • the cell passage conditions can be appropriately selected depending on the cell type.
  • the test substance is a substance that can be evaluated as to whether or not it is a candidate substance having an inhibitory effect on VGLL3, and is not particularly limited.
  • compounds, proteins, peptides, nucleic acids, lipids, sugars, glycolipids, glycoproteins, metals and the like can be mentioned.
  • the method of administering the test substance is also not particularly limited.
  • the measured value that reflects the expression level of VGLL3 is not particularly limited.
  • it may be a measured value of the amount of mRNA expressed from the VGLL3 gene (number of copies, number of reads, etc.), a measured value of a protein, and chemiluminescence intensity by a reporter assay.
  • the measured value of mRNA can be obtained by using a known method such as microarray, quantitative RT-PCR method, or RNA-Seq method.
  • the measured value of protein can be measured by protein chip, ELISA method, Western blotting method, etc.
  • reporter assay activation or inactivation of the transcriptional regulatory region can be detected by a reporter assay.
  • the reporter include unstable spiny oyster shrimp luciferase, firefly luciferase, sea urchin shiitake mushroom, GFP (Green Fluorescent Protein), ⁇ -galactosidase and the like.
  • the reporter assay can be performed according to a known method. When performing a reporter assay, for example, a reporter plasmid in which a transcriptional regulatory region of the VGLL3 gene is inserted is constructed upstream of the reporter gene, this reporter plasmid is introduced into the cell, and a reporter assay cell carrying the reporter plasmid is carried out. To make.
  • the reporter plasmid can be introduced into cells by lipofection, electroporation, calcium phosphate method or the like.
  • the introduction of the reporter plasmid into the cells may be transient or stable.
  • the reporter gene When the reporter gene is luciferase, a measured value that reflects the expression of the VGLL3 gene can be obtained by measuring the chemiluminescence from the substrate by luciferase.
  • the reporter gene When the reporter gene is GFP, a measured value that reflects the expression of the VGLL3 gene can be obtained by measuring the fluorescence intensity.
  • the reporter gene When the reporter gene is ⁇ -galactosidase, a measured value reflecting the expression of the VGLL3 gene can be obtained by measuring the fluorescence intensity derived from the substrate fluorescein di- ⁇ -D-glucopyranoside.
  • the reporter assay is preferably a luciferase assay using luciferase.
  • Inhibitors of VGLL3 are effective preventive or therapeutic agents for fibrotic diseases.
  • the VGLL3 protein having the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9 or 11 of the Sequence Listing, or SEQ ID NO: 1, 3, 5, 7, of the Sequence Listing, A mutant VGLL3 protein having the substitution, deletion, or addition of one or several amino acid residues in the VGLL3 protein having the amino acid sequence shown in 9 or 11, and having the same activity as the VGLL3 protein.
  • a method for screening a substance that inhibits the function of VGLL3 protein using a protein more specifically, (1) Substitution, deletion, or addition of one or several amino acid residues in the VGLL3 protein having the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 in the sequence listing, or in the VGLL3 protein.
  • the test substance is brought into contact with the mutant VGLL3 protein having the same activity as the VGLL3 protein, or the VGLL3 protein or cells expressing the mutant VGLL3 protein.
  • a method for screening a substance that inhibits the function of the VGLL3 protein which comprises determining whether or not the test substance inhibits the function of the VGLL3 protein or the mutant VGLL3 protein.
  • SEQ ID NOs: 1, 3 and 5 in the Sequence Listing are amino acid sequences of human VGLL3 protein, and SEQ ID NOs: 7, 9 and 11 are amino acid sequences of mouse VGLL3 protein.
  • both human VGLL3 protein and mouse VGLL3 protein can be used.
  • it is a human VGLL3 protein.
  • the marker protein By fusing the target protein with the marker protein in frame and expressing it, it is possible to confirm the expression, confirm the intracellular localization, purify, etc.
  • the marker protein include FLAG epitope, hexahistidine tag (Hexa-Histidine tag), hemagglutinin tag, myc epitope and the like.
  • a protease such as enterokinase, factor Xa, and thrombin
  • the screening method of the present invention quantifies the step of culturing myofibroblasts in the presence of a test substance and the expression level of VGLL3 gene mRNA or VGLL3 protein in the cultured myofibroblasts.
  • the test substance is used for the prevention or treatment of fibrotic disease when the step and the quantified expression level of the VGLL3 gene mRNA or the VGLL3 protein are reduced as compared with the expression level in the absence of the test substance.
  • a method for screening a prophylactic or therapeutic agent for a fibrotic disease which comprises a step of determining that the agent is an agent.
  • Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
  • Another aspect of the present invention is a method for determining the degree of fibrosis.
  • the reference amount of VGLL3 in myofibroblasts may be the amount of VGLL3 in myofibroblasts of the same subject measured prior to step (a10), and VGLL3 in healthy subjects. May be the amount of.
  • the present invention provides, in yet another aspect, a biomarker that is VGLL3 of myofibroblasts, which can determine the degree of fibrosis progression.
  • the present invention provides the use of myofibroblast VGLL3 as a biomarker capable of determining the degree of fibrosis progression.
  • VGLL3 is a myofibroblast-specific marker molecule and a molecule that promotes fibrosis, which is associated with fibrotic diseases, especially severe fibrotic diseases. It was.
  • the use of the methods, biomarkers and VGLL3 of the present invention makes it possible to predict the progression of fibrosis and is beneficial for the prevention or treatment of fibrotic diseases.
  • substances called extracellular matrix such as collagen fibers are highly increased in the skin and internal organs, and as a result, the skin and internal organs become hard, which leads to a risk of fibrotic disease. ..
  • the amount of VGLL3 in myofibroblasts may be the amount of mRNA or the amount of protein. If there is an antibody against VGLL3, it can be measured by immunological methods. For example, it can be measured by an ELISA method well known to those skilled in the art.
  • the detection of mRNA for VGLL3 can be performed, for example, by the RNA scoop method, which is a kind of super-sensitive in situ hybridization. In the RNAscope method, if the mRNA of the target molecule is expressed, it can be detected in dots by using a probe specific to that molecule. RNA can also be recovered from myofibroblasts and measured by real-time RT-PCR.
  • the myofibroblasts of the subject can be collected by biopsy and the amount of VGLL3 can be measured.
  • Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
  • the present invention comprises a primer set for amplifying VGLL3 cDNA, a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding agent for VGLL3 protein for detection of myofibroblasts.
  • a primer set for amplifying VGLL3 cDNA a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding agent for VGLL3 protein for detection of myofibroblasts.
  • VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in the living body, that is, hardly expressed in the heart, liver and kidney of the living body in the normal state. , It was clarified that the expression is significantly increased only when each organ is fibrotic. We also found that VGLL3 expression is specific for myofibroblasts that perform fibrosis. Therefore, VGLL3 is a novel myofibroblast-specific marker protein.
  • the primer set is not particularly limited as long as it can amplify the cDNA of the VGLL3 gene of the animal species to be detected.
  • the probe that specifically hybridizes to VGLL3 mRNA is not particularly limited as long as it specifically hybridizes to VGLL3 gene mRNA.
  • the probe may be immobilized on a carrier to form a DNA microarray or the like.
  • the specific binding substance is the same as that described above.
  • the specific binding substance may be immobilized on a carrier to form a protein chip or the like.
  • proteins such as ⁇ -SMA and periostin have been used as markers for myofibroblasts, which are cells that execute fibrosis. However, these were all intracellular proteins. That is, conventionally, a cell membrane protein specifically expressed in myofibroblasts has not been known.
  • VGLL3 is a cell membrane protein that is not expressed in the originating cell but whose expression increases when it differentiates into myofibroblasts. Since it is a cell membrane protein, for example, by labeling an antibody specific for VGLL3 with a fluorescent or radioisotope, myofibroblasts can be visualized invasively or non-invasively.
  • Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
  • VGLL3 is a molecule that promotes fibrosis and is associated with fibrotic diseases, especially severe fibrotic diseases. Since VGLL3 is not expressed in normal cells other than placenta, it is considered possible to prevent, prevent, and avoid fibrosis by knocking out its expression.
  • the present invention relates to cells, organs and / or tissues carrying a disrupted gene of VGLL3. For example, disruption is expected to be a gene that exhibits at least about 50%, 60%, 70%, 80%, 90%, 99% or 100% homology (at the nucleic acid or protein level). Gene suppression is also possible.
  • gene expression can be reduced by knockout, promoter modification of the gene, and / or administration of interfering RNA (knockdown).
  • knockdown it is preferable to knock out VGLL3 expression in myofibroblasts.
  • Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
  • One or more genes in non-human animals can be knocked out using any method known in the art. For example, it involves deleting the gene for VGLL3 from the genome of a non-human animal. Knockout can also include removing all or part of the VGLL3 gene sequence from non-human animals. Knockout can be performed in any cell, organ and / or tissue in a non-human animal. For example, the knockout can be a whole body knockout. For example, the expression of the VGLL3 gene is reduced in all cells of non-human animals. Knockouts can also be specific for one or more cells, tissues and / or organs of non-human animals.
  • Knockout technology can also include gene editing.
  • gene editing can be performed using nucleases that include CRISPR-related proteins (Cas proteins such as Cas9), zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and meganucleases.
  • the nuclease can be a naturally occurring nuclease, genetically modified, and / or recombinant.
  • the CRISPR / cas system may be suitable as a gene editing system.
  • the reagents used in this example were purchased from the following companies. Somnopentil (Kyoritsu Seiyaku Co., Ltd.) 10% Neutral Buffered Formalin Solution (Wako Pure Chemical Industries, Ltd.) Tissue-Tek TM OCTTM Compound (Sakura Finetek) FluorSave TM Reagent (Calbiochem) Red Blood Cell Lysis Buffer (Roche) Type II collagenase (Worthington Biochemical) Elastase (Worthington Biochemical) Isogen (Nippon Gene) RNeasy Plus Mini Kit (Qiagen) High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) Luna Universal qPCR Master Mix (New England BioLabs) Paraformaldehyde (nacalai tesque) Trypsin (Sigma-Aldrich) EDTA (Wako Pure Chemical Industries, Ltd.) Dulbecco's Modified Eagle's Medium (Gibco) Fetal bovine serum (Tissue Culture
  • Example 1 Search for transcription-related factors involved in fibrosis 1-1 Preparation of experimental animals and myocardial infarction model mice 8-10 week old C57BL / 6J wild male mice were purchased from Nippon SLC Co., Ltd. Mice were weighed and anesthetized with Somnopentyl injection (50 mg / kg pentobarbital sodium) intraperitoneally and then fixed on the operating table in the supine position. The neck and chest were shaved and an incision was made in the midline of the neck under surgical microscopy to expose the trachea.
  • Somnopentyl injection 50 mg / kg pentobarbital sodium
  • a cannula was inserted into the trachea, and artificial respiration was performed with a single respiration volume of 0.5 cc and a respiration rate of 120 times / minute.
  • the heart was then exposed by an incision in the second intercostal space on the left side of the ribs.
  • Myocardial infarction was performed by ligating the anterior descending branch of the left coronary artery of the heart with a 6 mm silk blade suture. Then, the incision was sutured with a suture.
  • Mice that underwent the above treatment were included in the myocardial infarction (MI) group, and mice that underwent the same treatment except for ligation of the coronary arteries were included in the pseudo-treatment (sham) group (Fig. 1A).
  • DNA synthase is activated at 95 ° C for 60 seconds according to the protocol of Luna Universal qPCR Master Mix (New England BioLabs), and cDNA denaturation at 95 ° C for 15 seconds and elongation reaction at 60 ° C for 60 seconds are performed. 45 cycles were performed. Analysis was performed using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or 18S rRNA as an internal standard.
  • primers and TaqMan probe were purchased from Sigma-Aldrich.
  • the sequence of primers and probes is as follows; Fw: Forward, Rv: Reverse: Mouse ⁇ -smooth muscle actin ( ⁇ SMA, mouse ACTA2) Fw: 5'-GTTCTCTTCAAGGGACAAGGCTG -3'(SEQ ID NO: 13) Rv: 5'-TCCTGGTATGAGATAGCAAATCGG -3'(SEQ ID NO: 14) Probe: 5'-TACGTGCTCCTCACCCACACCGTCA -3' (SEQ ID NO: 15)
  • Human type III collagen, alpha 1 (Collagen 3 ⁇ 1, human COL3A1)
  • Fw 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 31)
  • Rv 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 32)
  • GPDH Mouse glyceraldehyde-3-phosphate dehydrogenase
  • Fw 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 34)
  • Rv 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 35)
  • Probe 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 36)
  • 18S ribosomal RNA (18S rRNA, eukaryote 18S rRNA) Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 37) Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 38) Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 39)
  • VGLL3 with unknown function was found by sorting based on (Fig. 1B).
  • Myofibroblasts isolated from mouse hearts after myocardial infarction model treatment were cultured on cultured polystyrene plates, transferred to low-protein adsorption-treated plates, and cultured in suspension for 7 days.
  • SMA and periostin SMA and periostin
  • the expression of myofibroblast marker proteins such as FIG. 1D, E, F
  • the expression of the stem cell marker Oct-4 Fig. 1G
  • FIG. 1G the expression of the stem cell marker Oct-4
  • DNA microarray analysis was used to select genes that were significantly down-regulated (less than 1/16) in dedifferentiated myofibroblasts and significantly increased (> 16-fold) with redifferentiation (FIG. 1J).
  • genes whose expression levels were significantly increased in mouse hearts (2 to the 1.5th power or higher) by treatment for inducing myocardial infarction were further selected.
  • VGLL3 a molecule involved in transcription.
  • VGLL3 Expression distribution of VGLL3 found by search
  • the protein expression distribution of VGLL3 found by search in each human organ was investigated from The Human Protein ATLAS (http://www.proteinatlas.org/). ..
  • VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in vivo (Fig. 1K). This suggests that VGLL3 may be a target molecule for the development of innovative fibrotic therapies.
  • VGLL3 Changes in VGLL3 expression level in response to mechanical stimulation VGLL3 is similar to myofibroblast marker proteins SMA and periostin (Fig. 1D, E, F), myofibroblasts and redifferentiated myofibroblasts. It was investigated whether it was expressed in. As a result, the mRNA level of VGLL3 was reduced in myofibroblasts dedifferentiated by the disappearance of mechanical stimulation after being suspended and cultured for 7 days, and the cells were cultured again on a polystyrene plate and redifferentiated by mechanical stimulation. The expression level of myofibroblasts was significantly increased (Fig. 1L). This indicates that the expression level of VGLL3 is increased by mechanical stimulation.
  • VGLL1 5'-CTCTTCTTGGACAATTGCACAGCCAGCAGC-3' (SEQ ID NO: 52) and 5'-CTTTGGAGATGGGCTTCTTTCATCATCAAG-3' (SEQ ID NO: 53);
  • VGLL2 5'-CAAAGCACACAGAAGCTCTGGACCCTGGAG-3' (SEQ ID NO: 54) and 5'-CTGCATTCCCTCCTCCGCTTACCTGAGTCC-3' (SEQ ID NO: 55);
  • VGLL3 5'-GACATTGGGTCAGTAGTGGATGAACACTTC-3'(SEQ ID NO: 56) and 5'-GACT
  • VGLL4 is expressed in addition to VGLL3 in muscle fibroblasts isolated from the heart (Fig. 1M). Based on this result, the expression levels of VGLL3 and VGLL4 in myofibroblasts isolated from the heart were absolutely quantified (Fig. 1N).
  • VGLL3 Fw5'-TGCCCTACAGCCTGCTATCA-3'(SEQ ID NO: 60), Rv5 '-CTCCTCCTCCTCCTCCTCTTG-3' (SEQ ID NO: 61), Probe 5'-CTTGCTGTATACCGCTAACTTCTGCTGGC-3' (SEQ ID NO: 62); (SEQ ID NO: 64), probe 5'-CCTGTACGCATCTCTCCCCAGCCTCAT-3' (SEQ ID NO: 65)) was used to draw a standard curve.
  • CCD-18Co Changes in fibrosis-related factors and VGLL3 expression levels in response to mechanical stimulation in human intestinal myofibroblasts
  • CCD-18Co which is the cell line of human intestinal myofibroblasts instead of heart myofibroblasts Except for using cells (ATCC), the expression levels of ACTA2, various collagens, fibrillin-1 and VGLL3 mRNAs are measured in real time by substantially following the method described in 1-3: Search for transcription-related factors. Measured by (Fig. 1O).
  • Fig. 1O Search for transcription-related factors.
  • Example 2 Specific expression of VGLL3 in myofibroblasts during myocardial infarction
  • VGLL3 Using cardiac sections 3 days after MI treatment, co-staining of an antibody against VGLL3 and an antibody against ⁇ SMA, which is a marker molecule of myofibroblasts, was performed. As a result, it was found that the stained image of VGLL3 almost overlapped with the stained image of ⁇ SMA (Fig. 2A).
  • VGLL3 is specifically expressed in myofibroblasts, not in macrophages or cardiomyocytes, during myocardial infarction.
  • the immunohistochemical staining was performed as follows. The heart removed from the mice after MI treatment was fixed overnight with 4% PFA solution, the PFA solution was replaced with a sucrose solution, and then embedded with Tissue-Tek TM OCTTM Compound (Sakura Finetek) in liquid nitrogen. The frozen sample was used as a frozen sample. Frozen specimens were sliced into 6 ⁇ m with Cryostat CM1100 (Leica Biosystems) and air-dried for 1 hour. Frozen sections were subjected to membrane permeation treatment with 0.1% Triton X-100 / PBS for 5 minutes, blocked in 5% BSA / PBS for 1 hour at room temperature, and diluted with 5% BSA / PBS.
  • RNAscope is a type of highly sensitive in situ hybridization (28). RNAscope followed the protocol of Advanced Cell Diagnostics. First, the heart was removed from the mice treated with myocardial infarction (MI), the atrium was removed, and the mice were immediately immersed in 10% neutral buffered formalin solution and fixed at room temperature for 16 to 32 hours. Then, it was embedded in paraffin to prepare a tissue section of 5 ⁇ m. The staining of heart specimens of human myocardial infarction patients was conducted as a joint research with the cooperation of Professor Toru Tanaka of Jichi Medical University.
  • the tissue section was incubated at 60 ° C. for 1 hour, and then paraffin was removed using xylene and 100% ethanol. Then, a hydrogen peroxide solution was added dropwise to the tissue section and reacted at room temperature for 10 minutes to inactivate the endogenous peroxidase. After washing with distilled water, an antigen activation solution is further added dropwise, reacted at 98 to 102 ° C. for 30 minutes using a hot plate, washed with distilled water, then the protease solution is added dropwise, and the HybEZTM oven (Advanced Cell Diagnostics) is used. ) The reaction was carried out at 40 ° C. for 30 minutes.
  • a probe for VGLL3 was added dropwise to the tissue section, and the reaction was carried out in a HybEZTM oven at 40 ° C. for 2 hours, and then a signal amplification reaction was carried out with an Amp solution. Subsequently, the reaction was carried out with a Fast Red mixture or a fluorescently labeled Tyramide to detect a signal of VGLL3 mRNA.
  • 10% BSA / PBS was reacted at room temperature for 1 hour (blocking), and the primary antibody was reacted at 4 ° C. overnight.
  • the fluorescently labeled secondary antibody was reacted at room temperature for 1 hour, stained with DAPI nuclei, and then incubated at 60 ° C. for at least 15 minutes and dried.
  • the sections enclosed with EcoMount were imaged with a confocal microscope (Zeiss, LSM700).
  • VGLL3 mRNA detection of VGLL3 and immunostaining with ⁇ SMA antibody were performed on the same section.
  • co-detection of periostin mRNA and VGLL3 mRNA, which are famous as marker molecules for myofibroblasts other than ⁇ SMA was also performed.
  • periostin mRNA and VGLL3 mRNA which are famous as marker molecules for myofibroblasts other than ⁇ SMA
  • VGLL3 mRNA was found in both ⁇ SMA-positive and periostin-positive myofibroblasts present in the infarcted region (Fig. 2D, 2E).
  • VGLL3 mRNA expression was scarcely observed in the sham-treated (sham) group of hearts (Fig. 2F).
  • VGLL3 is hardly expressed in the normal heart, but is specifically expressed in myofibroblasts that appear during myocardial infarction. Furthermore, in the heart specimens of human myocardial infarction patients, VGLL3 mRNA signals were also observed in ⁇ SMA-positive myofibroblasts (Fig. 2G).
  • Example 3 Mechanical stimulus-dependent cytoplasmic to nuclear transfer ability in VGLL3
  • the infarcted area is 3 compared to the non-infarcted area due to the excessive accumulation of extracellular matrix such as collagen in the infarcted area of the heart that has undergone myocardial infarction. It has been reported to be twice to seven times as hard.
  • transcription-coupled factors such as MRTF (myocardin-related transcription factor) and YAP1 (Yes-associated protein 1) migrate from the cytoplasm into the nucleus in response to changes in pericellular hardness (mechanical stimulation) and are downstream. By regulating the activity of transcription factors, it promotes the expression of fibrosis-related factors such as collagen and CTGF (connective tissue growth factor) (Non-Patent Document 9).
  • VGLL3 is specifically expressed in myofibroblasts that appear in the fibrotic region of the infarcted region, and the expression transition of VGLL3 in the mouse heart after myocardial infarction is very similar to that of fibrosis-related factors. (Figs. 1 and 2). From these facts, like MRTF and YAP1, VGLL3 senses the hardness of the infarcted area, that is, the change in extracellular mechanical stimulus, transfers from the cytoplasm to the nucleus, and regulates the expression of fibrosis-related factors. I thought it might be.
  • Example 3-1 Localization of VGLL3 in the cytoplasm and nucleus First, it was examined in vitro whether the intracellular localization of VGLL3 was changed by mechanical stimulation. Myofibroblasts were isolated from the heart 3 days after MI treatment, and cultured in an adherent state and in a floating state. Isolation of myofibroblasts was performed as follows. On the 3rd day after MI treatment, the heart was removed from the mouse, the atrium was removed, and then the ventricles were divided into 15-20 pieces.
  • the heart pieces were shaken 10 times at 37 ° C for 10 minutes in a type II collagenase solution (0.1% type II collagenase / 0.01% elastase / PBS) to recover the extracellular fluid containing myofibroblasts.
  • a type II collagenase solution (0.1% type II collagenase / 0.01% elastase / PBS)
  • the erythrocyte component was removed by Red Blood Cell Lysis Buffer (Roche), the cells were suspended in 10% FBS / 1% penicillin / streptomycin / DMEM, and then the cells were seeded on a culture plate. After culturing in a CO 2 incubator overnight, non-adhesive cells were removed by PBS washing and trypsin-EDTA-adhered cells were collected.
  • the cells were reacted with a magnetic bead-labeled CD45 antibody for 30 minutes, and CD45-positive blood cells were removed by MACS magnetic cell separation using an MS column, and CD45 (-) cells were recovered.
  • the CD45 (-) cells were reacted with APC-labeled PDGFR ⁇ (myofibroblast membrane surface marker) antibody, then reacted with magnetic bead-labeled anti-APC antibody for 30 minutes, and by MACS magnetic cell separation method. , PDGFR ⁇ (+) myofibroblasts were collected.
  • Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining.
  • Myofibroblasts were seeded on a 35-cm diameter glass bottom dish (IWAKI) coated with poly-L-lysine (Sigma-Aldrich), cultured overnight in a CO 2 incubator, and adhered to the dish. Used as a sample of.
  • myofibroblasts cultured in a low-adsorption culture dish (Sumitomo Bakelite) in a CO 2 incubator for 2 hours in a floating state were used as samples under floating conditions.
  • immune cell staining was performed as follows.
  • Myofibroblasts cultured by the above method were fixed in 1% PFA / PBS for 15 minutes, washed with PBS, added with 6 ⁇ M CellTrace Far Red solution (Thermo Fisher Scientific), and incubated in a CO 2 incubator for 30 minutes. .. After that, membrane permeation treatment was performed using 0.5% saponin / PBS (sample of condition 1) or 0.1% Triton X-100 / PBS (sample of conditions 2 to 4), and 1 in 5% BSA / PBS at room temperature. After blocking for hours, it was allowed to stand overnight at 4 ° C. in a primary antibody solution diluted with 5% BSA / PBS.
  • the image was taken with a confocal microscope (LSM700, Carl Zeiss).
  • the nuclear / cytoplasmic ratio (Nuc / Cyt ratio), which is a parameter of nuclear localization of VGLL3 and YAP1, was calculated from the average brightness of each factor in the nucleus and cytoplasm by image analysis using ImageJ software (NIH). ..
  • VGLL3 was strongly localized in the nucleus in myofibroblasts cultured under the adhesive state, that is, under the condition of mechanical stimulation (Fig. 3A).
  • Fig. 3A myofibroblasts cultured in a floating state, that is, under conditions without mechanical stimulation
  • VGLL3 was strongly localized in the cytoplasm (Fig. 3A).
  • the abundance ratio (Nuc / Cyt ratio) of VGLL3 in the nucleus and cytoplasm was calculated.
  • the value of the Nuc / Cyt ratio approaches 1, and the more it is localized in the nucleus, the larger the value (29-31).
  • the Nuc / Cyt ratio of VGLL3 was significantly increased in the adhered state as compared with the floating state (Fig. 3A). From this, it was considered that VGLL3 was transferred into the nucleus by the adhesive stimulation.
  • myofibroblast YAP1 also migrated to the outside of the nucleus in a floating state and into the nucleus in an adherent state, similar to VGLL3 (Fig. 3B).
  • Example 3-2 Localization of VGLL3 in overexpression system
  • FLAG-labeled VGLL3 using a retrovirus was used. Overexpressed myofibroblasts were stained with anti-FLAG antibody and examined for intracellular localization in the floating and adherent states.
  • Gene transfer into myofibroblasts using a retrovirus is as follows. FLAG-tag-labeled VGLL3 / pMXs-puro or pMXs-puro as a control was gene-transduced into PLAT-E cells, which are retrovirus packaging cells, using X-treme GENE9 (Roche).
  • the cells were cultured for 24 hours and the medium was exchanged. After further culturing for 24 hours, the culture supernatant was centrifuged (1500 ⁇ rpm, 5 min, 4 ° C twice) to remove impurities, and used as a retrovirus solution.
  • a retrovirus solution and 10 ⁇ g / mL polybrene were added to myofibroblasts isolated from the heart 3 days after MI treatment, and gene transfer was performed. After 6 hours, an equal volume of 10% FBS / 1% penicillin / streptomycin / DMEM was added to the retrovirus solution, and the cells were cultured for another 18 hours.
  • the transcription factor VGLL3 changes the intracellular localization depending on the presence or absence of the adhesive state, as in the case of the endogenous case (Fig. 3C).
  • Example 3-3 Changes in localization of VGLL3 over time during adhesion time
  • Myofibroblasts cultured for 2 hours under floating conditions were seeded on a 35-cm diameter glass bottom dish coated with poly-L-lysine. , 30 minutes after seeding and 1 hour, 2 hours, 4 hours cells were used as samples.
  • the immune cell staining described in Example 3-1 was performed in the same manner. It was found that when myofibroblasts cultured in a floating state were reattached to the culture dish, VGLL3 localized in the cytoplasm was transferred into the nucleus (Fig. 3D). When this localization change was examined over time, the intranuclear translocation of VGLL3 increased significantly as the adhesion time increased (Fig. 3D). From the above results, it was clarified that VGLL3 is a factor that translocates from the cytoplasm to the nucleus depending on mechanical stimulation.
  • Example 4 Intranuclear migration of VGLL3, which depends on pericellular hardness In fibrotic organs, tissue becomes stiff due to excessive accumulation of extracellular matrix.
  • the elastic modulus (kPa) is widely used as an index of the hardness of an organ. For example, it has been reported that the elastic modulus of the heart under normal conditions is about 10 kPa, whereas the elastic modulus increases to about 35 to 70 kPa when myocardial infarction occurs (Fig. 4A).
  • the elastic modulus of plastic or glass culture dishes used for cell culture is 2 to 4 ⁇ 10 6 kPa, which is extremely hard compared to living organisms (Fig. 4A). Therefore, in order to clarify the physiological function of VGLL3, it is necessary to analyze the behavior of VGLL3 in an environment with hardness close to that of a living body.
  • myofibroblasts were cultured on a hydrogel plate of 1 kPa to 50 kPa that mimics the hardness of organs during normal or fibrosis, and how the localization of VGLL3 changes was investigated.
  • Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining.
  • Myofibroblasts were seeded on a 35-cm diameter Softview Easy coat hydrogel glass bottom dish (Matrigen) coated with poly-L-lysine, cultured overnight in a CO 2 incubator, and adhered to the dish as a sample. used.
  • the immune cell staining described in Example 3-1 was performed in the same manner.
  • VGLL3 was widely localized in the whole cell in the 1 kPa hide gel plate, whereas it was more localized in the nucleus in the 50 kPa hydrogel (Fig. 4B).
  • Fig. 4B From the quantitative results of the Nuc / Cyt ratio, it was clarified that the intranuclear translocation of VGLL3 is promoted as the elastic modulus of Hydegel increases (Fig. 4B). From the above results, it was clarified that VGLL3 is a factor that changes the intracellular localization even in an environment close to a living body and translocates into the nucleus depending on the hardness around the cell.
  • Example 5 Control by Cytoskeleton Polymerization in Cytoskeleton Translocation of VGLL3
  • Rho a low molecular weight G protein
  • spherical actin Globular, G-actin
  • filamentous actin filamentous, F
  • myofibroblasts were treated with an inhibitor of cytoskeleton formation and the localization of VGLL3 was observed.
  • Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining.
  • Myofibroblasts were seeded on a 35-cm diameter glass bottom dish coated with poly-L-lysine and cultured overnight in a CO 2 incubator. The following inhibitors were then added (inhibitor concentration indicates final concentration): Latrunculin A, Lat. A (2 ⁇ M, abcam), Blebbistatin, Blebbist.
  • Example 3-1 The stimulation time was 24 hours only for VS-4718, and 4 hours for the others.
  • the immune cell staining described in Example 3-1 was performed in the same manner.
  • Latrunculin A (Lat. A), which inhibits the polymerization of G-actin, and Blebbist. (37), which inhibits the association of actin with non-muscle myosin II, which is a constituent molecule of the cytoskeleton.
  • Y27632 (38), which is an inhibitor of ROCK, which is a kinase that controls phosphorylation of the myosin light chain required for cytoskeletal motility, and C3 transfer enzyme, which inhibits the low-molecular-weight G protein Rho that activates ROCK.
  • Example 6 Regulation of VGLL3 translocation into the nucleus by activation of focal adhesion spots
  • the Rho / ROCK pathway that regulates cytoskeleton formation and cell motility is controlled by various upstreams such as tyrosine kinase type receptors, G protein-coupled receptors, and focal adhesion spots. It is known that activity is regulated (40).
  • focal adhesion is an aggregate of adhesion proteins such as integrins, and by binding to extracellular matrix such as collagen and actin filaments, which are the cytoskeleton, the hardness of the extracellular environment and other factors are mechanical. It is thought to play a role in transmitting various stimuli into the cell (41, 42).
  • Example 7 Increased expression of fibrosis-related factors in myofibroblasts by VGLL3 From the results so far, VGLL3 is a molecule that is strongly expressed in myofibroblasts, accepts the hardness of extracellular matrix, and translocates into the nucleus. Became clear. It is known that myofibroblasts are activated by hardening the extracellular matrix during fibrosis, and the fibrotic ability is increased (Non-Patent Document 5). Therefore, in order to clarify whether VGLL3 translocated into the nucleus is involved in the production of fibrosis-related factors in myofibroblasts, siRNA was used to knock down VGLL3 in myofibroblasts, and a real-time RT-PCR method was performed. The mRNA expression level of fibrosis-related factors was measured by.
  • siRNAs used are:
  • siRNA into myofibroblasts was performed according to the protocol of lipofectamine RNAiMAX (invitrogen). Specifically, the optimal amount of siRNA and lipofectamine RNA iMAX when introducing siRNA into myofibroblasts isolated from the heart 3 days after MI treatment, or the human colon-derived myofibroblast line CCD-18co (ATCC). Evaluated and determined the amount of FAM-labeled siRNA introduced using FACS. SiRNA (Ambion) against VGLL3 or Silencer Select Negative Control no. 1 siRNA (Ambion) was introduced into myofibroblasts cultured in 10% FBS / 1% penicillin / streptomycin / DMEM using lipofectamine RNAiMAX. Four hours later, medium exchange (10% FBS / 1% penicillin / streptomycin / DMEM) was performed. 96 hours after siRNA introduction, total RNA was recovered from each cell using RNeasyPlusMiniKit.
  • VGLL3 is a factor that promotes fibrosis by controlling extracellular matrix production from myofibroblasts.
  • VGLL3 is a factor that promotes fibrosis by controlling extracellular matrix production not only in heart but also in mouse myofibroblasts in liver.
  • Example 8 Search for proteins that interact directly with VGLL3 FLAF-VGLL3 was forcibly expressed in muscle fibroblasts isolated from the heart, and immunoprecipitation was performed with an anti-FLAG antibody. The immunoprecipitate was then analyzed using mass spectrometry to identify VGLL3 binding protein candidates (FIG. 8A).
  • a binding protein candidate Tead3, which is a member of the Tead family protein, was discovered (Fig. 8A). It has been reported to interact with VGLL family proteins (Dev Genes Evol (2016) 226: 297-315 DOI 10.1007 / s00427-016-0546-3). Therefore, we investigated the effect of Tead3 on the expression of fibrosis-related genes. However, siRNA treatment of Tead3 did not affect the expression of fibrosis-related genes in myocardial fibroblasts (Fig. 9), which is related to Tead3's VGLL3-mediated fibrosis-related pathway. It suggests not.
  • DDX5 which is known to be involved in RNA editing and transcription of various genes
  • DDX17 which has an amino acid sequence similar to DDX5
  • DDX5A FLAG-VGLL3 was overexpressed in myofibroblasts and the cells were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation experiments showed that VGLL3 forms a complex with DDX5 (Fig. 8B).
  • DDX5 interacts directly with VGLL3.
  • a GST pull-down assay using recombinant GST-VGLL3 and His-DDX5 proteins revealed that DDX5 is a protein that binds directly to VGLL3 (Fig. 8C).
  • the N-terminal only mutant or C-terminal only mutant and HA-DDX5 shown in FIG. 8F were overexpressed in NIH3T3 cells using a retrovirus, and immunoprecipitation was performed with an anti-FLAG antibody. Then, Western blotting was performed with an anti-HA antibody. As a result, it was found that the domain of VGLL3 required for interaction with DDX5 is the C-terminal (124 to 325) (Fig. 8G).
  • Example 9 Regulation of DDX5 on Fibrotic Gene Expression in Myofibroblasts
  • DDX5 was knocked down in myocardial fibroblasts and the expression levels of intracellular collagen, periostin, fibrillin-1 and other fibrous genes were evaluated by real-time RT-PCR, these fibrous genes were evaluated. It was found that the expression level was significantly reduced (Fig. 10A). That is, DDX5 promotes the expression of these genes.
  • miR-29b (SEQ ID NO: 66: uagcaccauuugaaaucaguguu) is known to bind to various collagen mRNAs including Col1a1 and lead to degradation of the mRNA (Van Rooij, E. et al. Dysregulation). of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc. Natl. Acad. Sci. USA, 13027-13032 (2008)), expression of this miR29b in myocardial fibrosis cells knocked down by DDX5 It was examined by real-time RT-PCR (Fig. 10B).
  • RNA immunoprecipitation using DDX5 antibody was performed on myofibroblasts isolated from fibrotic heart, and a primer specific to Pri-miR29b-1 (ABI Mm03306189_pri) or Pri-miR29b-2 (ABI Mm03307196_pri). PCR was performed using the set (Fig. 10D). As a result, it was clarified that DDX5 binds to Pri-miR29b-1 and Pri-miR29b-2. This indicates that DDX5 may bind to Pri-miR29b-1, Pri-miR29b-2 and suppress the production of miR-29b, resulting in an increase in collagen content.
  • Example 10 Attenuation of cardiac fibrosis after MI in VGLL3 knockout mice The contribution of VGLL3 to fibrosis was investigated in vivo.
  • VGLL3 KO mice were established and underwent MI surgery (permanent occlusion of the left anterior descending artery) to induce cardiomyocyte death and subsequent fibrosis by myofibroblasts to replace the injured area.
  • MI surgery permanent occlusion of the left anterior descending artery
  • the infarcted heart was removed from the mouse and divided into an infarcted part and a non-infarcted part.
  • Total RNA was collected from each region and the mRNA expression level of fibrosis-related genes in each region was measured.
  • VGLL3 undergoes unilateral ureteral obstruction (UUO) model treatment in wild-type (WT) mice specifically expressed in fibrotic kidney myofibroblasts, and renal recovery 10 days after treatment is performed in real-time RT.
  • UUO unilateral ureteral obstruction
  • Fig. 12A The expression level of VGLL3 mRNA was evaluated by PCR (Fig. 12A). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the kidney by fibrosis.
  • unilateral ureteral obstruction (UUO) model treatment was performed, and immunostaining with ⁇ SMA antibody and in situ hybridization to VGLL3 were simultaneously performed on wild-type mouse kidney sections in which fibrosis was induced (Fig. 12B). As a result, it was clarified that the VGLL3 signal appears in ⁇ SMA-positive cells.
  • Example 12 Evaluation of VGLL3 mRNA expression in liver disease
  • Wild-type (WT) mice were fed a choline-deficient high-fat diet, methionine weight loss, and 0.1% methionine addition for 10 weeks to induce non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • the liver was collected and the expression level of VGLL3 mRNA was evaluated by real-time RT-PCR (Fig. 13A). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the liver by fibrosis.
  • mRNAs of Cyp7a1, Cd68, and ACTA2 which are marker molecules of activated stellate cells in the regenerated liver.
  • the mouse liver in which fibrosis was induced by administration of CCL4 (carbon tetrachloride) was enzymatically treated, centrifuged at 50 g, and the precipitated cells were collected as hepatocytes (HC).
  • CCL4 carbon tetrachloride
  • HC hepatocytes
  • magnetic cell separation was performed on non-precipitated cells using anti-F4 / 80 antibody, and F4 / 80 positive cells were activated as Kupffer cells (KC) and F4 / 80 negative cells were activated.
  • Collected as cells (HSC) myofibroblasts in the liver).
  • Example 13 Evaluation of VGLL3 mRNA expression in pulmonary fibrosis A 6-8 week old female mouse was cannulated under somnopentyl anesthesia and a single dose of bleomycin solution (1.5 ⁇ g / g) or saline was injected into the trachea using a syringe. It was administered. Mice treated as described above were classified into a bleomycin (BLM) -administered group and a saline (Saline) -administered group, respectively. On the 14th day after BLM administration, the abdomen was opened under anesthesia by intraperitoneal administration of somnopentyl injection, blood was removed by amputation of the abdominal aorta, and the lungs were removed. RNA was recovered from the removed lung and the expression level of collagen was evaluated by real-time RT-PCR (Fig. 14). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the lung by fibrosis.
  • Rho kinases critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis. Pharmacological reviews 67, 103-117, doi: 10.1124 / pr.114.009381 (2015) ). 34. Vitriol, E. A. et al. Two functionally distinct sources of actin communicating supply the leading edge of lamellipodia. Cell reports 11, 433-445, doi: 10.1016 / j.celrep. 2015.03.033 (2015). 35. Vishavkarma, R. et al. Role of actin filaments in correlating nuclear shape and cell spreading.
  • the present invention it is possible to identify a marker protein of myofibroblasts and provide a technique for preventing or treating fibrotic diseases.

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Abstract

The purpose of the present invention is to identify a marker protein for a myofibroblast and provide a technique for preventing or treating a fibrotic disease.　A pharmaceutical composition for preventing or treating a fibrotic disease, which contains a VGLL3-inhibiting substance.

Description

転写関連因子を標的とする線維化疾患の予防または治療Prevention or treatment of fibrotic diseases that target transcription-related factors
　本発明は、線維化疾患を予防または治療するための医薬組成物に関する。詳細には、本発明は、線維化疾患を予防または治療する物質および核酸分子、ならびにそれらをスクリーニングするための方法、およびそのためのキットに関する。 The present invention relates to a pharmaceutical composition for preventing or treating fibrotic diseases. In particular, the present invention relates to substances and nucleic acid molecules that prevent or treat fibrotic diseases, methods for screening them, and kits for them.
　線維化とは組織の間質にコラーゲンなどの細胞外マトリックスタンパク質が過剰に蓄積された状態であり、炎症や老化等によって心臓や肝臓、肺、腸、腎臓などほぼ全ての臓器において誘導される (非特許文献１、２)。組織が障害を受けると、心筋細胞や肝細胞などの実質細胞が細胞死を起こし、その実質細胞が脱落した箇所を補填するようにコラーゲンなどの細胞外マトリックスが蓄積する。線維化を起こした臓器の特徴として、伸縮性の無い細胞外マトリックスが蓄積するため、組織が非常に硬くなることが挙げられる。 Fibrosis is a condition in which extracellular matrix proteins such as collagen are excessively accumulated in the interstitium of tissues, and is induced in almost all organs such as the heart, liver, lungs, intestines, and kidneys due to inflammation and aging. Non-Patent Documents 1 and 2). When a tissue is damaged, parenchymal cells such as myocardial cells and hepatocytes undergo cell death, and extracellular matrix such as collagen accumulates to compensate for the loss of the parenchymal cells. A characteristic of fibrotic organs is the accumulation of non-stretchable extracellular matrix, which makes the tissue extremely stiff.
　過剰な線維化は、臓器の機能障害を引き起こし、放置すれば死に至る。また、全死亡原因のうちの約45%において線維化が関与するという報告もなされている (非特許文献３)。 Excessive fibrosis causes organ dysfunction and can lead to death if left untreated. It has also been reported that fibrosis is involved in about 45% of all causes of death (Non-Patent Document 3).
　臓器の線維化は、コラーゲン等を産生する、筋線維芽細胞という細胞群によって実行される。筋線維芽細胞は組織が正常な時には、ほとんど存在しない。しかしながら、炎症が起きると免疫細胞から分泌されるTGF-βなどの液性因子が刺激となって、常在性の線維芽細胞などが筋線維芽細胞へと分化することが知られている (非特許文献４)。一方で、近年では、TGF-βなどのサイトカインだけでなく、細胞周囲の硬さ、機械的な刺激 (Mechanical stress) が、筋線維芽細胞への分化に重要な働きを果たすことが報告されている (非特許文献５－８)。細胞が機械的な刺激を受容すると、転写関連因子であるMRTF (ミオカルジン関連転写因子 (Myocardin-related transcription factor) ) やYAP (Yes関連タンパク質 (Yes-associated protein) ) が細胞質から核内に移行し、コラーゲンなどの線維化関連因子の発現を増加させると考えられている。このように機械的な刺激の強さに応じて細胞質と核を行き来する転写関連因子としては、現在、MRTFとYAPのみが知られている (非特許文献９)。 Organ fibrosis is carried out by a group of cells called myofibroblasts that produce collagen and the like. Myofibroblasts are rarely present when the tissue is normal. However, it is known that when inflammation occurs, humoral factors such as TGF-β secreted from immune cells are stimulated to differentiate resident fibroblasts into myofibroblasts ( Non-Patent Document 4). On the other hand, in recent years, it has been reported that not only cytokines such as TGF-β, but also pericellular hardness and mechanical stress play important roles in differentiation into myofibroblasts. (Non-Patent Document 5-8). When cells receive mechanical stimuli, transcription-related factors MRTF (Myocardin-related transcription factor) and YAP (Yes-associated protein) are transferred from the cytoplasm into the nucleus. , Collagen and other fibrosis-related factors are thought to increase expression. Currently, only MRTF and YAP are known as transcription-related factors that move back and forth between the cytoplasm and the nucleus according to the strength of mechanical stimulation (Non-Patent Document 9).
　このように、これまで線維化を制御する転写関連因子について様々な研究が行われてきた。しかしながら、これらの研究が未だ創薬に結びついた例はない。すなわち、これまで線維化に有効な治療薬はほとんどなく、線維化に対する画期的で効果的な治療薬・治療法の開発が望まれている。 In this way, various studies have been conducted on transcription-related factors that control fibrosis. However, there are no examples of these studies leading to drug discovery. That is, there have been few effective therapeutic agents for fibrosis so far, and development of epoch-making and effective therapeutic agents and therapeutic methods for fibrosis is desired.
　現在、線維化の治療薬としてピレスパ錠 (一般名；ピルフェニドン) およびオフェブ錠 (一般名；ニンテダニブ) の2剤が上市されている。これらは共に特発性肺線維症の治療に使用されている。ピルフェニドンは、特発性肺線維症の患者において、抗線維化作用により、肺活量の低下を抑制することが臨床的に示されている (Margaritopoulos, G. A., Vasarmidi, E. & Antoniou, K. M. Pirfenidone in the treatment of idiopathic pulmonary fibrosis: an evidence-based review of its place in therapy. Core evidence 11, 11-22, doi:10.2147/ce.S76549 (2016))。しかし、ピルフェニドンは作用機序が詳しく解明されていない事や、光線過敏症や肝機能障害などの一定の副作用が認められる。ニンテダニブは、血小板由来増殖因子受容体 (PDGFR)、線維芽細胞増殖因子受容体 (FGFR) および血管内皮増殖因子受容体 (EGFR) を阻害し、特発性肺線維症の病態形成に関与する線維芽細胞の増殖と遊走を抑制するという作用機序だと考えられている (Wollin, L. et al. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. The European respiratory journal 45, 1434-1445, doi:10.1183/09031936.00174914 (2015))。ニンテダニブに関しても、肝機能障害や血栓塞栓症等の一定の副作用が認められる。以上のように、現在の線維化の治療薬は一定の副作用を招く危険性がある。副作用が多い原因として、正常時の組織に恒常的に発現しているPDGFR等の受容体をターゲットとしている事が考えられる。 Currently, two drugs, Piresupa Tablets (generic name; pirfenidone) and Ofeb Tablets (generic name: nintedanib), are on the market as therapeutic agents for fibrosis. Both of these are used in the treatment of idiopathic pulmonary fibrosis. Pirfenidone has been clinically shown to suppress the decline in vital capacity by its antifibrotic effect in patients with idiopathic pulmonary fibrosis (Margaritopoulos, G.A., Vasarmidi, E. & Antoniou, K. M. Pirfenidone in the treatment of idiopathic pulmonary fibrosis: an evidence-based review of its place in therapy. Core evidence 11, 11-22, doi: 10.2147 / ce.S76549 (2016)). However, the mechanism of action of pirfenidone has not been elucidated in detail, and certain side effects such as photosensitivity and hepatic dysfunction are observed. Nintedanib inhibits platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (EGFR), and is involved in the pathogenesis of idiopathic pulmonary fibrosis. It is thought to be a mechanism of action that suppresses cell proliferation and migration (Wollin, L. et al. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. The European respiratory journal 45, 1434-1445, doi 10.1183 / 09031936.00174914 (2015)). Nintedanib also has certain side effects such as liver dysfunction and thromboembolism. As described above, the current therapeutic agents for fibrosis have a risk of causing certain side effects. It is considered that the cause of many side effects is that it targets receptors such as PDGFR that are constantly expressed in normal tissues.
　本発明は、VGLL3を標的とした線維化に対する画期的で効果的な医薬組成物を提供することを課題とする。また、VGLL3を標的とした線維化疾患の予防または治療に有効な候補物質をスクリーニングする方法を提供することを課題とする。 An object of the present invention is to provide an epoch-making and effective pharmaceutical composition for fibrosis targeting VGLL3. Another object of the present invention is to provide a method for screening candidate substances effective for the prevention or treatment of fibrotic diseases targeting VGLL3.
　本発明者は、未だ十分な治療法が確立されていない、線維化病態に対する革新的な治療法・治療薬の開発を目指し、筋線維芽細胞に発現する線維化に関与する転写関連因子の探索を行ったところ、機能のほとんど分かっていないVGLL3を見出した。さらに研究を進め、VGLL3が、機械的な刺激依存的に核内に移行し、線維化関連因子の発現を制御することを初めて明らかにし、本発明は、当該知見に基づいて完成されたものである。 The present inventor seeks transcription-related factors involved in fibrosis expressed in myofibroblasts, aiming at the development of innovative therapeutic methods and agents for fibrotic pathologies for which sufficient therapeutic methods have not yet been established. I found VGLL3, whose function is almost unknown. Further research revealed for the first time that VGLL3 translocates into the nucleus in a mechanical stimulus-dependent manner and regulates the expression of fibrosis-related factors, and the present invention was completed based on this finding. is there.
　すなわち、本発明は以下の態様を含む。
＜線維化疾患を予防または治療するための医薬組成物＞
［１］
　VGLL3の阻害物質を含有する、線維化疾患を予防または治療するための医薬組成物。
［２］
　VGLL3の阻害物質が、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質である、［１］記載の医薬組成物。
［３］
　VGLL3発現の阻害物質が、以下の (a) ～ (d) のいずれかに示される遺伝子または核酸の発現を阻害する物質である、［２］記載の医薬組成物：
　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸。
［４］
　遺伝子または核酸の発現を阻害する物質が、siRNA、shRNA、miRNA、アンチセンスおよびリボザイムからなる群から選択される少なくとも１つである［３］記載の医薬組成物。
＜核酸等＞
［５］
　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸、
の発現を阻害する、siRNA、shRNA、miRNA、アンチセンスおよびリボザイムからなる群から選ばれる核酸。
［６］
　［５］記載の核酸を発現する、ベクター。
［７］
　［６］記載のベクターを含む、細胞。
［８］
　筋線維芽細胞である、［７］記載の細胞。
＜スクリーニング方法＞
［９］
　被験物質の存在下で、細胞中のVGLL3の発現量を測定する工程と、
　前記発現量が、前記被験物質の非存在下における発現量と比較して低下していた場合に、前記被験物質は線維化疾患の予防または治療に有効な候補物質であると判断する工程とを備える、
　線維化疾患の予防または治療に有効な候補物質のスクリーニング方法。
＜バイオマーカー等＞
［１０］
　線維化の進行度を判定する方法であって、
 (a10) 被検者の筋線維芽細胞のVGLL3の量 (被検バイオマーカー量) を測定する工程、
 (b10) 被検バイオマーカー量と、基準の筋線維芽細胞のVGLL3の量 (対照バイオマーカー量) とを比較する工程、および
 (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、線維化の進行度が高いと判定する方法。
［１１］
　前記基準の筋線維芽細胞のVGLL3の量が、前記 (a10) 工程より前に測定された同一被検者の筋線維芽細胞のVGLL3の量である、［１０］に記載の方法。
［１２］
　前記基準の筋線維芽細胞のVGLL3の量が、健常者の筋線維芽細胞のVGLL3の量である、［１０］に記載の方法。
［１３］
　筋線維芽細胞のVGLL3の、線維化の進行度を判定するためのバイオマーカーとしての使用。
＜キット＞
［１４］
　VGLL3のcDNAを増幅するためのプライマーセット、VGLL3のmRNAに特異的にハイブリダイズするプローブ、または、VGLL3タンパク質に対する特異的結合物質を含む、筋線維芽細胞の検出用キット。 That is, the present invention includes the following aspects.
<Pharmaceutical composition for preventing or treating fibrotic diseases>
[1]
A pharmaceutical composition containing an inhibitor of VGLL3 for preventing or treating fibrotic diseases.
[2]
The pharmaceutical composition according to [1], wherein the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5.
[3]
The pharmaceutical composition according to [2], wherein the inhibitor of VGLL3 expression is a substance that inhibits the expression of the gene or nucleic acid shown in any of the following (a) to (d):
(a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
(b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
(c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing A nucleic acid that hybridizes with a base sequence complementary to the VGLL3 gene under stringent conditions.
[4]
The pharmaceutical composition according to [3], wherein the substance that inhibits the expression of a gene or nucleic acid is at least one selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme.
<Nucleic acid, etc.>
[5]
(a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
(b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
(c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing Nucleic acid that hybridizes under stringent conditions with a base sequence complementary to the VGLL3 gene,
Nucleic acid selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme that inhibits the expression of.
[6]
A vector that expresses the nucleic acid according to [5].
[7]
A cell comprising the vector according to [6].
[8]
The cell according to [7], which is a myofibroblast.
<Screening method>
[9]
The step of measuring the expression level of VGLL3 in cells in the presence of the test substance,
When the expression level is lower than the expression level in the absence of the test substance, the step of determining that the test substance is an effective candidate substance for the prevention or treatment of fibrotic disease. Prepare, prepare
A screening method for candidate substances that are effective in the prevention or treatment of fibrotic diseases.
<Biomarkers, etc.>
[10]
A method of determining the degree of fibrosis
(a10) Step of measuring the amount of VGLL3 (test biomarker amount) in myofibroblasts of a subject,
(b10) A step of comparing the amount of the test biomarker with the amount of VGLL3 in the reference myofibroblast (control biomarker amount), and (c10) when the test biomarker amount is larger than the control biomarker amount. In addition, a method for determining a subject to have a high degree of fibrosis.
[11]
The method according to [10], wherein the reference amount of VGLL3 in myofibroblasts is the amount of VGLL3 in myofibroblasts of the same subject measured prior to step (a10).
[12]
The method according to [10], wherein the reference amount of VGLL3 in myofibroblasts is the amount of VGLL3 in healthy subjects.
[13]
Use of VGLL3 in myofibroblasts as a biomarker to determine the progression of fibrosis.
<Kit>
[14]
A kit for detecting myofibroblasts, which comprises a primer set for amplifying VGLL3 cDNA, a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding agent for VGLL3 protein.
　VGLL3を標的とした線維化疾患に対する画期的で効果的な医薬組成物を提供することができる。また、VGLL3を標的とした線維化疾患の予防または治療に有効な候補物質をスクリーニングする方法を提供することができる。VGLL3をノックダウンするとコラーゲンなどの細胞外マトリックスタンパク質の発現が抑制することからVGLL3が線維化に対して促進的に働く因子であることを見出した。本発明によれば、VGLL3の阻害物質に関連する、線維化疾患を予防または治療する種々の技術を提供することができる。 It is possible to provide an epoch-making and effective pharmaceutical composition for fibrotic diseases targeting VGLL3. In addition, a method for screening effective candidate substances for the prevention or treatment of fibrotic diseases targeting VGLL3 can be provided. We found that VGLL3 is a factor that promotes fibrosis because the expression of extracellular matrix proteins such as collagen is suppressed when VGLL3 is knocked down. According to the present invention, it is possible to provide various techniques for preventing or treating fibrotic diseases related to inhibitors of VGLL3.
図１は総じて、VGLL3の発現が線維化に関することを示す模式図である。図１Ａは、MI (心筋梗塞) 手術心臓の模式図である。MI手術の手順は、左前下行冠状動脈の閉塞である。梗塞心臓では、虚血による壊死領域が「梗塞」領域として認識され、梗塞領域の他の部分は「非梗塞」領域と認識される。FIG. 1 is a schematic diagram showing that the expression of VGLL3 is related to fibrosis as a whole. FIG. 1A is a schematic diagram of an MI (myocardial infarction) surgical heart. The procedure for MI surgery is occlusion of the left anterior descending coronary artery. In an infarcted heart, the ischemic necrotic region is recognized as the "infarcted" region and the rest of the infarcted region is recognized as the "non-infarcted" region. 図１Ｂは、線維化に関連している可能性があるVGLL3を同定する手順を示している。偽手術およびMI手術マウスの心臓における遺伝子の発現を、マイクロアレイ分析を用いて包括的に検出した。次に、偽手術心臓と比較して梗塞心臓において誘導されている遺伝子 (転写関連因子をコードしている) を選択した。さらに、選択した遺伝子の中で筋線維芽細胞において高発現している転写関連因子を検索した。これらの工程を経て、VGLL3が同定された。FIG. 1B shows the procedure for identifying VGLL3 that may be associated with fibrosis. Gene expression in the hearts of sham and MI operated mice was comprehensively detected using microarray analysis. Next, we selected genes that are induced in infarcted hearts (encoding transcription-related factors) compared to sham-surgery hearts. Furthermore, among the selected genes, transcription-related factors that are highly expressed in myofibroblasts were searched. Through these steps, VGLL3 was identified.
図１Ｃは、偽手術マウス心臓 (sham)、およびMI手術マウス心臓の非梗塞領域 (rem) および梗塞領域 (inf) におけるVGLL3および線維化関連遺伝子 (αSMA (α－平滑筋アクチン (α-Smooth Muscle Actin)、ペリオスチン (Periostin)、COL1A1、COL1A2およびCOL3A1) のmRNA発現量 (それぞれ、術後3、7および28日後) を示すグラフである。mRNA発現は、GAPDHの発現に対して正規化し、無傷のマウス (０日目) に対する倍数変化として示した。 n = 3 (0日)、3 (sham 3日)、5 (inf 3日)、5 (rem 3日)、4 (sham 7日)、5 (inf 7日)、5 (rem 7日)、3-4 (sham 28日)、6 (inf 28日)、および6 (rem 28日)。群間比較は、各時点における対応のない両側スチューデントのt検定によって行った。#P < 0.05, ###P < 0.001 (inf vs. sham); *P < 0.05, ***P < 0.001 (inf vs. rem).FIG. 1C shows VGLL3 and fibrosis-related genes (αSMA (α-Smooth Muscle)) in the non-infarcted region (rem) and infarcted region (inf) of the sham-operated mouse heart (sham) and the MI-operated mouse heart. It is a graph showing the mRNA expression level of Actin), periostin, COL1A1, COL1A2 and COL3A1) (3, 7 and 28 days after surgery, respectively). The mRNA expression is normalized to the expression of GAPDH and is intact. It is shown as a multiple change with respect to the mouse (day 0). N = 3 (0 days), 3 (sham 3 days), 5 (inf 3 days), 5 (rem 3 days), 4 (sham 7 days), 5 (inf 7 days), 5 (rem 7 days), 3-4 (sham 28 days), 6 (inf 28 days), and 6 (rem 28 days). Group comparisons are on both sides without correspondence at each time point. Performed by Student's t-test. #P <0.05, ### P <0.001 (inf vs. sham); * P <0.05, *** P <0.001 (inf vs. rem).
図１Ｄは、機械的刺激による筋線維芽細胞分化の調節のためのプロトコルの概略である。心筋線維芽細胞を接着皿上で７日間培養し、それらのいくつかを分離してさらに７日間非接着皿にプレートし (脱分化)、次いで幾つかの細胞を接着皿上で７日間再び培養した (再分化)。タンパク質およびRNAを各時点で筋線維芽細胞から収集した。FIG. 1D outlines a protocol for the regulation of myofibroblast differentiation by mechanical stimulation. Myocardial fibroblasts were cultured on an adhesive dish for 7 days, some of them were separated and plated on a non-adhesive dish for an additional 7 days (dedifferentiation), and then some cells were recultured on an adhesive dish for 7 days. (Redifferentiation). Protein and RNA were collected from myofibroblasts at each time point. 図１Ｅは、「接着」、「非接着」および「再接着」の心筋線維芽細胞における、αSMA、ペリオスチンおよびGAPDHのウエスタンブロット画像である。棒グラフは、GAPDHに対して正規化したウエスタンブロットシグナルの平均強度を示す。それぞれn ＝ 5。FIG. 1E is a Western blot image of αSMA, periostin and GAPDH in "adherent", "non-adherent" and "re-adhered" myocardial fibroblasts. The bar graph shows the average intensity of Western blot signals normalized to GAPDH. Each n = 5.
図１Ｆは、プレート上または懸濁液中で培養した心筋線維芽細胞における高純度心筋線維芽細胞の収集および線維症関連遺伝子の発現に関連し、MACS (Magnetic cell sorting) によるCD45〈+) 細胞除去前後のMI 3日目の心臓から単離された心臓細胞のFACSプロファイルを示す。除去前は、心臓細胞画分は52％のCD45 (+) 造血細胞および25.2％のαSMA (+) 筋線維芽細胞を含んでいた。MACSによるCD45 (+) 細胞除去とそれに続く7日間の培養の後、CD45 (+) 造血細胞は心臓細胞の画分ではほとんど検出されなかったが (1.1％)、同画分では高純度のαSMA (+) 筋線維芽細胞を含んでいた (99.8％)。青矢印または赤矢印と付したヒストグラムは、それぞれPEコンジュゲート化抗CD45抗体またはFITCコンジュゲート化抗αSMA抗体で染色された心臓細胞を表す。矢印の無いヒストグラムは、染色されていない対照を表す。FIG. 1F shows the collection of high-purity myocardial fibroblasts and the expression of fibrosis-related genes in myocardial fibroblasts cultured on a plate or suspension, and CD45 <+) cells by MACS (Magnetic cell sorting). The FACS profile of heart cells isolated from the heart on MI 3 days before and after removal is shown. Prior to removal, the cardiac cell fraction contained 52% CD45 (+) hematopoietic cells and 25.2% αSMA (+) myofibroblasts. After removal of CD45 (+) cells by MACS and subsequent culture for 7 days, CD45 (+) hematopoietic cells were scarcely detected in the cardiac cell fraction (1.1%), but in the same fraction, high-purity αSMA. (+) Containing myofibroblasts (99.8%). Histograms labeled with blue or red arrows represent heart cells stained with PE-conjugated anti-CD45 antibody or FITC-conjugated anti-αSMA antibody, respectively. Histograms without arrows represent unstained controls.
図１Ｇは、未分化細胞のマーカーであるOct4の、「接着 (Ad)」、「非接着 (Non)」および「再接着 (Re)」の心臓線維芽細胞におけるmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn = 3。FIG. 1G is a graph showing the mRNA expression of Oct4, which is a marker of undifferentiated cells, in “adherent”, “non-adherent” and “re-adhered” cardiac fibroblasts. mRNA expression was normalized to that of GAPDH. Each n = 3.
図１Ｈは、「接着 (Ad)」、「非接着 (Non)」および「再接着 (Re)」の心臓線維芽細胞における線維化関連遺伝子 (ACTA2、COL1A1、COL1A2、COL3A1、FN1、LOXおよびCTGF) のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn = 5。FIG. 1H shows fibrosis-related genes (ACTA2, COL1A1, COL1A2, COL3A1, FN1, LOX and CTGF) in cardiac fibroblasts of “adhesive”, “non-adherent” and “re-adherent” (Re). ) Is a graph showing mRNA expression. mRNA expression was normalized to that of GAPDH. Each n = 5.
図１Ｉは、「接着 (Ad)」、「非接着 (Non)」および「再接着 (Re)」の心臓線維芽細胞における線維化関連遺伝子 (インテグリナブ (Integrinav)、インテグリンブ1 (integrinb1) およびYap1) のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn = 3。FIG. 1I shows fibrosis-related genes (Integrinav), integrinb1 and Yap1 in "adhesive", "non-adherent" and "re-adherent" cardiac fibroblasts. ) It is a graph which shows the mRNA expression. mRNA expression was normalized to that of GAPDH. Each n = 3.
図１Ｊは、分化または再分化した筋線維芽細胞において、脱非分化型筋線維芽細胞と比較してアップレギュレート (16倍以上) した、または、非梗塞マウス心臓と比較して梗塞マウス心臓においてアップレギュレート (2の1.5乗以上) した、8つの重複転写産物を示すベン図である。FIG. 1J shows differentiated or redifferentiated myofibroblasts up-regulated (16-fold or more) compared to undifferentiated myofibroblasts or infarcted mouse hearts compared to non-infarcted mouse hearts. It is a Ben diagram showing eight duplicate transcripts up-regulated (2 to the 1.5th power or higher) in.
図１Ｋは、VGLL3のヒト各臓器におけるタンパク発現分布を示す。FIG. 1K shows the protein expression distribution of VGLL3 in each human organ.
図１Ｌは、「接着 (Ad)」、「非接着 (Non)」および「再接着 (Re)」の心臓線維芽細胞におけるVGLL3のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn = 5。FIG. 1L is a graph showing the mRNA expression of VGLL3 in “adhesive”, “non-adherent” and “re-adhered” cardiac fibroblasts. mRNA expression was normalized to that of GAPDH. Each n = 5.
図１Ｍは、心臓から単離した筋肉線維芽細胞におけるVGLLファミリーであるVGLL1、VGLL2、VGLL3およびVGLL4の発現量をPCR増幅として表している電気泳動の結果を示す。FIG. 1M shows the results of electrophoresis showing the expression levels of the VGLL families VGLL1, VGLL2, VGLL3 and VGLL4 in muscle fibroblasts isolated from the heart as PCR amplification.
図１Ｎは、心臓から単離した筋肉線維芽細胞におけるVGLLファミリーVGLL1、VGLL2、VGLL3およびVGLL4の発現量の絶対定量の結果を示すグラフである。FIG. 1N is a graph showing the results of absolute quantification of the expression levels of VGLL family VGLL1, VGLL2, VGLL3 and VGLL4 in muscle fibroblasts isolated from the heart.
図１Ｏは、「接着 (Ad)」、「非接着 (Non)」および「再接着 (Re)」のヒト結腸の筋線維芽細胞のセルラインであるCCD-18Coにおける線維化関連遺伝子 (ACTA2、COL1A1、COL1A2、COL6A1、COL14A1、FBN1、VGLL3) のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn = 5。FIG. 1O shows fibrosis-related genes in CCD-18Co, which are the cell lines of "adherent", "non-adherent" and "re-adhered" human colon myofibroblasts (ACTA2, It is a graph which shows the mRNA expression of COL1A1, COL1A2, COL6A1, COL14A1, FBN1, VGLL3). mRNA expression was normalized to that of GAPDH. Each n = 5.
図２Ａ－Ｇは総じて、VGLL3が線維化を呈する心臓の筋線維芽細胞において特異的に発現していることを示す、図面に代わる写真である。左端の写真における四角は、右側のパネルに拡大された領域を示す。矢印 (→) は重ね合わせにおいて、シグナルが重複している箇所を示す。スケールバーは３０μｍを示す。図２Ａは、VGLL3に対する抗体で染色したMI 3日目の心臓の左心室 (LV) の切片を、筋線維芽細胞マーカーαSMA (Ａ) に対する抗体で共染色した結果を示す。2A-G are photographs that replace the drawings, showing that VGLL3 is generally specifically expressed in fibrotic cardiac myofibroblasts. The square in the leftmost photo shows the enlarged area in the right panel. Arrows (→) indicate where the signals overlap in the overlay. The scale bar indicates 30 μm. FIG. 2A shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the myofibroblast marker αSMA (A). 図２Ｂは、VGLL3に対する抗体で染色したMI 3日目の心臓の左心室 (LV) の切片を、マクロファージマーカーCD68 (Ｂ) に対する抗体で共染色した結果を示す。FIG. 2B shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the macrophage marker CD68 (B). 図２Ｃは、VGLL3に対する抗体で染色したMI 3日目の心臓の左心室 (LV) の切片を、心筋細胞マーカーTNNI3 (Ｃ) に対する抗体で共染色した結果を示す。FIG. 2C shows the results of co-staining a section of the left ventricle (LV) of the heart on day 3 MI stained with an antibody against VGLL3 with an antibody against the cardiomyocyte marker TNNI3 (C). 図２Ｄは、MI 7日目のマウス心臓のLVにおけるVGLL3mRNA発現について評価したin situハイブリダイゼーション (RNAスコープ) 実験の代表的な画像である。心臓切片を、抗－αSMA抗体 (Ｄ) で共染色した。核をDAPIで染色した。灰色の四角は、右側のパネルに拡大された領域を示す。矢印 (→) は重ね合わせにおいて、シグナルが重複している箇所を示す。スケールバーは３０μｍを示す。FIG. 2D is a representative image of an in situ hybridization (RNA scope) experiment evaluating VGLL3 mRNA expression in LV of mouse heart on day 7 MI. Heart sections were co-stained with anti-αSMA antibody (D). Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows (→) indicate where the signals overlap in the overlay. The scale bar indicates 30 μm.
図２Ｅは、MI 7日目のマウス心臓のLVにおけるVGLL3mRNA発現について評価したin situハイブリダイゼーション (RNAスコープ) 実験の代表的な画像である。心臓切片を、ペリオスチンmRNAに対するRNAスコーププローブと共ハイブリダイズさせた (Ｅ)。核をDAPIで染色した。灰色の四角は、右側のパネルに拡大された領域を示す。矢印 (→) は重ね合わせにおいて、シグナルが重複している箇所を示す。スケールバーは３０μｍを示す。FIG. 2E is a representative image of an in situ hybridization (RNA scope) experiment evaluating VGLL3 mRNA expression in LV of mouse heart on day 7 of MI. Cardiac sections were co-hybridized with RNA scope probes for periostin mRNA (E). Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows (→) indicate where the signals overlap in the overlay. The scale bar indicates 30 μm. 図２Ｆは、sham7日目マウスのLVにおけるVGLL3mRNAおよびペリオスチンmRNA発現について評価したin situハイブリダイゼーション実験の代表的な画像である。心臓切片を抗－αSMA抗体で共染色した。核をDAPIで染色した。灰色の四角は、右側のパネルに拡大された領域を示す。スケールバーは３０μｍを示す。FIG. 2F is a representative image of an in situ hybridization experiment in which VGLL3 mRNA and periostin mRNA expression in LV of day 7 sham mice was evaluated. Cardiac sections were co-stained with anti-αSMA antibody. Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. The scale bar indicates 30 μm. 図２Ｇは、ヒト患者の梗塞心臓におけるVGLL3mRNA発現について評価したin situハイブリダイゼーション実験の代表的な画像である。心臓切片を抗－αSMA抗体で共染色した。核をDAPIで染色した。灰色の四角は、右側のパネルに拡大された領域を示す。矢印 (→) は重ね合わせにおいて、シグナルが重複している箇所を示す。スケールバーは３０μｍを示す。FIG. 2G is a representative image of an in situ hybridization experiment evaluating VGLL3 mRNA expression in an infarcted heart of a human patient. Cardiac sections were co-stained with anti-αSMA antibody. Nuclei were stained with DAPI. The gray squares indicate the enlarged area in the right panel. Arrows (→) indicate where the signals overlap in the overlay. The scale bar indicates 30 μm.
図３Ａ－Ｄは総じて、VGLL3の核内移行が機械的刺激により誘発されることを示す、図面に代わる写真である。MI 3日目の心臓から単離した心筋線維芽細胞におけるVGLL3および核 (DAPI) の代表的な共焦点免疫細胞化学画像である。細胞の全体像を観察するため、細胞をCellTraceで染色した。図３Ａは、VGLL3 (Ａ) の免疫細胞化学画像である。上のパネルは浮遊培養筋線維芽細胞であり、下のパネルは接着培養筋線維芽細胞を示す。グラフは、浮遊状態 (Float) および接着状態 (Adherent) で培養した筋線維芽細胞における細胞質VGLL3またはYAP1強度に対する核の比率を示す (各群n＞100細胞)。スケールバー：20μm。3A-D are photographs that replace the drawings, showing that the intranuclear translocation of VGLL3 is generally induced by mechanical stimulation. It is a representative confocal immunocytochemical image of VGLL3 and nucleus (DAPI) in myocardial fibroblasts isolated from the heart on the third day of MI. The cells were stained with Cell Trace to observe the overall picture of the cells. FIG. 3A is an immunocytochemical image of VGLL3 (A). The upper panel shows suspended cultured myofibroblasts and the lower panel shows adherent cultured myofibroblasts. The graph shows the ratio of nuclei to cytoplasmic VGLL3 or YAP1 strength in myofibroblasts cultured in the floating state (Float) and the adhesive state (Adherent) (n> 100 cells in each group). Scale bar: 20 μm. 図３Ｂは、YAP1 (Ｂ) の免疫細胞化学画像である。上のパネルは浮遊培養筋線維芽細胞であり、下のパネルは接着培養筋線維芽細胞を示す。グラフは、浮遊状態 (Float) および接着状態 (Adherent) で培養した筋線維芽細胞における細胞質VGLL3またはYAP1強度に対する核の比率を示す (各群n＞100細胞)。スケールバー：20μm。FIG. 3B is an immunocytochemical image of YAP1 (B). The upper panel shows suspended cultured myofibroblasts and the lower panel shows adherent cultured myofibroblasts. The graph shows the ratio of nuclei to cytoplasmic VGLL3 or YAP1 strength in myofibroblasts cultured in the floating state (Float) and the adhesive state (Adherent) (n> 100 cells in each group). Scale bar: 20 μm.
図３Ｃは、FLAGタグ化VGLL3を過剰発現している筋線維芽細胞の画像である。FLAGタグ化VGLL3を抗－FLAG抗体で染色した。グラフは、浮遊状態 (Float) および接着状態 (Adherent) で培養した筋線維芽細胞における細胞質FLAGタグ化VGLL3強度に対する核の比率を示す (各群n＞100細胞)。スケールバー：20μm。FIG. 3C is an image of myofibroblasts overexpressing FLAG-tagged VGLL3. FLAG-tagged VGLL3 was stained with anti-FLAG antibody. The graph shows the ratio of nuclei to cytoplasmic FLAG-tagged VGLL3 strength in myofibroblasts cultured in the floating state (Float) and the adhesive state (Adherent) (n> 100 cells in each group). Scale bar: 20 μm. 図３Ｄは、棒グラフに記載したそれぞれの時間における、ポリ-L-リジン被覆ガラス底皿に接着している筋線維芽細胞の画像である。グラフは、異なる時間 (0.5、1、2、および4時間；各群 n ＝ 100細胞) で皿に接着させた筋線維芽細胞における細胞質のVGLL3強度に対する核の比率を示す。スケールバー：20μm。2つの群間比較は、対応のない両側スチューデントのt検定によって行った; ***P < 0.001 (A-C)。 3つまたはそれ以上の群間比較は、1-way ANOVAとそれに続くNewman-Keuls分析によって計算した; **P < 0.01, ***P < 0.001 (D)。FIG. 3D is an image of myofibroblasts adhering to a poly-L-lysine coated glass bottom dish at each time shown in the bar graph. The graph shows the ratio of nuclei to cytoplasmic VGLL3 intensity in dish-adhered myofibroblasts at different times (0.5, 1, 2, and 4 hours; n = 100 cells in each group). Scale bar: 20 μm. Comparison between the two groups was performed by unpaired two-sided student's t-test; *** P <0.001 (A-C). Comparisons between three or more groups were calculated by 1-way ANOVA followed by Newman-Keuls analysis; ** P <0.01, *** P <0.001 (D).
図４は、VGLL3の核内移行が細胞外マトリックスの硬さによって調節されることを示す、図面に代わる写真である。図４Ａは、正常組織および線維性組織の硬さを表す概略図である。弾性率は、臓器や細胞の硬さである。図４Ｂは、異なる硬さ (1、8、25、および50kPaの弾性率) を有するハイドロゲルプレート上に播種した心筋線維芽細胞におけるVGLL3および核 (DAPI) の代表的な共焦点免疫細胞化学画像である。細胞の全体像を観察するため、細胞をCellTraceで染色した。グラフは、異なる硬さ (各群 ｎ > 100細胞) を有するハイドロゲルプレート上に播種した筋線維芽細胞における細胞質のVGLL3強度に対する核の比率を示す。群間比較は、1-way ANOVAとそれに続くNewman-Keuls分析によって計算した; ***P < 0.001。スケールバー：20μm。FIG. 4 is an alternative photograph showing that the intranuclear translocation of VGLL3 is regulated by the hardness of the extracellular matrix. FIG. 4A is a schematic diagram showing the hardness of normal tissue and fibrous tissue. Elastic modulus is the hardness of an organ or cell. FIG. 4B is a representative confocal immunocytochemical image of VGLL3 and nucleus (DAPI) in myocardial fibroblasts seeded on hydrogel plates with different hardnesses (moduli of 1, 8, 25, and 50 kPa). Is. The cells were stained with Cell Trace to observe the overall picture of the cells. The graph shows the ratio of nuclei to VGLL3 intensity of cytoplasm in myofibroblasts seeded on hydrogel plates with different hardness (n> 100 cells in each group). Intergroup comparisons were calculated by 1-way ANOVA followed by Newman-Keuls analysis; *** P <0.001. Scale bar: 20 μm.
図５は、VGLL3の核内移行がアクチン重合によって制御されることを示す、図面に代わる写真である。図５Ａは、細胞外環境の硬さなどの機械的刺激に応じた細胞骨格形成の模式図である。機械的刺激は、G‐アクチン重合とF‐アクチン形成を誘導した。図５Ｂは、軟質ハイドロゲル (弾性率１kPa) および硬質ハイドロゲル (弾性率５０kPa) 上にプレートした筋線維芽細胞の代表的な共焦点画像である。核をDAPIで染色し、細胞をCellTraceで染色して細胞の全体像を観察した。スケールバー：20μm。グラフは細胞の全画像の面積を示す (μm2、各群: n＞ 100細胞)。群間比較は、対応のない両側スチューデントのt検定によって計算した。***P < 0.001。図５Ｃは、DMSO (対照、0.5％)、F-アクチン阻害剤ラトランキュリンA (latrunculin A) (Lat.A、2μM)、非筋肉ミオシン阻害剤ブレビスタチン (blebbistatin) (Blebbist.、50μM)、ROCK阻害剤Y27632 (80μM)、およびRho阻害剤C3トランスフェラーゼ (C3、3μg / mL) で処理した心筋線維芽細胞におけるVGLL3および核 (DAPI) の代表的な共焦点免疫細胞化学画像である。ここに、Rhoとは低分子量Gタンパク質Rasホモローグファミリーメンバーである。細胞の全体像を観察するため、細胞をCellTraceで染色した。スケールバー：20μm。グラフは筋線維芽細胞における細胞質のVGLL3強度に対する核の比率を示す (各群: n＞ 100細胞)。群間比較は、1-way ANOVAとそれに続くNewman-Keuls分析によって計算した; ***P < 0.001。FIG. 5 is an alternative photograph showing that the nuclear translocation of VGLL3 is regulated by actin polymerization. FIG. 5A is a schematic diagram of cytoskeleton formation in response to mechanical stimuli such as the hardness of the extracellular environment. Mechanical stimulation induced G-actin polymerization and F-actin formation. FIG. 5B is a representative confocal image of myofibroblasts plated on a soft hydrogel (modulus of elasticity 1 kPa) and a hard hydrogel (modulus of elasticity 50 kPa). The nuclei were stained with DAPI and the cells were stained with Cell Trace to observe the whole picture of the cells. Scale bar: 20 μm. The graph shows the area of the entire image of cells (μm2, each group: n> 100 cells). Intergroup comparisons were calculated by unpaired two-sided student's t-test. *** P <0.001. FIG. 5C shows DMSO (control, 0.5%), F-actin inhibitor latrunculin A (Lat.A, 2 μM), non-muscle myosin inhibitor blebbistatin (Blebbist., 50 μM), ROCK inhibitor. It is a representative confocal immunocytochemical image of VGLL3 and nucleus (DAPI) in myocardial fibroblasts treated with the agent Y27632 (80 μM) and the Rho inhibitor C3 transferase (C3, 3 μg / mL). Here, Rho is a member of the Ras homologue family of low molecular weight G proteins. The cells were stained with Cell Trace to observe the overall picture of the cells. Scale bar: 20 μm. The graph shows the ratio of nuclei to VGLL3 intensity in the cytoplasm of myofibroblasts (each group: n> 100 cells). Intergroup comparisons were calculated by 1-way ANOVA followed by Newman-Keuls analysis; *** P <0.001.
図６は、VGLL3の核内移行が接着班の活性化によって調節されることを示す、図面に代わる写真である。図６Ａ－Ｂは、DMSO (対照、0.5％)、インテグリンβ1阻害剤BTT-3033 (30μM) (Ａ)、およびFAK阻害剤VS-4718 (50μM) (Ｂ)で処理した心筋線維芽細胞におけるVGLL3および核 (DAPI) の代表的な共焦点免疫細胞化学画像である。細胞の全体像を観察するため、細胞をCellTraceで染色した。スケールバー：20μm。グラフは筋線維芽細胞における細胞質のVGLL3強度に対する核の比率を示す (各群: n＞ 100細胞)。群間比較は、対応のない両側スチューデントのt検定によって計算した; ***P < 0.001。FIG. 6 is an alternative photograph showing that intranuclear translocation of VGLL3 is regulated by activation of focal adhesions. Figures 6A-B show VGLL3 in myocardial fibroblasts treated with DMSO (control, 0.5%), integrin β1 inhibitor BTT-3033 (30 μM) (A), and FAK inhibitor VS-4718 (50 μM) (B). And is a typical confocal immunocytochemical image of the nucleus (DAPI). The cells were stained with Cell Trace to observe the overall picture of the cells. Scale bar: 20 μm. The graph shows the ratio of nuclei to VGLL3 intensity in the cytoplasm of myofibroblasts (each group: n> 100 cells). Intergroup comparisons were calculated by unpaired two-sided student's t-test; *** P <0.001.
図７は、VGLL3が、筋線維芽細胞における線維化関連因子の発現を調節していることを示すグラフである。図７Ａは、対照siRNA (siCtrl) またはVGLL3を標的とするsiRNA (siVGLL3 #1 および #2) をトランスフェクトした心筋線維芽細胞におけるVGLL3および線維化関連遺伝子 (COL1A1、COL1A2、COL3A1) のmRNA発現を示すグラフである。それぞれn = 5。** P <0.01、*** P <0.001。 群間比較は、1-way ANOVAとそれに続くNewman-Keuls分析によって計算した; *P < 0.05, *P < 0.01, ***P < 0.001。図７Ｂは、対照siRNA (siCtrl) またはVGLL3を標的とするsiRNA (siVGLL3) をトランスフェクトしたヒト結腸筋線維芽細胞セルラインCCD-18coにおけるVGLL3および線維化関連遺伝子 (COL1A1、COL1A2、COL3A1) のmRNA発現を示すグラフである。それぞれn = 5。** P <0.01、*** P <0.001。 群間比較は、対応のない両側スチューデントのt検定によって計算した; *P < 0.05, *P < 0.01, ***P < 0.001。図７Ｃは、対照siRNA (siCtrl) またはVGLL3を標的とするsiRNA (siVGLL3) をトランスフェクトした、活性化した星細胞である肝臓筋線維芽細胞におけるVGLL3および線維化関連遺伝子 (COL1A1、COL1A2、COL14A1、Postn) のmRNA発現を示すグラフである。それぞれn = 5。** P <0.01、*** P <0.001。 群間比較は、対応のない両側スチューデントのt検定によって計算した; *P < 0.05, *P < 0.01, ***P < 0.001。FIG. 7 is a graph showing that VGLL3 regulates the expression of fibrosis-related factors in myofibroblasts. FIG. 7A shows the mRNA expression of VGLL3 and fibrosis-related genes (COL1A1, COL1A2, COL3A1) in myocardial fibroblasts transfected with control siRNA (siCtrl) or siRNA targeting VGLL3 (siVGLL3 # 1 and # 2). It is a graph which shows. Each n = 5. ** P <0.01, *** P <0.001. Intergroup comparisons were calculated by 1-way ANOVA followed by Newman-Keuls analysis; * P <0.05, * P <0.01, *** P <0.001. FIG. 7B shows mRNAs of VGLL3 and fibrosis-related genes (COL1A1, COL1A2, COL3A1) in human colonic myofibroblast cell line CCD-18co transfected with control siRNA (siCtrl) or VGLL3 targeting siRNA (siVGLL3). It is a graph which shows the expression. Each n = 5. ** P <0.01, *** P <0.001. Intergroup comparisons were calculated by unpaired two-sided student's t-test; * P <0.05, * P <0.01, *** P <0.001. FIG. 7C shows VGLL3 and fibrosis-related genes (COL1A1, COL1A2, COL14A1, COL1A1, COL1A2, COL14A1,) in activated stellate liver myofibroblasts transfected with control siRNA (siCtrl) or siRNA targeting VGLL3 (siVGLL3). It is a graph which shows the mRNA expression of Postn). Each n = 5. ** P <0.01, *** P <0.001. Intergroup comparisons were calculated by unpaired two-sided student's t-test; * P <0.05, * P <0.01, *** P <0.001.
図８Ａは、質量分析計を用いて同定した、VGLL3の結合タンパク質候補を示す。グラフの横軸は各種タンパク質の沈降物量、縦軸は信頼度を示す。FIG. 8A shows VGLL3 binding protein candidates identified using a mass spectrometer. The horizontal axis of the graph shows the amount of sediment of various proteins, and the vertical axis shows the reliability. 図８Ｂは、マウス筋線維芽細胞におけるFLAG-VGLL3とDDX5の相互作用を示す図面に代わる写真である。FLAG-VGLL3を、レトロウイルスを用いて発現させたマウス筋線維芽細胞からの細胞溶解物からFLAG-VGLL3を免疫沈降させた。DDX5は抗DDX5抗体によって検出した。FIG. 8B is an alternative photograph showing the interaction of FLAG-VGLL3 and DDX5 in mouse myofibroblasts. FLAG-VGLL3 was immunoprecipitated from cell lysates from mouse myofibroblasts expressed using a retrovirus. DDX5 was detected by anti-DDX5 antibody. 図８Ｃは、組換えGST-VGLL3および組換えHis-DDX5の間のインビトロ結合アッセイを示す、図面に代わる写真である。GST-VGLL3はグルタチオンセファロースビーズによって沈降させた。GST-VGLL3とHis-DDX5はそれぞれ抗GST抗体と抗DDX5抗体によって検出した。FIG. 8C is an alternative photograph showing the in vitro binding assay between recombinant GST-VGLL3 and recombinant His-DDX5. GST-VGLL3 was precipitated with glutathione sepharose beads. GST-VGLL3 and His-DDX5 were detected by anti-GST antibody and anti-DDX5 antibody, respectively. 図８Ｄは、野生型DDX5およびDDX5の欠失変異体の略図である。FIG. 8D is a schematic representation of wild-type DDX5 and deletion mutants of DDX5. 図８Ｅは、図８Ｄに示すFLAG-VGLL3およびHAタグ化DDX5の野生型またはDDX5の欠失変異体用レトロウイルスを用いて発現させたNIH3T3細胞を溶解し、抗FLAG抗体による免疫沈降に供した結果を示す図面に代わる写真である。免疫沈降物およびインプットは、抗HA抗体および抗FLAG抗体によって免疫ブロットした。FIG. 8E lysed NIH3T3 cells expressed using the FLAG-VGLL3 and HA-tagged DDX5 wild-type or DDX5 deletion mutant retroviruses shown in FIG. 8D and subjected to immunoprecipitation with anti-FLAG antibody. It is a photograph that replaces the drawing showing the result. Immunoprecipitates and inputs were immunoblotted with anti-HA and anti-FLAG antibodies. 図８Ｆは、VGLL3のN末端のみの変異体あるいはC末端のみの変異体の模式図である。FIG. 8F is a schematic diagram of a mutant having only the N-terminal or a mutant having only the C-terminal of VGLL3. 図８Ｇは、図８Ｆに示すVGLL3のN末端のみの変異体またはC末端のみの変異体とHA-DDX5をNIH3T3細胞においてレトロウイルスを用いて過剰発現させ、抗FLAG抗体で免疫沈降に供した結果を示す図面に代わる写真である。免疫沈降物およびインプットは、抗HA抗体および抗FLAG抗体によって免疫ブロットした。FIG. 8G shows the results of overexpressing the N-terminal only mutant or C-terminal only mutant of VGLL3 and HA-DDX5 shown in FIG. 8F in NIH3T3 cells using a retrovirus and subjecting them to immunoprecipitation with an anti-FLAG antibody. It is a photograph that replaces the drawing showing. Immunoprecipitates and inputs were immunoblotted with anti-HA and anti-FLAG antibodies.
図９は、Tead3を標的とするsiRNAでトランスフェクトされた心筋線維芽細胞におけるTead3および線維化関連遺伝子 (COL1A1、COL1A2、COL3A1、Postn、ACTA2、およびFN1) のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn ＝ 5。FIG. 9 is a graph showing the mRNA expression of Tead3 and fibrosis-related genes (COL1A1, COL1A2, COL3A1, Postn, ACTA2, and FN1) in myocardial fibroblasts transfected with Tead3 targeting siRNA. mRNA expression was normalized to that of GAPDH. Each n = 5.
図１０Ａは、DDX5を標的とするsiRNAでトランスフェクトした、線維化した心臓から単離した心筋線維芽細胞におけるDDX5および線維化関連遺伝子 (COL1A1、COL1A2、COL6A1、COL14A1、やペリオスチン、フィブリルリン-1 (fibrillin-1、FBV1) ) のmRNA発現を示すグラフである。mRNAの発現はGAPDHのそれに対して正規化した。それぞれn ＝ 5。FIG. 10A shows DDX5 and fibrosis-related genes (COL1A1, COL1A2, COL6A1, COL14A1, and periostin, fibrillin-1) in myocardial fibroblasts isolated from fibrotic hearts transfected with siRNA targeting DDX5. It is a graph which shows the mRNA expression of (fibrillin-1, FBV1)). mRNA expression was normalized to that of GAPDH. Each n = 5. 図１０Ｂは、DDX5を標的とするsiRNAでトランスフェクトした、線維化した心臓から単離した心筋線維芽細胞におけるmiR-29b (配列番号６６：uagcaccauuugaaaucaguguu) の発現量を示すグラフである。mRNAの発現はU6 snRNAの発現に対して正規化した。それぞれn ＝ 5。FIG. 10B is a graph showing the expression level of miR-29b (SEQ ID NO: 66: uagcaccauuugaaaucaguguu) in myocardial fibroblasts isolated from fibrotic hearts transfected with siRNA targeting DDX5. mRNA expression was normalized to U6 snRNA expression. Each n = 5. 図１０Ｃは、VGLL3を標的とするsiRNAでトランスフェクトした、線維化した心臓から単離した心筋線維芽細胞におけるmiR-29bの発現量を示すグラフである。miR-29bの発現はU6 snRNAの発現対して正規化した。それぞれn ＝ 5。FIG. 10C is a graph showing the expression level of miR-29b in myocardial fibroblasts isolated from fibrotic hearts transfected with siRNA targeting VGLL3. The expression of miR-29b was normalized to the expression of U6 snRNA. Each n = 5. 図１０Ｄは、線維化した心臓から単離した筋線維芽細胞に対し、DDX5抗体を用いたRNA免疫沈降を行い、Pri-miR29b-1あるいはPri-miR29b-2に特異的なprimerセットを 用いてPCRを行った結果を示す、図面に代わる写真である。RTは逆転写酵素（reverse transcriptase）であり、（-）、（+）はその有無を示す。In FIG. 10D, RNA immunoprecipitation using a DDX5 antibody was performed on myofibroblasts isolated from a fibrotic heart, and a primer set specific for Pri-miR29b-1 or Pri-miR29b-2 was used. It is a photograph instead of a drawing which shows the result of performing PCR. RT is reverse transcriptase, and (-) and (+) indicate its presence or absence.
図１１Ａは、MIの7日後の野生型 (WT) およびVGLL3 KOマウスの心臓における線維化関連遺伝子 (Col1α1、Col1α2、Col3α1、FN1およびFBN1) のmRNA発現を示すグラフである。mRNA発現はGAPDHのそれに正規化した。それぞれn ＝ 6。VGLL3の欠乏は、線維化を減少させ、心筋梗塞後の心臓の状態を改善した。FIG. 11A is a graph showing the mRNA expression of fibrosis-related genes (Col1α1, Col1α2, Col3α1, FN1 and FBN1) in the hearts of wild-type (WT) and VGLL3KO mice 7 days after MI. mRNA expression was normalized to that of GAPDH. Each n = 6. VGLL3 deficiency reduced fibrosis and improved cardiac condition after myocardial infarction. 図１１Ｂは、MIの7日後のWTおよびVGLL3 KOマウスの心臓におけるmir-29bの発現を示すグラフである。miRNAの発現はU6 snRNAの発現に対して正規化した。それぞれn ＝ 6。FIG. 11B is a graph showing the expression of mir-29b in the hearts of WT and VGLL3KO mice 7 days after MI. The expression of miRNA was normalized to the expression of U6 snRNA. Each n = 6. 図１１Ｃは、MI手術の28日後のWTおよびVGLL3マウスのピクロシリウスレッド染色した心臓切片の代表的な画像である。コラーゲン体積分率 (CVF) は、ピクロシリウスレッド陽性コラーゲンの沈着面積の百分率として計算した。スケールバー：１mm。それぞれn ＝ 6。FIG. 11C is a representative image of a picrosirius red-stained heart section of WT and VGLL3 mice 28 days after MI surgery. Collagen volume fraction (CVF) was calculated as a percentage of the deposited area of picrosirius red-positive collagen. Scale bar: 1 mm. Each n = 6. 図１１Ｄは、MI手術の28日後の心エコー検査を示すグラフである。短縮率 (FS)、駆出率 (EF) を表示している。n ＝ 5。FIG. 11D is a graph showing echocardiography 28 days after MI surgery. The shortening rate (FS) and ejection fraction (EF) are displayed. n = 5.
図１２Ａは、野生型（WT）マウスに片側尿管閉塞（UUO）モデル処置を施し、処置後10日目の腎臓を回収し、リアルタイムRT-PCRによって、VGLL3mRNAの発現量を評価した結果を示すグラフである。mRNA発現はGAPDHのそれに正規化した。それぞれn ＝ 4。FIG. 12A shows the results of unilateral ureteral obstruction (UUO) model treatment of wild-type (WT) mice, recovery of kidneys 10 days after treatment, and evaluation of VGLL3 mRNA expression level by real-time RT-PCR. It is a graph. mRNA expression was normalized to that of GAPDH. Each n = 4. 図１２Ｂは、VGLL3 mRNAをin situ ハイブリダイゼーションにより、筋線維芽細胞マーカーαSMAをそれに対する抗体により、さらにDAPIで染色した、片側尿管閉塞（UUO）モデル処置マウス腎臓 の代表的な画像である。矢印 (→) は、VGLL3を発現する筋線維芽細胞でのそれぞれのシグナルを示している。スケールバーは30μmである。FIG. 12B is a representative image of a unilateral ureteral obstruction (UUO) model-treated mouse kidney in which VGLL3 mRNA was in situ hybridized, myofibroblast marker αSMA was stained with an antibody against it, and further stained with DAPI. Arrows (→) indicate the respective signals in myofibroblasts expressing VGLL3. The scale bar is 30 μm. 図１２Ｃは、WTマウスおよびVGLL3KOマウスに片側尿管閉塞（UUO）モデル処置を施し、処置後10日目の腎臓を回収し、リアルタイムRT-PCRによって、コラーゲンの発現量を評価した結果を示すグラフである。WT: n＝14, KO: n=9FIG. 12C is a graph showing the results of unilateral ureteral obstruction (UUO) model treatment of WT and VGLL3KO mice, recovery of the kidney 10 days after the treatment, and evaluation of collagen expression level by real-time RT-PCR. Is. WT: n = 14, KO: n = 9 図１３Ａは、野生型（WT）マウスに非アルコール性脂肪性肝炎（NASH）を誘導した肝臓について、リアルタイムRT-PCRによってVGLL3mRNAの発現量を評価した結果を示すグラフである。mRNA発現はGAPDHのそれに正規化した。それぞれn ＝ 15。図１３Ｂは、4週間のCCl4（四塩化炭素）投与により肝障害が起こり、線維化した肝臓（CCl4）、4週間のCCl4投与により線維化を誘導した後、投与をやめて4週間が経過し、肝臓が再生したマウスの肝臓（Regene）について、リアルタイムRT-PCRによってVGLL3mRNAの発現量を評価した結果を示すグラフである。mRNA発現はGAPDHのそれに正規化した。それぞれビヒクル＝6、CCl4=6、Regene=5。図１３Ｃは、CCL4の投与により線維化誘導マウス肝臓の一定処理後における、遠心沈降した肝細胞（HC）、ならびに非沈降細胞におけるF4/80陽性のクッパー細胞（KC）およびF4/80陰性の星細胞（HSC）それぞれの細胞分画において、肝細胞、クッパー細胞、活性化した星細胞のマーカー分子であるCyp7a1、Cd68、Acta2のmRNAを測定した結果を示すグラフである。図１３Ｄは、図１３Ｃで回収したそれぞれの細胞画分におけるVGLL3mRNAの発現量をリアルタイムRT-PCRにより測定した結果を示すグラフである。FIG. 13A is a graph showing the results of evaluating the expression level of VGLL3 mRNA by real-time RT-PCR in a liver in which nonalcoholic steatohepatitis (NASH) was induced in wild-type (WT) mice. mRNA expression was normalized to that of GAPDH. Each n = 15. In FIG. 13B, liver damage was caused by administration of CCl4 (carbon tetrachloride) for 4 weeks, and fibrosis was induced by administration of fibrotic liver (CCl4) and CCl4 for 4 weeks, and then 4 weeks had passed since the administration was stopped. It is a graph which shows the result of having evaluated the expression level of VGLL3 mRNA by real-time RT-PCR about the liver (Regene) of the mouse which regenerated the liver. mRNA expression was normalized to that of GAPDH. Vehicle = 6, CCl4 = 6, Regene = 5, respectively. FIG. 13C shows centrifuge hepatocytes (HC) and F4 / 80-positive Kupffer cells (KC) and F4 / 80-negative stars in non-precipitated cells after constant treatment of fibrotic-induced mouse liver by administration of CCL4. It is a graph which shows the result of having measured the mRNA of Cyp7a1, Cd68, and Acta2 which are marker molecules of hepatocyte, Kupffer cell, and activated stellate cell in each cell fraction of a cell (HSC). FIG. 13D is a graph showing the results of measuring the expression level of VGLL3 mRNA in each cell fraction collected in FIG. 13C by real-time RT-PCR.
図１４は、線維化状態にある肺におけるVGLL3mRNA発現を示すグラフである。生理食塩水処理、n ＝ 4、BLM（ブレオマイシン）処理、n ＝ 3。FIG. 14 is a graph showing VGLL3 mRNA expression in fibrotic lungs. Saline treatment, n = 4, BLM (bleomycin) treatment, n = 3.
＜組成物＞
　本発明は一実施態様において、VGLL3の阻害物質、具体的にはVGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質を含有する組成物を提供する。前記組成物は医薬組成物、または食品組成物を含む。前記医薬組成物は、VGLL3の阻害物質、具体的にはVGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質を有効成分として含有する、線維化疾患を予防または治療するための医薬組成物である。また、前記食品組成物は、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質を含有する、線維化疾患の治療の補助、症状の緩和または予防用の食品組成物である。 <Composition>
The present invention provides, in one embodiment, a composition comprising an inhibitor of VGLL3, specifically an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5. .. The composition includes a pharmaceutical composition or a food composition. The pharmaceutical composition contains an inhibitor of VGLL3, specifically, an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5 as an active ingredient. It is a pharmaceutical composition for preventing or treating. In addition, the food composition contains an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5, and assists in the treatment of fibrotic diseases, alleviates or prevents symptoms. Food composition for use.
　本発明によって、心筋梗塞時の心臓で発現量が増加するVGLL3が、細胞外の硬さに依存して細胞内局在を変え、線維化を調節する因子であることがはじめて明らかとなった。心筋梗塞時には、梗塞によって心筋細胞が壊死する。心筋細胞が壊死した箇所は、心破裂を防ぐために筋線維芽細胞によってコラーゲンなどの細胞外マトリックスタンパク質が急激に産生される。すなわち、梗塞領域は細胞外マトリックスタンパク質と過剰に置換され、結果、線維化が強く誘導される (図１Ａ)。VGLL3の発現は、線維化が強く進行し組織が硬くなる梗塞領域において認められ、その発現細胞は、組織の線維化を担う筋線維芽細胞に特異的であった。さらに、筋線維芽細胞が焦点接着斑と呼ばれる集合体によって細胞外環境の硬さなどの機械的な刺激を受容し、Rho/ROCKの活性化による細胞骨格形成を促進させることで、VGLL3が細胞質から核内へ移行することを明らかにした。また、VGLL3をノックダウンすることで、コラーゲンなどの細胞外マトリックスタンパク質の発現が減少したことから、VGLL3が線維化に対して促進的に働く因子であることも見出した。本発明によれば、VGLL3の阻害物質に関連する、線維化疾患を予防または治療する種々の技術を提供することができる。 According to the present invention, it has been clarified for the first time that VGLL3, whose expression level is increased in the heart at the time of myocardial infarction, is a factor that changes intracellular localization depending on extracellular hardness and regulates fibrosis. During myocardial infarction, the infarction causes cardiomyocyte necrosis. Where cardiomyocytes are necrotic, myofibroblasts rapidly produce extracellular matrix proteins such as collagen to prevent cardiac rupture. That is, the infarcted region is excessively replaced with extracellular matrix protein, and as a result, fibrosis is strongly induced (Fig. 1A). Expression of VGLL3 was observed in the infarcted region where fibrosis progressed strongly and the tissue became hard, and the expressing cells were specific to myofibroblasts responsible for tissue fibrosis. Furthermore, VGLL3 is cytoplasmic by allowing myofibroblasts to receive mechanical stimuli such as the hardness of the extracellular environment by aggregates called focal adhesion spots and promote cytoskeleton formation by activating Rho / ROCK. It was clarified that it shifts from to into the nucleus. We also found that knockdown of VGLL3 reduced the expression of extracellular matrix proteins such as collagen, indicating that VGLL3 is a factor that promotes fibrosis. According to the present invention, it is possible to provide various techniques for preventing or treating fibrotic diseases related to inhibitors of VGLL3.
　VGLL3は、ショウジョウバエの羽の形成にかかわる遺伝子Vgl類似の分子であり、哺乳動物にはVgll1～4の4つあるVGLL (Vestigial-like) ファミリータンパク質の一つである (Williamsら, 1991, Genes Dev. 5:2481-95)。VGLLファミリータンパク質は、転写因子であるTEAD (TEA domain family member) に結合することが知られている。
　VGLL3の機能としては、これまで、VGLL３の男女での発現差が自己免疫病の発症頻度の差を決める可能性を示す報告 (JCI Insight.2019 Apr 18;4(8). pii: 127291. doi: 10.1172/jci.insight.127291.、Nat Immunol.2017 Feb;18(2):152-160. doi: 10.1038/ni.3643.)、脂肪の分化を抑制する (J Lipid Res.2013 Feb;54(2):473-81. doi: 10.1194/jlr.M032755.) との報告があるが、未だ不明な点が多い。 VGLL3 is a molecule similar to the gene Vgl involved in the formation of Drosophila wings, and is one of the four VGLL (Vestigial-like) family proteins in mammals (Williams et al., 1991, Genes Dev). . 5: 2481-95). VGLL family proteins are known to bind to the transcription factor TEAD (TEA domain family member).
As for the function of VGLL3, it has been reported that the difference in the expression of VGLL3 between men and women may determine the difference in the incidence of autoimmune diseases (JCI Insight.2019 Apr 18; 4 (8). Pii: 127291. Doi : 10.1172 / jci.insight.127291., Nat Immunol.2017 Feb; 18 (2): 152-160. Doi: 10.1038 / ni.3643.), Suppresses lipid differentiation (J Lipid Res.2013 Feb; 54 (2): 473-81. Doi: 10.1194 / jlr.M032755.), But there are still many unclear points.
　現在、国内外において線維化治療の標的となる分子が活発に探索されており、例えばTEADおよびYAP/TAZは筋線維芽細胞において線維化を促進することが知られている。しかし、TEADおよびYAP/TAZは、筋線維芽細胞のみならず、正常時の様々な組織の細胞に発現し、正常時の細胞機能にも密接に関与している。このことがTEAD、YAP/TAZを創薬標的とする上での障害となっていると考えられる。本発明により見出されたVGLL3のヒト各臓器におけるタンパク発現分布を、The Human Protein ATLAS (http://www.proteinatlas.org/)より調べた。結果、データベース上では、VGLL3は、生体内の正常な組織において妊娠時の胎盤にのみ多く発現すると報告されていることが判明した (図１Ｋ)。
　これは、VGLL3が革新的な線維化治療開発のための標的分子になり得る可能性を示唆している。
　なお、特表2015-501151には、VGLL3が前立腺癌の分類マーカーの一例として記載されているが、筋線維芽細胞や線維化疾患に関する具体的な記載はない。また、特開2011-019520には、VGLLが転写因子として記載されているが、筋線維芽細胞や線維化疾患に関する具体的な記載はない。 Currently, molecules that are targets for fibrosis treatment are being actively searched for in Japan and overseas, and for example, TEAD and YAP / TAZ are known to promote fibrosis in myofibroblasts. However, TEAD and YAP / TAZ are expressed not only in myofibroblasts but also in cells of various tissues during normal times, and are closely involved in cell functions during normal times. This is considered to be an obstacle to targeting TEAD and YAP / TAZ for drug discovery. The protein expression distribution of VGLL3 found by the present invention in each human organ was investigated from The Human Protein ATLAS (http://www.proteinatlas.org/). As a result, it was found on the database that VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in vivo (Fig. 1K).
This suggests that VGLL3 may be a target molecule for the development of innovative fibrotic therapies.
Although VGLL3 is described as an example of a classification marker for prostate cancer in Special Table 2015-501151, there is no specific description regarding myofibroblasts and fibrotic diseases. Further, although VGLL is described as a transcription factor in Japanese Patent Application Laid-Open No. 2011-019520, there is no specific description regarding myofibroblasts and fibrotic diseases.
　本発明において、VGLL3はヒトVGLL3およびマウスVGLL3を含む。さらにそれらのバリアントも含む。ヒトVGLL3のバリアント１、バリアント２およびバリアント３のアミノ酸配列および核酸配列をそれぞれ、バリアント１について配列表の配列番号１および２に、バリアント２について配列表の配列番号３および４に、そしてバリアント３について配列表の配列番号５および６に示す。マウスVGLL3のバリアント１、バリアント２およびバリアント３のアミノ酸配列および核酸配列をそれぞれ、バリアント１について配列表の配列番号７および８に、バリアント２について配列表の配列番号９および１０に、そしてバリアント３について配列表の配列番号１１および１２に示す。実施例において後述するように、VGLL3は、筋線維芽細胞に特異的に発現し、αSMA、ペリオスチン、COL1A1 (コラーゲン1α1)、COL1A2 (コラーゲン1α2) およびCOL3A1 (コラーゲン3α1) などの線維化関連因子の発現を促進し、そして周囲が硬くなると核に移行する性質を有する。したがって、VGLL3の阻害物質は、線維化疾患の予防または治療に用いることができる。 In the present invention, VGLL3 includes human VGLL3 and mouse VGLL3. It also includes those variants. The amino acid and nucleic acid sequences of variants 1, variant 2 and variant 3 of human VGLL3 are shown in SEQ ID NOs: 1 and 2 of the Sequence Listing for variant 1, SEQ ID NOs: 3 and 4 of the sequence listing for variant 2, and for variant 3, respectively. It is shown in SEQ ID NOs: 5 and 6 of the sequence listing. The amino acid and nucleic acid sequences of Variants 1, Variant 2 and Variant 3 of mouse VGLL3 are shown in SEQ ID NOs: 7 and 8 of the Sequence Listing for Variant 1, SEQ ID NOs: 9 and 10 of the Sequence Listing for Variant 2, and for Variant 3, respectively. It is shown in SEQ ID NO: 11 and 12 of the sequence listing. As will be described later in the examples, VGLL3 is specifically expressed in myofibroblasts and is associated with fibrosis-related factors such as αSMA, periostin, COL1A1 (collagen 1α1), COL1A2 (collagen 1α2) and COL3A1 (collagen 3α1). It promotes expression and has the property of translocating to the nucleus when the surroundings become hard. Therefore, inhibitors of VGLL3 can be used for the prevention or treatment of fibrotic diseases.
　本発明において、線維化とは皮膚や内臓に膠原線維 (コラーゲン) などの細胞外基質と呼ばれる物質が増加し、その結果、皮膚や内臓が硬くなる現象を指し、「硬化」とも呼ばれる。本発明における線維化疾患とは、心臓線維症、肝臓線維症、腎臓線維症、肺線維症、全身性強皮症、皮膚硬化症、さらには膵臓がんなどの難治性がんなど線維化による疾患を意味する。 In the present invention, fibrosis refers to a phenomenon in which substances called extracellular matrix such as collagen fibers increase in the skin and internal organs, and as a result, the skin and internal organs become hard, and is also called "hardening". The fibrotic disease in the present invention is due to fibrosis such as heart fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis, systemic scleroderma, skin sclerosis, and intractable cancer such as pancreatic cancer. Means disease.
　本発明において、「阻害物質」の「阻害」とは、VGLL3発現、VGLL3の活性、VGLL3の核内移行またはVGLL3とDDX5との結合、を阻害または抑制することを意味する。 In the present invention, "inhibition" of an "inhibitor" means inhibiting or suppressing VGLL3 expression, VGLL3 activity, nuclear translocation of VGLL3 or binding of VGLL3 to DDX5.
　本発明において、VGLL3発現の阻害物質として、以下の (a) ～ (d) のいずれかに示される遺伝子または核酸の発現を阻害する物質が挙げられる：
　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸。 In the present invention, examples of the VGLL3 expression inhibitor include substances that inhibit the expression of the gene or nucleic acid shown in any of the following (a) to (d):
(a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
(b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
(c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing A nucleic acid that hybridizes with a base sequence complementary to the VGLL3 gene under stringent conditions.
　本発明において、遺伝子または核酸の発現を阻害する態様において遺伝子または核酸 (以下「標的核酸」ということがある。) とは、筋線維芽細胞特異的なマーカー分子であり、線維化を促進する分子であることが本発明者らにより発見された配列番号２、４または６記載の塩基配列で表されるVGLL3遺伝子であるか、または配列表の配列番号２、４または６に示される塩基配列を含む核酸において、１若しくは数個の塩基が欠失、置換または付加された塩基配列を含む核酸、配列表の配列番号２、４または６に示される塩基配列と同一性が９０％以上の塩基配列の核酸、若しくは配列表の配列番号２、４または６に示す塩基配列を含む核酸と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸であって、配列番号１、３または５記載のアミノ酸配列で示されるタンパク質、または該タンパク質と同じ機能を有するタンパク質をコードする塩基配列を有する核酸であれば、何れでもよい。好ましくは、配列表の配列番号２、４または６で表されるVGLL3遺伝子である。 In the present invention, in the embodiment of inhibiting the expression of a gene or nucleic acid, the gene or nucleic acid (hereinafter, may be referred to as “target nucleic acid”) is a myofibroblast-specific marker molecule and a molecule that promotes fibrosis. The VGLL3 gene represented by the nucleotide sequence of SEQ ID NO: 2, 4 or 6 discovered by the present inventors, or the nucleotide sequence shown in SEQ ID NO: 2, 4 or 6 of the sequence listing. Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2, 4 or 6 in the sequence listing. , Or a nucleic acid that hybridizes under stringent conditions with a base sequence complementary to the nucleic acid containing the base sequence shown in SEQ ID NO: 2, 4 or 6 of the sequence listing, according to SEQ ID NO: 1, 3 or 5. Any nucleic acid may be used as long as it has a protein represented by an amino acid sequence or a nucleic acid having a base sequence encoding a protein having the same function as the protein. Preferably, it is the VGLL3 gene represented by SEQ ID NO: 2, 4 or 6 in the sequence listing.
　上記 (b) において、「１個若しくは数個」とは、例えば、前記 (b) の遺伝子がVGLL3機能を有する物質をコードする範囲であればよい。「１個若しくは数個」は、VGLL3遺伝子において、例えば１～１０個、好ましくは１～７個、より好ましくは１～５個、さらに好ましくは１～３個、特に好ましくは１または２個である。 In the above (b), "one or several" may be, for example, a range in which the gene of the above (b) encodes a substance having a VGLL3 function. "1 or several" is, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 1 or 2 in the VGLL3 gene. is there.
　上記 (c) において、「同一性」とは相同性または類似性と同義である。「９０％以上」は好ましくは、９３％以上、より好ましくは９５％以上、さらに好ましくは９８％以上である。 In (c) above, "identity" is synonymous with homology or similarity. "90% or more" is preferably 93% or more, more preferably 95% or more, still more preferably 98% or more.
　上記 (d) において、「ストリンジェントな条件」は、例えば低ストリンジェントな条件、中ストリンジェントな条件、高ストリンジェントな条件のいずれでもよい。「低ストリンジェントな条件」は、例えば、5×SSC、5×デンハルト溶液、0.5％SDS、50％ホルムアミド、32℃の条件である。「中ストリンジェントな条件」は、例えば、5×SSC、5×デンハルト溶液、0.5％SDS、50％ホルムアミド、42℃の条件である。「高ストリンジェントな条件」は、例えば、5×SSC、5×デンハルト溶液、0.5％SDS、50％ホルムアミド、50℃の条件である。ストリンジェンシーの程度は、当業者であれば、例えば、温度、塩濃度、プローブの濃度および長さ、イオン強度、時間等の条件を適宜選択することで、設定可能である。「ストリンジェントな条件」は、例えば、前述したザンブルーク (Sambrook) ら編「モレキュラー・クローニング：ア・ラボラトリーマニュアル第２版 (Molecular Cloning： A Laboratory Manual 2nd Ed．)」［Cold Spring Harbor Laboratory Press (1989) ］等に記載の条件を採用することもできる。 In the above (d), the "stringent condition" may be, for example, any of a low stringent condition, a medium stringent condition, and a high stringent condition. “Low stringent conditions” are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. "Stringent conditions" are, for example, "Molecular Cloning: A Laboratory Manual 2nd Ed." Edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989). )] Etc. can also be adopted.
　本発明においてmRNAとは、標的核酸によりコードされるmRNA、または標的核酸によりコードされるタンパク質をコードするものであればいずれでもよい。好ましくはVGLL3遺伝子でコードされるmRNAである。 In the present invention, the mRNA may be any mRNA encoded by the target nucleic acid or any one encoding a protein encoded by the target nucleic acid. It is preferably an mRNA encoded by the VGLL3 gene.
　本発明の一実施態様では、VGLL3発現の阻害物質として、例えば、siRNA、shRNA、miRNA、アンチセンス、リボザイム、化合物等が挙げられる。 In one embodiment of the present invention, examples of the inhibitor of VGLL3 expression include siRNA, shRNA, miRNA, antisense, ribozyme, and compounds.
　siRNA (small interfering RNA) は、RNA干渉による遺伝子サイレンシングのために用いられる21～23塩基対の低分子２本鎖ＲＮＡである。細胞内に導入されたsiRNAは、RNA誘導サイレンシング複合体 (RISC) と結合する。この複合体はsiRNAと相補的な配列を持つmRNAに結合し切断する。これにより、配列特異的に遺伝子の発現を抑制する。 SiRNA (small interfering RNA) is a 21-23 base pair low molecular weight double-stranded RNA used for gene silencing by RNA interference. The siRNA introduced into the cell binds to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that has a sequence complementary to siRNA. As a result, gene expression is suppressed in a sequence-specific manner.
　siRNAは、センス鎖及びアンチセンス鎖オリゴヌクレオチドをDNA/RNA自動合成機でそれぞれ合成し、例えば、適当なアニーリング緩衝液中、90～95℃で約1分程度変性させた後、30～70℃で約1～8時間アニーリングさせることにより調製することができる。 For siRNA, sense strand and antisense strand oligonucleotides are synthesized by a DNA / RNA automatic synthesizer, respectively, and denatured in an appropriate annealing buffer at 90 to 95 ° C. for about 1 minute, and then 30 to 70 ° C. It can be prepared by annealing in 1 to 8 hours.
　shRNA (short hairpin RNA) は、RNA干渉による遺伝子サイレンシングのために用いられるヘアピン型のRNA配列である。shRNAは、ベクターによって細胞に導入し、U6プロモーターまたはH1プロモーターで発現させてもよいし、shRNA配列を有するオリゴヌクレオチドをDNA/RNA自動合成機で合成し、siRNAと同様の方法によりセルフアニーリングさせることによって調製してもよい。細胞内に導入されたshRNAのヘアピン構造は、siRNAへと切断され、RNA誘導サイレンシング複合体 (RISC) と結合する。この複合体はsiRNAと相補的な配列を持つmRNAに結合し切断する。これにより、配列特異的に遺伝子の発現を抑制する。 ShRNA (short hairpin RNA) is a hairpin-type RNA sequence used for gene silencing by RNA interference. The shRNA may be introduced into cells by a vector and expressed by the U6 promoter or the H1 promoter, or an oligonucleotide having the shRNA sequence may be synthesized by a DNA / RNA automatic synthesizer and self-annealed by the same method as siRNA. May be prepared by. The hairpin structure of the shRNA introduced into the cell is cleaved into siRNA and binds to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that has a sequence complementary to siRNA. As a result, gene expression is suppressed in a sequence-specific manner.
　miRNA (microRNA、マイクロRNA) は、ゲノム上にコードされ、多段階的な生成過程を経て最終的に約２０塩基の微小ＲＮＡとなる機能性核酸である。miRNAは、機能性のncRNA (non-coding RNA、非コードRNA：タンパク質に翻訳されないＲＮＡの総称) に分類されており、他の遺伝子の発現を調節するという、生命現象において重要な役割を担っている。特定の塩基配列を有するmiRNAを生体に投与することにより、VGLL3の発現を阻害することができる。 MiRNA (microRNA, microRNA) is a functional nucleic acid that is encoded on the genome and finally becomes a microRNA of about 20 bases through a multi-step production process. miRNAs are classified as functional ncRNAs (non-coding RNAs, a general term for RNAs that are not translated into proteins), and play an important role in life phenomena by regulating the expression of other genes. There is. The expression of VGLL3 can be inhibited by administering a miRNA having a specific base sequence to a living body.
　本発明のリボザイムとはDNA制限エンドヌクレアーゼと類似の機構により他の一本鎖RNA分子を特異的に切断するRNA分子を意味する。既知の手法によりRNAの核酸配列を適宜修飾することにより、RNA一本鎖中の特定の塩基配列を認識し切断するリボザイムを作製することができる (Science，239，p．１412-1416，1988)。 The ribozyme of the present invention means an RNA molecule that specifically cleaves another single-stranded RNA molecule by a mechanism similar to that of a DNA-restricted endonuclease. By appropriately modifying the nucleic acid sequence of RNA by a known method, it is possible to prepare a ribozyme that recognizes and cleaves a specific base sequence in an RNA single strand (Science, 239, p. 1412-1416, 1988). ..
　アンチセンス核酸は、標的配列に相補的な核酸である。アンチセンス核酸は、三重鎖形成による転写開始阻害、RNAポリメラーゼによって局部的に開状ループ構造が形成された部位とのハイブリッド形成による転写抑制、合成の進みつつあるＲＮＡとのハイブリッド形成による転写阻害、イントロンとエクソンとの接合点でのハイブリッド形成によるスプライシング抑制、スプライソソーム形成部位とのハイブリッド形成によるスプライシング抑制、ｍＲＮＡとのハイブリッド形成による核から細胞質への移行抑制、キャッピング部位やポリ (A) 付加部位とのハイブリッド形成によるスプライシング抑制、翻訳開始因子結合部位とのハイブリッド形成による翻訳開始抑制、開始コドン近傍のリボソーム結合部位とのハイブリッド形成による翻訳抑制、ｍＲＮＡの翻訳領域やポリソーム結合部位とのハイブリッド形成によるペプチド鎖の伸長阻止、核酸とタンパク質との相互作用部位とのハイブリッド形成による遺伝子発現抑制等により、標的遺伝子の発現を抑制することができる。 Antisense nucleic acid is a nucleic acid complementary to the target sequence. Antisense nucleic acids inhibit transcription initiation by triple-strand formation, transcription inhibition by hybrid formation with a site where an open loop structure is locally formed by RNA polymerase, and transcription inhibition by hybrid formation with RNA whose synthesis is progressing. Suppression of splicing by hybrid formation at the junction of intron and exon, suppression of splicing by hybrid formation with sprisosome formation site, suppression of transcription from nucleus to cytoplasm by hybrid formation with mRNA, capping site and poly (A) addition site Suppressing splicing by hybrid formation with, translation initiation inhibition by hybrid formation with translation initiation factor binding site, translation inhibition by hybrid formation with ribosome binding site near the initiation codon, by hybrid formation with mRNA translation region or polysome binding site The expression of the target gene can be suppressed by inhibiting the elongation of the peptide chain, suppressing the gene expression by hybridizing the interaction site between the nucleic acid and the protein, and the like.
　本発明のsiRNAとは標的核酸の発現を抑制する二本鎖RNAを指し、「RNAi剤」、「短鎖干渉性RNA」、「短鎖干渉性核酸」、「siRNA」を意味し、配列特異的なRNA干渉 (RNAi) または遺伝子サイレンシングを介して遺伝子発現またはウイルス複製を阻害または下向き調節することのできる核酸分子である。これは、RNAのみからなるものであってもよいし、DNAとRNAとの融合体であってもよい。 The siRNA of the present invention refers to a double-stranded RNA that suppresses the expression of a target nucleic acid, and means "RNAi agent", "short-chain interfering RNA", "short-chain interfering nucleic acid", "siRNA", and is sequence-specific. A nucleic acid molecule capable of inhibiting or downwardly regulating gene expression or viral replication through RNA interference (RNAi) or gene silencing. It may consist only of RNA or it may be a fusion of DNA and RNA.
　siRNA、shRNA、miRNA、リボザイムおよびアンチセンス核酸は、安定性や活性を向上させるために、種々の化学修飾を含んでいてもよい。例えば、ヌクレアーゼ等の加水分解酵素による分解を防ぐために、リン酸残基を、例えば、ホスホロチオエート (PS)、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換してもよい。また、少なくとも一部をペプチド核酸 (PNA) 等の核酸類似体により構成してもよい。 SiRNAs, shRNAs, miRNAs, ribozymes and antisense nucleic acids may contain various chemical modifications to improve stability and activity. For example, in order to prevent degradation by a hydrolase such as a nuclease, the phosphate residue may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. Further, at least a part thereof may be composed of nucleic acid analogs such as peptide nucleic acid (PNA).
　本発明のsiRNA、shRNA、miRNA、リボザイムおよびアンチセンス核酸は標的核酸 (VGLL3遺伝子) の発現を阻害することから、標的核酸の発現を調節する物質として、線維化阻害剤、ひいては線維化疾患を予防または治療する医薬組成物として利用可能である。かかるsiRNAは例えば、配列表の配列番号４０～５１のいずれかに示されるRNA配列を有するsiRNAが挙げられる。好ましくは、配列表の配列番号４６～５１のいずれかに示されるRNA配列を有するsiRNAである。 Since the siRNA, shRNA, miRNA, ribozyme and antisense nucleic acid of the present invention inhibit the expression of the target nucleic acid (VGLL3 gene), they prevent fibrosis inhibitors and thus fibrotic diseases as substances that regulate the expression of the target nucleic acid. Alternatively, it can be used as a therapeutic pharmaceutical composition. Examples of such siRNA include siRNA having an RNA sequence shown in any of SEQ ID NOs: 40 to 51 in the sequence listing. Preferably, it is siRNA having the RNA sequence shown in any of SEQ ID NOs: 46 to 51 in the sequence listing.
　本発明のアンチセンス配列を有する核酸は標的核酸 (VGLL3遺伝子) の発現を阻害することから、標的核酸の発現を調節する物質として、線維化阻害剤、ひいては線維化疾患を予防または治療する医薬組成物として利用可能である。 Since the nucleic acid having the antisense sequence of the present invention inhibits the expression of the target nucleic acid (VGLL3 gene), as a substance that regulates the expression of the target nucleic acid, a fibrosis inhibitor, and thus a pharmaceutical composition for preventing or treating a fibrotic disease. It can be used as a thing.
　本発明において、VGLL3発現の阻害物質としての化合物等とは、VGLL3遺伝子の発現を阻害・抑制する化合物等であってもよいし、VGLL3タンパク質の発現を阻害・抑制する化合物等であってもよいし、VGLL3の活性を阻害・抑制する化合物等であってもよい。前記化合物等とは、VGLL3転写因子阻害物質、VGLL3タンパク質翻訳因子阻害物質、VGLL3タンパク質分解促進物質、VGLL3タンパク質に対する特異的結合物質 (抗体、抗体断片、ペプチド、アプタマー、低分子化合物等) が挙げられる。例えば、抗体は、マウス等の動物に、VGLL3タンパク質またはその断片を抗原として免疫することによって作製することができる。市販のものであってもよい。前記抗体は、モノクローナル抗体であるこが好ましい。 In the present invention, the compound or the like as an inhibitor of VGLL3 expression may be a compound or the like that inhibits or suppresses the expression of the VGLL3 gene, or may be a compound or the like that inhibits or suppresses the expression of the VGLL3 protein. However, it may be a compound or the like that inhibits or suppresses the activity of VGLL3. Examples of the compounds include VGLL3 transcription factor inhibitors, VGLL3 protein translation factor inhibitors, VGLL3 proteolysis promoters, and specific binding substances for VGLL3 proteins (antibodies, antibody fragments, peptides, aptamers, low molecular weight compounds, etc.). .. For example, an antibody can be produced by immunizing an animal such as a mouse with the VGLL3 protein or a fragment thereof as an antigen. It may be a commercially available product. The antibody is preferably a monoclonal antibody.
　簡潔には、VGLL3の阻害物質は、例えば、VGLL3の発現をmRNAレベルまたはタンパク質レベルで抑制する物質であってもよいし、VGLL3に対するアンタゴニスト等であってもよいし、VGLL3の活性を阻害する物質であってもよい。 Briefly, the inhibitor of VGLL3 may be, for example, a substance that suppresses the expression of VGLL3 at the mRNA level or the protein level, an antagonist against VGLL3, or the like, or a substance that inhibits the activity of VGLL3. It may be.
　VGLL3に対するアンタゴニストは、VGLL3に対する特異的結合物質のうち、VGLL3を活性化させない物質、VGLL3に対するリガンドの結合を阻害する物質等が挙げられる。 Examples of antagonists to VGLL3 include substances that do not activate VGLL3 and substances that inhibit the binding of ligands to VGLL3 among specific binding substances to VGLL3.
　本発明におけるVGLL3の阻害物質は、VGLL3を介した線維化経路を阻害し、結果として線維化を抑制する物質を広く包含することができ、その意味において、VGLL3の阻害物質として、G-アクチンの重合を阻害するラトランクリンA、細胞骨格の構成分子である非筋型のミオシンIIとアクチンの会合を阻害するブレビスタチン、細胞骨格の運動に必要なミオシン軽鎖のリン酸化を制御するキナーゼであるROCKの阻害剤であるY27632、ROCKを活性化させる低分子量Gタンパク質Rhoを阻害剤するC3転移酵素 (39)、インテグリンβ1の阻害剤BTT-303343、FAK (焦点接着班キナーゼ) の阻害剤であるVS-471844を挙げることができる。 The VGLL3 inhibitor in the present invention can broadly include a substance that inhibits the VGLL3-mediated fibrosis pathway and, as a result, suppresses fibrosis, and in that sense, as an inhibitor of VGLL3, G-actin Latrinclin A, which inhibits polymerization, blevisstatin, which inhibits the association of non-muscle myosin II, which is a constituent of the cytoskeleton, and actin, and kinase, which controls the phosphorylation of the myosin light chain required for cytoskeletal movement. Y27632, an inhibitor of ROCK, C3 transferase (39), which inhibits the low molecular weight G protein Rho that activates ROCK, BTT-303343, an inhibitor of integrin β1, and FAK (focal adhesion group kinase). One VS-471844 can be mentioned.
　本発明の一実施態様では、本発明の医薬組成物には、VGLL3の核内移行の阻害物質が含まれる。実施例において証明している通り、VGLL3は、転写共役因子YAP/TAZと同様、機械的刺激によって核外から核内へと移行し、それはアクチン重合に依存しており、過剰な線維化に寄与している。したがって、VGLL3の核内移行を阻害すれば、線維化を阻害、抑制、軽減できると期待される。 In one embodiment of the present invention, the pharmaceutical composition of the present invention contains an inhibitor of VGLL3 translocation into the nucleus. As demonstrated in the examples, VGLL3, like the transcriptional conjugation factor YAP / TAZ, migrates from extranuclear to intranuclear by mechanical stimulation, which is dependent on actin polymerization and contributes to excessive fibrosis. doing. Therefore, it is expected that inhibition of nuclear translocation of VGLL3 can inhibit, suppress, or reduce fibrosis.
　本発明の一実施態様では、本発明の医薬組成物には、VGLL3とDDX5との結合の阻害物質が含まれる。実施例において証明している通り、VGLL3は、Rho/ROCKの活性を介して核移行し、過剰な線維化に寄与している。したがって、VGLL3とDDX5との結合を阻害すれば、線維化を阻害、抑制、軽減できると期待される。VGLL3とDDX5との結合の阻害物質の具体例としては、VGLL3のDDX5との結合ドメインに相当するペプチド、あるいはDDX5のVGLL3との結合ドメインに相当する阻害ペプチドが考えられる。 In one embodiment of the present invention, the pharmaceutical composition of the present invention contains an inhibitor of the binding of VGLL3 and DDX5. As demonstrated in the examples, VGLL3 translocates via Rho / ROCK activity and contributes to excessive fibrosis. Therefore, it is expected that inhibition of the binding between VGLL3 and DDX5 can inhibit, suppress, and reduce fibrosis. Specific examples of the inhibitor of the binding between VGLL3 and DDX5 include a peptide corresponding to the binding domain of VGLL3 with DDX5 and an inhibitory peptide corresponding to the binding domain of DDX5 with VGLL3.
　本明細書において、「治療」とは、(１) 線維化疾患または線維化状態の発症を遅延させる； (２) 線維化疾患または線維化状態の症状の進行、増悪または悪化を減速または停止させる； (３) 線維化疾患または線維化状態の症状の寛解をもたらす；あるいは (４) 線維化疾患または線維化状態を治癒させることを目的とする方法またはプロセスを意味する。治療は、予防的措置として疾患または状態の発症前に施してもよいし、あるいはまた、治療は、疾患の発症後に施してもよい。 As used herein, "treatment" refers to (1) delaying the onset of fibrotic disease or fibrotic state; (2) slowing or stopping the progression, exacerbation or exacerbation of symptoms of fibrotic disease or fibrotic state. (3) Brings remission of the symptoms of fibrotic disease or fibrotic state; or (4) means a method or process aimed at curing fibrotic disease or fibrotic state. Treatment may be given as a precautionary measure before the onset of the disease or condition, or treatment may be given after the onset of the disease.
　本明細書において、「予防」とは、線維化疾患または線維化状態の発症を予防することを意味する。 As used herein, "prevention" means preventing the onset of fibrotic disease or fibrotic state.
　本発明において医薬組成物とは、通常、疾患の治療もしくは予防、あるいは検査・診断のための薬剤を意味する。 In the present invention, the pharmaceutical composition usually means a drug for treating or preventing a disease, or for testing / diagnosis.
　本発明の医薬組成物は、当業者に公知の方法で製剤化することが可能である。例えば、水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、または懸濁液剤の注射剤の形で非経口的に使用できる。例えば、薬理学上許容される担体もしくは媒体、具体的には、滅菌水や生理食塩水、植物油、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、賦形剤、ベヒクル、防腐剤、結合剤などと適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することが考えられる。これら製剤における有効成分量は、指示された範囲の適当な容量が得られるように設定する。 The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injectable suspension. For example, pharmacologically acceptable carriers or vehicles, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavors, excipients, vehicles, preservatives. , It is conceivable to formulate by appropriately combining with a binder and the like and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set so as to obtain an appropriate volume in the specified range.
　注射のための無菌組成物は注射用蒸留水のようなビヒクルを用いて通常の調剤に従って処方することができる。 The sterile composition for injection can be formulated according to the usual preparation using a vehicle such as distilled water for injection.
　注射用の水溶液としては、例えば生理食塩水、ブドウ糖やその他の補助薬 (例えばD-ソルビトール、D-マンノース、D-マンニトール、塩化ナトリウム) を含む等張液が挙げられる。適当な溶解補助剤、例えばアルコール (エタノール等)、ポリアルコール (プロピレングリコール、ポリエチレングリコール等)、非イオン性界面活性剤 (ポリソルベート80 (TM)、HCO-50等) を併用してもよい。 Examples of the aqueous solution for injection include isotonic solutions containing physiological saline, glucose and other auxiliary agents (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) may be used in combination.
　油性液としてはゴマ油、大豆油があげられ、溶解補助剤として安息香酸ベンジルおよび／またはベンジルアルコールを併用してもよい。また、緩衝剤 (例えば、リン酸塩緩衝液および酢酸ナトリウム緩衝液)、無痛化剤 (例えば、塩酸プロカイン)、安定剤 (例えば、ベンジルアルコールおよびフェノール)、酸化防止剤と配合してもよい。調製された注射液は通常、適当なアンプルに充填する。 Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent. It may also be blended with a buffer (eg, phosphate buffer and sodium acetate buffer), a soothing agent (eg, procaine hydrochloride), a stabilizer (eg, benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled in a suitable ampoule.
　本発明の医薬組成物は、好ましくは非経口投与により投与される。例えば、注射剤型、経鼻投与剤型、経肺投与剤型、経皮投与型の組成物とすることができる。例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより全身または局部的に投与することができる。 The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, it can be an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type composition. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
　投与方法は、患者の年齢、症状により適宜選択することができる。ポリペプチドを含有する医薬組成物の投与量は、例えば、１回につき体重1kgあたり0.0001mgから1000mgの範囲に設定することが可能である。または、例えば、患者あたり0.001～100000mgの投与量とすることもできるが、本発明はこれらの数値に必ずしも制限されるものではない。投与量および投与方法は、患者の体重、年齢、症状などにより変動するが、当業者であればそれらの条件を考慮し適当な投与量および投与方法を設定することが可能である。 The administration method can be appropriately selected depending on the patient's age and symptoms. The dose of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose may be 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptom and the like of the patient, but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.
　本発明は別の態様として、線維化疾患を予防または治療するための方法であって、VGLL3の阻害物質を、そのような処置または予防を必要としている患者に投与することを含む方法に関する。好ましくは、VGLL3の阻害物質が、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質である、線維化疾患を予防または治療するための方法である。 The present invention, in another aspect, relates to a method for preventing or treating a fibrotic disease, comprising administering an inhibitor of VGLL3 to a patient in need of such treatment or prevention. Preferably, the inhibitor of VGLL3 is a method for preventing or treating fibrotic disease, which is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5. ..
　さらに、本発明は別の態様として、線維化疾患を予防または治療するための、VGLL3の阻害物質に関する。好ましくは、VGLL3の阻害物質が、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質である。
　本発明はさらなる別の態様として、線維化疾患を予防または治療する医薬を製造するための、VGLL3の阻害物質の使用に関する。好ましくは、VGLL3の阻害物質が、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質である、使用である。
　ここに、上記態様において、VGLL3の阻害物質には、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質およびVGLL3とDDX5との結合の阻害物質、ならびにそれらの具体的な化合物、遺伝子または核酸が包含される。 Furthermore, the present invention relates to, in another aspect, an inhibitor of VGLL3 for preventing or treating fibrotic diseases. Preferably, the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of the binding of VGLL3 to DDX5.
In yet another aspect, the invention relates to the use of an inhibitor of VGLL3 to produce a medicament for preventing or treating fibrotic disease. Preferably, the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding of VGLL3 to DDX5.
Here, in the above embodiment, the VGLL3 inhibitor includes an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3 and an inhibitor of binding between VGLL3 and DDX5, and specific compounds, genes or nucleic acids thereof. Is included.
＜核酸分子等＞
　本発明は、別の実施態様として、
　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸、
の発現を阻害する、siRNA、shRNA、miRNA、アンチセンス、リボザイムからなる群から選択される少なくとも１つの核酸分子を提供する。上記の通り、本発明のsiRNA、shRNA、miRNA、アンチセンスおよびリボザイムからなる群から選択される少なくとも１つ核酸分子は標的核酸 (VGLL3遺伝子) の発現を阻害することから、標的核酸の発現を調節する物質として、線維化阻害剤、ひいては線維化疾患を予防または治療する医薬組成物として利用可能である。 <Nucleic acid molecules, etc.>
The present invention, as another embodiment,
(a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
(b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
(c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing Nucleic acid that hybridizes under stringent conditions with a base sequence complementary to the VGLL3 gene,
Provided is at least one nucleic acid molecule selected from the group consisting of siRNA, shRNA, miRNA, antisense, and ribozyme that inhibits the expression of. As described above, at least one nucleic acid molecule selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme of the present invention inhibits the expression of the target nucleic acid (VGLL3 gene), thus regulating the expression of the target nucleic acid. As a substance to be used, it can be used as a fibrosis inhibitor, and thus as a pharmaceutical composition for preventing or treating a fibrosis disease.
　好ましくは、本発明の核酸分子は、ベクターにて発現される。前記ベクターが遺伝子治療ベクターの場合、遺伝子治療とは、多くの場合、遺伝的障害に対する治療を指すが、本発明においては、線維化疾患を予防または治療、あるいは線維化疾患の進行を抑止するための治療を意味する。本発明において、遺伝子治療は、線維化疾患を有する患者の細胞内へインビボでの遺伝子のコピー挿入を含んでもよい。遺伝子治療はまた、遺伝子を停止させることを含んでもよい。遺伝子組換えがまた、エキソビボ遺伝子治療において使用されてもよい。例えば、ヒト幹細胞、免疫細胞または癌細胞は、種々の用途のために遺伝的に修飾されてもよい。細胞は、分化、分化転換または再プログラミングを誘導するように修飾される。また、細胞は治療タンパク質を送達するビヒクルとして役立つように修飾されてもよい。 Preferably, the nucleic acid molecule of the present invention is expressed in a vector. When the vector is a gene therapy vector, gene therapy often refers to treatment for a genetic disorder, but in the present invention, in order to prevent or treat a fibrotic disease or suppress the progression of the fibrotic disease. Means the treatment of. In the present invention, gene therapy may include in vivo copy insertion of a gene into the cell of a patient with a fibrotic disease. Gene therapy may also include stopping the gene. Genetic recombination may also be used in exobibo gene therapy. For example, human stem cells, immune cells or cancer cells may be genetically modified for a variety of uses. Cells are modified to induce differentiation, transdifferentiation or reprogramming. The cells may also be modified to serve as vehicles that deliver therapeutic proteins.
　本発明はさらに、本発明の前記核酸分子を発現するベクターを含む細胞を提供する。本発明の細胞は、線維化疾患を予防または治療あるいは線維化疾患の進行を抑止するための細胞療法に利用することができる。典型的には、本発明の細胞は、本発明のベクターを含む心臓、肺、腎臓、肝臓または腸由来の筋線維芽細胞である。本明細書の実施例１０にて、VGLL3ノックアウトマウスにおけるMI後の心臓線維化の減弱が、実施例１１においてVGLL3ノックアウトマウスにおける片側尿管閉塞後の腎臓線維化の減弱が証明されており、VGLL3の発現を阻害する効果が確認できている。 The present invention further provides a cell containing the vector expressing the nucleic acid molecule of the present invention. The cells of the present invention can be used for cell therapy for preventing or treating fibrotic diseases or suppressing the progression of fibrotic diseases. Typically, the cells of the invention are myofibroblasts from the heart, lung, kidney, liver or intestine containing the vectors of the invention. Example 10 of the present specification demonstrates the attenuation of cardiac fibrosis after MI in VGLL3 knockout mice, and Example 11 demonstrates the attenuation of renal fibrosis after unilateral ureteral obstruction in VGLL3 knockout mice. The effect of inhibiting the expression of is confirmed.
＜スクリーニング方法＞
　本発明は別の実施態様において、VGLL3の阻害物質の候補物質をスクリーニングする方法を提供する。より具体的には、被験物質の存在下で、細胞中のVGLL3の発現量を測定する工程と、前記発現量が、前記被験物質の非存在下における発現量と比較して低下していた場合に、前記被験物質はVGLL3阻害作用を有する候補物質であると判断する工程とを備える、VGLL3の阻害物質の候補物質のスクリーニング方法を提供する。また、上述または実施例において後述するとおり、VGLL3の阻害物質は線維化疾患を予防または治療に有効であることから、線維化疾患の予防または治療に有効な候補物質のスクリーニング方法を提供する。具体的には、被験物質の存在下で、細胞中のVGLL3の発現量を測定する工程と、前記発現量が、前記被験物質の非存在下における発現量と比較して低下していた場合に、前記被験物質は線維化疾患の予防または治療に有効な候補物質であると判断する工程とを備える、線維化疾患の予防または治療に有効な候補物質のスクリーニング方法を提供する。 <Screening method>
The present invention provides, in another embodiment, a method of screening for candidate substances for inhibitors of VGLL3. More specifically, the step of measuring the expression level of VGLL3 in cells in the presence of the test substance, and the case where the expression level is lower than the expression level in the absence of the test substance. The present invention provides a method for screening a candidate substance for a VGLL3 inhibitor, which comprises a step of determining that the test substance is a candidate substance having a VGLL3 inhibitory action. Further, as described above or described later in Examples, since an inhibitor of VGLL3 is effective in preventing or treating fibrotic diseases, a screening method for candidate substances effective in preventing or treating fibrotic diseases is provided. Specifically, in the step of measuring the expression level of VGLL3 in cells in the presence of the test substance, and when the expression level is lower than the expression level in the absence of the test substance. Provided is a method for screening a candidate substance effective for prevention or treatment of fibrotic disease, which comprises a step of determining that the test substance is an effective candidate substance for prevention or treatment of fibrotic disease.
　細胞は、培養細胞である限り制限されない。細胞は好ましくは筋線維芽細胞であり、ヒトの筋線維芽細胞であり、より好ましくは心臓、肺、腎臓、肝臓または腸由来の筋線維芽細胞である。細胞の継代条件は、細胞種に応じて適宜選択することができる。 The cells are not limited as long as they are cultured cells. The cells are preferably myofibroblasts, human myofibroblasts, and more preferably heart, lung, kidney, liver or intestinal myofibroblasts. The cell passage conditions can be appropriately selected depending on the cell type.
　被験物質とは、VGLL3の阻害作用を有する候補物質であるか否かを評価する対象となりうる物質であり、特に制限されない。例えば、化合物、タンパク質、ペプチド、核酸、脂質、糖質、糖脂質、糖タンパク、金属等を挙げることができる。被験物質の投与方法も特に制限されない。 The test substance is a substance that can be evaluated as to whether or not it is a candidate substance having an inhibitory effect on VGLL3, and is not particularly limited. For example, compounds, proteins, peptides, nucleic acids, lipids, sugars, glycolipids, glycoproteins, metals and the like can be mentioned. The method of administering the test substance is also not particularly limited.
　VGLL3の発現量を反映する測定値は、特に制限されない。例えば、VGLL3遺伝子から発現されるmRNA量 (コピー数またはリード数等) の測定値、タンパク質の測定値、及びレポーターアッセイによる化学発光強度等であり得る。 The measured value that reflects the expression level of VGLL3 is not particularly limited. For example, it may be a measured value of the amount of mRNA expressed from the VGLL3 gene (number of copies, number of reads, etc.), a measured value of a protein, and chemiluminescence intensity by a reporter assay.
　mRNAの測定値は、マイクロアレイ、定量的RT-PCR法、またはRNA-Seq法等の公知の方法を使用して取得することができる。 The measured value of mRNA can be obtained by using a known method such as microarray, quantitative RT-PCR method, or RNA-Seq method.
　タンパク質の測定値は、プロテインチップ、ELISA法、ウエスタンブロッティング法等によって測定することができる。 The measured value of protein can be measured by protein chip, ELISA method, Western blotting method, etc.
　さらに、転写調節領域の活性化又は不活性化はレポーターアッセイによって検出することができる。レポーターとしては、不安定型トゲオキヒオドシエビルシフェラーゼ、ホタルルシフェラーゼ、ウミシイタケルシフェラーゼ、GFP (Green Fluorescent Protein)、β－ガラクトシダーゼ等を挙げることができる。レポーターアッセイは、公知の方法に従って行うことができる。レポーターアッセイを行う場合には、例えば、レポーター遺伝子の上流に、VGLL3遺伝子の転写調節領域を挿入したレポータープラスミドを構築し、このレポータープラスミドを前記細胞に導入し、レポータープラスミドを保有するレポーターアッセイ用細胞を作製する。細胞へのレポータープラスミドの導入は、リポフェクション、エレクトロポレーション、リン酸カルシウム法等で行うことができる。 Furthermore, activation or inactivation of the transcriptional regulatory region can be detected by a reporter assay. Examples of the reporter include unstable spiny oyster shrimp luciferase, firefly luciferase, sea urchin shiitake mushroom, GFP (Green Fluorescent Protein), β-galactosidase and the like. The reporter assay can be performed according to a known method. When performing a reporter assay, for example, a reporter plasmid in which a transcriptional regulatory region of the VGLL3 gene is inserted is constructed upstream of the reporter gene, this reporter plasmid is introduced into the cell, and a reporter assay cell carrying the reporter plasmid is carried out. To make. The reporter plasmid can be introduced into cells by lipofection, electroporation, calcium phosphate method or the like.
　細胞へのレポータープラスミドの導入は、一過性であってもステイブルであってもよい。 The introduction of the reporter plasmid into the cells may be transient or stable.
　レポーター遺伝子がルシフェラーゼである場合には、ルシフェラーゼによる基質からの化学発光を測定することによりVGLL3遺伝子の発現を反映する測定値を得ることができる。レポーター遺伝子がＧＦＰである場合には、蛍光強度を測定することによりVGLL3遺伝子の発現を反映する測定値を得ることができる。レポーター遺伝子がβ－ガラクトシダーゼである場合には、基質であるフルオレセインジ-β－Ｄ－グルコピラノシドに由来する蛍光強度を測定することによりVGLL3遺伝子の発現を反映する測定値を得ることができる。レポーターアッセイとして好ましくはルシフェラーゼを使用するルシフェラーゼアッセイである。 When the reporter gene is luciferase, a measured value that reflects the expression of the VGLL3 gene can be obtained by measuring the chemiluminescence from the substrate by luciferase. When the reporter gene is GFP, a measured value that reflects the expression of the VGLL3 gene can be obtained by measuring the fluorescence intensity. When the reporter gene is β-galactosidase, a measured value reflecting the expression of the VGLL3 gene can be obtained by measuring the fluorescence intensity derived from the substrate fluorescein di-β-D-glucopyranoside. The reporter assay is preferably a luciferase assay using luciferase.
　VGLL3の阻害物質は、効果的な線維化疾患の予防剤又は治療剤となる。 Inhibitors of VGLL3 are effective preventive or therapeutic agents for fibrotic diseases.
　本発明はさらなる別の実施態様において、配列表の配列番号１、３、５、７、９または１１に示されるアミノ酸配列を有するVGLL3タンパク質、または配列表の配列番号１、３、５、７、９または１１に示されるアミノ酸配列を有するVGLL3タンパク質において１個または数個のアミノ酸残基の置換、欠失、付加を有する変異VGLL3タンパク質であって、該VGLL3タンパク質と同等の活性を有する該変異VGLL3タンパク質を用いる、VGLL3タンパク質の機能を阻害する物質のスクリーニング方法、より具体的には、
　(１) 配列表の配列番号１、３、５、７、９または１１に示されるアミノ酸配列を有するVGLL3タンパク質、または該VGLL3タンパク質において１個または数個のアミノ酸残基の置換、欠失、付加を有する変異VGLL3タンパク質であって、該VGLL3タンパク質と同等の活性を有する該変異VGLL3タンパク質、または該VGLL3タンパク質もしくは該変異VGLL3タンパク質を発現する細胞に被験物質を接触させ、
　(２) 該被験物質が該VGLL3タンパク質または該変異VGLL3タンパク質の機能を阻害するか否かを判定することを含む、VGLL3タンパク質の機能を阻害する物質をスクリーニングする方法を提供する。
　配列表の配列番号１、３および５は、ヒトVGLL3タンパク質のアミノ酸配列であり、配列番号７、９および１１はマウスVGLL3タンパク質のアミノ酸配列である。本発明におけるVGLL3の阻害物質をスクリーニングする方法では、ヒトVGLL3タンパク質、マウスVGLL3タンパク質のいずれも使用することができる。好ましくは、ヒトVGLL3タンパク質である。 In yet another embodiment of the invention, the VGLL3 protein having the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9 or 11 of the Sequence Listing, or SEQ ID NO: 1, 3, 5, 7, of the Sequence Listing, A mutant VGLL3 protein having the substitution, deletion, or addition of one or several amino acid residues in the VGLL3 protein having the amino acid sequence shown in 9 or 11, and having the same activity as the VGLL3 protein. A method for screening a substance that inhibits the function of VGLL3 protein using a protein, more specifically,
(1) Substitution, deletion, or addition of one or several amino acid residues in the VGLL3 protein having the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 in the sequence listing, or in the VGLL3 protein. The test substance is brought into contact with the mutant VGLL3 protein having the same activity as the VGLL3 protein, or the VGLL3 protein or cells expressing the mutant VGLL3 protein.
(2) Provided is a method for screening a substance that inhibits the function of the VGLL3 protein, which comprises determining whether or not the test substance inhibits the function of the VGLL3 protein or the mutant VGLL3 protein.
SEQ ID NOs: 1, 3 and 5 in the Sequence Listing are amino acid sequences of human VGLL3 protein, and SEQ ID NOs: 7, 9 and 11 are amino acid sequences of mouse VGLL3 protein. In the method for screening VGLL3 inhibitors in the present invention, both human VGLL3 protein and mouse VGLL3 protein can be used. Preferably, it is a human VGLL3 protein.
　標的タンパク質はマーカータンパク質とインフレームで融合して発現させることで、発現の確認、細胞内局在の確認、精製等が可能になる。マーカータンパク質としては、例えば、FLAG エピトープ、ヘキサヒスチジンタグ (Hexa-Histidine tag)、ヘマグルチニンタグ、mycエピトープ等がある。また、マーカータンパク質と標的タンパク質のアミノ酸配列の間にエンテロキナーゼ、ファクターXa、トロンビンなどの、プロテアーゼが認識する特異的なアミノ酸配列を挿入することにより、マーカータンパク質部分をこれらのプロテアーゼにより切断除去することが可能である。例えば、ムスカリンアセチルコリン受容体とヘキサヒスチジンタグとをトロンビン認識配列で連結した報告がある (J. Biochem., 120, p.1232-1238, 1996)。これらマーカータンパク質をコードする塩基配列と標的核酸とをインフレームで融合させたものをベクターに組み込んだ形質転換細胞は、標的核酸または標的タンパク質の機能を調節する物質のスクリーニングに用いることができる。 By fusing the target protein with the marker protein in frame and expressing it, it is possible to confirm the expression, confirm the intracellular localization, purify, etc. Examples of the marker protein include FLAG epitope, hexahistidine tag (Hexa-Histidine tag), hemagglutinin tag, myc epitope and the like. In addition, by inserting a specific amino acid sequence recognized by a protease such as enterokinase, factor Xa, and thrombin between the amino acid sequences of the marker protein and the target protein, the marker protein portion is cleaved and removed by these proteases. Is possible. For example, there is a report in which a muscarinic acetylcholine receptor and a hexahistidine tag are linked by a thrombin recognition sequence (J. Biochem., 120, p.1232-1238, 1996). Transformed cells in which a base sequence encoding these marker proteins and a target nucleic acid are fused in frame and incorporated into a vector can be used for screening a target nucleic acid or a substance that regulates the function of the target protein.
　本発明のスクリーニング方法は、さらに具体的には、被験物質の存在下で筋線維芽細胞を培養する工程と、培養した該筋線維芽細胞におけるVGLL3遺伝子のmRNAまたはVGLL3タンパク質の発現量を定量する工程と、定量した該VGLL3遺伝子のmRNAまたは該VGLL3タンパク質の発現量が、該被験物質非存在下における発現量と比較して低下していた場合に、該被験物質は線維化疾患の予防または治療剤であると判定する工程と、を備える、線維化疾患の予防または治療剤のスクリーニング方法を提供する。筋線維芽細胞は好ましくはヒトの筋線維芽細胞であり、より好ましくはヒト心臓、ヒト肺、ヒト腎臓、ヒト肝臓またはヒト腸由来の筋線維芽細胞である。 More specifically, the screening method of the present invention quantifies the step of culturing myofibroblasts in the presence of a test substance and the expression level of VGLL3 gene mRNA or VGLL3 protein in the cultured myofibroblasts. The test substance is used for the prevention or treatment of fibrotic disease when the step and the quantified expression level of the VGLL3 gene mRNA or the VGLL3 protein are reduced as compared with the expression level in the absence of the test substance. Provided is a method for screening a prophylactic or therapeutic agent for a fibrotic disease, which comprises a step of determining that the agent is an agent. Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
＜バイオマーカー等＞
　本発明は別の態様として、線維化の進行度を判定する方法であって、
 (a10) 被検者の筋線維芽細胞のVGLL3の量 (被検バイオマーカー量) を測定する工程、
 (b10) 被検バイオマーカー量と、基準の筋線維芽細胞のVGLL3の量 (対照バイオマーカー量) とを比較する工程、および
 (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、線維化の進行度が高いと判定する方法、を提供する。
　基準の筋線維芽細胞のVGLL3の量は、(a10) 工程より前に測定された同一被検者の筋線維芽細胞のVGLL3の量であってもよく、健常者の筋線維芽細胞のVGLL3の量であってもよい。
　これに関連し、本発明はさらに別の態様として、線維化の進行度を判定することができる、筋線維芽細胞のVGLL3であるバイオマーカーを提供する。さらに、本発明は、線維化の進行度を判定することができるバイオマーカーとしての筋線維芽細胞VGLL3の使用を提供する。 <Biomarkers, etc.>
Another aspect of the present invention is a method for determining the degree of fibrosis.
(a10) Step of measuring the amount of VGLL3 (test biomarker amount) in myofibroblasts of a subject,
(b10) A step of comparing the amount of the test biomarker with the amount of VGLL3 in the reference myofibroblast (control biomarker amount), and (c10) when the test biomarker amount is larger than the control biomarker amount. To provide a method for determining a subject to have a high degree of fibrosis.
The reference amount of VGLL3 in myofibroblasts may be the amount of VGLL3 in myofibroblasts of the same subject measured prior to step (a10), and VGLL3 in healthy subjects. May be the amount of.
In this regard, the present invention provides, in yet another aspect, a biomarker that is VGLL3 of myofibroblasts, which can determine the degree of fibrosis progression. Furthermore, the present invention provides the use of myofibroblast VGLL3 as a biomarker capable of determining the degree of fibrosis progression.
　本発明者らは、VGLL3が筋線維芽細胞特異的なマーカー分子であり、線維化を促進する分子であり、それが線維化疾患、特に重篤な線維化疾患に関連していることを見出した。本発明の方法、バイオマーカーおよびVGLL3の使用により、線維化の進行度を予測することが可能となり、線維化疾患の予防または治療にとって有益である。線維化の進行度が高いと、皮膚や内臓に膠原線維 (コラーゲン) などの細胞外基質と呼ばれる物質が高度に増加し、その結果、皮膚や内臓が硬くなり、ひいては線維化疾患のおそれを招く。 We have found that VGLL3 is a myofibroblast-specific marker molecule and a molecule that promotes fibrosis, which is associated with fibrotic diseases, especially severe fibrotic diseases. It was. The use of the methods, biomarkers and VGLL3 of the present invention makes it possible to predict the progression of fibrosis and is beneficial for the prevention or treatment of fibrotic diseases. When the degree of fibrosis is high, substances called extracellular matrix such as collagen fibers are highly increased in the skin and internal organs, and as a result, the skin and internal organs become hard, which leads to a risk of fibrotic disease. ..
　筋線維芽細胞のVGLL3の量は、mRNA量であってもタンパク質量であっても良い。VGLL3に対する抗体があれば、免疫学的手法により測定することができる。例えば、当業者に周知のELISA法によって測定することができる。VGLL3に対するmRNAの検出を、例えば、超好感度なin situ ハイブリダイゼーションの一種であるRNAｓcｏｐｅ法によって行うことができる。RNAscope法では、目的分子のmRNAが発現していれば、その分子に特異的なプローブを用いる事により、ドット状に検出することができる。筋線維芽細胞からRNAを回収し、リアルタイムRT-PCR法により測定することもできる。 The amount of VGLL3 in myofibroblasts may be the amount of mRNA or the amount of protein. If there is an antibody against VGLL3, it can be measured by immunological methods. For example, it can be measured by an ELISA method well known to those skilled in the art. The detection of mRNA for VGLL3 can be performed, for example, by the RNA scoop method, which is a kind of super-sensitive in situ hybridization. In the RNAscope method, if the mRNA of the target molecule is expressed, it can be detected in dots by using a probe specific to that molecule. RNA can also be recovered from myofibroblasts and measured by real-time RT-PCR.
　被検者の筋線維芽細胞は生検 (バイオプシー) によって採取し、VGLL3量を測定することもできる。筋線維芽細胞は好ましくはヒトの筋線維芽細胞であり、より好ましくはヒト心臓、ヒト肺、ヒト腎臓、ヒト肝臓またはヒト腸由来の筋線維芽細胞である。 The myofibroblasts of the subject can be collected by biopsy and the amount of VGLL3 can be measured. Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
＜筋線維芽細胞の検出用キット＞
　実施態様において、本発明は、VGLL3のcDNAを増幅するためのプライマーセット、VGLL3のmRNAに特異的にハイブリダイズするプローブ、または、VGLL3タンパク質に対する特異的結合物質を備える、筋線維芽細胞の検出用キットを提供する。 <Myofibroblast detection kit>
In embodiments, the present invention comprises a primer set for amplifying VGLL3 cDNA, a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding agent for VGLL3 protein for detection of myofibroblasts. Provide a kit.
　実施例において後述するように、発明者らは、VGLL3が生体内の正常な組織においては妊娠時の胎盤にのみ多く発現し、すなわち正常時の生体の心臓、肝臓、腎臓にはほとんど発現せず、それぞれの臓器が線維化した時にのみ発現が顕著に増加することを明らかにした。また、VGLL3の発現が線維化を実行する筋線維芽細胞に特異的であることを明らかにした。したがって、VGLL3は、新規の筋線維芽細胞特異的マーカータンパク質である。 As will be described later in the examples, the inventors have found that VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in the living body, that is, hardly expressed in the heart, liver and kidney of the living body in the normal state. , It was clarified that the expression is significantly increased only when each organ is fibrotic. We also found that VGLL3 expression is specific for myofibroblasts that perform fibrosis. Therefore, VGLL3 is a novel myofibroblast-specific marker protein.
　本実施態様のキットにおいて、プライマーセットとしては、検出対象の動物種のVGLL3遺伝子のcDNAを増幅することができるものであれば特に限定されない。また、VGLL3のmRNAに特異的にハイブリダイズするプローブとしては、VGLL3遺伝子のmRNAに特異的にハイブリダイズするものであれば特に限定されない。プローブは、担体上に固定されてDNAマイクロアレイ等を構成していてもよい。また、特異的結合物質については上述したものと同様である。特異的結合物質は、担体上に固定されてプロテインチップ等を構成していてもよい。 In the kit of this embodiment, the primer set is not particularly limited as long as it can amplify the cDNA of the VGLL3 gene of the animal species to be detected. The probe that specifically hybridizes to VGLL3 mRNA is not particularly limited as long as it specifically hybridizes to VGLL3 gene mRNA. The probe may be immobilized on a carrier to form a DNA microarray or the like. Further, the specific binding substance is the same as that described above. The specific binding substance may be immobilized on a carrier to form a protein chip or the like.
　線維化の実行細胞である筋線維芽細胞のマーカーとして、従来、α-SMA、ペリオスチン等のタンパク質が用いられてきた。しかしながら、これらは全て細胞内タンパク質であった。すなわち、従来、筋線維芽細胞に特異的に発現する細胞膜タンパク質は知られていなかった。 Conventionally, proteins such as α-SMA and periostin have been used as markers for myofibroblasts, which are cells that execute fibrosis. However, these were all intracellular proteins. That is, conventionally, a cell membrane protein specifically expressed in myofibroblasts has not been known.
　これに対し、実施例において後述するように、VGLL3は、起源細胞には発現せず、筋線維芽細胞に分化すると発現が上昇する細胞膜タンパク質である。細胞膜タンパク質であることから、例えば、VGLL3に特異的な抗体等を蛍光または放射性同位元素で標識することにより、筋線維芽細胞を侵襲的または非侵襲的に可視化することが可能となる。筋線維芽細胞は好ましくはヒトの筋線維芽細胞であり、より好ましくはヒト心臓、ヒト肺、ヒト腎臓、ヒト肝臓またはヒト腸由来の筋線維芽細胞である。 On the other hand, as will be described later in Examples, VGLL3 is a cell membrane protein that is not expressed in the originating cell but whose expression increases when it differentiates into myofibroblasts. Since it is a cell membrane protein, for example, by labeling an antibody specific for VGLL3 with a fluorescent or radioisotope, myofibroblasts can be visualized invasively or non-invasively. Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
＜遺伝子治療＞
　本発明は、VGLL3が、線維化を促進する分子であり、線維化疾患、特に重篤な線維化疾患に関連していることを見出した。VGLL3は、胎盤以外の正常細胞では発現が認められないので、その発現はノックアウトすることで、線維化の防止、予防、回避が可能と考えられる。ここに、本発明は、VGLL3の破壊された遺伝子を有する細胞、臓器および／または組織に関する。例えば、破壊とは、（核酸レベルまたはタンパク質レベルで）少なくとも約５０％、６０％、７０％、８０％、９０％、９９％または１００％の相同性を示す遺伝子となることが想定される。遺伝子抑制もまた、可能である。例えば、遺伝子発現は、ノックアウト、遺伝子のプロモーター変更、および／または干渉ＲＮＡを投与すること（ノックダウン）によって低減し得る。特に、筋線維芽細胞におけるVGLL3発現をノックアウトするのが、好ましい。筋線維芽細胞は好ましくはヒトの筋線維芽細胞であり、より好ましくはヒト心臓、ヒト肺、ヒト腎臓、ヒト肝臓またはヒト腸由来の筋線維芽細胞である。 <Gene therapy>
The present invention has found that VGLL3 is a molecule that promotes fibrosis and is associated with fibrotic diseases, especially severe fibrotic diseases. Since VGLL3 is not expressed in normal cells other than placenta, it is considered possible to prevent, prevent, and avoid fibrosis by knocking out its expression. Here, the present invention relates to cells, organs and / or tissues carrying a disrupted gene of VGLL3. For example, disruption is expected to be a gene that exhibits at least about 50%, 60%, 70%, 80%, 90%, 99% or 100% homology (at the nucleic acid or protein level). Gene suppression is also possible. For example, gene expression can be reduced by knockout, promoter modification of the gene, and / or administration of interfering RNA (knockdown). In particular, it is preferable to knock out VGLL3 expression in myofibroblasts. Myofibroblasts are preferably human myofibroblasts, more preferably human heart, human lung, human kidney, human liver or human intestinal myofibroblasts.
　非ヒト動物中の１つまたは複数の遺伝子は、当該分野で公知の任意の方法を使用してノックアウトし得る。例えば、非ヒト動物のゲノムからVGLL3の遺伝子を欠失させることを含む。ノックアウトすることは、非ヒト動物からVGLL3遺伝子配列の全てまたは一部を除去することもまた含み得る。ノックアウトは、非ヒト動物中の任意の細胞、臓器および／または組織において実施され得る。例えば、ノックアウトは、全身ノックアウトであり得る。例えば、VGLL3の遺伝子の発現が、非ヒト動物の全ての細胞において低減される。ノックアウトはまた、非ヒト動物の１つまたは複数の細胞、組織および／または臓器に対して特異的であり得る。 One or more genes in non-human animals can be knocked out using any method known in the art. For example, it involves deleting the gene for VGLL3 from the genome of a non-human animal. Knockout can also include removing all or part of the VGLL3 gene sequence from non-human animals. Knockout can be performed in any cell, organ and / or tissue in a non-human animal. For example, the knockout can be a whole body knockout. For example, the expression of the VGLL3 gene is reduced in all cells of non-human animals. Knockouts can also be specific for one or more cells, tissues and / or organs of non-human animals.
　ノックアウトテクノロジーの任意の組合せが可能である。例えば、組織特異的ノックアウトは、誘導性テクノロジーと組み合わせ、組織特異的な誘導性ノックアウトを創出し得る。さらに、他の系、例えば発生特異的プロモーターが、組織特異的プロモーターおよび／または誘導性ノックアウトと組み合わせて使用され得る。ノックアウトテクノロジーは、遺伝子編集もまた含み得る。例えば、遺伝子編集は、ＣＲＩＳＰＲ関連タンパク質（Ｃａｓタンパク質、例えばＣａｓ９）、ジンクフィンガーヌクレアーゼ（ＺＦＮ）、転写アクチベーター様エフェクターヌクレアーゼ（ＴＡＬＥＮ）およびメガヌクレアーゼが含まれるヌクレアーゼを使用して実施され得る。ヌクレアーゼは、天然に存在するヌクレアーゼ、遺伝子改変された、および／または組換えであり得る。例えば、ＣＲＩＳＰＲ／ｃａｓ系は、遺伝子編集系として適切であり得る。 Any combination of knockout technology is possible. For example, tissue-specific knockouts can be combined with inducible technology to create tissue-specific inducible knockouts. In addition, other systems, such as development-specific promoters, can be used in combination with tissue-specific promoters and / or inducible knockouts. Knockout technology can also include gene editing. For example, gene editing can be performed using nucleases that include CRISPR-related proteins (Cas proteins such as Cas9), zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and meganucleases. The nuclease can be a naturally occurring nuclease, genetically modified, and / or recombinant. For example, the CRISPR / cas system may be suitable as a gene editing system.
　以下、本発明を実施例により、詳細に説明するが、これらは本発明の範囲を限定するものでなく、単なる例示であることに留意すべきである。 Hereinafter, the present invention will be described in detail by way of examples, but it should be noted that these do not limit the scope of the present invention and are merely examples.
　本実施例で使用する試薬などは以下の会社より購入した。
ソムノペンチル (共立製薬株式会社) 
10% 中性緩衝ホルマリン液 (和光純薬工業株式会社) 
Tissue-TekTM O.C.TTM Compound (Sakura Finetek) 
FluorSaveTM Reagent (Calbiochem) 
Red Blood Cell Lysis Buffer (Roche) 
II型コラゲナーゼ (Worthington Biochemical) 
エラスターゼ (Worthington Biochemical) 
Isogen (アイソジェン) (ニッポンジーン) 
RNeasy Plus Mini Kit (Qiagen) 
High Capacity cDNA Reverse Transcription Kit (Applied Biosystems)  
Luna Universal qPCR Master Mix (New England BioLabs) 
パラホルムアルデヒド (nacalai tesque) 
トリプシン (Sigma-Aldrich) 
EDTA (和光純薬工業株式会社) 
ダルベッコ改変イーグル培地 (Gibco) 
ウシ胎仔血清 (Tissue Culture Biologicals) 
ペニシリン／ストレプトマイシン (nacalai tesque) 
ポリ-L-リジン (Sigma-Aldrich) 
X-tremeGENE9 (Roche)
抗－VGLL3抗体 (Human Atlas) (免疫組織染色用) 
抗－VGLL3抗体 (abcam) (免疫細胞染色用) 
抗－αSMA 抗体 (abcam)
抗－CD68 抗体 (AbD Serotec) 
抗－TNNI3 抗体 (abcam)
抗－FLAG 抗体 (Novus Biologicals) 
Cy3-コンジュゲート化 抗－αSMA 抗体 (Sigma-Aldrich) 
Alexa Fluor 488TM-コンジュゲート化 ヤギ 抗－ウサギ IgG (Invitrogen) 
Alexa Fluor 488TM-コンジュゲート化 ロバ 抗－ウサギ IgG (Invitrogen) 
Alexa Fluor 594TM-コンジュゲート化 ヤギ 抗－ウサギ IgG (Invitrogen) 
Alexa Fluor 488TM-コンジュゲート化 ヤギ 抗－ラット IgG (Invitrogen) 
Alexa Fluor 594TM-コンジュゲート化 ロバ 抗－ヤギ IgG (Jackson Immunoresearch) 
Alexa Fluor 546TM-コンジュゲート化 ヤギ 抗－マウス IgG (Invitrogen) 
APC-コンジュゲート化 抗－PDGFRα 抗体 (BioLegend) 
CD45 マイクロビーズ (Miltenyi Biotec) 
APC マイクロビーズ (Miltenyi Biotec) 
DAPI 溶液 (Dojindo) 
CellTrace Far Red (Thermo Fisher Scientific) 
RNAscopeTM Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) 
RNAscopeTM 2.5HD Reagent Kit-RED (Advanced Cell Diagnostics) 
HybEZTMオーブン (Advanced Cell Diagnostics) (Advanced Cell Diagnostics) 
RNAscopeTM プローブ-C1 マウス VGLL3 (Advanced Cell Diagnostics) 
RNAscopeTM プローブ-C1 human VGLL3 (Advanced Cell Diagnostics) 
RNAscopeTM プローブ-C2 マウス Periostin (Advanced Cell Diagnostics) 
TSA Plus Cyanine 3/Cyanine 5 System (Perkin Elmer)
DMSO (和光純薬工業株式会社) 
Latrunculin A (abcam) 
Blebbistatin (和光純薬工業株式会社) 
Y27632 (和光純薬工業株式会社) 
C3 トランスフェラーゼ (Cytoskeleton) 
BTT-3033 (TOCRIS) 
VS-4718 (Chemie Tek) 
パラホルムアルデヒド (nacalai tesque) 
トリトン X-100 (ポリオキシエチレン (10) オクチルフェニル エーテル) (和光純薬工業株式会社) 
BSA (Sigma-Aldrich) 
VGLL3に対するsiRNA #1、#2 (Ambion) 
Silencer Select ネガティブ対照 no. 1 siRNA (Ambion)  The reagents used in this example were purchased from the following companies.
Somnopentil (Kyoritsu Seiyaku Co., Ltd.)
10% Neutral Buffered Formalin Solution (Wako Pure Chemical Industries, Ltd.)
Tissue-Tek TM OCTTM Compound (Sakura Finetek)
FluorSave TM Reagent (Calbiochem)
Red Blood Cell Lysis Buffer (Roche)
Type II collagenase (Worthington Biochemical)
Elastase (Worthington Biochemical)
Isogen (Nippon Gene)
RNeasy Plus Mini Kit (Qiagen)
High Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
Luna Universal qPCR Master Mix (New England BioLabs)
Paraformaldehyde (nacalai tesque)
Trypsin (Sigma-Aldrich)
EDTA (Wako Pure Chemical Industries, Ltd.)
Dulbecco's Modified Eagle's Medium (Gibco)
Fetal bovine serum (Tissue Culture Biologicals)
Penicillin / streptomycin (nacalai tesque)
Poly-L-lysine (Sigma-Aldrich)
X-tremeGENE9 (Roche)
Anti-VGLL3 antibody (Human Atlas) (for immunohistochemical staining)
Anti-VGLL3 antibody (abcam) (for immune cell staining)
Anti-αSMA antibody (abcam)
Anti-CD68 antibody (AbD Serotec)
Anti-TNNI3 antibody (abcam)
Anti-FLAG antibody (Novus Biologicals)
Cy3-conjugated anti-αSMA antibody (Sigma-Aldrich)
Alexa Fluor 488 TM -Conjugated Goat Anti-Rabbit IgG (Invitrogen)
Alexa Fluor 488 TM -Conjugated Donkey Anti-Rabbit IgG (Invitrogen)
Alexa Fluor 594 TM -Conjugated Goat Anti-Rabbit IgG (Invitrogen)
Alexa Fluor 488 TM -Conjugated Goat Anti-Rat IgG (Invitrogen)
Alexa Fluor 594 TM -Conjugated Donkey Anti-Goat IgG (Jackson Immunoresearch)
Alexa Fluor 546 TM -Conjugated Goat Anti-Mouse IgG (Invitrogen)
APC-conjugated anti-PDGFRα antibody (BioLegend)
CD45 Microbeads (Miltenyi Biotec)
APC Microbeads (Miltenyi Biotec)
DAPI solution (Dojindo)
CellTrace Far Red (Thermo Fisher Scientific)
RNAscope TM Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics)
RNAscope TM 2.5HD Reagent Kit-RED (Advanced Cell Diagnostics)
HybEZTM Oven (Advanced Cell Diagnostics) (Advanced Cell Diagnostics)
RNAscope TM Probe-C1 Mouse VGLL3 (Advanced Cell Diagnostics)
RNAscope TM Probe-C1 human VGLL3 (Advanced Cell Diagnostics)
RNAscope TM Probe-C2 Mouse Periostin (Advanced Cell Diagnostics)
TSA Plus Cyanine 3 / Cyanine 5 System (Perkin Elmer)
DMSO (Wako Pure Chemical Industries, Ltd.)
Latrunculin A (abcam)
Blebbistatin (Wako Pure Chemical Industries, Ltd.)
Y27632 (Wako Pure Chemical Industries, Ltd.)
C3 transferase (Cytoskeleton)
BTT-3033 (TOCRIS)
VS-4718 (Chemie Tek)
Paraformaldehyde (nacalai tesque)
Triton X-100 (Polyoxyethylene (10) Octylphenyl Ether) (Wako Pure Chemical Industries, Ltd.)
BSA (Sigma-Aldrich)
SiRNA # 1, # 2 (Ambion) for VGLL3
Silencer Select Negative Control no. 1 siRNA (Ambion)
実施例１
線維化に関与する転写関連因子の探索
１－１：実験動物および心筋梗塞モデルマウスの作製
　8-10週齢のC57BL/6J野性型雄性マウスは日本エスエルシー株式会社より購入した。マウスの体重を測定し、ソムノペンチル注射液 (50 mg/kgペントバルビタールナトリウム) を腹腔内投与で麻酔後、仰臥位にて手術台に固定した。頚部及び胸部を剃毛し、手術用顕微鏡観察下で頚部の正中を切開し、気管を露出させた。カニューレを気管に挿入し、一回呼吸量0.5 cc、呼吸回数120回/分にて人工呼吸を行った。その後、肋骨左側の第二肋間の切開によって心臓を露出させた。心筋梗塞施術は、心臓の左冠動脈前下行枝を6 mm絹ブレード縫合糸で結紮することにより行った。その後、縫合糸で切開箇所を縫合した。以上の処置を施したマウスを心筋梗塞 (MI) 群とし、冠動脈の結紮以外は同様の処置を施したマウスを偽処置 (シャム、sham) 群とした (図１Ａ)。
　ここに、線維化に関与する新たな因子を同定するため、偽処置群 (sham) および心筋梗塞 (MI) 処置後３日目のマウス心臓からRNAを抽出し、それらRNAを用いてマイクロアレイ解析を行った。 Example 1
Search for transcription-related factors involved in fibrosis 1-1: Preparation of experimental animals and myocardial infarction model mice 8-10 week old C57BL / 6J wild male mice were purchased from Nippon SLC Co., Ltd. Mice were weighed and anesthetized with Somnopentyl injection (50 mg / kg pentobarbital sodium) intraperitoneally and then fixed on the operating table in the supine position. The neck and chest were shaved and an incision was made in the midline of the neck under surgical microscopy to expose the trachea. A cannula was inserted into the trachea, and artificial respiration was performed with a single respiration volume of 0.5 cc and a respiration rate of 120 times / minute. The heart was then exposed by an incision in the second intercostal space on the left side of the ribs. Myocardial infarction was performed by ligating the anterior descending branch of the left coronary artery of the heart with a 6 mm silk blade suture. Then, the incision was sutured with a suture. Mice that underwent the above treatment were included in the myocardial infarction (MI) group, and mice that underwent the same treatment except for ligation of the coronary arteries were included in the pseudo-treatment (sham) group (Fig. 1A).
Here, in order to identify new factors involved in fibrosis, RNA was extracted from the mouse heart 3 days after the sham treatment group (sham) and myocardial infarction (MI) treatment, and microarray analysis was performed using these RNAs. went.
１－２：mRNAの定量
　MI処置後のマウスから摘出した心臓を梗塞領域と非梗塞領域とに切り分け、液体窒素を用いて速やかに凍結させ、-80℃に保存した。-80℃に保存していたマウスの心臓からIsogen (アイソジェン) (ニッポンジーン) のプロトコルに従い、全RNAを抽出した。その後、RNeasy Plus Mini Kit (Qiagen) を用いてRNAを精製し、精製したRNAは微量分光光度計DS-11 (DeNovix) により濃度を決定し、High Capacity cDNA Reverse Transcription Kit (Applied Biosystems)  を用いてcDNAへの逆転写反応を行った。mRNAの定量は、Luna Universal qPCR Master Mix (New England BioLabs) のプロトコルに従い、95℃でDNA合成酵素の活性化を60秒行い、95℃ 15秒によるcDNAの変性と60℃ 60秒の伸長反応を45サイクル行った。内部標準としてGAPDH (グリセルアルデヒド-3-リン酸 デヒドロゲナーゼ) または18S rRNAを用いて解析を行った。 1-2: Quantification of mRNA The heart removed from the mouse after MI treatment was divided into an infarcted region and a non-infarcted region, rapidly frozen using liquid nitrogen, and stored at -80 ° C. Total RNA was extracted from the hearts of mice stored at -80 ° C according to the Isogen (Nippon Gene) protocol. After that, RNA was purified using the RNeasy Plus Mini Kit (Qiagen), the concentration of the purified RNA was determined by the microspectrophotometer DS-11 (DeNovix), and the concentration was determined using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A reverse transcription reaction to cDNA was performed. For mRNA quantification, DNA synthase is activated at 95 ° C for 60 seconds according to the protocol of Luna Universal qPCR Master Mix (New England BioLabs), and cDNA denaturation at 95 ° C for 15 seconds and elongation reaction at 60 ° C for 60 seconds are performed. 45 cycles were performed. Analysis was performed using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or 18S rRNA as an internal standard.
　以下のプライマーおよびTaqManプローブはSigma-Aldrichから購入した。プライマーおよびプローブの配列は下記の通りである；Fw: フォワード (Forward)、Rv: リバース (Reverse)：
マウス α-平滑筋アクチン (αSMA, mouse ACTA2) 
Fw: ５’- GTTCTCTTCAAGGGACAAGGCTG -３’ (配列番号１３) 
Rv: ５’- TCCTGGTATGAGATAGCAAATCGG -３’ (配列番号１４) 
プローブ: 5’- TACGTGCTCCTCACCCACACCGTCA -３’ (配列番号１５)  The following primers and TaqMan probe were purchased from Sigma-Aldrich. The sequence of primers and probes is as follows; Fw: Forward, Rv: Reverse:
Mouse α-smooth muscle actin (αSMA, mouse ACTA2)
Fw: 5'-GTTCTCTTCAAGGGACAAGGCTG -3'(SEQ ID NO: 13)
Rv: 5'-TCCTGGTATGAGATAGCAAATCGG -3'(SEQ ID NO: 14)
Probe: 5'-TACGTGCTCCTCACCCACACCGTCA -3' (SEQ ID NO: 15)
マウスI型コラーゲン, アルファ 1 (COL1A1, mouse COL1A1) 
Fw: 5’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号１６) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号１７) 
プローブ:５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号１８)  Mouse type I collagen, alpha 1 (COL1A1, mouse COL1A1)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 16)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 17)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 18)
ヒトI型コラーゲン, アルファ 1 (COL1A1, human COL1A1) 
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号１９) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号２０) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号２１)  Human type I collagen, alpha 1 (COL1A1, human COL1A1)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 19)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 20)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 21)
マウスI型コラーゲン, アルファ 2 (Collagen 1α2, mouse COL1A2) 
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号２２) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号２３) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号２４)  Mouse type I collagen, alpha 2 (Collagen 1α2, mouse COL1A2)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 22)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 23)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 24)
ヒトI型コラーゲン, アルファ 2 (Collagen 1α2, human COL1A2) 
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号２５) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号２６) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号２７)  Human type I collagen, alpha 2 (Collagen 1α2, human COL1A2)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 25)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 26)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 27)
マウスIII型コラーゲン, アルファ 1 (Collagen 3α1, mouse COL3A1) 
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号２８) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号２９) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号３０)  Mouse type III collagen, alpha 1 (Collagen 3α1, mouse COL3A1)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 28)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 29)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 30)
ヒトIII型コラーゲン, アルファ 1 (Collagen 3α1, human COL3A1) 
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号３１) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号３２) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号３３)  Human type III collagen, alpha 1 (Collagen 3α1, human COL3A1)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 31)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 32)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 33)
マウス グルセルアルデヒド-3-リン酸 デヒドロゲナーゼ (GAPDH, mouse Gapdh)
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号３４) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号３５) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号３６)  Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH, mouse Gapdh)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 34)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 35)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 36)
18S リボゾーム RNA (18S rRNA, eukaryote 18S rRNA)
Fw: ５’- AATTCCTGGCGTTACCTTGGT -３’ (配列番号３７) 
Rv: ５’- TGTATTCCGTCTCCTTGGTTCA -３’ (配列番号３８) 
プローブ: ５’- CCGGCTGCTGACCCCCACTGATA -３’ (配列番号３９)  18S ribosomal RNA (18S rRNA, eukaryote 18S rRNA)
Fw: 5'-AATTCCTGGCGTTACCTTGGT-3'(SEQ ID NO: 37)
Rv: 5'-TGTATTCCGTCTCCTTGGTTCA -3'(SEQ ID NO: 38)
Probe: 5'-CCGGCTGCTGACCCCCCACTGATA -3' (SEQ ID NO: 39)
１－３：転写関連因子の探索
　次いで、１) MI処置群において発現量が顕著に増加する転写関連因子、2) 線維化の実行細胞である筋線維芽細胞に顕著に発現するという２つの基準をもとに選別を行い、機能未知のVGLL3を見出した (図１Ｂ)。 1-3: Search for transcription-related factors Next, there are two criteria: 1) transcription-related factors whose expression levels increase significantly in the MI treatment group, and 2) significant expression in myofibroblasts, which are fibrotic cells. VGLL3 with unknown function was found by sorting based on (Fig. 1B).
　詳細には、リアルタイムRT-PCR法により、心臓におけるMI処置後のVGLL3の発現量変化を経時的に測定した。
　結果、VGLL3は正常時にはほとんど発現せず、MI処置後7日目をピークとして顕著に発現量が増加することが明らかとなった (図１Ｃ)。一方、筋線維芽細胞のマーカー分子であるαSMAや、線維化のマーカーであるペリオスチン、COL1A1、COL1A2およびCOL3A1の発現量も同様に測定したところ、それらの発現推移は、VGLL3の発現推移と類似していた (図１Ｃ)。したがって、VGLL3が線維化に関連する因子である可能性が考えられた。 Specifically, real-time RT-PCR was used to measure changes in VGLL3 expression in the heart over time after MI treatment.
As a result, it was clarified that VGLL3 was hardly expressed under normal conditions, and the expression level increased remarkably after peaking on the 7th day after MI treatment (Fig. 1C). On the other hand, when the expression levels of αSMA, which is a marker molecule of myofibroblast, and periostin, COL1A1, COL1A2, and COL3A1, which are markers of fibrosis, were also measured in the same manner, their expression transitions were similar to those of VGLL3. It was (Fig. 1C). Therefore, it is possible that VGLL3 is a factor related to fibrosis.
　心筋梗塞モデル処置後のマウス心臓から単離した心筋線維芽細胞を培養ポリスチレンプレート上で培養した後、低タンパク質吸着処理したプレートに移し、懸濁液中で7日間培養したところ、SMAやペリオスチン(図１Ｄ、Ｅ、Ｆ)などの筋線維芽細胞マーカータンパク質の発現がほとんど消失した。代わりに、それら細胞において幹細胞マーカーであるOct-4(図１Ｇ)の発現が増加し、筋線維芽細胞が懸濁培養により脱分化する可能性が示唆された。興味深いことに、これらの「脱分化した筋線維芽細胞」を培養ポリスチレンプレート上に再びまきなおし、7日間培養すると、細胞内の筋線維芽細胞マーカータンパク質の発現が回復した(図１Ｄ、Ｅ)。すなわち、培養プレートに付着するだけで筋線維芽細胞に再分化することが示唆された。これらのデータと一致して、線維症に関与する種々の遺伝子のmRNAレベルは、7日間、浮遊させて培養した筋線維芽細胞で減少し、細胞を再びポリスチレンプレート上で培養すると、その発現量は大きく増加した(図１Ｈ、１Ｉ)。これらの結果から、機械的刺激による筋線維芽細胞の分化に関与する遺伝子は、筋線維芽細胞、「脱分化筋線維芽細胞」および「再分化筋線維芽細胞」に発現する遺伝子を比較することにより同定できるという考えに至った。DNAマイクロアレイ解析を用いて、脱分化筋線維芽細胞において大きくダウンレギュレートされ(1/16未満)、かつ再分化(図１Ｊ)とともに大きく増加した(16倍以上)遺伝子を選別した。得られた113の遺伝子の中で、心筋梗塞(図１Ｊ)を誘導する処置によりマウス心臓で発現レベルが著しく増加した(2の1.5乗以上)遺伝子をさらに選択した。選択した8つの遺伝子のうち、転写に関わる分子であるVGLL3に焦点を当てた。 Myofibroblasts isolated from mouse hearts after myocardial infarction model treatment were cultured on cultured polystyrene plates, transferred to low-protein adsorption-treated plates, and cultured in suspension for 7 days. As a result, SMA and periostin (SMA and periostin) The expression of myofibroblast marker proteins such as FIG. 1D, E, F) almost disappeared. Instead, the expression of the stem cell marker Oct-4 (Fig. 1G) was increased in these cells, suggesting that myofibroblasts may be dedifferentiated by suspension culture. Interestingly, these "dedifferentiated myofibroblasts" were re-sown on cultured polystyrene plates and cultured for 7 days to restore intracellular myofibroblast marker protein expression (FIGS. 1D, E). .. That is, it was suggested that they redifferentiate into myofibroblasts just by adhering to the culture plate. Consistent with these data, mRNA levels of various genes involved in fibrosis were reduced in myofibroblasts suspended and cultured for 7 days, and when the cells were cultured again on polystyrene plates, their expression levels. Increased significantly (Fig. 1H, 1I). From these results, the genes involved in the differentiation of myofibroblasts by mechanical stimulation are compared with the genes expressed in myofibroblasts, "dedifferentiated myofibroblasts" and "redifferentiated myofibroblasts". This led to the idea that it could be identified. DNA microarray analysis was used to select genes that were significantly down-regulated (less than 1/16) in dedifferentiated myofibroblasts and significantly increased (> 16-fold) with redifferentiation (FIG. 1J). Among the 113 genes obtained, genes whose expression levels were significantly increased in mouse hearts (2 to the 1.5th power or higher) by treatment for inducing myocardial infarction (Fig. 1J) were further selected. Of the eight genes selected, we focused on VGLL3, a molecule involved in transcription.
１－４：探索により見出されたVGLL3の発現分布
　探索により見出されたVGLL3のヒト各臓器におけるタンパク発現分布を、The Human Protein ATLAS (http://www.proteinatlas.org/)より調べた。
　結果、データベース上では、VGLL3は、生体内の正常な組織において妊娠時の胎盤にのみ多く発現すると報告されていることが判明した (図１Ｋ)。
　これは、VGLL3が革新的な線維化治療開発のための標的分子になり得る可能性を示唆している。 1-4: Expression distribution of VGLL3 found by search The protein expression distribution of VGLL3 found by search in each human organ was investigated from The Human Protein ATLAS (http://www.proteinatlas.org/). ..
As a result, it was found on the database that VGLL3 is highly expressed only in the placenta during pregnancy in normal tissues in vivo (Fig. 1K).
This suggests that VGLL3 may be a target molecule for the development of innovative fibrotic therapies.
１－５：機械的刺激に対するVGLL3発現量の変化
　VGLL3が、筋線維芽細胞マーカータンパク質であるSMAやペリオスチンと同様(図１Ｄ、Ｅ、Ｆ)、筋線維芽細胞および再分化した筋線維芽細胞に発現するか調べた。結果、VGLL3のmRNAレベルは、7日間、浮遊させて培養し、機械的刺激の消失により脱分化した筋線維芽細胞で減少し、細胞を再びポリスチレンプレート上で培養し、機械的刺激により再分化した筋線維芽細胞において、その発現量は大きく増加した（図１Ｌ）。これは、VGLL3が機械的刺激により発現量が増加することを示している。 1-5: Changes in VGLL3 expression level in response to mechanical stimulation VGLL3 is similar to myofibroblast marker proteins SMA and periostin (Fig. 1D, E, F), myofibroblasts and redifferentiated myofibroblasts. It was investigated whether it was expressed in. As a result, the mRNA level of VGLL3 was reduced in myofibroblasts dedifferentiated by the disappearance of mechanical stimulation after being suspended and cultured for 7 days, and the cells were cultured again on a polystyrene plate and redifferentiated by mechanical stimulation. The expression level of myofibroblasts was significantly increased (Fig. 1L). This indicates that the expression level of VGLL3 is increased by mechanical stimulation.
１－６：心臓から単離した筋線維芽細胞におけるVGLLファミリーの発現
　心筋梗塞後3日目の心臓から単離した筋線維芽細胞からRNAを回収し、逆転写によってcDNAを作成した。その後、VGLL1、VGLL2、VGLL3およびVGLL4に特異的なプライマーセットでPCRをかけ、PCRバンドが認められるか、すなわち発現しているかを検討した：
プライマーセット
VGLL1　5'-CTCTTCTTGGACAATTGCACAGCCAGCAGC-3'（配列番号５２）および5'-CTTTGGAGATGGGCTTCTTTCATCATCAAG-3'（配列番号５３）;
VGLL2　5'-CAAAGCACACAGAAGCTCTGGACCCTGGAG-3'（配列番号５４）および5'-CTGCATTCCCTCCTCCGCTTACCTGAGTCC-3'（配列番号５５）;
VGLL3　5'-GACATTGGGTCAGTAGTGGATGAACACTTC-3'（配列番号５６）および5'-GACTGTAGTCAGATCTGTCTTGGTGATGTC-3'（配列番号５７）;
VGLL4　5'-GAATAAGACTGTCAACGGAGACTGCCGCAG-3'（配列番号５８）および5'-CAGGAGACCACAGAGGGGGAGTGACTATGG-3'（配列番号５９）. 1-6: Expression of VGLL family in myofibroblasts isolated from heart RNA was recovered from myofibroblasts isolated from heart 3 days after myocardial infarction, and cDNA was prepared by reverse transcription. Then, PCR was performed with primer sets specific for VGLL1, VGLL2, VGLL3 and VGLL4 to examine whether the PCR band was observed, that is, expressed:
Primer set
VGLL1 5'-CTCTTCTTGGACAATTGCACAGCCAGCAGC-3' (SEQ ID NO: 52) and 5'-CTTTGGAGATGGGCTTCTTTCATCATCAAG-3' (SEQ ID NO: 53);
VGLL2 5'-CAAAGCACACAGAAGCTCTGGACCCTGGAG-3' (SEQ ID NO: 54) and 5'-CTGCATTCCCTCCTCCGCTTACCTGAGTCC-3' (SEQ ID NO: 55);
VGLL3 5'-GACATTGGGTCAGTAGTGGATGAACACTTC-3'(SEQ ID NO: 56) and 5'-GACTGTAGTCAGATCTGTCTTGGTGATGTC-3' (SEQ ID NO: 57);
VGLL4 5'-GAATAAGACTGTCAACGGAGACTGCCGCAG-3' (SEQ ID NO: 58) and 5'-CAGGAGACCACAGAGGGGGAGTGACTATGG-3' (SEQ ID NO: 59).
　その結果、心臓から単離した筋肉線維芽細胞においては、VGLL3以外にVGLL4が発現することが明らかとなった（図１M）。この結果を受け、心臓から単離した筋線維芽細胞におけるVGLL3とVGLL4の発現量の絶対定量を行った（図１Ｎ）。具体的には、マウスVGLL3およびVGLL4を組み込んだベクターの希釈系列を作成し、VGLL3およびVGLL4に特異的なリアルタイムPCRプローブセット（VGLL3: Fw 5'-TGCCCTACAGCCTGCTATCA-3'（配列番号６０）, Rv 5'-CTCCTCCTCCTCCTCCTCTTG-3' （配列番号６１）, プローブ 5'-CTTGCTGTATACCGCTAACTTCTGCTGGC-3' （配列番号６２）； VGLL4: Fw 5'-GTGAGCTTGCATGGTGGC-3'（配列番号６３）, Rv 5'-CACTGCTGTTCTTAGTCAGGG-3' （配列番号６４）,プローブ 5'-CCTGTACGCATCTCTCCCCAGCCTCAT-3'（配列番号６５））を用いて標準曲線を描いた。その後、その標準曲線を基に筋肉線維芽細胞におけるVGLL3、VGLL4のコピー数を算出した。その結果、心臓から単離した筋線維芽細胞においては、VGLL3がVGLL4に比べ、かなり多く存在することが明らかとなった。 As a result, it was clarified that VGLL4 is expressed in addition to VGLL3 in muscle fibroblasts isolated from the heart (Fig. 1M). Based on this result, the expression levels of VGLL3 and VGLL4 in myofibroblasts isolated from the heart were absolutely quantified (Fig. 1N). Specifically, a dilution series of vectors incorporating mouse VGLL3 and VGLL4 was created, and a real-time PCR probe set specific for VGLL3 and VGLL4 (VGLL3: Fw5'-TGCCCTACAGCCTGCTATCA-3'(SEQ ID NO: 60), Rv5 '-CTCCTCCTCCTCCTCCTCTTG-3' (SEQ ID NO: 61), Probe 5'-CTTGCTGTATACCGCTAACTTCTGCTGGC-3' (SEQ ID NO: 62); (SEQ ID NO: 64), probe 5'-CCTGTACGCATCTCTCCCCAGCCTCAT-3' (SEQ ID NO: 65)) was used to draw a standard curve. Then, the copy numbers of VGLL3 and VGLL4 in myofibroblasts were calculated based on the standard curve. As a result, it was clarified that VGLL3 is considerably more abundant than VGLL4 in myofibroblasts isolated from the heart.
１－７：ヒト腸の筋線維芽細胞における機械的刺激に対する線維化関連因子およびVGLL3発現量の変化
　心臓の筋線維芽細胞の代わりにヒト腸の筋線維芽細胞のセルラインであるCCD-18Co細胞(ATCC)を用いる以外は、１－３：転写関連因子の探索に記載している手法に実質的に従い、ACTA2や各種コラーゲン、fibrillin-1さらにはVGLL3のmRNAの発現量をリアルタイムRT-PCRによって測定した（図１Ｏ）。その結果、CCD-18Coにおいても心臓の筋線維芽細胞の場合と同様に機械的刺激によって線維化関連因子およびVGLL3 mRNAの発現が上昇し、機械的刺激の消失によってそれら因子の発現量が減少することが明らかとなった。 1-7: Changes in fibrosis-related factors and VGLL3 expression levels in response to mechanical stimulation in human intestinal myofibroblasts CCD-18Co, which is the cell line of human intestinal myofibroblasts instead of heart myofibroblasts Except for using cells (ATCC), the expression levels of ACTA2, various collagens, fibrillin-1 and VGLL3 mRNAs are measured in real time by substantially following the method described in 1-3: Search for transcription-related factors. Measured by (Fig. 1O). As a result, in CCD-18Co, the expression of fibrosis-related factors and VGLL3 mRNA is increased by mechanical stimulation as in the case of cardiac myofibroblasts, and the expression level of these factors is decreased by the disappearance of mechanical stimulation. It became clear.
実施例２
心筋梗塞時の筋線維芽細胞におけるVGLL3の特異的な発現
　VGLL3の発現細胞について詳細に検討した。MI処置後3日目の心臓切片を用い、VGLL3に対する抗体と、筋線維芽細胞のマーカー分子であるαSMAに対する抗体との共染色を行った。その結果、VGLL3の染色像はαSMAの染色像とほとんど重なり合うことが判明した (図２Ａ)。一方で、マクロファージのマーカー分子であるCD68や心臓の実質細胞である心筋細胞のマーカー分子TNNI3とも共染色を行ったが、VGLL3の染色像はCD68やTNNI3の染色像とは一致しなかった (図２Ｂ、２Ｃ)。これらの結果から、心筋梗塞時において、VGLL3はマクロファージや心筋細胞ではなく、筋線維芽細胞特異的に発現することが明らかとなった。 Example 2
Specific expression of VGLL3 in myofibroblasts during myocardial infarction We investigated in detail the cells expressing VGLL3. Using cardiac sections 3 days after MI treatment, co-staining of an antibody against VGLL3 and an antibody against αSMA, which is a marker molecule of myofibroblasts, was performed. As a result, it was found that the stained image of VGLL3 almost overlapped with the stained image of αSMA (Fig. 2A). On the other hand, co-staining was performed with CD68, which is a marker molecule for macrophages, and TNNI3, which is a marker molecule for cardiomyocytes, which are parenchymal cells of the heart, but the stained image of VGLL3 did not match the stained image of CD68 and TNNI3 (Fig.). 2B, 2C). From these results, it was clarified that VGLL3 is specifically expressed in myofibroblasts, not in macrophages or cardiomyocytes, during myocardial infarction.
　なお、免疫組織染色は次の通りに行った。
　MI処置後のマウスから摘出した心臓を4% PFA溶液で一晩固定し、PFA溶液をスクロース溶液に置換した後、Tissue-TekTM O.C.TTM Compound (Sakura Finetek) を用いて包埋し液体窒素にて凍結させたものを凍結標本とした。凍結標本は、クライオスタットCM1100 (Leica Biosystems) で6μmに薄切後1時間風乾させた。凍結切片は0.1% トリトン X-100/PBSを用いて5分間膜透過処理をした後、5 % BSA/PBS中で室温にて1時間ブロッキングし、5% BSA/PBSを用いて希釈した各種タンパク質抗体溶液中で4℃にて一晩静置した。翌日、PBS溶液による5分間、3回の洗浄後、5% BSA/PBSで希釈した各種蛍光標識二次抗体溶液中で室温にて1時間インキュベートした。PBS溶液による5分間、3回の洗浄後、DAPIにより核を染色し、FluorSaveTM Reagent を用いて封入した。染色像は共焦点顕微鏡 (LSM700, Carl Zeis) を用いて撮像した。 The immunohistochemical staining was performed as follows.
The heart removed from the mice after MI treatment was fixed overnight with 4% PFA solution, the PFA solution was replaced with a sucrose solution, and then embedded with Tissue-Tek TM OCTTM Compound (Sakura Finetek) in liquid nitrogen. The frozen sample was used as a frozen sample. Frozen specimens were sliced into 6 μm with Cryostat CM1100 (Leica Biosystems) and air-dried for 1 hour. Frozen sections were subjected to membrane permeation treatment with 0.1% Triton X-100 / PBS for 5 minutes, blocked in 5% BSA / PBS for 1 hour at room temperature, and diluted with 5% BSA / PBS. It was allowed to stand overnight at 4 ° C. in the antibody solution. The next day, after washing 3 times with PBS solution for 5 minutes, it was incubated for 1 hour at room temperature in various fluorescently labeled secondary antibody solutions diluted with 5% BSA / PBS. After washing 3 times for 5 minutes with PBS solution, the nuclei were stained with DAPI and encapsulated with FluorSaveTM Reagent. The stained image was imaged using a confocal microscope (LSM700, Carl Zeiss).
　上記の免疫染色の結果を確かめるため、RNAscope法を用いて、心筋梗塞処置後の線維化したマウス心臓におけるVGLL3mRNAの検出を行った。RNAscope法とは高感度なin situ ハイブリダイゼーションの一種である (28)。
　RNAscopeは、Advanced Cell Diagnostics社のプロトコルに従った。まず、心筋梗塞 (MI) 処置後のマウスから心臓を摘出し、心房を除いた後、速やかに10 % 中性緩衝ホルマリン液に浸し、室温で16～32時間固定した。その後、パラフィンで包埋し、5 μmの組織切片を作成した。なお、ヒト心筋梗塞患者の心臓標本の染色は、自治医科大学の田中亨教授の協力のもと、共同研究として実験を行った。 To confirm the results of the above immunostaining, VGLL3 mRNA was detected in the fibrotic mouse heart after myocardial infarction treatment using the RNAscope method. The RNAscope method is a type of highly sensitive in situ hybridization (28).
RNAscope followed the protocol of Advanced Cell Diagnostics. First, the heart was removed from the mice treated with myocardial infarction (MI), the atrium was removed, and the mice were immediately immersed in 10% neutral buffered formalin solution and fixed at room temperature for 16 to 32 hours. Then, it was embedded in paraffin to prepare a tissue section of 5 μm. The staining of heart specimens of human myocardial infarction patients was conducted as a joint research with the cooperation of Professor Toru Tanaka of Jichi Medical University.
　組織切片は60℃、1時間インキュベートした後に、キシレンおよび100%エタノールを用いてパラフィンを除去した。その後、組織切片に過酸化水素溶液を滴下し、室温で10分間反応させ、内在性ペルオキシダーゼを不活化させた。蒸留水にて洗浄後、さらに抗原賦活化溶液を滴下し、ホットプレートを用いて98～102℃で30分間反応させ、蒸留水により洗浄した後、プロテアーゼ溶液を滴下し、HybEZTMオーブン (Advanced Cell Diagnostics) 内にて40℃で30分間反応させた。その後、VGLL3に対するプローブを組織切片に滴下し、HybEZTMオーブンにおいて40℃、2時間反応させ、その後、Amp溶液によるシグナル増幅反応を行った。続いて、Fast Red混合液、または蛍光標識Tyramideで反応させてVGLL3mRNAのシグナルを検出した。抗体との共染色を行う場合は、シグナル検出反応の後、10% BSA/PBSを室温、1時間反応 (ブロッキング) させ、一次抗体を4℃で一晩反応させた。翌日、蛍光標識された二次抗体を室温で1時間反応させ、DAPIによる核の染色を行った後に、60℃で15分以上インキュベートし、乾燥させた。EcoMountを用いて封入した切片は共焦点顕微鏡 (Zeiss, LSM700) により撮像を行った。 The tissue section was incubated at 60 ° C. for 1 hour, and then paraffin was removed using xylene and 100% ethanol. Then, a hydrogen peroxide solution was added dropwise to the tissue section and reacted at room temperature for 10 minutes to inactivate the endogenous peroxidase. After washing with distilled water, an antigen activation solution is further added dropwise, reacted at 98 to 102 ° C. for 30 minutes using a hot plate, washed with distilled water, then the protease solution is added dropwise, and the HybEZTM oven (Advanced Cell Diagnostics) is used. ) The reaction was carried out at 40 ° C. for 30 minutes. Then, a probe for VGLL3 was added dropwise to the tissue section, and the reaction was carried out in a HybEZTM oven at 40 ° C. for 2 hours, and then a signal amplification reaction was carried out with an Amp solution. Subsequently, the reaction was carried out with a Fast Red mixture or a fluorescently labeled Tyramide to detect a signal of VGLL3 mRNA. When co-staining with the antibody, after the signal detection reaction, 10% BSA / PBS was reacted at room temperature for 1 hour (blocking), and the primary antibody was reacted at 4 ° C. overnight. The next day, the fluorescently labeled secondary antibody was reacted at room temperature for 1 hour, stained with DAPI nuclei, and then incubated at 60 ° C. for at least 15 minutes and dried. The sections enclosed with EcoMount were imaged with a confocal microscope (Zeiss, LSM700).
　VGLL3の発現量がピークに達するMI処置後7日目の心臓切片を用い、VGLL3のmRNA検出と、αSMA抗体による免疫染色を同じ切片上で行った。また、αSMA以外の筋線維芽細胞のマーカー分子として有名なペリオスチンのmRNAとVGLL3のmRNAの共検出も行った。
　その結果、梗塞領域に存在するαSMA陽性およびペリオスチン陽性の筋線維芽細胞の両方で、VGLL3のmRNAが認められた (図２Ｄ、２Ｅ)。一方で、偽処置 (sham) 群の心臓では、VGLL3のmRNA発現はほとんど認められなかった (図２Ｆ)。
　以上の結果から、VGLL3は正常時の心臓にはほとんど発現せず、心筋梗塞時に出現する筋線維芽細胞に特異的に発現することが明らかとなった。
　さらに、ヒト心筋梗塞患者の心臓標本においても、αSMA陽性の筋線維芽細胞にVGLL3のmRNAのシグナルが観察された (図２Ｇ)。 Using a cardiac section 7 days after MI treatment when the expression level of VGLL3 reached its peak, mRNA detection of VGLL3 and immunostaining with αSMA antibody were performed on the same section. In addition, co-detection of periostin mRNA and VGLL3 mRNA, which are famous as marker molecules for myofibroblasts other than αSMA, was also performed.
As a result, VGLL3 mRNA was found in both αSMA-positive and periostin-positive myofibroblasts present in the infarcted region (Fig. 2D, 2E). On the other hand, VGLL3 mRNA expression was scarcely observed in the sham-treated (sham) group of hearts (Fig. 2F).
From the above results, it was clarified that VGLL3 is hardly expressed in the normal heart, but is specifically expressed in myofibroblasts that appear during myocardial infarction.
Furthermore, in the heart specimens of human myocardial infarction patients, VGLL3 mRNA signals were also observed in αSMA-positive myofibroblasts (Fig. 2G).
実施例３
VGLL3における機械的刺激依存的な細胞質から核への移行能
　心筋梗塞を起こした心臓の梗塞領域には、コラーゲンなどの細胞外マトリックスが過剰に蓄積するため、梗塞領域は非梗塞領域に比べて3倍から7倍の硬さになることが報告されている。また、MRTF (myocardin-related transcription factor) やYAP1 (Yes-associated protein 1) といった転写共役因子は、細胞周囲の硬さ (機械的刺激) の変化に応じて、細胞質から核内へ移行し、下流の転写因子の活性を調節することで、コラーゲンやCTGF(結合組織成長因子 (connective tissue growth factor) )など線維化関連因子の発現を促進する (非特許文献９)。 Example 3
Mechanical stimulus-dependent cytoplasmic to nuclear transfer ability in VGLL3 The infarcted area is 3 compared to the non-infarcted area due to the excessive accumulation of extracellular matrix such as collagen in the infarcted area of the heart that has undergone myocardial infarction. It has been reported to be twice to seven times as hard. In addition, transcription-coupled factors such as MRTF (myocardin-related transcription factor) and YAP1 (Yes-associated protein 1) migrate from the cytoplasm into the nucleus in response to changes in pericellular hardness (mechanical stimulation) and are downstream. By regulating the activity of transcription factors, it promotes the expression of fibrosis-related factors such as collagen and CTGF (connective tissue growth factor) (Non-Patent Document 9).
　VGLL3は、梗塞領域の線維化領域に出現する、筋線維芽細胞に特異的に発現し、また、心筋梗塞後のマウス心臓における、VGLL3の発現推移は線維化関連因子のそれらとよく似ている (図１、図２)。これらのことから、MRTFやYAP1のように、VGLL3は梗塞領域の硬さ、すなわち細胞外の機械的な刺激の変化を感知して細胞質から核へ移行し、線維化関連因子の発現を調節しているのではないかと考えた。 VGLL3 is specifically expressed in myofibroblasts that appear in the fibrotic region of the infarcted region, and the expression transition of VGLL3 in the mouse heart after myocardial infarction is very similar to that of fibrosis-related factors. (Figs. 1 and 2). From these facts, like MRTF and YAP1, VGLL3 senses the hardness of the infarcted area, that is, the change in extracellular mechanical stimulus, transfers from the cytoplasm to the nucleus, and regulates the expression of fibrosis-related factors. I thought it might be.
実施例３－１：VGLL3の細胞質および核における局在
　まず、機械的な刺激によってVGLL3の細胞内の局在が変化するかをin vitroで検討した。MI処置後3日目の心臓から筋線維芽細胞を単離し、接着状態での培養と浮遊状態での培養を行った。
　筋線維芽細胞の単離は、次の通りに行った。
　MI処置後3日目にマウスから心臓を摘出し、心房を取り除いた後、心室を15-20片に分割した。その後、心臓片をII型コラゲナーゼ溶液 (0.1% II型コラゲナーゼ/0.01% エラスターゼ/PBS) 中で37°C 10分間の振盪を10回行うことにより、筋線維芽細胞を含む細胞液を回収した。その後、Red Blood Cell Lysis Buffer (Roche) により赤血球成分を除去し、10%FBS/1%ペニシリン/ ストレプトマイシン/DMEMで細胞を懸濁した後に、培養プレートに細胞を播種した。一晩、CO2インキュベータで培養させた後に、非接着系の細胞をPBS洗浄によって取り除き、トリプシン-EDTAにより接着した細胞を回収した。磁気ビーズ標識されたCD45抗体で30分間反応させ、MSカラムを用いたMACS磁気細胞分離法により、CD45陽性の血球系細胞を除去し、CD45(-)細胞を回収した。そのCD45(-)細胞をAPCで標識されたPDGFRα (筋線維芽細胞の膜表面マーカー) 抗体と反応させた後、磁気ビーズ標識された抗APC抗体で30分間反応させ、MACS磁気細胞分離法により、PDGFRα (+)である筋線維芽細胞を回収した。 Example 3-1: Localization of VGLL3 in the cytoplasm and nucleus First, it was examined in vitro whether the intracellular localization of VGLL3 was changed by mechanical stimulation. Myofibroblasts were isolated from the heart 3 days after MI treatment, and cultured in an adherent state and in a floating state.
Isolation of myofibroblasts was performed as follows.
On the 3rd day after MI treatment, the heart was removed from the mouse, the atrium was removed, and then the ventricles were divided into 15-20 pieces. Then, the heart pieces were shaken 10 times at 37 ° C for 10 minutes in a type II collagenase solution (0.1% type II collagenase / 0.01% elastase / PBS) to recover the extracellular fluid containing myofibroblasts. Then, the erythrocyte component was removed by Red Blood Cell Lysis Buffer (Roche), the cells were suspended in 10% FBS / 1% penicillin / streptomycin / DMEM, and then the cells were seeded on a culture plate. After culturing in a CO 2 incubator overnight, non-adhesive cells were removed by PBS washing and trypsin-EDTA-adhered cells were collected. The cells were reacted with a magnetic bead-labeled CD45 antibody for 30 minutes, and CD45-positive blood cells were removed by MACS magnetic cell separation using an MS column, and CD45 (-) cells were recovered. The CD45 (-) cells were reacted with APC-labeled PDGFRα (myofibroblast membrane surface marker) antibody, then reacted with magnetic bead-labeled anti-APC antibody for 30 minutes, and by MACS magnetic cell separation method. , PDGFRα (+) myofibroblasts were collected.
　接着条件と浮遊条件での培養は、次の通りに行った。
　MI処置後3日目の心臓から単離した筋線維芽細胞を機械的刺激の異なる下記の条件で培養したものを、免疫細胞染色のサンプルに使用した。
　ポリ-L-リジン (Sigma-Aldrich) でコーティングした径35-cm ガラス底皿 (IWAKI) に筋線維芽細胞を播種し、一晩、CO2インキュベータで培養させ、皿に接着したものを接着条件のサンプルとして使用した。また、細胞低吸着培養皿 (住友ベークライト) に筋線維芽細胞をCO2インキュベータで2時間、浮遊状態で培養したものを浮遊条件のサンプルとして使用した。
　次に、免疫細胞染色は、次の通りに行った。
　上記の方法で培養した筋線維芽細胞を1% PFA/PBSで15分間固定し、PBSで洗浄した後、6 μM CellTrace Far Red溶液 (Thermo Fisher Scientific) を加え、CO2インキュベータで30分間インキュベートした。その後、0.5% saponin/PBS (条件1のサンプル) または0.1% トリトン X-100/PBS (条件2~4のサンプル) を用いて膜透過処理を行い、5 % BSA/PBS中で室温にて1時間ブロッキングした後、5% BSA/PBSを用いて希釈した一次抗体溶液中で4℃にて一晩静置した。翌日、PBS溶液による5分間、3回の洗浄後、5% BSA/PBSで希釈した各種蛍光標識二次抗体溶液中で室温にて1時間インキュベートした。PBS溶液による5分間、3回の洗浄後、DAPIにより核を染色し、FluorSaveTM Reagent を用いて封入した。浮遊条件のサンプルは、全ての染色操作を1.5 mL tube中で行い、染色操作が終わった後、細胞懸濁液を径35-cm ガラス底皿にロードし、遠心によりディッシュ底面に細胞を貼りつけ、FluorSaveTM Reagent を用いて封入した。像は共焦点顕微鏡 (LSM700, Carl Zeis) を用いて撮像した。VGLL3およびYAP1の核内局在のパラメータである核/細胞質比 (Nuc/Cyt ratio) は、Image Jソフトウェア (NIH) を用いた画像解析により、核内と細胞質における各因子の平均輝度から算出した。 Culturing under adhesive and floating conditions was carried out as follows.
Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining.
Myofibroblasts were seeded on a 35-cm diameter glass bottom dish (IWAKI) coated with poly-L-lysine (Sigma-Aldrich), cultured overnight in a CO 2 incubator, and adhered to the dish. Used as a sample of. In addition, myofibroblasts cultured in a low-adsorption culture dish (Sumitomo Bakelite) in a CO 2 incubator for 2 hours in a floating state were used as samples under floating conditions.
Next, immune cell staining was performed as follows.
Myofibroblasts cultured by the above method were fixed in 1% PFA / PBS for 15 minutes, washed with PBS, added with 6 μM CellTrace Far Red solution (Thermo Fisher Scientific), and incubated in a CO 2 incubator for 30 minutes. .. After that, membrane permeation treatment was performed using 0.5% saponin / PBS (sample of condition 1) or 0.1% Triton X-100 / PBS (sample of conditions 2 to 4), and 1 in 5% BSA / PBS at room temperature. After blocking for hours, it was allowed to stand overnight at 4 ° C. in a primary antibody solution diluted with 5% BSA / PBS. The next day, after washing 3 times with PBS solution for 5 minutes, it was incubated for 1 hour at room temperature in various fluorescently labeled secondary antibody solutions diluted with 5% BSA / PBS. After washing 3 times for 5 minutes with PBS solution, the nuclei were stained with DAPI and encapsulated with FluorSaveTM Reagent. For samples under floating conditions, all staining operations are performed in a 1.5 mL tube, and after the staining operation is completed, the cell suspension is loaded on a glass bottom dish with a diameter of 35-cm, and the cells are attached to the bottom of the dish by centrifugation. , FluorSaveTM Reagent was used for encapsulation. The image was taken with a confocal microscope (LSM700, Carl Zeiss). The nuclear / cytoplasmic ratio (Nuc / Cyt ratio), which is a parameter of nuclear localization of VGLL3 and YAP1, was calculated from the average brightness of each factor in the nucleus and cytoplasm by image analysis using ImageJ software (NIH). ..
　それぞれの条件で培養した細胞を、VGLL3に対する抗体で染色し、核をDAPIで標識した。また、細胞の全体像を観察するために細胞膜透過性の蛍光分子であるCellTraceを用いて細胞全体を標識し、共焦点顕微鏡を用いて、VGLL3の細胞内局在を観察した。なお、実験系のポジティブ対照として、筋線維芽細胞のYAP1を染色し、その局在も観察した。その結果、接着状態、すなわち機械的刺激のある条件で培養した筋線維芽細胞では、VGLL3は核内に強く局在していた (図３Ａ)。一方で、浮遊状態、すなわち機械的刺激の無い条件で培養した筋線維芽細胞では、VGLL3は細胞質に強く局在していた (図３Ａ)。 Cells cultured under each condition were stained with an antibody against VGLL3, and the nuclei were labeled with DAPI. In addition, in order to observe the whole cell image, the whole cell was labeled with CellTrace, which is a fluorescent molecule that is permeable to the cell membrane, and the intracellular localization of VGLL3 was observed using a confocal microscope. As a positive control of the experimental system, YAP1 of myofibroblasts was stained and its localization was also observed. As a result, VGLL3 was strongly localized in the nucleus in myofibroblasts cultured under the adhesive state, that is, under the condition of mechanical stimulation (Fig. 3A). On the other hand, in myofibroblasts cultured in a floating state, that is, under conditions without mechanical stimulation, VGLL3 was strongly localized in the cytoplasm (Fig. 3A).
　このVGLL3の局在を定量的に評価するために、核内と細胞質内のVGLL3の存在比 (Nuc/Cyt 比率) を算出した。VGLL3の局在が細胞質と核内で等しい場合はNuc/Cyt 比率の値は1に近づき、核内に多く局在するほどその値は大きくなる (29-31)。定量の結果、VGLL3のNuc/Cyt 比率は、浮遊状態と比較して接着状態で顕著に増加していた (図３Ａ)。このことから、接着刺激によってVGLL3が核内に移行することが考えられた。また、筋線維芽細胞のYAP1も、VGLL3と同様に浮遊状態で核外に、接着状態で核内に移行していた (図３Ｂ)。 In order to quantitatively evaluate the localization of VGLL3, the abundance ratio (Nuc / Cyt ratio) of VGLL3 in the nucleus and cytoplasm was calculated. When the localization of VGLL3 is equal in the cytoplasm and in the nucleus, the value of the Nuc / Cyt ratio approaches 1, and the more it is localized in the nucleus, the larger the value (29-31). As a result of quantification, the Nuc / Cyt ratio of VGLL3 was significantly increased in the adhered state as compared with the floating state (Fig. 3A). From this, it was considered that VGLL3 was transferred into the nucleus by the adhesive stimulation. In addition, myofibroblast YAP1 also migrated to the outside of the nucleus in a floating state and into the nucleus in an adherent state, similar to VGLL3 (Fig. 3B).
実施例３－２：過剰発現系におけるVGLL3の局在
　次に、内在性だけでなく、過剰発現系においてもVGLL3の局在が変化するかを確かめるため、レトロウイルスを用いてFLAG標識したVGLL3を過剰発現させた筋線維芽細胞を抗FLAG抗体で染色し、浮遊状態と接着状態での細胞内局在を調べた。
　レトロウイルスを用いた筋線維芽細胞への遺伝子導入は次の通りである。
　レトロウイルスのパッケージング細胞であるPLAT-E細胞にX-tremeGENE9 (Roche) を用いて、FLAG-tag標識VGLL3/pMXs-puroあるいは対照としてpMXs-puroを遺伝子導入した。その後、24時間培養し、培地交換を行った。さらに24時間培養した後の培養上清を遠心 (1500×rpm, 5min ,4℃を2回) し、不純物を除いたものをレトロウイルス溶液とした。MI処置後3日目の心臓から単離した筋線維芽細胞にレトロウイルス溶液及び10μg/mL polybreneを加え、遺伝子導入を行った。6時間後にレトロウイルス溶液と等量の10%FBS/1%ペニシリン/ ストレプトマイシン/DMEMを加え、さらに18時間培養した。その後、培地交換を行い、さらに24時間培養 (ウイルス感染開始から48時間後) したものを実験に使用した。
　その結果、内在性の場合と同様に、転写因子VGLL3は接着状態の有無で細胞内の局在を変化させることを見出した (図３Ｃ)。 Example 3-2: Localization of VGLL3 in overexpression system Next, in order to confirm whether the localization of VGLL3 changes not only in the endogenous system but also in the overexpression system, FLAG-labeled VGLL3 using a retrovirus was used. Overexpressed myofibroblasts were stained with anti-FLAG antibody and examined for intracellular localization in the floating and adherent states.
Gene transfer into myofibroblasts using a retrovirus is as follows.
FLAG-tag-labeled VGLL3 / pMXs-puro or pMXs-puro as a control was gene-transduced into PLAT-E cells, which are retrovirus packaging cells, using X-treme GENE9 (Roche). Then, the cells were cultured for 24 hours and the medium was exchanged. After further culturing for 24 hours, the culture supernatant was centrifuged (1500 × rpm, 5 min, 4 ° C twice) to remove impurities, and used as a retrovirus solution. A retrovirus solution and 10 μg / mL polybrene were added to myofibroblasts isolated from the heart 3 days after MI treatment, and gene transfer was performed. After 6 hours, an equal volume of 10% FBS / 1% penicillin / streptomycin / DMEM was added to the retrovirus solution, and the cells were cultured for another 18 hours. After that, the medium was exchanged, and the culture medium was further cultured for 24 hours (48 hours after the start of virus infection) and used in the experiment.
As a result, it was found that the transcription factor VGLL3 changes the intracellular localization depending on the presence or absence of the adhesive state, as in the case of the endogenous case (Fig. 3C).
実施例３－３：VGLL3の接着時間における経時的な局在変化
　筋線維芽細胞を浮遊条件で2時間培養したものを、ポリ-L-リジンでコーティングした径35-cm ガラス底皿に播種し、播種後30分および1時間、2時間、4時間の細胞をサンプルとして使用した。次に、実施例３－１に記載の免疫細胞染色を同様に行った。
　浮遊状態で培養した筋線維芽細胞を再び培養皿に接着させると、細胞質に局在していたVGLL3が核内に移行することを見出した (図３Ｄ)。この局在変化を継時的に調べたところ、接着時間が長くなるにしたがってVGLL3の核内移行が有意に増加していた (図３Ｄ)。
　以上の結果から、VGLL3は機械的な刺激に依存して細胞質から核内へ移行する因子であることが明らかとなった。 Example 3-3: Changes in localization of VGLL3 over time during adhesion time Myofibroblasts cultured for 2 hours under floating conditions were seeded on a 35-cm diameter glass bottom dish coated with poly-L-lysine. , 30 minutes after seeding and 1 hour, 2 hours, 4 hours cells were used as samples. Next, the immune cell staining described in Example 3-1 was performed in the same manner.
It was found that when myofibroblasts cultured in a floating state were reattached to the culture dish, VGLL3 localized in the cytoplasm was transferred into the nucleus (Fig. 3D). When this localization change was examined over time, the intranuclear translocation of VGLL3 increased significantly as the adhesion time increased (Fig. 3D).
From the above results, it was clarified that VGLL3 is a factor that translocates from the cytoplasm to the nucleus depending on mechanical stimulation.
実施例４
細胞周囲の硬さに依存するVGLL3の核内移行
　線維化を起こした臓器では、細胞外マトリックスの過剰な蓄積のために組織が硬くなる。臓器の硬さの指標として弾性率 (elastic modulus, kPa) が広く用いられている。例えば正常時の心臓の弾性率は約10 kPaであるのに対し、心筋梗塞を起こすと弾性率は約35～70 kPaまで高くなることが報告されている (図４Ａ)。一方で、細胞培養に使用するプラスティック製やガラス製の培養皿の弾性率は2～4×106 kPaであり、生体と比べると極めて硬い (図４Ａ)。そのため、VGLL3の生理的機能を明らかにするためには、生体に近い硬さの環境下におけるVGLL3の挙動を解析する必要があると考えられる。 Example 4
Intranuclear migration of VGLL3, which depends on pericellular hardness In fibrotic organs, tissue becomes stiff due to excessive accumulation of extracellular matrix. The elastic modulus (kPa) is widely used as an index of the hardness of an organ. For example, it has been reported that the elastic modulus of the heart under normal conditions is about 10 kPa, whereas the elastic modulus increases to about 35 to 70 kPa when myocardial infarction occurs (Fig. 4A). On the other hand, the elastic modulus of plastic or glass culture dishes used for cell culture is 2 to 4 × 10 6 kPa, which is extremely hard compared to living organisms (Fig. 4A). Therefore, in order to clarify the physiological function of VGLL3, it is necessary to analyze the behavior of VGLL3 in an environment with hardness close to that of a living body.
　そこで、正常時や線維化時の臓器の硬さを模倣した1 kPaから50 kPaのハイドロゲルプレート上で筋線維芽細胞を培養し、VGLL3の局在がどのように変化するかについて検討した。
　MI処置後3日目の心臓から単離した筋線維芽細胞を機械的刺激の異なる下記の条件で培養したものを、免疫細胞染色のサンプルに使用した。ポリ-L-リジンでコーティングした径35-cm Softview Easy coat ハイドロゲルガラス底皿 (Matrigen) に筋線維芽細胞を播種し、一晩、CO2インキュベータで培養させて皿に接着したものをサンプルとして使用した。次に、実施例３－１に記載の免疫細胞染色を同様に行った。 Therefore, myofibroblasts were cultured on a hydrogel plate of 1 kPa to 50 kPa that mimics the hardness of organs during normal or fibrosis, and how the localization of VGLL3 changes was investigated.
Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining. Myofibroblasts were seeded on a 35-cm diameter Softview Easy coat hydrogel glass bottom dish (Matrigen) coated with poly-L-lysine, cultured overnight in a CO 2 incubator, and adhered to the dish as a sample. used. Next, the immune cell staining described in Example 3-1 was performed in the same manner.
　その結果、1 kPaのハイドゲルプレートでは、VGLL3は細胞全体に広く局在しているのに対し、50 kPaのハイドロゲルでは、核に多く局在していた (図４Ｂ)。また、Nuc/Cyt 比率の定量結果から、ハイドゲルの弾性率が増加するにしたがって、VGLL3の核内移行が促進されることが明らかとなった (図４Ｂ)。
　以上の結果から、VGLL3は生体に近い環境でも細胞内の局在を変化させ、細胞周囲の硬さに依存して核内移行する因子であることが明らかとなった。 As a result, VGLL3 was widely localized in the whole cell in the 1 kPa hide gel plate, whereas it was more localized in the nucleus in the 50 kPa hydrogel (Fig. 4B). In addition, from the quantitative results of the Nuc / Cyt ratio, it was clarified that the intranuclear translocation of VGLL3 is promoted as the elastic modulus of Hydegel increases (Fig. 4B).
From the above results, it was clarified that VGLL3 is a factor that changes the intracellular localization even in an environment close to a living body and translocates into the nucleus depending on the hardness around the cell.
実施例５
VGLL3の核内移行における細胞骨格の重合による制御
　周囲の環境が硬くなると、低分子量Gタンパク質であるRhoが活性化され、球形アクチン (Globular、G-アクチン) が重合し、糸状アクチン (filamentous、F-アクチン) と呼ばれる細胞骨格の形成が促進される (32、33)。細胞骨格の形成が促進されると、細胞は仮足を伸ばすため、細胞の面積が増加する (34、35) (図５Ａ)。実際に、筋線維芽細胞をハイドロゲルプレートで培養したとき、硬いハイドロゲル (50 kPa) で培養した筋線維芽細胞は、軟らかいハイドロゲル (1 kPa) で培養したものと比較して、細胞の面積が増大していた (図５Ｂ)。このことから、細胞周囲の硬さに依存したVGLL3の核内移行は、細胞面積の増大、すなわち細胞骨格の形成との相関が考えられた。 Example 5
Control by Cytoskeleton Polymerization in Cytoskeleton Translocation of VGLL3 When the surrounding environment becomes hard, Rho, a low molecular weight G protein, is activated, spherical actin (Globular, G-actin) is polymerized, and filamentous actin (filamentous, F) -The formation of a cytoskeleton called actin) is promoted (32, 33). When the formation of the cytoskeleton is promoted, the cells extend their pseudopodia, resulting in an increase in cell area (34, 35) (Fig. 5A). In fact, when myofibroblasts were cultured on a hydrogel plate, myofibroblasts cultured on a hard hydrogel (50 kPa) were more cellular than those cultured on a soft hydrogel (1 kPa). The area was increasing (Fig. 5B). From this, it was considered that the nuclear translocation of VGLL3, which depends on the hardness around the cell, correlates with the increase in cell area, that is, the formation of the cytoskeleton.
　そこで、VGLL3の核内移行が、細胞骨格の形成によって制御されているかを明らかにするため、筋線維芽細胞を細胞骨格形成の阻害剤で処置し、VGLL3の局在を観察した。
　MI処置後3日目の心臓から単離した筋線維芽細胞を機械的刺激の異なる下記の条件で培養したものを、免疫細胞染色のサンプルに使用した。ポリ-L-リジンでコーティングした径35-cm ガラス底皿に筋線維芽細胞を播種し、一晩、CO2インキュベータで培養した。その後、次の阻害剤を添加した (阻害剤の濃度は最終濃度を示している) ： ラトランクリンA (Latrunculin A、Lat. A) (2 μM、abcam)、ブレビスタチン (Blebbistatin、Blebbist.) (50 μM、wako)、Y27632 (80 μM、wako)、C3 トランスフェラーゼ (3 μg/mL、Cytoskeleton)、BTT-3033 (30 μM、TOCRIS)、VS-4718 (50 μM、Chemie Tek)。 刺激時間は、VS-4718のみ24時間で、その他は4時間で刺激を行った。次に、実施例３－１に記載の免疫細胞染色を同様に行った。 Therefore, in order to clarify whether the nuclear translocation of VGLL3 is regulated by the formation of cytoskeleton, myofibroblasts were treated with an inhibitor of cytoskeleton formation and the localization of VGLL3 was observed.
Myofibroblasts isolated from the heart 3 days after MI treatment were cultured under the following conditions with different mechanical stimuli, and used as samples for immune cell staining. Myofibroblasts were seeded on a 35-cm diameter glass bottom dish coated with poly-L-lysine and cultured overnight in a CO 2 incubator. The following inhibitors were then added (inhibitor concentration indicates final concentration): Latrunculin A, Lat. A (2 μM, abcam), Blebbistatin, Blebbist. (50 μM, wako), Y27632 (80 μM, wako), C3 transferase (3 μg / mL, Cytoskeleton), BTT-3033 (30 μM, TOCRIS), VS-4718 (50 μM, Chemie Tek). The stimulation time was 24 hours only for VS-4718, and 4 hours for the others. Next, the immune cell staining described in Example 3-1 was performed in the same manner.
　結果、G-アクチンの重合を阻害するラトランクリンA (Lat. A) や、細胞骨格の構成分子である非筋型のミオシンIIとアクチンの会合を阻害するブレビスタチン (Blebbist.) (37) を処置すると、対照群 (DMSO) と比較してVGLL3の核内移行が有意に抑制された (図５Ｃ)。また、細胞骨格の運動に必要なミオシン軽鎖のリン酸化を制御するキナーゼであるROCKの阻害剤であるY27632 (38) や、ROCKを活性化させる低分子量Gタンパク質Rhoを阻害剤するC3転移酵素 (39) を筋線維芽細胞に処置した場合においても、VGLL3の核内移行が有意に抑制された (図５Ｃ)。
　以上の結果から、VGLL3の核内移行は、Rho/ROCK経路を介した細胞骨格の重合によって制御されていることが明らかとなった。 As a result, Latrunculin A (Lat. A), which inhibits the polymerization of G-actin, and Blebbist. (37), which inhibits the association of actin with non-muscle myosin II, which is a constituent molecule of the cytoskeleton. Treatment significantly suppressed the nuclear translocation of VGLL3 compared to the control group (DMSO) (Fig. 5C). In addition, Y27632 (38), which is an inhibitor of ROCK, which is a kinase that controls phosphorylation of the myosin light chain required for cytoskeletal motility, and C3 transfer enzyme, which inhibits the low-molecular-weight G protein Rho that activates ROCK. When (39) was treated on myofibroblasts, the nuclear translocation of VGLL3 was also significantly suppressed (Fig. 5C).
From the above results, it was clarified that the nuclear translocation of VGLL3 is regulated by the polymerization of the cytoskeleton via the Rho / ROCK pathway.
実施例６
焦点接着斑の活性化によるVGLL3の核内移行制御
　細胞骨格の形成や細胞運動を調節するRho/ROCK経路は、チロシンキナーゼ型受容体やGタンパク質共役型受容体、焦点接着斑など様々な上流によって活性が制御されていることが知られている (40)。特に、焦点接着斑 (Focal adhesion) はインテグリンなどの接着タンパク質の集合体であり、コラーゲンなどの細胞外基質や細胞骨格であるアクチンフィラメントと結合することで、細胞外環境の硬さなど、機械的な刺激を細胞内に伝える役割を持つと考えられている (41、42)。 Example 6
Regulation of VGLL3 translocation into the nucleus by activation of focal adhesion spots The Rho / ROCK pathway that regulates cytoskeleton formation and cell motility is controlled by various upstreams such as tyrosine kinase type receptors, G protein-coupled receptors, and focal adhesion spots. It is known that activity is regulated (40). In particular, focal adhesion is an aggregate of adhesion proteins such as integrins, and by binding to extracellular matrix such as collagen and actin filaments, which are the cytoskeleton, the hardness of the extracellular environment and other factors are mechanical. It is thought to play a role in transmitting various stimuli into the cell (41, 42).
　そこで、この焦点接着斑の活性化によって、VGLL3の核内移行が制御されるかを明らかにするため、筋線維芽細胞を焦点接着斑の阻害剤で処理し、VGLL3の局在を観察した。
　まず、焦点接着斑の主たる構成分子であるインテグリンβ1の阻害剤BTT-303343を処理したところ、VGLL3の核内移行は、対称群 (DMSO) と比較して有意に抑制された (図６Ａ)。さらに、インテグリンの下流分子でありRhoの活性化を制御する非受容体型チロシンキナーゼであるFAK (焦点接着班キナーゼ) の阻害剤であるVS-471844を処理したところ、VGLL3の核内移行は有意に抑制された (図６Ｂ)。
　以上の結果から、VGLL3の核内移行を制御する最上流の因子は、インテグリンβ1から構成される焦点接着斑であることが明らかとなった。 Therefore, in order to clarify whether the activation of focal adhesion plaques regulates the nuclear translocation of VGLL3, myofibroblasts were treated with an inhibitor of focal adhesion plaques and the localization of VGLL3 was observed.
First, when the inhibitor BTT-303343 of integrin β1, which is the main constituent molecule of focal adhesion spots, was treated, the nuclear translocation of VGLL3 was significantly suppressed as compared with the symmetric group (DMSO) (Fig. 6A). Furthermore, when VS-471844, an inhibitor of FAK (focal adhesion adhesion kinase), a non-receptor tyrosine kinase that controls the activation of Rho, which is a downstream molecule of integrin, was treated, the nuclear translocation of VGLL3 was significantly significant. It was suppressed (Fig. 6B).
From the above results, it was clarified that the most upstream factor controlling the nuclear translocation of VGLL3 is focal adhesion lesion composed of integrin β1.
実施例７
VGLL3による筋線維芽細胞における線維化関連因子の発現増加
　これまでの結果から、VGLL3は筋線維芽細胞に強く発現し、細胞外マトリックスの硬さを受容して核内に移行する分子であることが明らかとなった。線維化時において、細胞外マトリックスが硬くなることによって筋線維芽細胞は活性化し、線維化能が上昇することが知られている (非特許文献５)。そこで、核内に移行したVGLL3が筋線維芽細胞の線維化関連因子の産生に関与するかを明らかにするため、siRNAを用いて筋線維芽細胞のVGLL3をノックダウンし、リアルタイムRT-PCR法によって線維化関連因子のmRNA発現量を測定した。 Example 7
Increased expression of fibrosis-related factors in myofibroblasts by VGLL3 From the results so far, VGLL3 is a molecule that is strongly expressed in myofibroblasts, accepts the hardness of extracellular matrix, and translocates into the nucleus. Became clear. It is known that myofibroblasts are activated by hardening the extracellular matrix during fibrosis, and the fibrotic ability is increased (Non-Patent Document 5). Therefore, in order to clarify whether VGLL3 translocated into the nucleus is involved in the production of fibrosis-related factors in myofibroblasts, siRNA was used to knock down VGLL3 in myofibroblasts, and a real-time RT-PCR method was performed. The mRNA expression level of fibrosis-related factors was measured by.
　使用したsiRNAは、次の通りである：
Figure JPOXMLDOC01-appb-T000001
 The siRNAs used are:
Figure JPOXMLDOC01-appb-T000001
　筋線維芽細胞へのsiRNAの導入は、リポフェクタミン RNAiMAX (invitrogen) のプロトコルに従って行った。詳細には、MI処置後3日目の心臓から単離した筋線維芽細胞、またはヒト大腸由来筋線維芽細胞株CCD-18co (ATCC) にsiRNAを導入する際のsiRNAおよびリポフェクタミン RNAiMAXの至適量は、FAM標識されたsiRNAの導入量をFACSを用いて評価し、決定した。10%FBS/1%ペニシリン/ストレプトマイシン/DMEMで培養している筋線維芽細胞にリポフェクタミン RNAiMAXを用いてVGLL3に対するsiRNA (Ambion) あるいは対照としてSilencer Select Negative Control no. 1 siRNA (Ambion) を導入し、その4時間後に培地交換 (10%FBS/1%ペニシリン/ストレプトマイシン/DMEM) を行った。siRNA導入から96時間後に、RNeasy Plus Mini Kitを用いて、それぞれの細胞から全RNAを回収した。 The introduction of siRNA into myofibroblasts was performed according to the protocol of lipofectamine RNAiMAX (invitrogen). Specifically, the optimal amount of siRNA and lipofectamine RNA iMAX when introducing siRNA into myofibroblasts isolated from the heart 3 days after MI treatment, or the human colon-derived myofibroblast line CCD-18co (ATCC). Evaluated and determined the amount of FAM-labeled siRNA introduced using FACS. SiRNA (Ambion) against VGLL3 or Silencer Select Negative Control no. 1 siRNA (Ambion) was introduced into myofibroblasts cultured in 10% FBS / 1% penicillin / streptomycin / DMEM using lipofectamine RNAiMAX. Four hours later, medium exchange (10% FBS / 1% penicillin / streptomycin / DMEM) was performed. 96 hours after siRNA introduction, total RNA was recovered from each cell using RNeasyPlusMiniKit.
　その結果、VGLL3をノックダウンすることで、COL1A1、COL1A2、COL3A1といった細胞外マトリックスの発現が有意に抑制されることを見出した (図７Ａ)。また、ヒト大腸由来筋線維芽細胞の細胞株であるCCD-18co細胞に対しても同様に、siRNAを用いてVGLL3をノックダウンしたところ、COL1A1、COL1A2、COL3A1の発現は有意に抑制されていた (図７Ｂ)。これらの結果から、VGLL3は、筋線維芽細胞からの細胞外マトリックス産生を制御することで、線維化を促進させる因子であることが明らかになった。 As a result, it was found that the expression of extracellular matrix such as COL1A1, COL1A2, and COL3A1 was significantly suppressed by knocking down VGLL3 (Fig. 7A). Similarly, when VGLL3 was knocked down using siRNA in CCD-18co cells, which are cell lines of human colon-derived myofibroblasts, the expression of COL1A1, COL1A2, and COL3A1 was significantly suppressed. (Fig. 7B). From these results, it was clarified that VGLL3 is a factor that promotes fibrosis by controlling extracellular matrix production from myofibroblasts.
　さらに、CCL4（四塩化炭素）の投与により線維化を誘導したマウス肝臓を酵素処理し、50gで遠心分離を行い、沈降しなかった細胞を回収した。その後、回収した細胞に対して抗F4/80抗体を用いて磁気細胞分離を行い、F4/80陰性の細胞を回収し、活性化した星細胞(肝臓における筋線維芽細胞)として用いた。この活性化した星細胞に対して、VGLL3のsiRNA処置し、各種コラーゲンやペリオスチンの発現量を評価した（図７Ｃ）。これらの結果から、VGLL3は、心臓のみならず肝臓のマウス筋線維芽細胞においても細胞外マトリックス産生を制御することで、線維化を促進させる因子であることが明らかになった。 Furthermore, the mouse liver in which fibrosis was induced by administration of CCL4 (carbon tetrachloride) was treated with an enzyme and centrifuged at 50 g, and the cells that did not settle were collected. Then, magnetic cell separation was performed on the collected cells using an anti-F4 / 80 antibody, and F4 / 80 negative cells were collected and used as activated stellate cells (myofibroblasts in the liver). These activated stellate cells were treated with VGLL3 siRNA, and the expression levels of various collagens and periostin were evaluated (Fig. 7C). From these results, it was clarified that VGLL3 is a factor that promotes fibrosis by controlling extracellular matrix production not only in heart but also in mouse myofibroblasts in liver.
実施例８
VGLL3と直接的に相互作用するタンパク質の探索
　心臓から単離した筋肉線維芽細胞においてFLAF-VGLL3を強制発現させ、抗FLAG抗体で免疫沈降を行った。その後、免疫沈降物を、質量分析を用いて解析し、VGLL3の結合タンパク質候補を同定した (図８Ａ)。結合タンパク質候補として、Teadファミリータンパク質のメンバーであるTead3を発見した (図８Ａ)。これはVGLLファミリータンパク質と相互作用することが報告されている (Dev Genes Evol (2016) 226:297-315 DOI 10.1007/s00427-016-0546-3)。そこで、線維化関連遺伝子の発現に対するTead3の効果を調べた。しかし、Tead3に対してsiRNA処理を行っても、心筋線維芽細胞における線維化関連遺伝子の発現に影響を与えなかった (図９)、このことは、Tead3がVGLL3を介する線維化関連経路に関連しないことを示唆している。 Example 8
Search for proteins that interact directly with VGLL3 FLAF-VGLL3 was forcibly expressed in muscle fibroblasts isolated from the heart, and immunoprecipitation was performed with an anti-FLAG antibody. The immunoprecipitate was then analyzed using mass spectrometry to identify VGLL3 binding protein candidates (FIG. 8A). As a binding protein candidate, Tead3, which is a member of the Tead family protein, was discovered (Fig. 8A). It has been reported to interact with VGLL family proteins (Dev Genes Evol (2016) 226: 297-315 DOI 10.1007 / s00427-016-0546-3). Therefore, we investigated the effect of Tead3 on the expression of fibrosis-related genes. However, siRNA treatment of Tead3 did not affect the expression of fibrosis-related genes in myocardial fibroblasts (Fig. 9), which is related to Tead3's VGLL3-mediated fibrosis-related pathway. It suggests not.
　そこで次に、RNA編集および様々な遺伝子の転写に関与することが知られているDDX5およびDDX5と類似したアミノ酸配列を持つDDX17に注目した (図８Ａ)。VGLL3およびDDX5の相互作用を確認するため、FLAG-VGLL3を筋線維芽細胞で過剰発現させ、細胞を抗FLAG抗体で免疫沈降させた。免疫沈降実験は、VGLL3がDDX5と複合体を形成することを示した (図８Ｂ)。次に、DDX5がVGLL3と直接相互作用するかどうかを調べた。組換えGST-VGLL3およびHis-DDX5タンパク質を使用するGSTプルダウンアッセイは、DDX5がVGLL3に直接結合するタンパク質であることを明らかにした (図８Ｃ)。さらに、DDX5の3つの欠失変異体を作製することにより、VGLL3との結合に関与するDDX5の領域を調べた。NIH3T3細胞においてDDX5欠失変異体を用いることによる免疫沈降アッセイは、DDX5の領域 (430～615) がVGLL3との結合に必要であることを示した (図８Ｄ、Ｅ)。 Next, we focused on DDX5, which is known to be involved in RNA editing and transcription of various genes, and DDX17, which has an amino acid sequence similar to DDX5 (Fig. 8A). To confirm the interaction between VGLL3 and DDX5, FLAG-VGLL3 was overexpressed in myofibroblasts and the cells were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation experiments showed that VGLL3 forms a complex with DDX5 (Fig. 8B). Next, we investigated whether DDX5 interacts directly with VGLL3. A GST pull-down assay using recombinant GST-VGLL3 and His-DDX5 proteins revealed that DDX5 is a protein that binds directly to VGLL3 (Fig. 8C). Furthermore, by creating three deletion mutants of DDX5, the region of DDX5 involved in binding to VGLL3 was investigated. Immunoprecipitation assays using DDX5 deletion mutants in NIH3T3 cells showed that the DDX5 region (430-615) was required for binding to VGLL3 (FIGS. 8D, E).
　図８Ｆに示すVGLL3のN末端のみの変異体またはC末端のみの変異体とHA-DDX5をNIH3T3細胞においてレトロウイルスを用いて過剰発現させ、抗FLAG抗体で免疫沈降を行った。その後、抗HA抗体でウエスタンブロッティングを行った。その結果、DDX5との相互作用に必要なVGLL3のドメインがC末端(124～325)であることを見出した（図８G）。 The N-terminal only mutant or C-terminal only mutant and HA-DDX5 shown in FIG. 8F were overexpressed in NIH3T3 cells using a retrovirus, and immunoprecipitation was performed with an anti-FLAG antibody. Then, Western blotting was performed with an anti-HA antibody. As a result, it was found that the domain of VGLL3 required for interaction with DDX5 is the C-terminal (124 to 325) (Fig. 8G).
実施例９
筋線維芽細胞における線維化遺伝子の発現に対するDDX5の調節
　次に、DDX5が線維性遺伝子の発現を調節する経路に関与しているかどうかを調べた。心筋線維芽細胞においてDDX5をノックダウンし、リアルタイムRT-PCRによって細胞内のコラーゲンやペリオスチン、フィブリルリン-1 (fibrillin-1)などの線維性遺伝子の発現量を評価したところ、それら線維性遺伝子の発現レベルが有意に低下することがわかった (図１０Ａ)。すなわち、DDX5はこれらの遺伝子の発現を促進している。また、miR-29b (配列番号６６：uagcaccauuugaaaucaguguu) は、Col1a1をはじめ、様々なコラーゲンのmRNAに結合し、そのmRNAを分解へと導くことが知られており (Van Rooij, E. et al. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc. Natl. Acad. Sci. U. S. A., 13027-13032 (2008))、このmiR29bの発現を、DDX5をノックダウンした心筋線維芽細胞においてリアルタイムRT-PCRによって調べた (図１０Ｂ)。結果、DDX5をノックダウンすると、対照と比べ、miR29bのmRNA発現が上方制御されることが判明した。これはDDX5の抑制により、コラーゲン量が減少し、繊維化を軽減する方向に働くことを示している。さらに、心筋線維芽細胞においてVGLL3をノックダウンし、同様に、リアルタイムRT-PCRによってmiR29bのmRNA発現を評価したところ(図１０Ｃ)、VGLL3をノックダウンすると、対照と比べ、miR29bのmRNA発現が上方制御され、コラーゲン量が減少し、線維化を軽減する方向に働くことが分かった。 Example 9
Regulation of DDX5 on Fibrotic Gene Expression in Myofibroblasts Next, we investigated whether DDX5 is involved in the pathway that regulates fibrotic gene expression. When DDX5 was knocked down in myocardial fibroblasts and the expression levels of intracellular collagen, periostin, fibrillin-1 and other fibrous genes were evaluated by real-time RT-PCR, these fibrous genes were evaluated. It was found that the expression level was significantly reduced (Fig. 10A). That is, DDX5 promotes the expression of these genes. In addition, miR-29b (SEQ ID NO: 66: uagcaccauuugaaaucaguguu) is known to bind to various collagen mRNAs including Col1a1 and lead to degradation of the mRNA (Van Rooij, E. et al. Dysregulation). of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc. Natl. Acad. Sci. USA, 13027-13032 (2008)), expression of this miR29b in myocardial fibrosis cells knocked down by DDX5 It was examined by real-time RT-PCR (Fig. 10B). As a result, it was found that knockdown of DDX5 upregulated the mRNA expression of miR29b as compared with the control. This indicates that the suppression of DDX5 reduces the amount of collagen and works to reduce fibrosis. Furthermore, when VGLL3 was knocked down in myocardial fibroblasts and the mRNA expression of miR29b was similarly evaluated by real-time RT-PCR (Fig. 10C), when VGLL3 was knocked down, the mRNA expression of miR29b was higher than that of the control. It was found to be controlled, reduce collagen content, and work to reduce fibrosis.
　線維化した心臓から単離した筋線維芽細胞に対し、DDX5抗体を用いたRNA免疫沈降を行い、Pri-miR29b-1(ABI Mm03306189_pri)あるいはPri-miR29b-2(ABI Mm03307196_pri)に特異的なprimerセットを用いてPCRを行った（図１０Ｄ）。その結果、DDX5がPri-miR29b-1およびPri-miR29b-2に結合することが明らかとなった。このことは、DDX5がPri-miR29b-1, Pri-miR29b-2に結合し、miR-29bの産生を抑制することで結果的に、コラーゲン量を増加させる可能性を示す。 RNA immunoprecipitation using DDX5 antibody was performed on myofibroblasts isolated from fibrotic heart, and a primer specific to Pri-miR29b-1 (ABI Mm03306189_pri) or Pri-miR29b-2 (ABI Mm03307196_pri). PCR was performed using the set (Fig. 10D). As a result, it was clarified that DDX5 binds to Pri-miR29b-1 and Pri-miR29b-2. This indicates that DDX5 may bind to Pri-miR29b-1, Pri-miR29b-2 and suppress the production of miR-29b, resulting in an increase in collagen content.
実施例１０
VGLL3ノックアウトマウスにおけるMI後の心臓線維化の減弱
　in vivoで線維化に対するVGLL3の寄与を調べた。VGLL3 KOマウスを確立し、損傷領域を補うために心筋細胞死およびその後の筋線維芽細胞による線維化を誘発するMI手術 (左前下行動脈の永久閉塞) を行った。MI手術の3日後、マウスから梗塞心臓を取り出し、梗塞部分と非梗塞部分の2つに分けた。各領域から全RNAを収集し、そして各領域における線維化関連遺伝子のmRNA発現レベルを測定した。野生型 (WT) マウスでは、梗塞領域においてCOL1A1、1A2、3A1、FN1およびFBN1の発現が著しく増加した (図１１Ａ)。しかし、梗塞領域におけるこれらの遺伝子のアップレギュレーション (上方制御) は、WTマウスと比較してVGLL3 KOマウスにおいて有意に減弱していた (図１１Ａ)。一貫して、miR-29bの発現は、VGLL3 KOマウスの梗塞領域において有意に上方制御され (図１１Ｂ)、これは、VGLL3/DDX5軸によるmiR-29b発現の抑制がインビボで機能することを示唆する。さらに、MIの28日後のKOマウスとWTマウスの心臓の状態を比較した。マウス心臓切片のピクロシリウスレッド染色は、MIから28日後の線維化の程度が、VGLL3 KOマウスにおいて有意に減少したことを示している (図１１Ｃ)。このデータと一致して、心エコー検査は、MIの28日後の心機能 (駆出率および短縮率) がVGLL3 KOマウスにおいて有意に改善されたことを実証した (図１１Ｄ)。 Example 10
Attenuation of cardiac fibrosis after MI in VGLL3 knockout mice The contribution of VGLL3 to fibrosis was investigated in vivo. VGLL3 KO mice were established and underwent MI surgery (permanent occlusion of the left anterior descending artery) to induce cardiomyocyte death and subsequent fibrosis by myofibroblasts to replace the injured area. Three days after MI surgery, the infarcted heart was removed from the mouse and divided into an infarcted part and a non-infarcted part. Total RNA was collected from each region and the mRNA expression level of fibrosis-related genes in each region was measured. In wild-type (WT) mice, the expression of COL1A1, 1A2, 3A1, FN1 and FBN1 was significantly increased in the infarcted area (Fig. 11A). However, the up-regulation of these genes in the infarcted region was significantly attenuated in VGLL3 KO mice compared to WT mice (Fig. 11A). Consistently, miR-29b expression was significantly upregulated in the infarcted region of VGLL3 KO mice (Fig. 11B), suggesting that suppression of miR-29b expression by the VGLL3 / DDX5-axis functions in vivo. To do. Furthermore, the cardiac conditions of KO and WT mice 28 days after MI were compared. Picrosirius red staining of mouse heart sections shows that the degree of fibrosis 28 days after MI was significantly reduced in VGLL3 KO mice (Fig. 11C). Consistent with this data, echocardiography demonstrated that cardiac function (jection fraction and shortening rate) after 28 days of MI was significantly improved in VGLL3 KO mice (Fig. 11D).
実施例１１
VGLL3は線維化を呈する腎臓の筋線維芽細胞において特異的に発現する
　野生型（WT）マウスに片側尿管閉塞（UUO）モデル処置を施し、処置後10日目の腎臓を回収し、リアルタイムRT-PCRによって、VGLL3mRNAの発現量を評価した（図１２Ａ）。その結果、VGLL3mRNAの発現量が腎臓においても線維化によって発現上昇することが明らかとなった。また、片側尿管閉塞（UUO）モデル処置を施し、線維化を誘導した野生型マウス腎臓切片に対してαSMA抗体を用いた免疫染色、VGLL3に対するin situ ハイブリダイゼーションを同時に行った（図１２Ｂ）。その結果、VGLL3のシグナルはαSMA陽性の細胞に出現することが明らかとなった。 Example 11
VGLL3 undergoes unilateral ureteral obstruction (UUO) model treatment in wild-type (WT) mice specifically expressed in fibrotic kidney myofibroblasts, and renal recovery 10 days after treatment is performed in real-time RT. -The expression level of VGLL3 mRNA was evaluated by PCR (Fig. 12A). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the kidney by fibrosis. In addition, unilateral ureteral obstruction (UUO) model treatment was performed, and immunostaining with αSMA antibody and in situ hybridization to VGLL3 were simultaneously performed on wild-type mouse kidney sections in which fibrosis was induced (Fig. 12B). As a result, it was clarified that the VGLL3 signal appears in αSMA-positive cells.
次に、WTマウスおよびVGLL3KOマウスに片側尿管閉塞（UUO）モデル処置を施し、処置後10日目の腎臓を回収し、リアルタイムRT-PCRによって、コラーゲンの発現量を評価した（図１２Ｃ）。その結果、VGLL3KOマウスにおいてUUO後の腎臓におけるコラーゲンの発現量が有意に減少していることが明らかとなった。 Next, WT mice and VGLL3KO mice were treated with a unilateral ureteral obstruction (UUO) model, and the kidney was collected 10 days after the treatment, and the collagen expression level was evaluated by real-time RT-PCR (Fig. 12C). As a result, it was clarified that the expression level of collagen in the kidney after UUO was significantly reduced in VGLL3KO mice.
実施例１２
肝臓疾患におけるVGLL3mRNA発現の評価
　非アルコール性脂肪性肝炎におけるVGLL3mRNA発現を評価した。野生型（WT）マウスにコリン欠乏高脂肪飼料、メチオニン減量、0.1%メチオニン添加を10週間給餌し、非アルコール性脂肪性肝炎（NASH）を誘導した。その後、肝臓を回収し、リアルタイムRT-PCRによって、VGLL3mRNAの発現量を評価した（図１３Ａ）。その結果、VGLL3mRNAの発現量が肝臓においても線維化によって発現上昇することが明らかとなった。 Example 12
Evaluation of VGLL3 mRNA expression in liver disease We evaluated VGLL3 mRNA expression in nonalcoholic steatohepatitis. Wild-type (WT) mice were fed a choline-deficient high-fat diet, methionine weight loss, and 0.1% methionine addition for 10 weeks to induce non-alcoholic steatohepatitis (NASH). Then, the liver was collected and the expression level of VGLL3 mRNA was evaluated by real-time RT-PCR (Fig. 13A). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the liver by fibrosis.
　次に、再生肝におけるVGLL3mRNA発現を評価した。CCL4（四塩化炭素）を4週間投与し、線維化を誘導したマウス肝臓（CCL4）およびコントロールの非投与群のマウス肝臓（ビヒクル）、さらにはCCL4を4週間投与後、投与をやめ、４週間飼育し、肝臓が再生したマウスの肝臓（Regene）からRNAを回収し、それぞれの肝臓におけるVGLL3mRNAの発現量をリアルタイムRT-PCRにより測定した（図１３Ｂ）。その結果、VGLL3KOマウスにおいてNASH誘導後の腎臓におけるコラーゲンの発現量が有意に減少していることが明らかとなった。 Next, the expression of VGLL3 mRNA in the regenerated liver was evaluated. CCL4 (carbon tetrachloride) was administered for 4 weeks to induce fibrosis of mouse liver (CCL4), control non-administered mouse liver (vehicle), and CCL4 was administered for 4 weeks and then discontinued for 4 weeks. RNA was recovered from the liver (Regene) of mice that had been bred and regenerated, and the expression level of VGLL3 mRNA in each liver was measured by real-time RT-PCR (FIG. 13B). As a result, it was clarified that the expression level of collagen in the kidney after NASH induction was significantly reduced in VGLL3KO mice.
　また、再生肝における活性化した星細胞のマーカー分子であるCyp7a1、Cd68、ACTA2のmRNAを測定した。CCL4（四塩化炭素）の投与により線維化を誘導したマウス肝臓を酵素処理し、50gで遠心分離を行い、沈降した細胞を肝細胞（HC）として回収した。一方で、沈降しなかった細胞に対してanti-F4/80抗体を用いて磁気細胞分離を行い、F4/80陽性の細胞をクッパー細胞（KC）、F4/80陰性の細胞を活性化した星細胞（HSC）(肝臓における筋線維芽細胞)として回収した。それぞれの細胞分画が純度よく回収できているかについて肝細胞、クッパー細胞、活性化した星細胞のマーカー分子である、Cyp7a1、Cd68、ACTA2のmRNAを測定することにより評価した（図１３Ｃ）。その結果、それぞれの分画が高純度で回収できていることが明らかとなった。
　次いで、図１３Ｃで回収したそれぞれの細胞画分におけるVGLL3mRNAの発現量をリアルタイムRT-PCRにより測定した（図１３Ｄ）。その結果、VGLL3は、活性化した星細胞（HSC）(肝臓における筋線維芽細胞)にのみ発現することが明らかとなった。 In addition, the mRNAs of Cyp7a1, Cd68, and ACTA2, which are marker molecules of activated stellate cells in the regenerated liver, were measured. The mouse liver in which fibrosis was induced by administration of CCL4 (carbon tetrachloride) was enzymatically treated, centrifuged at 50 g, and the precipitated cells were collected as hepatocytes (HC). On the other hand, magnetic cell separation was performed on non-precipitated cells using anti-F4 / 80 antibody, and F4 / 80 positive cells were activated as Kupffer cells (KC) and F4 / 80 negative cells were activated. Collected as cells (HSC) (myofibroblasts in the liver). Whether each cell fraction was recovered with high purity was evaluated by measuring the mRNAs of Cyp7a1, Cd68, and ACTA2, which are marker molecules of hepatocytes, Kupffer cells, and activated stellate cells (Fig. 13C). As a result, it was clarified that each fraction could be recovered with high purity.
Next, the expression level of VGLL3 mRNA in each cell fraction collected in FIG. 13C was measured by real-time RT-PCR (FIG. 13D). As a result, it was revealed that VGLL3 is expressed only in activated stellate cells (HSC) (myofibroblasts in the liver).
実施例１３
肺の線維化状態におけるVGLL3mRNA発現の評価
　6-8週齢の雌性マウスにソムノペンチル麻酔下でカニューレを挿入し、シリンジを用いてブレオマイシン溶液（1.5 μg/g）または生理食塩水を気管内に単回投与した。以上の処置を施したマウスをそれぞれブレオマイシン（BLM）投与群および生理食塩水（Saline）投与群とした。BLM投与後、14日目にソムノペンチル注射液の腹腔投与による麻酔下で開腹し、腹部大動脈の切断により脱血を行い、肺の摘出を行った。摘出した肺からRNAを回収し、リアルタイムRT-PCRによって、コラーゲンの発現量を評価した（図１４）。その結果、VGLL3mRNAの発現量が肺においても線維化によって発現上昇することが明らかとなった。 Example 13
Evaluation of VGLL3 mRNA expression in pulmonary fibrosis A 6-8 week old female mouse was cannulated under somnopentyl anesthesia and a single dose of bleomycin solution (1.5 μg / g) or saline was injected into the trachea using a syringe. It was administered. Mice treated as described above were classified into a bleomycin (BLM) -administered group and a saline (Saline) -administered group, respectively. On the 14th day after BLM administration, the abdomen was opened under anesthesia by intraperitoneal administration of somnopentyl injection, blood was removed by amputation of the abdominal aorta, and the lungs were removed. RNA was recovered from the removed lung and the expression level of collagen was evaluated by real-time RT-PCR (Fig. 14). As a result, it was clarified that the expression level of VGLL3 mRNA is also increased in the lung by fibrosis.
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　本発明によれば、筋線維芽細胞のマーカータンパク質を同定し、線維化疾患を予防または治療する技術を提供することができる。 According to the present invention, it is possible to identify a marker protein of myofibroblasts and provide a technique for preventing or treating fibrotic diseases.

Claims (14)

  1. 　VGLL3の阻害物質を含有する、線維化疾患を予防または治療するための医薬組成物。 A pharmaceutical composition containing an inhibitor of VGLL3 for preventing or treating fibrotic diseases.
  2. 　VGLL3の阻害物質が、VGLL3発現の阻害物質、VGLL3の核内移行の阻害物質、またはVGLL3とDDX5との結合の阻害物質である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the inhibitor of VGLL3 is an inhibitor of VGLL3 expression, an inhibitor of nuclear translocation of VGLL3, or an inhibitor of binding between VGLL3 and DDX5.
  3. 　VGLL3発現の阻害物質が、以下の (a) ～ (d) のいずれかに示される遺伝子または核酸の発現を阻害する物質である、請求項２に記載の医薬組成物：
    　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
    　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
    　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
    　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸。     The pharmaceutical composition according to claim 2, wherein the inhibitor of VGLL3 expression is a substance that inhibits the expression of the gene or nucleic acid shown in any of the following (a) to (d):
    (a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
    (b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
    (c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing A nucleic acid that hybridizes with a base sequence complementary to the VGLL3 gene under stringent conditions.
  4. 　遺伝子または核酸の発現を阻害する物質が、siRNA、shRNA、miRNA、アンチセンスおよびリボザイムからなる群から選択される少なくとも１つである請求項３に記載の医薬組成物。 The pharmaceutical composition according to claim 3, wherein the substance that inhibits the expression of a gene or nucleic acid is at least one selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme.
  5. 　(a) 配列表の配列番号２、４または６に示されるVGLL3遺伝子、
    　(b) 配列表の配列番号２、４または６に示されるVGLL3遺伝子において１個若しくは数個の塩基が欠失、置換または付加されている塩基配列を含む核酸、
    　(c) 配列表の配列番号２、４または６に示されるVGLL3遺伝子の塩基配列と同一性が９０％以上の塩基配列の核酸
    　(d) 配列表の配列番号２、４または６で表されるVGLL3遺伝子と相補的な塩基配列とストリンジェントな条件でハイブリダイズする核酸、
    の発現を阻害する、siRNA、shRNA、miRNA、アンチセンスおよびリボザイムからなる群から選択される少なくとも１つである核酸。     (a) The VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the Sequence Listing,
    (b) Nucleic acid containing a base sequence in which one or several bases are deleted, substituted or added in the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing.
    (c) Nucleic acid having a base sequence of 90% or more identity with the base sequence of the VGLL3 gene shown in SEQ ID NO: 2, 4 or 6 in the sequence listing (d) Represented by SEQ ID NO: 2, 4 or 6 in the sequence listing Nucleic acid that hybridizes under stringent conditions with a base sequence complementary to the VGLL3 gene,
    A nucleic acid that is at least one selected from the group consisting of siRNA, shRNA, miRNA, antisense and ribozyme that inhibits the expression of.
  6. 　請求項５に記載の核酸を発現する、ベクター。 A vector that expresses the nucleic acid according to claim 5.
  7. 　請求項６に記載のベクターを含む、細胞。 A cell containing the vector according to claim 6.
  8. 　筋線維芽細胞である、請求項７に記載の細胞。 The cell according to claim 7, which is a myofibroblast.
  9. 　被験物質の存在下で、細胞中のVGLL3の発現量を測定する工程と、
    　前記発現量が、前記被験物質の非存在下における発現量と比較して低下していた場合に、前記被験物質は線維化疾患の予防または治療に有効な候補物質であると判断する工程とを備える、
    　線維化疾患の予防または治療に有効な候補物質のスクリーニング方法。     The step of measuring the expression level of VGLL3 in cells in the presence of the test substance,
    When the expression level is lower than the expression level in the absence of the test substance, the step of determining that the test substance is an effective candidate substance for the prevention or treatment of fibrotic disease. Prepare, prepare
    A screening method for candidate substances that are effective in the prevention or treatment of fibrotic diseases.
  10. 　線維化の進行度を判定する方法であって、
     (a10) 被検者の筋線維芽細胞のVGLL3の量 (被検バイオマーカー量) を測定する工程、
     (b10) 被検バイオマーカー量と、基準の筋線維芽細胞のVGLL3の量 (対照バイオマーカー量) とを比較する工程、および
     (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、線維化の進行度が高いと判定する方法。     A method of determining the degree of fibrosis
    (a10) Step of measuring the amount of VGLL3 (test biomarker amount) in myofibroblasts of a subject,
    (b10) A step of comparing the amount of the test biomarker with the amount of VGLL3 in the reference myofibroblast (control biomarker amount), and (c10) when the test biomarker amount is larger than the control biomarker amount. In addition, a method for determining a subject to have a high degree of fibrosis.
  11. 　前記基準の筋線維芽細胞のVGLL3の量が、前記 (a10) 工程より前に測定された同一被検者の筋線維芽細胞のVGLL3の量である、請求項１０に記載の方法。 The method according to claim 10, wherein the amount of VGLL3 of myofibroblasts of the reference is the amount of VGLL3 of myofibroblasts of the same subject measured before the step (a10).
  12. 　前記基準の筋線維芽細胞のVGLL3の量が、健常者の筋線維芽細胞のVGLL3の量である、請求項１０に記載の方法。 The method according to claim 10, wherein the amount of VGLL3 in myofibroblasts of the reference is the amount of VGLL3 in myofibroblasts of a healthy person.
  13. 　筋線維芽細胞のVGLL3の、線維化の進行度を判定するためのバイオマーカーとしての使用。 Use of VGLL3 in myofibroblasts as a biomarker to determine the degree of fibrosis.
  14. 　VGLL3のcDNAを増幅するためのプライマーセット、VGLL3のmRNAに特異的にハイブリダイズするプローブ、または、VGLL3タンパク質に対する特異的結合物質を含む、筋線維芽細胞の検出用キット。 A kit for detecting myofibroblasts, which contains a primer set for amplifying VGLL3 cDNA, a probe that specifically hybridizes to VGLL3 mRNA, or a specific binding substance for VGLL3 protein.
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