DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.
S-Nitrosylation
of DNA methyltransferase (DNMT) inhibits its enzymatic activity, resulting in
DNA hypomethylation and aberrant gene expression related to its pathogenesis.
The authors demonstrated that nitric oxide epigenetically induces CA9
expression in human small airway epithelial cells through pharmacological
evaluation using DBIC, a specific inhibitor of DNMT3B S-nitrosylation. Hypoxia-inducible
factor 1 alpha (HIF1α) is recruited to the CA9 promoter region via
nitric oxide-induced epigenetic regulation. These findings indicate that nitric
oxide is a key epigenetic regulator in normal human cells.
Transcriptional activation, based on Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and known as CRISPR activation (CRISPRa), is a specific and safe tool to upregulate endogenous genes. Therefore, CRISPRa is valuable not only for analysis of molecular mechanisms of cellular events, but also for treatment of various diseases. Regulating autophagy has been proposed to enhance effects of some therapies. In this study, we upregulated genes for phosphoinositide phosphatases, SACM1L, PIP4P1, and PIP4P2, using CRISPRa, and their effects on autophagy were examined. Our results suggested that TMEM55A/PIP4P2, a phosphatidylinositol-4,5-bisphosphate 4-phosphatase, positively regulates basal autophagy in 293A cells. Furthermore, it was also suggested that SAC1, a phosphatidylinositol 4-phosphatase, negatively regulates basal autophagic degradation.
Transcriptional
activation of endogenous genes using clustered regularly interspaced short
palindromic repeats activation (CRISPRa) is an excellent tool not only for biological
research but also for treatment of diseases. The authors have successfully upregulated
three endogenous genes encoding phosphoinositide phosphatases using the CRISPRa
system targeting multiple promoter sites. The effects of gene upregulation on
autophagy, a potential therapeutic target for various diseases, were investigated.
The results showed that TMEM55A/PIP4P2, a phosphatidylinositol-4,5-bisphosphate
4-phosphatase, promotes autophagosome formation. It was also revealed that TMEM55B/PIP4P1
and SAC1 are involved in autolysosome formation.
The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5′nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.
The increasing
number of patients with depressive disorder is a serious socioeconomic problem
worldwide, and effectiveness of several therapeutic agents used clinically is
insufficient and thus discovery of novel therapeutic targets is desired. Focusing
on dysregulation of neuronal purinergic signaling in depressive-like behavior, Nishioka
et al. revealed that in astrocytes derived from cerebral cortex of
chronic social defeat stress-susceptible mice, the expression levels of mRNAs
for connexin 43 and P2X7 receptors were inversely correlated with mouse
sociability. Together with recent findings, it is suggested that ATP channels
expressed by cortical astrocytes might be potential therapeutic targets for
depressive disorder.
A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.
[Highlighted Paper selected
by Editor-in-Chief]
This
study revealed the gene
expression profiles of bicellular and tricellular tight junction components in
different segments of the human intestinal tract. Claudin-8, angulin-1
and -2 could be potential targets for intestinal permeation enhancers in the
rectum. Claudin-2 and -15 may serve as targets for drug absorption enhancers in
the upper intestine. Claudin-7, occludin, and tricellulin appear to be suitable
targets for enhancing drug absorption throughout all intestinal segments.
Furthermore, claudin-3, -4, and -7 modulators seem to be the most potent
intestinal permeation enhancers. Thus, this study provides valuable insights
for the development of intestinal drug permeation enhancers.
Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug–drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.
Understanding the
mechanisms behind the induction or inhibition of CYP enzymes, which are pivotal
for drug metabolism, is essential for predicting drug-drug interactions (DDI). In
this study, the authors demonstrate that omeprazole, a well-known inducer of
CYP1A2, not only increased CYP1A2 mRNA expression but also
elevated CYP3A4 mRNA levels. However,
omeprazole treatment did not lead to an increase in CYP3A4 protein levels
because it caused the CYP3A4 protein to degrade more quickly. These
findings suggest that evaluating CYP protein degradation, in addition to CYP
induction and inhibition, is crucial for more accurate DDI predictions.
Total Purine and Purine Base Content of Common Foodstuffs for Facilitating Nutritional Therapy for Gout and Hyperuricemia
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Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport
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Effect of Psilocin on Extracellular Dopamine and Serotonin Levels in the Mesoaccumbens and Mesocortical Pathway in Awake Rats
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Yuichi Sakashita, Kenji Abe, Nobuyuki Katagiri, Toshie Kambe, Toshiaki Saitoh, Iku Utsunomiya, Yoshie Horiguchi, Kyoji Taguchi
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Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography
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Kozo Takeuchi, Osamu Yasuhiko
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Effects of Hura crepitans and Its Active Ingredient, Daphne Factor F3, on Dihydrotestosterone-Induced Neurotrophin-4 Activation and Hair Retardation
Released on J-STAGE: January 05, 2012 | Volume 35 Issue 1 Pages 42-47
Chiyoko Uchiyama, Kazuhiro Ishida, Takuya Tsutsui, Atsushi Naito, Kei Kurita, Hiroyuki Hanihara, Tetsushi Serizawa, Masami Fujiwara, Motoyasu Ohdera
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