The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation
<p>Calreticulin (CALR) mutant proteins are faintly expressed in the transfected and primary cells. (<b>A</b>) HEK293T cells were transfected with plasmids to express wild type (WT) or mutant CALR proteins. Forty-eight hours later, total cell lysates (TCL) and supernatant precipitates were subjected to western-blot analysis using anti-total CALR antibody. Actin serves as a loading control for cell lysates. (<b>B</b>) Quantification of signals measured on western-blot analysis of total cell lysates of transfected HEK293T cells performed in (A). Results are expressed as the ratio of CALR to actin signal normalized on the “Empty Vector” condition. The histogram represents the mean ± standard error of the mean (SEM) of six independent experiments. * <span class="html-italic">p</span> < 0.05 Platelets (<b>C</b>) and polymorphonuclear cells (PMN, (<b>D</b>) were purified from peripheral blood of 11 <span class="html-italic">CALR</span> mutated (patient numbers 1 to 11) and one <span class="html-italic">JAK2</span>V617F mutated MPN patients. CALR protein expression was assessed by western blotting using antitotal CALR antibody. Actin was used as loading control. The white arrowhead indicates WT CALR, the black one the position of the type-1 (del52) mutant CALR. All patients present a type 1 mutation (del52), except patient 6, who exhibits a homozygous type 2 (ins5) mutation. (<b>E</b>) Quantification of <span class="html-italic">CALR</span> mutant ratio on RNA of HEK293T 48 h after transfection. Results are expressed as the ratio of the copy numbers of mutant to total <span class="html-italic">CALR</span>. The histograms represent the mean +/− SEM of three independent experiments. (<b>F</b>) Quantification of <span class="html-italic">CALR</span> mutant ratio on RNA purified from peripheral PMN. Results are expressed as the ratio of the copy numbers of mutant to total <span class="html-italic">CALR</span>.</p> "> Figure 2
<p>Calreticulin (CALR) mutant proteins are substrates of Endoplasmic Reticulum Associated Degradation (ERAD)-proteasome and autophagy degradation pathways. (<b>A</b>) HEK293T were transfected with plasmids to express non-tagged mutant CALR proteins. Twenty-four hours later, cells were treated by MG-132 (10 μM, Santa Cruz Biotechnology, Heidelberg, Germany), chloroquine (CQ, 50 μM, Sigma-Aldrich, Lyon, France), or Bafilomycin 1A (Bafilo, 100 nM, Sigma-Aldrich, Lyon, France) for another 24 h. The expression of the proteins was assessed by western blotting using the CAL2 antibody specific of the neoepitope generated by the frameshift mutations. Actin serves as a loading control. (<b>B</b>) Quantification results are expressed as a ratio of CAL2 signal to actin signal and then normalized against the “not treated” or “Dimethyl Sulfoxide” (DMSO) condition. Data are expressed as mean ± SEM from five experiments. * <span class="html-italic">p</span> < 0.05 (<b>C</b>) HEK293T were transfected with pcDNA6 plasmids encoding Myc-tagged WT or mutant CALR. Forty-eight hours later, total cell lysates (TCL) and supernatant precipitates were subjected to western blot analysis. Actin serves as a loading control for cell lysates. (<b>D</b>) Quantification of signals measured on western blot analysis of total cell lysates of transfected HEK293T cells performed in (C). Results are expressed as the ratio of Myc to actin signal, normalized on the CALR WT level. The histogram represents the mean ± SEM of three independent experiments. *** <span class="html-italic">p</span> < 0.001 (<b>E</b>,<b>F</b>) HEK293T were transfected with pcDNA6 plasmids to express Myc-6His tagged WT or mutant CALR proteins. Cells were treated 24h by (<b>E</b>) MG-132 (10 μM) or (<b>F</b>) chloroquine (CQ, 50 μM) or Bafilomycin A1 (Bafilo, 100 nM) and CALR expression was assessed by western-blotting with anti-Myc antibody. Actin was used as a loading control. Data presented are representative of three to ten experiments.</p> "> Figure 3
<p>Impact of Calreticulin (CALR) mutant expression on endoplasmic reticulum (ER) homeostasis. Hematopoietic cell lines were transduced with pCW57.1 vector enabling tetracyclin-inducible expression of Myc-6His tagged wild type (WT) or mutant CALR. (<b>A</b>) The expression of Unfolded Protein Response (UPR) target genes was assessed in DAMI cells by RT-qPCR before and 48 h after treatment by doxycyclin (+Dox). GAPDH was used as control gene. The histograms represent the mean ± standard error of the mean (SEM) of three independent experiments. ** <span class="html-italic">p</span> < 0.01. (<b>B</b>) Leucocytes were obtained from peripheral blood samples of <span class="html-italic">CALR</span> mutated or patients presenting with reactive hyperleucocytosis and/or thrombocytosis (RHL). After extraction of RNA, XBP1 splicing was assessed by RT-PCR and densitometry analysis. The boxes represent the dispersion from minimum to maximum values. Ten patients were tested in each group. (<b>C</b>) Cell mortality induced by Tunicamycin (Tc, 10 μg/mL) was assessed by Annexin V- 4’,6-diamidino-2-phénylindole (DAPI) labeling in UT-7-MPL cells 24 h after CALR transgene expression induction by Doxycyclin (Dox). These experiments were performed in the presence or in the absence of thrombopoietin (TPO). Dimethyl Sulfoxide (DMSO) was used as a vehicle control. Results are expressed as the mean proportion of cells positive for Annexin V in four independent experiments.</p> "> Figure 4
<p>Expression of Calreticulin (CALR) mutants does not alter the expression of endogenous CALR protein. (<b>A</b>) HEK293T were transfected with pcDNA6 plasmids to express Myc-6His tagged wild type (WT) or mutant CALR proteins. Forty-eight hours later, endogenous WT CALR expression was assessed by western blotting using the antitotal CALR antibody. Actin was used as a loading control. Note that because of the presence of the tag, the CALR proteins present with an increased apparent molecular weight (MW) compared to the nontagged forms. Hence, WT and ins5 proteins (black arrowhead) present a MW superior to the endogenous nontagged WT CALR protein (white arrowhead), while the tagged form of del52 protein (the Molecular Weight of which is lower than that of WT and ins5) present the same MW as the WT endogenous protein. (<b>B</b>) The expression of endogenous CALR was quantified and expressed as the ratio of CALR to actin signal and normalized against the “empty vector” (EV) condition. For Myc-6His tagged del52 mutant CALR (which presents the same MW as the endogenous CALR) a correction was applied by subtracting the signal generated by the transgene estimated from the comparison of the signal measured for WT and ins5 CALR with anti-Myc and anti-CALR antibodies. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. (<b>C</b>) UT-7 cells were transduced with pCW57.1 vector enabling tetracyclin-inducible expression of Myc-6His tagged WT or mutant <span class="html-italic">CALR</span>. Expression of WT, type 1 (del52), and type 2 (ins5) <span class="html-italic">CALR</span> mutants was induced by Doxycyclin treatment. Forty-eight hours later, endogenous WT CALR expression was assessed by western blotting using the antitotal CALR antibody. Actin was used as a loading control. For Myc labeling, * denotes the specific signal, as determined by the molecular weight. (<b>D</b>) Expression of WT, type 1 (del52), and type 2 (ins5) CALR mutants was induced in UT-7 cells using a tetracyclin-inducible vector. Forty-eight hours later, Calnexin (CANX) expression and phosphorylation were assessed by western blotting. Actin was used as a loading control.</p> "> Figure 5
<p>Endoplasmic reticulum Degradation-Enhancing α-Mannosidase-like protein 3 (EDEM3) is a key player for targeting Calreticulin (CALR) mutants to the Endoplasmic Reticulum Associated Degradation (ERAD)-proteasome pathway. (<b>A</b>) HEK293T were transfected with pcDNA6 plasmids encoding Myc-6His tagged wild type (WT) or mutant CALR proteins. Twenty-four hours post transfection, the cells were treated with the indicated pharmacological agents at the concentration indicated in the Materials and Methods section. CALR expression was then assessed by western blotting using anti-Myc antibody. Actin serves as a loading control. Each lane corresponds to a drug as indicated in (B) and with the same order. (<b>B</b>) The expression of WT and type 1 (del52) mutant of CALR was assessed and expressed as the ratio of Myc signal to actin signal normalized against the “not treated” or “Dimethyl Sulfoxide” (DMSO) condition. The results presented represent mean +/− standard error of the mean (SEM) determined in two independent experiments. (<b>C</b>) HEK293T cells were transfected with siRNA directed against different actors of the ERAD pathway. Forty-eight hours later, they were transfected with pcDNA6-CALR plasmids. Twenty-four hours later, cells were lysed before analysis by western blotting. CALR proteins were detected via their Myc epitope and actin was used as a loading control. Each lane corresponds to a siRNA as indicated in (D) and with the same order. (<b>D</b>) The expression of the different forms of CALR secondary to ERAD actors silencing was quantified as in (B). The histograms represent mean ± SEM determined in three to six independent experiments. * <span class="html-italic">p</span> < 0.05 (<b>E</b>) Hierarchical clustering was performed on gene expression microarray data from Rampal et al. [<a href="#B25-cancers-11-01921" class="html-bibr">25</a>]. The expression of Endoplasmic Reticulum (ER) quality control and ERAD actors in <span class="html-italic">CALR</span> mutated Myeloproliferative Neoplasm (MPN) patients was compared to matched normal patients. Blue denotes decreased expression, while red represents increased expression. NT: Not treated, 4-PBA: 4-phenylbutyrate, TUDCA: Tauroursodesoxycholic acid, Prot inh: Protease inhibitors, KIF: Kifunensin, CST: Castanospermin, DNJ: 1-Deoxynojirimycin, LCT: Lactacystin, RCV: Roscovitin, CQ: Chloroquine, Bafilo: Bafilomycin A1. DMSO was used as a control for all drugs except for 4-PBA, protease inhibitors, and chloroquine.</p> "> Figure 6
<p>Calreticulin (CALR) mutant proteins are subjected to an interaction with a network involving different proximal Endoplasmic Reticulum Associated Degradation (ERAD) actors. (<b>A</b>) Graphical representation of the actors involved in a network leading to the disposal of mutant CALR proteins based on results observed in <a href="#cancers-11-01921-f005" class="html-fig">Figure 5</a>, available databases (<a href="https://string-db.org/" target="_blank">https://string-db.org/</a>; <a href="https://thebiogrid.org/" target="_blank">https://thebiogrid.org/</a>; <a href="https://www.ebi.ac.uk/intact/" target="_blank">https://www.ebi.ac.uk/intact/</a>), and literature. (<b>B</b>) HEK293T cells were transfected with siRNA directed against TXNDC11 or a scramble control. Twenty-four hours later, cells were transfected with pcDNA6-CALR plasmids. Twenty-four hours later, cells were lysed before analysis by western blotting. CALR proteins were detected via their Myc epitope and actin was used as a loading control. The expression of the different forms of CALR secondary to TXNDC11 silencing was expressed as in <a href="#cancers-11-01921-f005" class="html-fig">Figure 5</a>D. The histograms represent mean ± standard error of the mean (SEM) determined in four independent experiments. (<b>C</b>) Upper panel: HEK293T were cotransfected with pcDNA6 plasmids to express Myc-6His tagged wild type (WT) or mutant CALR proteins together with a plasmid allowing the expression of FLAG-tagged Endoplasmic reticulum Degradation-Enhancing α-Mannosidase-like protein 3 (EDEM3) protein. Cell lysates were subjected to immunoprecipitation with an anti-Myc antibody and then subjected to western-blot analysis. IP: Immunoprecipitation, IB: Immunoblot. Lower panel: Interaction of CALR with EDEM3 was quantified as the ratio of EDEM3 and CALR A.U.: Arbitrary Unit. (<b>D</b>,<b>E</b>) HEK293T were cotransfected with pcDNA6 plasmids to express Myc-6His tagged WT or mutant CALR proteins together with a plasmid allowing the expression of FLAG-tagged EDEM3 (<b>D</b>) or HA-tagged EDEM1 (<b>E</b>) proteins. Cell lysates were subjected to immunoprecipitation with an anti-Myc antibody and then subjected to western-blot analysis using an anticalnexin (CANX) antibody. Shown are representative results observed in three independent experiments. IP: Immunoprecipitation, IB: Immunoblot, WCE: Whole cell extracts.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Mutant CALR are Faintly Expressed in Transfected and Primary Cells
2.2. Proteasome-Mediated Degradation Participates in Low CALR Mutant Expression
2.3. Mutant CALR Expression Does Not Alter ER Homeostasis
2.4. ERAD-Dependent Mechanisms of CALR Variants Degradation
2.5. Identification of a Network Involved in the ERAD-Dependent Degradation of CALR Variants
3. Discussion
4. Materials and Methods
4.1. Patients and DNA Constructs
4.2. Cell Culture and Lentiviral Transduction
4.3. Chemicals and Antibodies
4.4. RNA Interference
4.5. Coimmunoprecipitation and Western Blotting
4.6. RT-qPCR and ddPCR
4.7. Gene Expression Microarray Analysis and Statistical Selection
4.8. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Mansier, O.; Prouzet-Mauléon, V.; Jégou, G.; Barroso, K.; Raymundo, D.P.; Chauveau, A.; Dumas, P.-Y.; Lagarde, V.; Turcq, B.; Pasquet, J.-M.; et al. The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation. Cancers 2019, 11, 1921. https://doi.org/10.3390/cancers11121921
Mansier O, Prouzet-Mauléon V, Jégou G, Barroso K, Raymundo DP, Chauveau A, Dumas P-Y, Lagarde V, Turcq B, Pasquet J-M, et al. The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation. Cancers. 2019; 11(12):1921. https://doi.org/10.3390/cancers11121921
Chicago/Turabian StyleMansier, Olivier, Valérie Prouzet-Mauléon, Gwénaële Jégou, Kim Barroso, Diana Pelizzari Raymundo, Aurélie Chauveau, Pierre-Yves Dumas, Valérie Lagarde, Béatrice Turcq, Jean-Max Pasquet, and et al. 2019. "The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation" Cancers 11, no. 12: 1921. https://doi.org/10.3390/cancers11121921