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• W2606694411 abstract "•Calreticulin and TGF-β signaling intersect downstream of TGF-β receptor I and II•Calreticulin permits TGF-β-mediated inactivation of GSK3β•Calreticulin promotes Snail2/Slug-regulated repression of E-cadherin•Calreticulin is required for TGF-β mediated EMT and cardiomyogenesis from mESCs Calreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs. Calreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs. Ca2+-mediated signaling is essential for differentiation of embryonic stem cells (ESCs) into functional cardiomyocytes (Tonelli et al., 2012Tonelli F.M. Santos A.K. Gomes D.A. da Silva S.L. Gomes K.N. Ladeira L.O. Resende R.R. Stem cells and calcium signaling.Adv. Exp. Med. Biol. 2012; 740: 891-916Crossref PubMed Scopus (101) Google Scholar). Previous studies in embryos, cardiomyocytes in vitro, and ESCs show the regulatory role of Ca2+ in various stages of heart development and cardiomyogenesis (Liu et al., 2002Liu W. Yasui K. Opthof T. Ishiki R. Lee J.K. Kamiya K. Yokota M. Kodama I. Developmental changes of Ca(2+) handling in mouse ventricular cells from early embryo to adulthood.Life Sci. 2002; 71: 1279-1292Crossref PubMed Scopus (73) Google Scholar, Porter et al., 2003Porter Jr., G.A. Makuck R.F. Rivkees S.A. Intracellular calcium plays an essential role in cardiac development.Dev. Dyn. 2003; 227: 280-290Crossref PubMed Scopus (42) Google Scholar, Yanagida et al., 2004Yanagida E. Shoji S. Hirayama Y. Yoshikawa F. Otsu K. Uematsu H. Hiraoka M. Furuichi T. Kawano S. Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells.Cell Calcium. 2004; 36: 135-146Crossref PubMed Scopus (60) Google Scholar, Puceat and Jaconi, 2005Puceat M. Jaconi M. Ca(2+) signalling in cardiogenesis.Cell Calcium. 2005; 38: 383-389Crossref PubMed Scopus (46) Google Scholar, Itzhaki et al., 2006Itzhaki I. Schiller J. Beyar R. Satin J. Gepstein L. Calcium handling in embryonic stem cell-derived cardiac myocytes: of mice and men.Ann. N. Y. Acad. Sci. 2006; 1080: 207-215Crossref PubMed Scopus (38) Google Scholar, Janowski et al., 2006Janowski E. Cleemann L. Sasse P. Morad M. Diversity of Ca2+ signaling in developing cardiac cells.Ann. N. Y. Acad. Sci. 2006; 1080: 154-164Crossref PubMed Scopus (31) Google Scholar). The expression of Ca2+ pumps, channels, and regulatory and handling proteins is tightly controlled as cardiac differentiation progresses (Imanaka-Yoshida et al., 1996Imanaka-Yoshida K. Amitani A. Ioshii S.O. Koyabu S. Yamakado T. Yoshida T. Alterations of expression and distribution of the Ca2+-storing proteins in endo/sarcoplasmic reticulum during differentiation of rat cardiomyocytes.J. Mol. Cell. 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Groenendyk J. Szabo E. Gold L.I. Opas M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.Biochem. J. 2009; 417: 651-666Crossref PubMed Scopus (530) Google Scholar), is highly expressed in the early embryonic heart. It is silenced in the adult heart, highlighting its importance as a key regulator of cardiac differentiation (Michalak et al., 2002aMichalak M. Lynch J. Groenendyk J. Guo L. Parker J.M.R. Opas M. Calreticulin in cardiac development and pathology.Biochim. Biophys. Acta. 2002; 1600: 32-37Crossref PubMed Scopus (42) Google Scholar, Michalak et al., 2004Michalak M. Guo L. Robertson M. Lozak M. Opas M. Calreticulin in the heart.Mol. Cell. Biochem. 2004; 263: 137-142Crossref PubMed Scopus (25) Google Scholar, Papp et al., 2009bPapp S. Dziak E. Opas M. Embryonic stem cell derived cardiomyogenesis: a novel role for Calreticulin as a regulator.Stem Cells. 2009; 27: 1507-1515Crossref PubMed Scopus (15) Google Scholar, Faustino et al., 2016Faustino R.S. Behfar A. Groenendyk J. Wyles S.P. Niederlander N. Reyes S. Puceat M. Michalak M. Terzic A. Perez-Terzic C. Calreticulin secures calcium-dependent nuclear pore competency required for cardiogenesis.J. Mol. Cell. Cardiol. 2016; 92: 63-74Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The crucial role of calreticulin during cardiogenesis has been demonstrated by the lethal phenotype of calreticulin-deficient mice due to cardiac defects (Mesaeli et al., 1999Mesaeli N. Nakamura K. Zvaritch E. Dickie P. Dziak E. Krause K.H. Opas M. MacLennan D.H. Michalak M. Calreticulin is essential for cardiac development.J. Cell Biol. 1999; 144: 857-868Crossref PubMed Scopus (424) Google Scholar, Rauch et al., 2000Rauch F. Prud'homme J. Arabian A. Dedhar S. St-Arnaud R. Heart, brain, and body wall defects in mice lacking Calreticulin.Exp. Cell Res. 2000; 256: 105-111Crossref PubMed Scopus (114) Google Scholar) and disorganized cardiomyogenesis in calreticulin-deficient ESCs (Li et al., 2002Li J. Puceat M. Perez-Terzic C. Mery A. Nakamura K. Michalak M. Krause K.H. Jaconi M.E. Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis.J. Cell Biol. 2002; 158: 103-113Crossref PubMed Scopus (78) Google Scholar, Papp et al., 2009aPapp S. Dziak E. Kabir G. Backx P. Clement S. Opas M. Evidence for cardiac hypertrophy attenuation by Calreticulin induction using pressure overload and soluble agonists.Am. J. Pathol. 2009; 176: 1113-1121Abstract Full Text Full Text PDF Scopus (12) Google Scholar, Papp et al., 2009bPapp S. Dziak E. Opas M. Embryonic stem cell derived cardiomyogenesis: a novel role for Calreticulin as a regulator.Stem Cells. 2009; 27: 1507-1515Crossref PubMed Scopus (15) Google Scholar, Faustino et al., 2010Faustino R.S. Chiriac A. Niederlander N.J. Nelson T.J. Behfar A. Mishra P.K. Macura S. Michalak M. Terzic A. Perez-Terzic C. Decoded Calreticulin-deficient embryonic stem cell transcriptome resolves latent cardiophenotype.Stem Cells. 2010; 28: 1281-1291PubMed Google Scholar). On the other hand, overexpression of calreticulin is also lethal due to conductivity problems (Nakamura et al., 2001Nakamura K. Robertson M. Liu G. Dickie P. Nakamura K. Guo J.Q. Duff H.J. Opas M. Kavanagh K. Michalak M. Complete heart block and sudden death in mice overexpressing Calreticulin.J. Clin. Invest. 2001; 107: 1245-1253Crossref PubMed Scopus (110) Google Scholar, Mery et al., 2005Mery A. Aimond F. Menard C. Mikoshiba K. Michalak M. Puceat M. Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling.Mol. Biol. Cell. 2005; 16: 2414-2423Crossref PubMed Scopus (91) Google Scholar, Hattori et al., 2007Hattori K. Nakamura K. Hisatomi Y. Matsumoto S. Suzuki M. Harvey R.P. Kurihara H. Hattori S. Yamamoto T. Michalak M. Endo F. Arrhythmia induced by spatiotemporal overexpression of Calreticulin in the heart.Mol. Genet. Metab. 2007; 91: 285-293Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), underscoring the importance of calreticulin in both cardiac development and function (Papp et al., 2008Papp S. Zhang X. Szabo E. Michalak M. Opas M. Expression of endoplasmic reticulum chaperones in cardiac development.Open Cardiovasc. Med. J. 2008; 2: 31-35Crossref PubMed Google Scholar, Wang et al., 2012Wang W.A. Groenendyk J. Michalak M. Calreticulin signaling in health and disease.Int. J. Biochem. Cell Biol. 2012; 44: 842-846Crossref PubMed Scopus (149) Google Scholar). The molecular mechanisms underlying calreticulin's effects on cardiac development continue to be elucidated. It is known that intranuclear translocation of cardiogenic transcription factors is affected by calcineurin, a Ca2+∕calmodulin-regulated cytosolic phosphatase (Crabtree, 1999Crabtree G.R. Generic signals and specific outcomes: signaling through Ca2+, Calcineurin, and NF-AT.Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, Frey et al., 2000Frey N. McKinsey T.A. Olson E.N. Decoding calcium signals involved in cardiac growth and function.Nat. Med. 2000; 6: 1221-1227Crossref PubMed Scopus (286) Google Scholar, Crabtree and Schreiber, 2009Crabtree G.R. Schreiber S.L. SnapShot: Ca2+-Calcineurin-NFAT signaling.Cell. 2009; 138 (210.e1): 210Abstract Full Text PDF PubMed Scopus (87) Google Scholar). The Ca2+ influx required to activate calcineurin depends on the sustained release of Ca2+ from ER stores (Crabtree, 2001Crabtree G.R. Calcium, Calcineurin, and the control of transcription.J. Biol. 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Commun. 2003; 311: 1173-1179Crossref PubMed Scopus (28) Google Scholar and Lynch et al., 2005Lynch J. Guo L. Gelebart P. Chilibeck K. Xu J. Molkentin J.D. Agellon L.B. Michalak M. Calreticulin signals upstream of Calcineurin and MEF2C in a critical Ca2+-dependent signaling cascade.J. Cell Biol. 2005; 170: 37-47Crossref PubMed Scopus (66) Google Scholar elegantly demonstrated that calreticulin is an upstream regulator of calcineurin, thus providing a mechanism for the control of early cardiogenesis. Gene regulation, cellular migration, and cell-cell and cell-substratum communication during cardiogenesis involve a variety of intrinsic and extrinsic factors (Maltsev et al., 1993Maltsev V.A. Rohwedel J. Hescheler J. Wobus A.M. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types.Mech. Dev. 1993; 44: 41-50Crossref PubMed Scopus (390) Google Scholar, Hescheler et al., 1997Hescheler J. Fleischmann B.K. Lentini S. 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Also, pleiotropic growth factors, most notably transforming growth factor β (TGF-β), induce cardiac development in general (Schneider and Parker, 1991Schneider M.D. Parker T.G. Cardiac growth factors.Prog. Growth Factor Res. 1991; 3: 1-26Abstract Full Text PDF PubMed Scopus (53) Google Scholar, MacLellan et al., 1993MacLellan W.R. Brand T. Schneider M.D. Transforming growth factor-beta in cardiac ontogeny and adaptation.Circ. Res. 1993; 73: 783-791Crossref PubMed Scopus (76) Google Scholar, Schneider et al., 1994Schneider M.D. Kirshenbaum L.A. Brand T. MacLellan W.R. Control of cardiac gene transcription by fibroblast growth factors.Mol. Reprod. Dev. 1994; 39: 112-117Crossref PubMed Scopus (7) Google Scholar) and cardiac differentiation from ESCs in particular (Behfar et al., 2002Behfar A. Zingman L.V. Hodgson D.M. Rauzier J.M. Kane G.C. Terzic A. Puceat M. Stem cell differentiation requires a paracrine pathway in the heart.FASEB J. 2002; 16: 1558-1566Crossref PubMed Scopus (427) Google Scholar, Sachinidis et al., 2003Sachinidis A. Fleischmann B.K. Kolossov E. Wartenberg M. Sauer H. Hescheler J. Cardiac specific differentiation of mouse embryonic stem cells.Cardiovasc. Res. 2003; 58: 278-291Crossref PubMed Scopus (194) Google Scholar, Lim et al., 2007Lim J.Y. Kim W.H. Kim J. Park S.I. Involvement of TGF-beta1 signaling in cardiomyocyte differentiation from P19CL6 cells.Mol. Cells. 2007; 24: 431-436PubMed Google Scholar, Faustino et al., 2008Faustino R.S. Behfar A. Perez-Terzic C. Terzic A. Genomic chart guiding embryonic stem cell cardiopoiesis.Genome Biol. 2008; 9: R6Crossref PubMed Scopus (62) Google Scholar), reviewed in (Puceat, 2007Puceat M. TGFbeta in the differentiation of embryonic stem cells.Cardiovasc. Res. 2007; 74: 256-261Crossref PubMed Scopus (57) Google Scholar, Kovacic et al., 2012Kovacic J.C. Mercader N. Torres M. Boehm M. Fuster V. Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: from cardiovascular development to disease.Circulation. 2012; 125: 1795-1808Crossref PubMed Scopus (291) Google Scholar). It has also been established that TGF-β induces EMT (Zavadil and Böttinger, 2005Zavadil J. Böttinger E.P. TGF-ß and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1383) Google Scholar, Xu et al., 2009Xu J. Lamouille S. Derynck R. TGF-beta-induced epithelial to mesenchymal transition.Cell Res. 2009; 19: 156-172Crossref PubMed Scopus (1941) Google Scholar, Lamouille et al., 2014Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (5235) Google Scholar), thereby promoting exit from pluripotency in ESCs (Acloque et al., 2009Acloque H. Adams M.S. Fishwick K. Bronner-Fraser M. Nieto M.A. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.J. Clin. Invest. 2009; 119: 1438-1449Crossref PubMed Scopus (1042) Google Scholar, Lamouille et al., 2014Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (5235) Google Scholar, Kim et al., 2014Kim Y.S. Yi B.R. Kim N.H. Choi K.C. Role of the epithelial-mesenchymal transition and its effects on embryonic stem cells.Exp. Mol. Med. 2014; 46: e108Crossref PubMed Scopus (83) Google Scholar). Importantly, Joanne Murphy-Ullrich's lab has demonstrated that calreticulin-regulated Ca2+ signaling controls TGF-β-induced deposition of extracellular matrix by mouse embryonic fibroblasts (Zimmerman et al., 2013Zimmerman K.A. Graham L.V. Pallero M.A. Murphy-Ullrich J.E. Calreticulin (CRT) regulates transforming growth factor-beta (TGF-beta) stimulated extracellular matrix production.J. Biol. Chem. 2013; 288: 14584-14598Crossref PubMed Scopus (44) Google Scholar) and of collagen I by vascular smooth muscle cells both in vitro and in vivo (Zimmerman et al., 2015Zimmerman K.A. Xing D. Pallero M.A. Lu A. Ikawa M. Black L. Hoyt K.L. Kabarowski J.H. Michalak M. Murphy-Ullrich J.E. Calreticulin regulates neointima formation and collagen deposition following carotid artery ligation.J. Vasc. Res. 2015; 52: 306-320Crossref PubMed Scopus (14) Google Scholar). This has been extended to pathological settings such as fibrosis (Kypreou et al., 2008Kypreou K.P. Kavvadas P. Karamessinis P. Peroulis M. Alberti A. Sideras P. Psarras S. Capetanaki Y. Politis P.K. Charonis A.S. Altered expression of Calreticulin during the development of fibrosis.Proteomics. 2008; 8: 2407-2419Crossref PubMed Scopus (56) Google Scholar, Van Duyn et al., 2010Van Duyn G.L. Sweetwyne M.T. Pallero M.A. Murphy-Ullrich J.E. Intracellular Calreticulin regulates multiple steps in fibrillar collagen expression, trafficking, and processing into the extracellular matrix.J. Biol. Chem. 2010; 285: 7067-7078Crossref PubMed Scopus (44) Google Scholar, Prakoura et al., 2013Prakoura N. Politis P.K. Ihara Y. Michalak M. Charonis A.S. Epithelial Calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.Am. J. Pathol. 2013; 183: 1474-1487Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). By controlling intracellular Ca2+ homeostasis, calreticulin has been firmly placed at the crossroads of several signaling pathways (Michalak et al., 2009Michalak M. Groenendyk J. Szabo E. Gold L.I. Opas M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.Biochem. J. 2009; 417: 651-666Crossref PubMed Scopus (530) Google Scholar). The aim of the present paper was to elucidate interconnections between calreticulin's role as a Ca2+ homeostasis regulator and TGF-β signaling in the context of cardiomyogenesis of mouse ESCs. To examine if the absence of calreticulin impairs cardiac differentiation of mouse ESCs, we counted contracting (“beating”) EBs during differentiation from day 3 to day 14 (D3-D14) in wild-type (WT), CRT-KO, and constitutively activated calcineurin-overexpressing CRT-KO (CN) cultures. The percentage of beating EBs was higher in WT versus CRT-KO EBs; the difference was already substantial at D7, became 2-fold at D10, and 3-fold by D14. In addition, the percentage of beating in CN EBs was significantly higher than in CRT-KO EBs (Figure 1A). Flow cytometry analysis on D14 WT, CRT-KO, and CN EBs using anti-cardiac troponin I antibody Alexa Fluor 488 conjugated showed higher cardiac troponin I in WT and CN EBs compared with CRT-KO EBs (Figure 1B). WT EBs showed increasing levels of cardiac myosin heavy chain (MHC) as differentiation progressed, while in CRT-KO cells MHC levels remained low (Figures 1C and 1D). During cardiomyogenesis of mouse ESCs, expression of the mesodermal and cardiac markers Mesp1, Gata4, and Nkx2-5 was induced in the WT cells but not in the CRT-KO cells, in which all markers remained at very low levels (Figure 1E). Immunoblot analysis was carried out to examine the expression levels of E- and N-cadherins in WT, CRT-KO, and CN cells throughout the differentiation period (Figures 2A and 2B ). The expression of E-cadherin in WT EBs progressively decreased as differentiation progressed, while remaining abundant in CRT-KO EBs. In contrast, the N-cadherin expression increased significantly starting at D7 in WT EBs and continued to increase up to D14. N-Cadherin expression remained very low in CRT-KO EBs. CN cells showed a similar trend as the WT cells for both cadherins. We also analyzed the mRNA expression level of E-Cadherin, N-Cadherin and their upstream transcription factors Snail1, Snail2/Slug, and Twist1 (Figure 2C). Similar to the protein expression pattern, E-Cadherin mRNA was higher overall in CRT-KO cells, peaking at D10. Snail2 expression, a repressor of E-Cadherin, remained very low throughout differentiation in these CRT-KO cells. On the other hand, N-Cadherin mRNA expression was very low in CRT-KO cells, while it was high and increased with time in WT cells. Twist1, an upstream regulator of N-Cadherin, also increased during differentiation in WT cells. Snail1 expression did not show any significant difference between CRT-KO and WT cells, and was thus not further examined. TGF-β regulates GSK3β activity and SNAIL2/SLUG nuclear translocation, thereby affecting E-Cadherin expression (Zhou et al., 2004Zhou B.P. Deng J. Xia W. Xu J. Li Y.M. Gunduz M. Hung M.C. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.Nat. Cell Biol. 2004; 6: 931-940Crossref PubMed Scopus (1319) Google Scholar, Kim et al., 2012Kim J.Y. Kim Y.M. Yang C.H. Cho S.K. Lee J.W. Cho M. Functional regulation of Slug/Snail2 is dependent on GSK-3beta-mediated phosphorylation.FEBS J. 2012; 279: 2929-2939Crossref PubMed Scopus (62) Google Scholar, Wakefield and Hill, 2013Wakefield L.M. Hill C.S. Beyond TGFß: roles of other TGFß superfamily members in cancer.Nat. Rev. Cancer. 2013; 13: 328-341Crossref PubMed Scopus (287) Google Scholar). We thus examined the expression of TGF-β receptor genes in WT and CRT-KO EBs throughout cardiac differentiation. The expression of all three TGF-β receptors was suppressed in CRT-KO EBs (Figure 3A). We then evaluated E- and N-cadherin expression, phosphorylation status of AKT on serine 473 (S473), indicative of AKT activation, and GSK3β phosphorylation on serine 9 (S9), indicative of GSK3β inactivation (Fang et al., 2000Fang X. Yu S.X. Lu Y. Bast R.C.J. Woodgett J.R. Mills G.B. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A.Proc. Natl. Acad. Sci. USA. 2000; 97: 11960-11965Crossref PubMed Scopus (639) Google Scholar). Figure 3B shows that, in contrast to WT cells, N-cadherin was significantly lower in CRT-KO cells, while E-cadherin was elevated. The CN cells followed a similar trend to that of the WT cells (Figure 3B). Phosphorylation of AKT on S473 and GSK3β on S9 was higher in WT and CN cells at D14 compared with CRT-KO cells. Total AKT and GSK3β levels were similar in all three cell lines. Since S9 phosphorylation renders GSK3β inactive, this indicates that the enzyme is more active in the absence of calreticulin. A detailed phosphorylation pattern for AKT and GSK3β in earlier days of cardiac differentiation is provided in Figure S1. We also assessed the nuclear localization of SNAIL2/SLUG in WT and CRT-KO cells by subcellular fractionation. Figure 3C shows that SNAIL2/SLUG expression is high in the nucleus of WT cells throughout cardiomyocyte differentiation, while the protein is almost undetectable in the nucleus of the CRT-KO cells. Next, we examined the expression levels of E-cadherin, N-cadherin, and GSK3β (S9) phosphorylation in the presence or absence of the GSK3β inhibitor SB415286 and the TGF-β inhibitor SB431542 by western blot analysis (Figures 4A and 4B ). GSK3β inhibition reduced E-cadherin levels in both WT and KO cells, with no significant effect on N-cadherin in WT cells. This is in stark contrast to CRT-KO cells, where N-cadherin abundance increased by 2-fold following GSK3β inhibition. TGF-β inhibition caused significant effects only in WT cells and was without effect on the CRT-KO cells. Specifically, TGF-β inhibition caused an increase in E-cadherin expression and a decrease in N-cadherin expression in WT cells. GSK3β inhibition did not appreciably affect the level of S9 phosphorylation of GSK3β in WT cells, while it increased S9 phosphorylation of GSK3β in CRT-KO cells. Finally, TGF-β inhibition effectively abolished S9 phosphorylation of GSK3β in the calreticulin-containing WT cells, while it did not affect the already low level of S9 phosphorylation of GSK3β in CRT-KO cells. Double-label confocal immunocolocalization of EBs (Figure 4C) shows that in control WT EBs (DMSO only), the E-cadherin signal was weak compared with the N-cadherin signal. In the untreated CRT-KO EBs (DMSO only), the E-cadherin signal was much stronger than the N-cadherin signal and it was predominantly cytoplasmic with a weak junctional component. GSK3β inhibition (GSK3β inh.) in WT EBs further lowers the weak E-cadherin signal. In contrast, GSK3β inhibition in WT EBs had a similar effect on the intensity of N-cadherin fluorescence as WT DMSO EBs. In CRT-KO EBs treated with GSK3β inhibitor (GSK3β inh.), the E-cadherin signal became very weak while there was a visible increase the N-cadherin signal. TGF-β inhibition in WT EBs increased the intensity of E-cadherin labeling in comparison with the control EBs; however, it substantially reduced N-cadherin fluorescence labeling, again in comparison with the untreated EBs. In CRT-KO EBs, TGF-β inhibition did not appear to bring about any substantial changes in labeling with either antibody in comparison with the control CRT-KO EBs. Finally, we examined GATA4 protein level and its nuclear localization. GATA4 was more abundant in WT and CN cells compared with CRT-KO cells (Figure 5A). Cell fractionation showed that GATA4 was substantially enriched in the nucleus of WT and CN cells at D10 and D14 of differentiation, while it was absent from the nuclei of the CRT-KO cells (Figure 5B). qPCR showed that, in WT cells, GSK3β inhibition did not affect Gata4 mRNA expression but TGF-β inhibition reduced Gata4 mRNA expression significantly (Figure 5C). In contrast, GSK3β inhibition significantly increased mRNA expression of Gata4 in CRT-KO cells, and TGF-β inhibition significantly decreased Gata4 mRNA expression (Figure 5D). The effect of TGF-β and GSK3β inhibition on mRNA expression of Gata4 in earlier days of differentiation is shown in Figure S2. Ca2+ has been identified as a major second messenger in directing stem cells toward cardiomyocyte differentiation (Puceat and Jaconi, 2005Puceat M. Jaconi M. Ca(2+) signalling in cardiogenesis.Cell Calcium. 2005; 38: 383-389Crossref PubMed Scopus (46) Google Scholar). In vitro cardiomyocyte differentiation of EBs revealed that CRT-KO cells failed to differentiate and beat properly (Mesaeli et al., 1999Mesaeli N. Nakamura K. Zvaritch E. Dickie P. Dziak E. Krause K.H. Opas M. MacLennan D.H. Michalak M. Calreticulin is essential for cardiac development.J. Cell Biol. 1999; 144: 857-868Crossref PubMed Scopus (424)" @default.
• W2606694411 created "2017-04-28" @default.
• W2606694411 creator A5071277216 @default.
• W2606694411 creator A5085815453 @default.
• W2606694411 date "2017-05-01" @default.
• W2606694411 modified "2023-10-09" @default.
• W2606694411 title "Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells" @default.
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