The molecular mechanisms underlying calreticulin s effects o

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, 1999; Frey et al., 2000; Crabtree and Schreiber, 2009). The Ca2+ influx required to activate calcineurin depends on the sustained release of Ca2+ from ER stores (Crabtree, 2001), which is dependent on calreticulin abundance (Michalak et al., 2002b). Importantly, overexpression of constitutively activated calcineurin specifically targeted to the heart rescues the lethal cardiac phenotype in calreticulin-null (CRT-KO) mice (Guo et al., 2002). Lynch and Michalak (2003) and Lynch et al. (2005) 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., 1993; Hescheler et al., 1997; Sachinidis et al., 2003). Epithelial-to-mesenchymal transition (EMT), has been identified as one of the first steps of cardiac differentiation (Thiery and Sleeman, 2006; Thiery et al., 2009; Kovacic et al., 2012). Expression of N-cadherin and the transcription factors SNAIL1 (SNAIL), SNAIL2 (SLUG), TWIST1, delta-EF1/ZEB1, and SIP1/ZEB2 are increased during EMT, while E-cadherin ck1 inhibitor is decreased (Lee et al., 2006; Zeisberg and Neilson, 2009). A multitude of transcription factors play a role in cardiomyocyte specification, including NKX2-5, MEF2c, GATA 4,5,6, MESP1, MYOCARDIN, TBX5, and TBX20, which work in a combinatorial manner to turn on the transcriptional activity of cardiac gene promoters (Durocher and Nemer, 1998; Brand, 2003; Olson, 2006). Also, pleiotropic growth factors, most notably transforming growth factor β (TGF-β), induce cardiac development in general (Schneider and Parker, 1991; MacLellan et al., 1993; Schneider et al., 1994) and cardiac differentiation from ESCs in particular (Behfar et al., 2002; Sachinidis et al., 2003; Lim et al., 2007; Faustino et al., 2008), reviewed in (Puceat, 2007; Kovacic et al., 2012). It has also been established that TGF-β induces EMT (Zavadil and Böttinger, 2005; Xu et al., 2009; Lamouille et al., 2014), thereby promoting exit from pluripotency in ESCs (Acloque et al., 2009; Lamouille et al., 2014; Kim et al., 2014). 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., 2013) and of collagen I by vascular smooth muscle cells both in vitro and in vivo (Zimmerman et al., 2015). This has been extended to pathological settings such as fibrosis (Kypreou et al., 2008; Van Duyn et al., 2010; Prakoura et al., 2013). By controlling intracellular Ca2+ homeostasis, calreticulin has been firmly placed at the crossroads of several signaling pathways (Michalak et al., 2009). 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.
Results
Discussion Ca2+ has been identified as a major second messenger in directing stem cells toward cardiomyocyte differentiation (Puceat and Jaconi, 2005). In vitro cardiomyocyte differentiation of EBs revealed that CRT-KO cells failed to differentiate and beat properly (Mesaeli et al., 1999; Papp et al., 2009a, 2009b; Faustino et al., 2010, 2016), but its mechanism has remained elusive. Depleting Ca2+ using BAPTA mimics the CRT-KO phenotype in ESC-generated EBs, which can be rescued by treatment with the Ca2+ ionophore, ionomycin (Li et al., 2002; Papp et al., 2009b; Faustino et al., 2016). Therefore, calreticulin as a regulator of Ca2+ homeostasis has a fundamental role in the early stages of ESC differentiation and cardiomyogenesis (Michalak et al., 2004; Bedard et al., 2005; Puceat and Jaconi, 2005). Events downstream of calreticulin mediated by Ca2+ involve calcineurin (Lynch and Michalak, 2003; Lynch et al., 2005). While it is generally accepted that multiple TGF-β-dependent pathways affect Ca2+ homeostasis, the reverse is also true as calcineurin affects TGF-β-dependent effects (Alevizopoulos et al., 1997; Gooch et al., 2006; Cobbs and Gooch, 2007; Zhao et al., 2013). In the present work, we show that calreticulin is essential for the induction of TGF-β-mediated EMT during cardiac differentiation of mouse ESCs. Using calreticulin-containing WT ESCs, we show increased N-cadherin expression and very low E-cadherin expression, known as the cadherin switch, which is a hallmark of EMT (Maeda et al., 2005; Wheelock et al., 2008). The mRNA and protein expression of the EMT biomarkers Snail1, Snail2/Slug, Twist1, and N-Cadherin were all low in the absence of calreticulin throughout cardiomyocyte differentiation. This is in contrast to the expression of E-Cadherin mRNA and protein, which are both highly expressed in CRT-KO ESCs. We thus conclude that the cadherin switch is impaired in the absence of calreticulin. A corresponding effect has been observed in a different cell system, the Madin-Darby canine kidney epithelial cells, where EMT is induced by calreticulin overexpression (Hayashida et al., 2006; Ihara et al., 2011).