In the presence of calreticulin

In the presence of calreticulin, a cadherin switch occurs between E- and N-cadherins during the course of cardiomyocyte differentiation. This is exhibited by a decrease in E-cadherin abundance with a progressive increase in (and replacement by) N-cadherin. The presence of N-cadherin at the cell surface is necessary for the proper formation and function, as demonstrated by contraction, of cardiomyocytes (Radice et al., 1997; Ong et al., 1998; Luo and Radice, 2003). Accordingly, we show an induction of TWIST1, a transcriptional regulator of N-cadherin (Derycke and Bracke, 2004), and increases in both N-Cadherin mRNA and protein, as the calreticulin-containing ESCs begin to express cardiac MHC and EBs start to beat. The expression of E-cadherin is downregulated by the SNAIL2/SLUG transcription factor, which suppresses the E-cadherin promoter (Batlle et al., 2000; Kim et al., 2012). GSK3β phosphorylates SNAIL2/SLUG exposing its nuclear export sequence (Dominguez et al., 2003; Kim et al., 2012). In calreticulin-containing WT cells, GSK3β is inactivated by phosphorylation at S9, whereby it is unable to facilitate export of SNAIL2/SLUG from the nucleus to the cytoplasm. SNAIL2/SLUG remains in the nucleus where it suppresses E-cadherin expression (Cano et al., 2000; Dominguez et al., 2003; Zhou et al., 2004; Ko et al., 2007); our current findings are in full accordance with these reports. Thus, in the absence of calreticulin, the expression of SNAIL2/SLUG is low and its nuclear translocation is impaired as well. This explains the absence of a cadherin switch in CRT-KO order GSK461364 and the abundant expression of E-cadherin throughout their dysregulated cardiomyocyte differentiation period. TGF-β is a potent inducer of EMT during differentiation (Derynck et al., 2014). We thus asked if the effect of calreticulin on EMT may be due to impaired TGF-β signaling in the absence of calreticulin. We show that the expression of TGF-β receptor mRNA is downregulated in the absence of calreticulin. Moreover, we analyzed AKT activation by phosphorylation on S473 downstream of TGF-β receptor activation (Alessi et al., 1996; Shin et al., 2001) and we show that AKT activation is impaired in the absence of calreticulin. Normally, activated, S473-phosphorylated AKT inhibits GSK3β activity by phosphorylating it on S9 (Fang et al., 2000). In the absence of calreticulin, GSK3β phosphorylation on S9 is diminished, and thus activity of this kinase is increased. In brief, we show here that inhibition of signaling from AKT/GSK3β causes downstream inhibition of nuclear accumulation of SNAIL2/SLUG and, in the end result, E-cadherin overexpression. Furthermore, we show that overexpression of calcineurin in CRT-KO cells rescues AKT activation. It is important to note here that calcineurin is an effector of calreticulin, in that Ca2+ influx required to activate calcineurin depends on Ca2+ stored in the ER by calreticulin (Michalak et al., 2002b). Conversely, it has been shown that inhibition of calcineurin impacts negatively, signaling downstream of AKT/GSK3β (De Windt et al., 2000; Soleimanpour et al., 2010). Collectively, in the absence of calreticulin, phosphorylation of AKT on S473 and GSK3β on S9 are diminished, thus rendering the TGF-β signaling pathway dysregulated. Our results show that inhibition of TGF-β during mesodermal formation in WT EBs from D3 to D5 with SB431542-compound, which interferes with Tgfbr1-mediated TGF-β signaling, significantly decreases the abundance of N-cadherin and increases E-cadherin levels to that seen in CRT-KO EBs. Inhibition of TGF-β does not have any significant effect on either E-cadherin or N-cadherin levels in CRT-KO EBs, as TGF-β signaling is already impaired in the absence of calreticulin. Consequently, we infer that the impairment in TGF-β signaling is the cause of the impaired EMT in the absence of calreticulin and, as a result, impaired cardiomyogenesis. TGF-β is one of the main regulators of GSK3β, whereby TGF-β decreases activity of this kinase (Yoshino et al., 2007; Millet et al., 2009; Byun et al., 2014; Cheng et al., 2014; Lal et al., 2014; see Guo and Wang, 2009; Xu et al., 2015 for reviews). This is in agreement with our findings that GSK3β S9 phosphorylation is high (i.e., activity is low) in the calreticulin-containing WT EBs, while GSK3β S9 phosphorylation is lower in the absence of calreticulin. SNAIL2/SLUG expression is higher in WT EBs and it is enriched in the nucleus, which may account for the lower levels of E-cadherin in WT EBs compared with CRT-KO EBs. Furthermore, GSK3β is phosphorylated on S9 in calreticulin-containing WT EBs. Hence, in these EBs, inhibition of GSK3β lacks substantial effect on either E- or N-cadherin expression as GSK3β is already inactive in the presence of calreticulin. Conversely, in CRT-KO cells, inhibition of GSK3β induces EMT as demonstrated by reduced levels of E-cadherin and increased levels of N-cadherin. TGF-β signaling is impaired in CRT-KO EBs where GSK3β remains active; hence GSK3β inhibition can rescue EMT in CRT-KO EBs. Finally, low mRNA expression levels of the cardiac transcription factor Gata4 in CRT-KO EBs can be rescued by inhibition of GSK3β during mesodermal formation. TGF-β inhibition completely impairs Gata4 mRNA expression in both WT and CRT-KO EBs. Thus, we conclude that the absence of calreticulin impairs TGF-β-mediated EMT and also impairs expression and nuclear translocation of cardiac transcription factors such as GATA4.