Abstract
Calreticulin, the major Ca2+ buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt-/- cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt-/- cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis. Most importantly, we demonstrated that calreticulin-containing cells had lower c-Src kinase activity, and this was accomplished via the Ca2+-homeostatic function of calreticulin. Specifically, lowering cytosolic [Ca2+] in calreticulin-containing osteogenic WT cells with BAPTA-AM, activated c-Src and impaired osteogenic differentiation. Conversely, increasing cytosolic [Ca2+] in crt-/- cells with ionomycin deactivated c-Src kinase and restored osteogenesis. The immediate effector of calreticulin, the Ser/Thr phosphatase calcineurin, was less active in crt-/- cells, however, its activity was rescued upon inhibition of c-Src activity by small molecule inhibitors. Finally, we showed that higher activity of calcineurin correlated with increased level of nuclear Runx2, a transcription factor that is the master regulator of osteogenesis. Collectively, our work has identified a novel pathway involving calreticulin regulated Ca2+ signalling via c-Src in osteogenic differentiation of embryonic stem cells.
Highlights
Calreticulin (CRT) is an endoplasmic reticulum (ER)-residing protein that regulates the intracellular Ca2+ ([Ca2+]i) homeostasis via its high Ca2+ storage capacity (Nishida, 1983; Toyoshima et al, 1986; Michalak et al, 2009)
We demonstrate that in the absence of calreti culin, osteogenic differentiation of calreticulin-deficient mouse ES cell (ESC) is impaired compared to wild type (WT) mESCs. c-Src activity is increased in crt−/− mESCs and, importantly, c-Src activity is shown to be regulated by [Ca2+]I, such that lowering of [Ca2+]i results in increased c-Src activity
Calreticulin WT, crt−/−, and crt−/− transfected with calreticulin expressing vectors mESCs were subjected to osteogenic differentiation
Summary
Calreticulin (CRT) is an endoplasmic reticulum (ER)-residing protein that regulates the intracellular Ca2+ ([Ca2+]i) homeostasis via its high Ca2+ storage capacity (Nishida, 1983; Toyoshima et al, 1986; Michalak et al, 2009). Gain- and loss-of-function experiments indicated that higher cal cineurin expression is positively correlated with osteoblastic differentiation and bone formation in mice, the potential mechanisms have not been fully elucidated (Pilquil et al, 2020; Sun, 2005; Tomita et al, 2002; Winslow, 2006) Together these findings underscore the significance of the calreticulin/Ca2+/calcineurin signalling axis in osteogenic differentiation. The absence of calreticulin inhibits the ac tivity of calcineurin and activates c-Src, which results in inhibition of the nuclear translocation of the master osteogenic transcription factor, runtdomain related transcription factor 2 (Runx2) This suggests a novel mechanism in which c-Src regulates mESCs osteogenic differentiation via the effects on Runx subcellular trafficking, downstream of calreticulin
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