Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells

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

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice

BACKGROUND: Calreticulin is a Ca(2+ )binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene

Functional specialization of calreticulin domains

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions