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.

钙网蛋白是内质网的主要 Ca ²⁺缓冲液，在胚胎干细胞的命运选择中起重要作用。使用器官发生的胚状体方法，我们显示crt ^-/-细胞相对于含有钙网蛋白的成骨WT细胞中的成骨受损。在非成骨crt ^-/-细胞中，c-Src-一种非受体酪氨酸激酶-被激活，其抑制作用挽救了成骨。最重要的是，我们证明了含有钙网蛋白的细胞具有较低的 c-Src 激酶活性，这是通过钙网蛋白的 Ca ²⁺ -稳态功能实现的。具体而言，降低细胞溶质 [Ca ²⁺] 在含有钙网蛋白的成骨 WT 细胞中，具有 BAPTA-AM、活化的 c-Src 和受损的成骨分化。相反，用离子霉素增加crt ^{-/-细胞中的胞质[Ca 2+} ]使c-Src激酶失活并恢复成骨。钙网蛋白的直接效应物，丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶，在crt ^-/-细胞中的活性较低，然而，在小分子抑制剂抑制 c-Src 活性后，其活性得以恢复。最后，我们发现钙调神经磷酸酶的更高活性与核 Runx2 水平的增加相关，核 Runx2 是一种转录因子，是成骨的主要调节因子。总的来说，我们的工作已经确定了一种涉及钙网蛋白调节的 Ca ^{2+的新途径}通过 c-Src 信号传导在胚胎干细胞的成骨分化中。