Regulation of tendon formation by Ca2+ signaling through CaV1.2 L-type voltage-gated calcium channel

项目总结 细胞内钙离子影响主要的肌腱信号通路。尽管已经研究了钙信号转导 因其在肌肉收缩、免疫细胞激活、激素分泌、细胞增殖、 神经调节、基因激活、肌腱钙信号转导细节及钙通道 进入肌腱成纤维细胞的情况在很大程度上是未知的。使用具有功能增益CaV1.2的新型小鼠模型 突变通道(CaV1.2TS)，我们观察到通过CaV1.2的钙内流对肌腱有很强的调节作用。 CaV1.2TS通道在致孔CaV1.2α1C亚基中携带功能增益G406R突变，该突变损害 电压依赖性失活，并允许更多的钙离子流入细胞。我们观察到，驾驶表情 CaV1.2TS合并肌腱硬化轴-CRE(ScxCre)导致肌腱质量明显增加。使用 记者老鼠发现，在肌腱发育过程中，CaV1.2在肌腱成纤维细胞中广泛表达。 因此，我们推测，肌腱成纤维细胞中的钙离子通过CaV1.2内流调节肌腱的形成。 与我们之前的论证相吻合的假设，即CaV1.2也在成骨细胞前体细胞中发挥作用 调节骨形成和动态平衡。在这项提议中，我们试图确定细胞和分子 CaV1.2调节肌腱形成的钙信号转导机制我们的具体假设是 CA2信号通过CaV1.2调节控制肌腱细胞的肌腱转录因子的表达 肌腱发育和力学过程中的增殖和/或肌腱细胞外基质合成 超负荷诱导的成人肌腱生长。在目标1中，我们将通过以下方式上调肌腱成纤维细胞的钙信号 我们的CaV1.2TS功能获得小鼠模型并测定细胞增殖、细胞外基质胶原 肌腱转录因子的合成和表达。我们将确定中介的信号级联 尾部肌腱成纤维细胞增殖和细胞外基质合成的钙信号增强 成纤维细胞培养体系。在目标2中，我们将使用我们新开发的Cav1.2可诱导条件淘汰赛 小鼠模型通过CaV1.2确定钙信号在肌腱发育过程中的生理作用 出生后肌腱形成，并在机械超负荷诱导成人肌腱生长。成功 这项研究的完成将从根本上了解钙信号在肌腱中的作用 形成和一个平台，以确定制定肌腱疾病治疗战略的新目标。

PROJECT SUMMARY Intracellular Ca2+ influences major tendon signaling pathways. Although Ca2+ signaling has been studied extensively for its roles in muscle contraction, immune cell activation, hormone secretion, cell proliferation, neuronal regulation, and gene activation, Ca2+ signaling details in tendon and the channels responsible for Ca2+ influx into tendon fibroblasts are largely unknown. Using novel mouse models with a gain-of-function CaV1.2 mutant channel (CaV1.2TS), we observed potent regulatory effects of Ca2+ influx through CaV1.2 on tendons. CaV1.2TS channels carry a gain-of-function G406R mutation in the pore-forming CaV1.2 α1C subunit that impairs voltage-dependent inactivation and allows more Ca2+ influx into the cell. We observed that driving expression of CaV1.2TS specifically in tendon with Scleraxis-Cre (ScxCre) leads to a marked increase in tendon mass. Using a reporter mouse, we found that CaV1.2 is extensively expressed in tendon fibroblasts during tendon development. We therefore postulate that Ca2+ influx through CaV1.2 in tendon fibroblasts regulates tendon formation, a hypothesis that fits with our previous demonstration that CaV1.2 also functions in osteoblast precursor cells to regulate bone formation and homeostasis. In this proposal we seek to determine the cellular and molecular mechanisms of Ca2+ signaling through CaV1.2 that regulate tendon formation. Our specific hypothesis is that Ca2+ signaling through CaV1.2 regulates expression of tendon transcription factors that control tendon cell proliferation and/or tendon extracellular matrix (ECM) synthesis during tendon development and mechanical overload-induced adult tendon growth. In Aim 1, we will upregulate Ca2+ signals in tendon fibroblasts by using our CaV1.2TS gain-of-function mouse model and determine cell proliferation, extracellular matrix collagen synthesis, and expression of tendon transcription factors. We will determine the signaling cascades that mediate increased Ca2+ signals to upregulated tendon fibroblast proliferation and ECM synthesis in vitro in a tail tendon fibroblast culture system. In Aim 2, we will use our newly-developed Cav1.2 inducible conditional knockout mouse model to determine the physiological role of Ca2+ signaling through CaV1.2 during tendon development, postnatal tendon formation, and during mechanical overload-induced adult tendon growth. Successful completion of this study will provide a fundamental understanding of the role of Ca2+ signaling on tendon formation and a platform to identify new targets for developing therapeutic strategies for tendon diseases.