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

Ca2信号通过CaV1.2 L型电压门控钙通道调节肌腱形成

• 批准号: 9896147
• 负责人: Chike Cao
• 金额: $ 23.19万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896147
• 关键词: Acute; Address; Adult; Anabolism; Automobile Driving; Biochemical; Biology; Blood Vessels; Calcium Channel; Cardiac; Cell Culture System; Cell Proliferation; Cell membrane; Cells; Chronic; Cicatrix; Collagen; Cre driver; Data; Development; Disease; Elderly; Extracellular Matrix; Fibroblasts; Future; Gene Activation; Gene Expression; Genes; Goals; High Prevalence; Histology; Homeostasis; Hormone secretion; Hyperplasia; Immunohistochemistry; Impairment; In Vitro; Injury; Knowledge; Mechanics; Mediating; Modeling; Molecular; Molecular Analysis; Mus; Muscle Contraction; Mutation; Neurons; Osteoblasts; Osteogenesis; Pharmacological Treatment; Pharmacology; Physiological; Physiological Processes; Process; Regulation; Reporter; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Source; System; Tail; Tamoxifen; Tendinopathy; Tendon Injuries; Tendon structure; Testing; Therapeutic; Transgenic Organisms; Transmission Electron Microscopy; chronic pain; conditional knockout; disability; gain of function; healing; immune activation; improved; in vivo; loss of function; mechanical load; mechanotransduction; mouse model; mutant; neuronal excitability; novel; novel therapeutic intervention; postnatal; precursor cell; scleraxis; tendon development; therapeutic development; transcription factor; transcriptome sequencing; voltage

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.

项目摘要 细胞内Ca 2+影响主要的肌腱信号通路。虽然Ca 2+信号已经被研究， 广泛用于肌肉收缩、免疫细胞活化、激素分泌、细胞增殖 神经元调节和基因激活，肌腱中的Ca 2+信号细节和负责Ca 2+的通道 流入肌腱成纤维细胞在很大程度上是未知的。使用具有功能获得性CaV1.2的新型小鼠模型 突变通道（CaV1.2TS），我们观察到Ca 2+通过CaV1.2内流对肌腱的有效调节作用。 CaV1.2TS通道在成孔CaV1.2 α1C亚基中携带功能获得性G406 R突变， 电压依赖性失活，并允许更多的Ca 2+流入细胞。我们观察到， CaV1.2TS特异性地在具有Scleraxis-Cre（ScxCre）的肌腱中导致肌腱质量的显著增加。使用 报告小鼠，我们发现CaV1.2广泛表达在肌腱发育过程中的肌腱成纤维细胞。 因此，我们推测，在肌腱成纤维细胞中，Ca 2+通过CaV1.2内流调节肌腱形成， 这一假设符合我们先前的证明，即CaV1.2也在成骨细胞前体细胞中起作用， 调节骨形成和体内平衡。在这个建议中，我们试图确定细胞和分子 通过CaV1.2调节肌腱形成的Ca 2+信号传导机制。我们的假设是， CaV1.2介导的Ca ~（2+）信号调节肌腱细胞转录因子的表达 增殖和/或肌腱细胞外基质（ECM）合成的研究 过载引起的成年肌腱生长。在目标1中，我们将通过使用 我们的CaV1.2TS功能获得性小鼠模型，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。我们将确定信号级联介导 增加Ca 2+信号以上调体外尾腱中腱成纤维细胞增殖和ECM合成 成纤维细胞培养系统在目标2中，我们将使用我们新开发的Cav1.2诱导型条件性敲除 小鼠模型以确定在肌腱发育期间通过CaV1.2的Ca 2+信号传导的生理作用， 出生后的肌腱形成，并在机械过载诱导的成年肌腱生长。成功 这项研究的完成将提供一个基本的理解的作用，钙信号对肌腱 形成和一个平台，以确定新的目标，为肌腱疾病的治疗策略。