Regulation & Function of Calponin

描述（由申请人提供）：细胞通过细胞骨架中的结构和功能修饰与机械环境相互作用。高度动态的肌动蛋白细胞骨架通过结构重组和功能改变来响应力，介导各种细胞活动。钙钙蛋白是一种肌动蛋白相关蛋白，被认为可以调节平滑肌细胞和非肌肉细胞中肌动蛋白丝的功能。钙钙蛋白的生物化学性质已被广泛研究，但其生理功能尚不清楚。我们最近证明钙钙蛋白h2亚型在上皮细胞和成纤维细胞中表现出张力调节的表达和降解（Hossain等人，JBC, 280:42442- 53,2005），并将这项研究扩展到其他细胞类型，包括肺泡细胞、成肌细胞、巨噬细胞和癌细胞。这些初步研究提出了一种假设，即h2-钙钙蛋白在肌动蛋白细胞骨架的机械张力反应中起细胞调节作用。本研究项目将阐明h2-钙钙蛋白在细胞骨架活动中的功能和调控，重点是张力反应。本文提出了三个具体目的：1)研究血管骨架张力的变化如何调节h2-钙钙蛋白基因的转录。这将通过在可变张力条件下培养的细胞中测试截断和突变的启动子结构来研究。最终目标是了解细胞如何将力信号转化为基因调控。2)研究张力信号对h2-钙钙蛋白降解的调控作用。我们已经证明h2-钙钙蛋白在细胞分裂期间或在张力降低的细胞中迅速降解。我们将确定在细胞骨架重塑过程中启动h2-钙钙蛋白降解过程的蛋白酶，并研究机械张力变化对h2-钙钙蛋白水解的调节。3)表征h2-钙钙蛋白在受张力调节的细胞活动中的作用。我们已经证明h2-钙钙蛋白在稳定肌动蛋白丝和抑制细胞增殖中的作用。我们将通过使用钙钙蛋白减少的癌细胞和h2-钙钙蛋白条件敲除小鼠的组织/细胞，进一步研究h2-钙钙蛋白在响应机械张力变化的细胞结构、运动性和细胞分裂中的功能。除了了解钙钙蛋白的调控和功能外，这些研究还解决了机械力如何影响活细胞生化过程的基本问题。研究结果将为细胞增殖、细胞迁移、伤口愈合、肌肉形成、肺泡力学和肿瘤转移等重要的生理和病理过程提供新的见解。

DESCRIPTION (provided by applicant): Cells interact with their mechanical environment via structural and functional modifications in the cytoskeleton. The highly dynamic actin cytoskeleton responds to force by structural reorganization and functional changes that mediate various cellular activities. Calponin is an actin-associated protein that is thought to regulate the function of actin filaments in both smooth muscle and non-muscle cells. While the biochemical properties of calponin have been extensively characterized, its physiological function remains unclear. We recently demonstrated that the h2 isoform of calponin exhibits tension-regulated expression and degradation in epithelial cells and fibroblasts (Hossain et al., JBC, 280:42442-53, 2005) and have extended this study to other cell types, including lung alveolar cells, myoblasts, macrophages, and cancer cells. These preliminary studies lead to a hypothesis that h2-calponin functions as a cellular regulator in response to mechanical tension in the actin cytoskeleton. This research project will elucidate the function and regulation of h2-calponin in cytoskeletal activities, focusing on tension responses. Three Specific Aims are proposed: 1) To investigate how changes in cvtoskeleton tension regulate the transcription of h2-calponin gene. This will be studied by testing truncated and mutated promoter constructs in cells cultured under variable tension conditions. The ultimate goal is to understand how cells translate force signals into gene regulation. 2) To investigate how tension signals regulate the proteolvtic degradation of h2-calponin. We have shown that h2-calponin is rapidly degraded during cytokinesis or in cells that are under reduced tension. We shall identify the protease(s) that initiate this process of h2-calponin degradation during cytoskeletal remodeling and investigate the regulation of h2-calponin proteolysis under mechanical tension changes. 3) To characterize the function of h2-calponin in cellular activities that are regulated by tension. We have demonstrated the role of h2-calponin in stabilizing actin filaments and inhibiting cell proliferation. We shall further examine the function of h2-calponin in cell structure, motility, and cytokinesis in response to mechanical tension changes by using cancer cells with reduced calponin and tissues/cells from h2-calponin conditional knockout mice. In addition to understanding the regulation and function of calponin, these studies address the fundamental question of how mechanical force affects biochemical processes in living cells. The results will provide new insights into many important physiological and pathological processes, such as cell proliferation, cell migration, wound healing, myogenesis, lung alveolar mechanics, and tumor metastasis.