Regulation & Function of Calponin

描述（由申请人提供）：细胞通过细胞骨架的结构和功能修饰与其机械环境相互作用。高度动态的肌动蛋白细胞骨架通过介导各种细胞活动的结构重组和功能变化来响应力。钙调蛋白是一种肌动蛋白相关蛋白，被认为可以调节平滑肌和非肌肉细胞中肌动蛋白丝的功能。虽然钙调蛋白的生化特性已被广泛表征，但其生理功能仍不清楚。我们最近证明，钙调蛋白的 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。这些初步研究得出了这样一个假设：h2-钙调蛋白作为细胞调节剂，响应肌动蛋白细胞骨架中的机械张力。该研究项目将阐明 h2-calponin 在细胞骨架活动中的功能和调节，重点关注张力反应。提出了三个具体目标： 1）研究细胞骨架张力的变化如何调节 h2-calponin 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短和突变的启动子构建体来研究。最终目标是了解细胞如何将力信号转化为基因调控。 2) 研究张力信号如何调节h2-钙调蛋白的蛋白降解。我们已经证明，h2-钙调蛋白在胞质分裂过程中或在张力降低的细胞中迅速降解。我们将鉴定在细胞骨架重塑过程中启动 h2-calponin 降解过程的蛋白酶，并研究机械张力变化下 h2-calponin 蛋白水解的调节。 3) 表征 h2-钙调蛋白在受张力调节的细胞活动中的功能。我们已经证明了 h2-钙调蛋白在稳定肌动蛋白丝和抑制细胞增殖方面的作用。我们将通过使用钙调蛋白减少的癌细胞和来自 h2-钙调蛋白条件性敲除小鼠的组织/细胞，进一步检查 h2-钙调蛋白在响应机械张力变化的细胞结构、运动和胞质分裂中的功能。除了了解钙调蛋白的调节和功能之外，这些研究还解决了机械力如何影响活细胞中的生化过程的基本问题。这些结果将为许多重要的生理和病理过程提供新的见解，例如细胞增殖、细胞迁移、伤口愈合、肌生成、肺泡力学和肿瘤转移。

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.