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. ? ? ?
