Visceral smooth muscle-specific gene expression

内脏平滑肌特异性基因表达

• 批准号: 6446620
• 负责人: BRIAN Paul HERRING
• 金额: $ 29.8万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6446620
• 关键词: cadherins; developmental genetics; gene expression; genetic promoter element; genetically modified animals; laboratory mouse; myogenesis; myosin light chain kinase; smooth muscle; tissue /cell culture; transcription factor; transfection

DESCRIPTION (Provided by Applicant): Our long-term goal is to determine the mechanisms regulating smooth muscle development. Unraveling these mechanisms is crucial to our understanding of the pathology of many different, intestinal, urogenital vascular and pulmonary diseases that are associated with altered contractile protein expression and contractility. Experiments are proposed to use the smooth muscle-specific telokin promoter to examine the molecular pathways controlling visceral smooth muscle development. We have previously identified several transcription factors that regulate the telokin promoter activity in vitro. In this proposal we will determine the role played by these factors in regulating telokin expression during intestinal development. In addition, experiments will begin to investigate the signaling pathways that regulate the expression and activity of these proteins. These studies will test the hypothesis that visceral smooth muscle-specific expression of telokin is modulated in part by signals derived from intestinal epithelial cells that regulate smooth muscle differentiation. In this proposal experiments will specifically focus on the role of SRF, Hox and Fox proteins in regulating telokin expression during gut development. Co-transfection studies together with DNA binding experiments and in vivo analysis of mutant promoters that are unable to bind Hox and Fox genes will be used to elucidate the role of these proteins in regulating telokin expression during gut development. SRF is known to be a key regulator of all smooth muscle genes thus far examined, although it is not known if it is required for basal expression of smooth muscle genes or if it is involved in mediating their tissue specific expression. Experiments using mutant chimeric promoters harnessed to beta-galactosidase transgenes will allow us to determine if SRF is required for the visceral smooth muscle-specific expression of telokin. It is likely that the tissue specific functions of SRF result from its interaction with other cell-type specific factors, hence the ability of several cell-restricted, SRF associated transcription factors to regulate telokin expression will be examined. Preliminary results have also suggested that duplin, a beta-catenin binding protein, can interact with SRF. These data suggest that wnt signaling may regulate expression of smooth muscle specific genes through SRF via its interaction with duplin and beta-catenin. The importance of this regulatory pathway will be evaluated in vitro by transfection assays in cultured cells and direct in vitro protein and DNA binding experiments.

描述(由申请人提供)： 我们的长期目标是确定调节平滑肌的机制 发展。解开这些机制对于我们理解 多种不同的肠道、泌尿生殖系统血管和肺的病理 与收缩蛋白表达改变和 伸缩性。实验建议使用特定于平滑肌的 端粒酶启动子研究控制内脏平滑的分子途径 肌肉发育。我们之前已经鉴定了几个转录因子 在体外调节端粒蛋白启动子的活性。在本提案中，我们将 确定这些因素在调节端粒蛋白表达中所起的作用 在肠道发育过程中。此外，实验将开始 研究调控蛋白表达和活性的信号通路 这些蛋白质。这些研究将检验内脏平滑的假设 端粒蛋白的肌肉特异性表达在一定程度上受信号的调节 来自调节平滑肌分化的肠道上皮细胞。 在本提案中，实验将专门关注SRF、HOX的作用 以及Fox蛋白质在肠道发育过程中调节端粒蛋白的表达。 结合DNA结合实验和体内实验的共转染研究 对不能结合Hox和Fox基因的突变启动子的分析将是 用于阐明这些蛋白在调节端粒蛋白表达中的作用 在肠道发育过程中。已知SRF是所有平滑肌的关键调节因子 到目前为止已经检测到的基因，尽管还不知道它是否是基本的 平滑肌基因的表达或者它是否参与了它们的 组织特异性表达。使用突变嵌合启动子的实验 利用β-半乳糖苷酶转基因将使我们能够确定SRF是否 内脏平滑肌特异性表达端粒酶所必需的。它是 SRF的组织特异性功能可能是其相互作用的结果 与其他细胞类型的特定因子一起，因此几个 细胞受限、SRF相关转录因子调节端粒蛋白 我们将对表情进行检查。初步结果还表明， Duplin是一种β-连环蛋白结合蛋白，可以与SRF相互作用。这些数据 提示WNT信号转导通路可能参与调节SMA的表达 基因通过SRF与Duplin和β-catenin相互作用。这个 这一调节途径的重要性将通过以下方法在体外进行评估 体外培养细胞中的转染法及其对蛋白质和DNA的直接作用 绑定实验。