Nuclear Transport and Transcription in Smooth Muscle

平滑肌中的核运输和转录

• 批准号: 6629158
• 负责人: KAREN M LOUNSBURY
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-05 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629158
• 关键词: animal tissue; biological signal transduction; cAMP response element binding protein; calcium flux; calcium indicator; cell nucleus; cell proliferation; genetic regulatory element; genetic transcription; guanosinetriphosphatases; immunofluorescence technique; intracellular transport; protein protein interaction; vascular smooth muscle

DESCRIPTION (Verbatim from the application): The proliferative response of vascular smooth muscle cells (VSMCs) plays a key role in the recovery of blood vessels from injury as well as in atherosclerotic lesion formation and postangioplasty restenosis. The long-term goal of this research is to understand pathways important for transmission of proliferative signals from the cytoplasm to the nucleus of VSMCs. A relationship between Ca2+ and proliferation has been implicated, but the mechanism(s) have not been identified. In addition, whereas transmission of Ca2+ and mitogenic signals across the nuclear membrane is necessary to elicit specific changes in gene expression, the regulation of signaling in the cytoplasm versus the nucleus is not known. We propose a novel mechanism for Ca2+-mediated gene transcription in smooth muscle, in which Ran-mediated nuclear transport is central to the signaling pathway leading from a rise in [Ca2+]i to phosphorylation of the transcription factor CREB. The goal of this proposal is to define compartmentalization of Ca2+-mediated signaling molecules in VSMCs as they relate to phosphorylation of CREB and transcription through the Ca2+ and cAMP response element (CRE). Specifically, we will pursue the following Aims: 1) To elucidate the role of intracellular Ca2+ stores and nuclear Ca2+ in CREB activation pathways; 2) To explore the mechanisms and structural elements regulating nuclear import of CREB; 3) To understand the function that nucleocytoplasmic shuttling of CREB has in both its phosphorylation and capacity to mediate transcription through the CRE; Methods will include immunofluorescence to detect phosphorylated CREB, in vitro and live cell nuclear transport assays, Ca2+ imaging, protein interaction assays and CRE-luciferase reporter assays. The combination of in vitro molecular approaches and live cell transport models will drive support for new paradigms in the regulation of CREB-mediated gene transcription by Ca2+ and the Ran GTPase in VSMCs.

描述（逐字化的应用程序）： 血管平滑肌细胞（VSMC）在恢复血液中起关键作用 受伤以及动脉粥样硬化病变形成和 后血管成形术再狭窄。这项研究的长期目标是 了解对于从 VSMC核的细胞质。 Ca2+和 增殖已被牵涉，但机制尚未 确定。另外，虽然CA2+和有丝分裂信号的传播 整个核膜对于引起基因的特定变化是必要的 表达，细胞质中信号传导的调节与细胞核的调节 不知道。我们提出了一种用于Ca2+介导的基因转录的新机制 平滑肌，其中运行介导的核运输是 从[Ca2+] i上升到磷酸化的信号传导途径 转录因子Creb。该提议的目的是定义 Ca2+介导的VSMC中Ca2+介导的信号分子的分室化。 与CREB和通过CA2+和CAMP转录的磷酸化有关 响应元素（CRE）。具体来说，我们将追求以下目的：1） 阐明细胞内Ca2+存储和核Ca2+在CREB中的作用 激活途径； 2）探索机制和结构元素 调节Creb的核进口； 3）了解该功能 CREB的核质穿梭物具有其磷酸化和 通过CRE介导转录的能力； 方法将包括免疫荧光以检测磷酸化的Creb，体外 和活细胞核转运测定，CA2+成像，蛋白质相互作用 测定和CRE-卢西斯酶报告基因测定。体外的组合 分子方法和活细胞传输模型将推动新的支持 CA2+和CREB介导的基因转录调节中的范例 在VSMC中运行GTPase。