REGULATION OF CELL PROLIFERATION

本研究旨在建立血管平滑肌(VSM) α -肌动蛋白基因在血管损伤和疾病的纤维增殖反应中的调控的分子基础。我们实验室之前的研究表明，在成纤维细胞和未分化成肌细胞中，小鼠VSM α -肌动蛋白基因的转录抑制是由序列特异性单链DNA （ssDNA）结合蛋白与TEF-1增强元件的相反链相互作用引起的。通过cDNA表达文库的结合位点筛选，这些蛋白被克隆并鉴定为MSY1 （Y-box核酸结合蛋白家族成员）、Puralpha（最初在Hela细胞中发现的一种视网膜母细胞瘤（Rb）结合蛋白）和Purbeta（一种性质未知的相关蛋白）。对人类冠状动脉粥样硬化的初步研究表明，这些蛋白在心血管疾病中具有功能作用。本申请中提出的实验将验证一个中心假设，即转录抑制是由TEF-1增强子内的ssdna结合蛋白依赖的碱基配对中断引起的，并且该过程的调节部分取决于高度特异性的蛋白质-蛋白质相互作用。该模型将通过描述蛋白质-蛋白质相互作用所必需的ssdna结合蛋白的功能区域，通过确定这些区域内突变对这些蛋白质调节增强子拓扑和功能的能力的影响，以及通过在VSM α -肌动蛋白表达细胞类型中对增强子结构的体内足迹进行测试。最后，这些蛋白在人类动脉粥样硬化中的作用将使用抗pur和抗msyl肽特异性抗体进行研究。这些研究将扩展我们对平滑肌肌动蛋白合成调控新机制的理解，这可能对冠状动脉疾病的发病机制有重要意义。

This research seeks to establish the molecular basis for the regulation of the vascular smooth muscle (VSM) alpha-actin gene during fibroproliferative responses characteristic of vascular injury and disease. Previous studies in our laboratories point to a model in which transcriptional repression of the mouse VSM alpha-actin gene in fibroblasts and undifferentiated myoblasts results from the interaction of sequence-specific single-stranded DNA (ssDNA) binding proteins with opposite strands of an essential TEF-1 enhancer element. Binding site screening of cDNA expression libraries has now resulted in the cloning of these proteins and their subsequent identification as MSY1, a member of the Y-box family of nucleic acid binding proteins, Puralpha, a retinoblastoma (Rb)-binding protein initially identified in Hela cells, and Purbeta, a related protein of unknown properties. Preliminary studies of human atherosclerotic coronary arteries suggest a functional role for these proteins in cardiovascular disease. Experiments proposed in this application will test a central hypothesis that transcriptional repression results from a ssDNA-binding protein-dependent disruption of base pairing within the TEF-1 enhancer and that regulation of this process depends, in part, upon highly specific protein-protein interactions. This model will be tested by delineating functional domains within the ssDNA-binding proteins essential for protein-protein interactions, by determining the effects of mutations within these domains on the ability of these proteins to modulate enhancer topology and function, and by in vivo footprinting of enhancer structure in VSM alpha-actin expressing cell types. Lastly, the involvement of these proteins in human atherogenesis will be studied using a repertoire of anti-Pur and anti-MSYl peptide-specific antibodies. These studies will extend our understanding of a novel mechanism for the regulation of smooth muscle actin synthesis which may be important to the pathogenesis of coronary artery disease.