ID3 REGULATION OF SMOOTH MUSCLE CELL PROLIFERATION

描述（改编自研究者摘要）：平滑肌细胞（SMC） 增殖在许多血管增殖性疾病中起关键作用， 包括动脉粥样硬化和球囊血管成形术后的再狭窄。的 该项目的长期目标是确定分子机制， 调节SMC的生长状态以响应血管损伤。主要研究者有 证明Id 3（DNA结合抑制剂），一种螺旋-环-螺旋（HLH） 在血管壁中表达的因子，增强培养中SMC的生长 潜在地通过抑制细胞周期调节因子p21的表达。 他最近从大鼠主动脉SMC文库中克隆了一种新的Id3同种型（Id3a 并证明了相对于ID3，该同种型的独特血管表达。 Id3a mRNA保留了一个"编码内含子"，产生了一个ID3a蛋白， 独特的羧基末端。他进一步表明，ID3的C-末端是 其阻断p21基因表达的能力至关重要。因此，他假设 血管损伤调节Id2亚型表达，导致功能性 独特的蛋白质协同调节SMC增殖 反应目的1是确定Id3增强SMC的具体机制 增殖，并确定Id3a对SMC生长的影响。研究将 包括：a）共转染研究，以确定Id3和Id3a是否具有作用 对SMC中p21转录的影响。确定Pan-1是否可以反式激活SMC中的p21 背景以及Id3和Id3a是否对Pan-1介导的p21反式激活有影响; B）Western分析以确定SMC中Id3 a蛋白表达是否受到调节 c）Id3a过表达实验，以确定 Id3a对SMC增殖，特别是对G1-S进程的影响; d） 在p21缺失细胞中进行共转染研究，以确定是否存在Id3和Id3a 对p21非依赖性增殖的影响; e）共转染 实验以确定Id3a是否是Id3的显性负调节剂;以及 f）结构域交换和诱变研究，以确定所述结构域中的氨基酸。 Id3的C-末端，这是其显性负功能所必需的， 阻断SMC中基因的反式激活，促进SMC增殖。目标二是 确定造成基本人权差异的机制， Id3和Id3a之间的功能特性。研究将包括： 结合测定以确定是否存在二聚化相互作用的差异， Id3和Id3a对反式激活具有不同的作用; B）使用哺乳动物双杂交系统的共转染研究，以确定 另外的细胞因子参与调节二聚化伴侣 选择性; c）电泳迁移率变动测定，以确定上述 Id3/E-蛋白二聚化足以抑制E-蛋白结合至 含有Ebox的寡核苷酸;和d）结构域交换和诱变 确定Id3的C-末端中的氨基酸的研究， 对于其改变阻断E蛋白与E盒结合的能力至关重要 含有寡核苷酸。

DESCRIPTION (Adapted from Investigator's Abstract): Smooth muscle cell (SMC) proliferation plays a key role in a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. The long-term goal of this project is to identify the molecular mechanisms that regulate the growth state of SMC in response to vascular injury. The PI has demonstrated that Id3 (inhibitor of DNA binding), a helix-loop-helix (HLH) factor expressed in the vessel wall, enhances growth of SMC in culture potentially through inhibition of expression of the cell cycle regulator p21. He has recently cloned a novel Id3 isoform (Id3a) from a rat aortic SMC library and demonstrated unique vascular expression of this isoform relative to ID3. The Id3a mRNA retains a "coding intron," generating an ID3a protein with a unique carboxyl terminus. He has further shown that the C-terminus of ID3 is essential for its ability to block p21 gene expression. Thus,he hypothesizes that vascular injury regulates Id2 isoform expression resulting in functionally unique proteins that work in concert to regulate the SMC proliferative response. Aim 1 is to identify specific mechanisms by which Id3 enhances SMC proliferation and to determine the effect of Id3a on SMC growth. Studies will include: a) co-transfection studies to determine if Id3 and Id3a have effects on p21 transcription in SMC. Determine if Pan-1 can transactivate p21 in a SMC context and if Id3 and Id3a have effects on Pan-1 mediated p21 transactivation; b) Western analysis to determine if Id3a protein expression in SMC is regulated by serum or other mitogens; c) Id3a over-expression experiments to determine effects of Id3a on SMC proliferation and specifically on G1-S progression; d) cotransfection studies in p21 null cells to determine if there are Id3 and Id3a effects on proliferation that are p21 independent; e) cotransfection experiments to determine if Id3a is a dominant negative regulator of Id3; and f) domain swapping and mutagenesis studies to determine the amino acids in the C-terminus of Id3 that are essential for its dominant negative function in blocking gene transactivation in SMC and enhancing SMC proliferation. Aim 2 is to identify the mechanisms responsible for the differences in the basic functional properties between Id3 and Id3a. Studies will include: a) in vitro binding assays to determine if differences in dimerization interactions between Id3 and Id3a are responsible for their differential effects on transactivation; b) cotransfection studies using the mammalian two-hybrid system to determine if additional cellular factors are involved in regulating dimerization partner selectivity; c) electrophoretic mobility shift assays to determine if the above Id3/E-protein dimerizations are sufficient for inhibiting E-protein binding to an Ebox containing oligonucleotide; and d) domain swapping and mutagenesis studies to determine the amino acids in the C-terminus of Id3 that are essential for its altered ability to block E-protein binding to E-box containing oligonucleotides.