ID3 REGULATION OF SMOOTH MUSCLE CELL PROLIFERATION

描述(摘自《调查者摘要》)：平滑肌细胞(SMC) 增殖在许多血管增生性疾病中起着关键作用， 包括动脉粥样硬化和球囊血管成形术后的再狭窄。这个 该项目的长期目标是确定 调节SMC的生长状态以应对血管损伤。少年派有 证明了ID3(DNA结合抑制物)，一种螺旋-环-螺旋(HLH) 血管壁上表达的因子促进培养的SMC生长 可能是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亚型(Id3a) 并且证明了该亚型相对于ID3的独特的血管表达。 Id3a的mrna保留了一个“编码内含子”，产生了一个带有 独特的羧基末端。他进一步证明了ID3的C-末端是 对其阻断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 协同作用调节SMC增殖的独特蛋白质 回应。目标1是确定ID3增强SMC的具体机制 并检测id3a对SMC生长的影响。研究将会 包括：a)共转染研究，以确定id3和id3a是否有作用 在SMC中的p21转录。确定PAN-1是否可以激活SMC中的p21 上下文以及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id3a蛋白的表达是否受调控 通过血清或其他有丝分裂原；c)id3a过表达实验确定 Id3a对SMC增殖的影响，特别是对G1-S进展的影响；d) 对p21缺失细胞的共转染研究以确定是否存在id3和id3a P21非依赖性对细胞增殖的影响；e)共转染 确定id3a是否是id3的显性负调控因子的实验；以及 F)结构域交换和突变研究，以确定 ID3的C末端是其显性负功能所必需的 阻断SMC基因反式激活，促进SMC增殖。目标2是 找出造成基本情况差异的机制 ID3和ID3a之间的功能特性。研究将包括：a)体外研究 结合分析以确定二聚化相互作用是否存在差异 ID3和ID3a对反式激活有不同的影响； B)利用哺乳动物双杂交系统进行共转染研究，以确定 更多的细胞因子参与调节二聚体伙伴 选择性；c)电泳迁移率改变分析以确定上述 ID3/E蛋白二聚体足以抑制E蛋白与 含有寡核苷酸的Ebox；和d)结构域交换和诱变 测定ID3 C-末端氨基酸的研究 其阻断E-蛋白与E-box结合的能力改变是必不可少的 含有寡核苷酸的。

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.