Id3 Regulation of Smooth Muscle Cell Proliferation

描述(申请人提供)：螺旋-环-螺旋(HLH)因子ID3已成为血管平滑肌细胞(VSMC)生长的重要调节因子。ID3被认为是通过抑制细胞周期蛋白依赖性激酶抑制因子(CDKI)P21cip1的表达而促进细胞增殖，而细胞周期蛋白依赖性激酶抑制因子P21被认为是VSMC增殖和血管病变形成的重要抑制因子。然而，最近的数据表明，ID3诱导的生长调节有其他机制。ID3受细胞周期蛋白依赖性蛋白激酶2(CDK2)的磷酸化调控。转录因子的磷酸化是信号转导和基因表达整合的主要途径。CDK2使丝氨酸5上的ID3磷酸化，增强MyoD/E12复合体对EMSA的ID3拮抗作用，并抑制大鼠胚胎成纤维细胞(REF)的S期进入。我们在VSMC的初步数据证实了ID3上这个磷酸化位点的重要性，但表明在VSMC中，IDs的丝氨酸5磷酸化通过其他机制调节生长。我们提供了第一个证据，即磷酸化提供了一个调节开关，控制ID3对VSMC中p21Clp1启动子激活的调节作用。此外，我们还提供了首次证据表明，ID3通过p21dp1依赖和p21cip1非依赖的机制在VSMC中作为G1-S进程的调节因子。因此，我们假设IDs在丝氨酸5上的CDK2磷酸化是一个关键的调节事件，它决定了IDs作为导致VSMC增殖的p21cip1基因表达的诱导者或抑制者的独特功能。我们假设非磷酸化的ID3与一个抑制p21cip1基因转录的因子发生二聚化，从而导致非磷酸化IDs的增加导致p21cip表达的增加。当ID3在丝氨酸5上被磷酸化时，它不再结合这个阻遏子，而是与E47相互作用，并抑制E47介导的p21cip1的转录，使其进入S期。我们进一步假设，ID3的CDK2磷酸化也调节ID3对其他重要的G1-S进程调节因子的作用，如细胞周期蛋白D、CDK2和Rb。为了验证这些假说，我们建议：确定Ids和Ids在丝氨酸5处的CDK2磷酸化对VSMC细胞周期进程和生长的影响；确定丝氨酸5磷酸化调节Ids诱导的p21cip1表达和VSMC细胞周期进程的分子机制；并将体外研究结果扩展到体内模型，研究Id3对血管损伤形成的影响。

DESCRIPTION (provided by applicant): The helix-loop-helix (HLH) factor Id3 has emerged as an important regulator of vascular smooth muscle cell (VSMC) growth. Id3 is thought to promote proliferation by inhibiting the expression of the cyclin-dependent kinase inhibitor (cdki) P21cip1, which has been implicated as an important inhibitor of VSMC proliferation and vascular lesion formation. However recent data suggest additional mechanisms of Id3-induced growth regulation. Id3 is regulated by cyclin-dependent kinase 2 (cdk2) phosphorylation. Phosphorylation of transcription factors represents a major means by which signal transduction and gene expression are integrated. Cdk2 phosphorylation of Id3 on serine 5 enhances Id3 antagonism of a MyoD/E12 complex on EMSA and inhibited S-phase entry in rat embryonic fibroblasts (REF). Our preliminary data in VSMC confirm the importance of this phosphorylation site on Id3, but suggest that in VSMC serine 5 phosphorylation of IdS regulates growth through other mechanisms. We provide the first evidence that phosphorylation provides a regulatory switch controlling the effects of Id3 on regulation of p21Clp1 promoter activation in VSMC. In addition, we provide the first evidence that Id3 functions as a regulator of G1-S progression in VSMC through both p21dp1-dependent and p21cip1- independent mechanisms. Accordingly, we hypothesize that cdk2 phosphorylation of IdS on serine 5 is a key regulatory event that determines the unique functional role of IdS as either an inducer or inhibitor of p21cip1 gene expression leading to VSMC proliferation. We hypothesize that nonphosphorylated Id3 dimerizes with a factor that acts as a repressors of p21cip1 gene transcription such that an increase in nonphosphorylated IdS results in an increase in the expression of p21cip. When Id3 is phosphorylated on serine 5, it no longer binds this repressor and instead interacts with E47 and inhibits E47-mediated transcription of p21cip1 allowing S-phase entry. We further hypothesize that cdk2 phosphorylation of Id3 also regulates Id3-induced effects on other important regulators of G1-S progression such as cyclin D, cdk2 and Rb. To test these hypotheses we propose: to determine the effects of IdS and cdk2-phosphorylation of IdS at serine 5 on cell cycle progression and growth in VSMC; to identify the molecular mechanisms whereby serine 5 phosphorylation regulates IdS-induced effects on p21cip1 expression and VSMC cell cycle progression; and to extend in vitro findings into an in vivo model to study the effects of Id3 on vascular lesion formation.