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被认为通过抑制细胞周期蛋白依赖性激酶抑制剂（cdki） P21cip1的表达来促进增殖，P21cip1被认为是VSMC增殖和血管病变形成的重要抑制剂。然而，最近的数据显示了id3诱导的生长调节的其他机制。Id3受细胞周期蛋白依赖性激酶2 （cdk2）磷酸化调控。转录因子的磷酸化是信号转导与基因表达相结合的主要手段。Cdk2在丝氨酸5上磷酸化Id3增强了Id3对MyoD/E12复合物对EMSA的拮抗作用，并抑制了大鼠胚胎成纤维细胞（REF）的s期进入。我们在VSMC中的初步数据证实了Id3磷酸化位点的重要性，但表明在VSMC中，丝氨酸5的IdS磷酸化通过其他机制调节生长。我们提供了第一个证据，证明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，我们提供了第一个证据，证明Id3通过p21dp1依赖性和p21cip1非依赖性机制作为VSMC中G1-S进展的调节剂。因此，我们假设cdk2在丝氨酸5上磷酸化IdS是一个关键的调控事件，决定了IdS作为诱导或抑制p21cip1基因表达导致VSMC增殖的独特功能作用。我们假设非磷酸化的Id3与一种抑制p21cip1基因转录的因子二聚，使得非磷酸化的Id3的增加导致p21cip表达的增加。当Id3在丝氨酸5上磷酸化时，它不再与该抑制因子结合，而是与E47相互作用并抑制E47介导的p21cip1转录，从而允许进入s期。我们进一步假设，Id3的cdk2磷酸化也调节了Id3对G1-S进展的其他重要调节因子（如cyclin D、cdk2和Rb）的诱导作用。为了验证这些假设，我们提出：确定IdS和cdk2在丝氨酸5处的磷酸化对VSMC细胞周期进程和生长的影响；鉴定丝氨酸5磷酸化调控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，以研究Id3对血管病变形成的影响。

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.