Smooth muscle cell integration of differentiation signals

描述(申请人提供)：血管平滑肌细胞(SMC)在病理性血管重塑过程中改变其分子和表型特征。Notch和转化生长因子β信号通路都促进成熟SMC的分化、收缩表型特征。然而，这些途径相互作用的机制才刚刚开始被理解。任何一种途径的突变都是人类心血管疾病的原因，针对这些途径的治疗目前正在进行临床试验。我们的实验室在SMC中发现了Notch和Smad之间的调控相互作用，它连接着Notch和转化生长因子β/BMP/Smad信号。Notch和转化生长因子β信号转导导致磷酸化Smad(PSmad)转录活性和SMC标志物表达协同激活。本项目的目标是明确导致SMC分化的整合分子信号机制。该项目利用人类原代血管细胞的分子和生化信号传递方法，以及小鼠转基因模型来研究体内基因调控和功能。我们提出以下假设：1)Jagged1激活SMC中的Notch信号，通过典型的CBF-1介导的途径转录抑制转化生长因子受体endoglin。2)协调Notch和转化生长因子β信号，通过含CBF1和pSmad的转录复合体相互作用，协同激活SMC收缩基因。3)在血管损伤前激活SMC中的Notch，可维持分化表型，抑制新生内膜病变的形成和动脉生成。具体目的1：验证SMC中转化生长因子受体Endoglin受Notch信号转录调控的假说。本研究的目的是利用体外分子检测和体内转基因小鼠模型研究Notch对SMC中endoglin表达和转化生长因子信号转导的调控。特定目的2：研究CBF1和Smad转录复合体与SMC收缩基因的相互作用。我们假设CBF1和pSmad转录复合体相互作用，在转录上调节收缩基因，包括平滑肌肌动蛋白(SM-肌动蛋白)和钙蛋白1。具体目标3：确定Notch对Endoglin和TGFβ信号的调节如何影响病理性血管重构。已建立的小鼠转基因品系将在血管疾病模型中进行评估。

DESCRIPTION (provided by applicant): The vascular smooth muscle cell (SMC) alters its molecular and phenotypic characteristics during pathological vascular remodeling. Both Notch and TGF¿ signaling pathways promote a differentiated, contractile phenotype characteristic of mature SMC. However, mechanisms by which these pathways interact with each other are only beginning to be understood. Mutations in either pathway are causal for human cardiovascular disease, and therapies targeting these pathways are currently in clinical trials. Our laboratory discovered a regulatory interaction between Notch and Smad in SMC, which links Notch and TGF¿/BMP/Smad signaling. Concomitant Notch and TGF¿ signaling leads to synergistic activation of phosphoSmad (pSmad) transcriptional activity and SMC marker expression. The goal of this project is to define mechanisms of integrative molecular signaling leading to SMC differentiation. This project utilizes molecular and biochemical signaling approaches in human primary vascular cells, and mouse transgenic models to study in vivo gene regulation and function. We propose the following hypotheses: 1) Jagged1 activation of Notch signaling in SMC transcriptionally represses the TGF¿ co-receptor, endoglin, via the canonical CBF-1 mediated pathway. 2) Coordinate Notch and TGF¿ signaling leads to synergistic activation of SMC contractile genes via interaction of CBF1- and pSmad- containing transcriptional complexes. 3) Activation of Notch in vivo in SMC prior to vascular injury will maintain the differentiated phenotype, leading to suppressed neointimal lesion formation and arteriogenesis. Specific Aim 1: Test the hypothesis that the TGF¿ co-receptor, endoglin, is transcriptionally regulated by Notch signaling in SMC. This aim will utilize molecular assays in vitro and transgenic mouse models in vivo to characterize Notch regulation of endoglin expression and TGF¿ signaling in SMC. Specific Aim 2: Characterize the interaction of CBF1 and Smad transcriptional complexes on SMC contractile genes. We hypothesize that CBF1 and pSmad transcriptional complexes interact to transcriptionally regulate contractile genes including smooth muscle ¿-actin (SM actin) and calponin1. Specific Aim 3: Determine how the regulation of endoglin and TGF¿ signaling by Notch affects pathological vascular remodeling. Established mouse transgenic strains will be evaluated in vascular disease models.