PPARG-dependent Mechanisms Control Endothelial-Smooth Muscle Coordination, Arterial Pressure, Vasomotor Function and Arterial Stiffness

摘要/摘要 血管在动脉血压的调节中起着重要作用。精确的BP调节 需要血管扩张剂和血管收缩信号在内皮(EC)和平滑之间的协调 肌肉(SMC)。血管内皮细胞衍生的一氧化氮(NO)是指示SMC扩张或 合同。我们的研究表明，NO途径是通过转录和后转录调节的。 核受体转录因子PPARγ启动的翻译途径我们的数据支持 PPARγ的概念：1)在EC中作为传感器来调节氧化还原状态，并通过这一点，NO的生物可利用性， 2)通过独立控制a)RhoA/Rho激酶来调节SMC对NO的反应性 (岩石)促进收缩的活动，以及b)环状GMP(CGMP)的产生和稳定性，这是一个关键的 血管舒张剂。在两种细胞类型中，依赖于PPARγ的分子机制的范围是 令人惊讶的复杂；需要新的转录辅助因子(例如视黄醇结合蛋白7；RBP7)才能形成 具有PPARγ的转录调控中心，以及关键的SMC介体(RhoA)的翻译后调控 和磷酸二酯酶5(PDE5)由cullin-3 E3泛素连接酶介导的蛋白质周转。重要的是，这 PPARγ启动的“最终共同通路”对血管运动功能、血压和血管有深远影响 僵硬，以及在此提出的研究对这些疾病的治疗具有潜在的意义。 然而，启动和调节这些反应的信号和分子靶标的范围仍然存在 人们对此知之甚少。这项提案将侧重于两条不同的PPARγ调节通路。我们将研究 PPARγ-RhoBTB_1-CULLIN-3信号转导途径的研究 通过评估RhoBTB1是否可以保护和逆转RhoBTB1，可以开发为未来的潜在治疗药物 高血压模型或其他以血管功能障碍为特征的疾病模型的表型 共病，2)确定RhoBTB1在包括内皮在内的其他细胞类型中是否重要，以及3)使用 利用蛋白质组学方法寻找新的RhoBTB1结合伙伴和血管内皮细胞中的cullin-3底物 肌肉。我们还将检测内皮细胞中的PPARγ-RBP7-抗氧化途径，并将执行1) 结构功能分析，以确定调控PPARγ转录活性的关键机制，以及 2)全基因组转录组和染色质免疫沉淀研究以评估RBP7的贡献 介导PPARγ及其专性异源二聚体RXR的转录活性。这一计划将带来新的 对于实地调查的概念和方向，不仅会加深我们对 血管系统中的两条基本重要的途径，但也将涉及基本的转录和 翻译后机制在许多细胞类型中具有相关性。

Summary/Abstract Blood vessels play an important role in the regulation of arterial blood pressure (BP). Precise BP regulation requires coordination between vasodilator and vasoconstrictor signals in the endothelium (EC) and smooth muscle (SMC). EC-derived nitric oxide (NO) is among the key signals which instruct the SMC to dilate or contract. Our studies show that the NO pathway is coordinately regulated through transcriptional and post- translational pathways initiated by PPARγ, a nuclear receptor transcription factor. Our data support the concepts that PPARγ: 1) acts as a sensor in EC to regulate redox state, and through this, bioavailability of NO, and 2) regulates the responsiveness of SMC to NO by independently controlling a) a RhoA/Rho kinase (ROCK) activity that promotes constriction, and b) production and stability of cyclic GMP (cGMP), a critical mediator of vasodilation. The range of PPARγ-dependent molecular mechanisms in both cell types is surprisingly complex; requiring novel transcriptional co-factors (e.g. retinol binding protein 7; RBP7) which form a transcriptional regulatory hub with PPARγ, and post-translational regulation of critical SMC mediators (RhoA and phosphodiesterase 5, PDE5) by Cullin-3 E3 ubiquitin ligase-mediated protein turnover. Importantly, this PPARγ initiated “final common pathway” has profound effects on vasomotor function, BP and vascular stiffness, and the studies proposed herein have potential implications for the treatment of these disorders. However, the signals which initiate and mediate these responses and the range of molecular targets remain poorly understood. This proposal will focus on two distinct PPARγ-regulated pathways. We will examine the PPARγ-RhoBTB1-Cullin-3 pathway in smooth muscle and will 1) determine if the RhoBTB1-Cullin-3 pathway can be exploited as a potential future therapeutic by assessing if RhoBTB1 can protect and reverse phenotypes in models of hypertension or in other disease models in which vascular dysfunction is a comorbidity, 2) determine if RhoBTB1 is important in other cells types including endothelium, and 3) employ a proteomic strategy to identify novel RhoBTB1 binding partners and Cullin-3 substrates in vascular smooth muscle. We will also examine the PPARγ-RBP7-anti-oxidant pathway in endothelium and will perform 1) structure function analysis to identify key mechanisms regulating PPARγ transcriptional activity by RBP7, and 2) genome wide transcriptome and chromatin immunoprecipitation studies to assess the contribution of RBP7 to mediated transcriptional activity of PPARγ and its obligate heterodimer RXR. This program will lead to new concepts and directions of investigation for the field and will not only deepen our understanding of the role of two fundamentally important pathways in the vasculature, but will also address fundamental transcriptional and post-translational mechanisms that are of relevance in many cell types.