【作者】 谢平；

【导师】 李汇华；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 正常血管平滑肌细胞(SMC)高度分化,但是在血管损伤效应发生时,SMC则向增殖表型转换。研究表明许多因素包括生长因子、细胞因子、转录因子等可调节血管SMC表型转化。其中心肌素(myocardin)是近年来在平滑肌细胞分化和表型转换方面具有重要意义的一个发现。目前已知,心肌素是最有效的SRF(serumresponse factor)共同激活因子,通过Q-富集功能区与SRF结合形成复合物,激活CArG盒子依赖的平滑肌细胞标志基因的表达,是平滑肌细胞发育和分化所必需的共激活转录因子。但是到目前为止,调节心肌素蛋白水平和活性的分子机制尚未完全阐明。近年来,人们发现转录后修饰特别是磷酸化、Sumoylation和泛素化对底物蛋白和酶的活性具有重要的调节作用。其中,泛素E3连接酶可有效地识别和泛素化蛋白底物,促进其被泛素-蛋白酶体系统(ubiquitin-proteasome system)降解。CHIP(Hsc70羧基末端反应蛋白)是一个重要的控制蛋白质量的泛素E3连接酶,可促进多种蛋白底物被泛素-蛋白酶体系统降解和清除,在心肌细胞损伤、细胞凋亡、肿瘤、热应急等方面起着非常重要的作用。但是CHIP调节SMC表型转换和动脉血管收缩性的分子机制尚未见报道。因此,本研究主要探讨了CHIP调节心肌素的蛋白水平和活性,促进SMC表型转化的分子机制。我们发现:1)过度表达CHIP不仅抑制心肌素依赖的SMC标志基因如SMα-actin,SM-MHC和SM22α的表达,显著降低SM22α-luc或ANF-luc荧光素酶报告基因活性和心肌素的蛋白水平,而且这种抑制效应依赖于CHIP U-box结构域的存在。2)转染siRNA-CHIP不仅促进SMC标志基因的表达,而且使SM22α报告基因活性明显增加。3) GST-pull down和免疫共沉淀实验证实CHIP在体内外均可与心肌素直接相互结合,而不是通过Hsp70和Hsp90。CHIP正负电荷区是结合心肌素的结构域,而心肌素C末端的TAD转录激活区是结合CHIP的结构域。免疫组化染色显示它们共定位于SMC胞浆中。4) Pulse-chase实验结果表明CHIP能明显下调心肌素的蛋白水平,而且这种作用可以被蛋白酶体抑制剂MG132阻断。体内外泛素化实验进一步证明CHIP可以促进心肌素泛素化和降解,而且这种泛素化过程依赖于CHIP E3连接酶活性。同时,心肌素的TAD结构域是被CHIP泛素化所必需的。5) CHIP介导的心肌素泛素化依赖GSK-3β诱导的磷酸化修饰。6)在动脉环中过度表达CHIP可明显降低血管心肌素蛋白水平及其所依赖的SMC标志基因的表达,而且动脉环收缩性明显降低,舒张性明显增加。本研究结果表明CHIP可通过泛素—蛋白酶体系统降低心肌素的蛋白水平和转录活性,抑制SMC表型转换和血管收缩性。这一机制的阐明可能为动脉粥样硬化、高血压和老年痴呆症等重大疾病的防治提供了重要的新思路。更多还原

【Abstract】 The phenotypic modulation of vascular muscle cells(SMCs) from differentiation to dedifferentiation is a critical feature of the onset and progression of the vascular remodeling under vessels injury conditions.There has been much progress in recent years to identify mechanisms including growth factors,cytokines and transcription factors that control expression of the repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function.One of the most exciting recent discoveries was the identification of the serum response factor(SRF) coactivator myocardin that appears to be required for expression of many SMC differentiation marker genes and for initial differentiation of SMC during development.During the process of SMC development and differentiation,myocardin plays a pivotal role in activating CArG-dependent genes expression through its combination with SRF with its Q-rich domain.However,it remains unclear about the molecular mechanism by which the protein level and activity of myocardin can be modulated.It is an important role for post-transcription modifications in modulating protein level and activity,especially as phosphorylation,sumoylation and ubiquitination.As referred to ubiquitination,E3 ligase can effectively and specifically recognize and ubiquitinate substrates,enhancing them degradation through proteasome.CHIP,known as an E3 ligase,can degradate multiple protein substrates through proteasome,playing an important role in pathological progress and ailments conditions like cardiomyocyte injury, apoptosis,neoplasm and thermo-stress.However,there is no report about the molecular mechanism by which CHIP can modulate SMC phenotype transformation and aortic contractility.Therefore,we focus on the molecular mechanism about CHIP modulating myocardin protein level and its activity as well as SMC phenotype modulation and we found that:1) CHIP overexpression not only inhibits myocardin-dependent SMC marker genes expression such as SMα-actin,SM-MHC and SM22α,but also dramatically decreases the SM22α-luc and ANF-luc luciferase report activity in U-box domain dependent manner.2) Knockout of CHIP by siRNA can promote SMC contractile marker genes and increase the SM22αluciferase reporter activity.3) GST-pull down and Co-ip assay demonstrate the direct interaction between CHIP and myocardin in vivo and in vitro and this interaction is not dependent on the existence of hsp70 or hsp90.CHIP charged domain and myocardin TAD domain are responsible for their interaction.Immunostaining indicates that CHIP is colocalized with myocardin in SMC.4) Pulse-chase assay indicates that CHIP rapidly decreases the level of myocardin protein,whereas proteasome inhibitor MG132 can attenuate this effect.The ubiquitination reactions in vivo and in vitro reveal that myocardin ubiquitination is dependent on E3 ligase activity of CHIP,and the myocardin TAD domain is required for its interaction with CHIP and its ubiquitination.5) CHIP specifically promotes phosphorylated myocardin ubiquitination-degradation in vivo and in vitro.6) In ex vivo aortic ring,CHIP overexpression downregulates the myocardin protein level and its dependent contractile gene transcription.Moreover,Rat aortic rings transduced with Ad-CHIP show significantly reduced contraction and increased relaxation.In conclusion,CHIP plays a critical role in modulating myocardin protein level and transactivity,and controlling the SMC phenotype and arterial tone through ubiquitin-proteosome system.This proposed mechanism may be associated with atherosclerosis,hypertension,and Alzheimer’s disease,opening a new therapeutic approach to these diseases’ prevention and treatment.更多还原