CHIP Represses Myocardin-induced Smooth Muscle Cell Differentiation Via Ubiquitin-Mediated Proteasomal Degradation

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.