P49/STRAP Regulates The Mechanism Of Myocardin-SRF-mediated Contractile Gene Transcription In Vascular Smooth Muscle Cells

Clinical complications such as myocardial infarction,stroke and peripheral arterial disease caused by atherosclerosis have become a major public health problem in today’s society.Transition from contracted phenotype to synthetic phenotype of vascular smooth muscle cells is an important cytological event of atherosclerosis or arterial vascular stenosis,which is the primary cause of vascular remodeling and occlusion.Therefore,exploring the molecular mechanism of VSMCs phenotypic transformation has important theoretical and practical significance.Myocardin(MYOCD)is a co-transcriptional activator for serum response factor(SRF).SRF is a transcription factor that specifically binds to the CArG box of target gene and activates the expression of the target gene.MYOCD interacts with N-terminal MADS box of SRF through the Q-rich domain,and combines on the CArG box of the target gene with SRF,forming the MYOCD-SRF-CArG box complex,which activates the expression of cardiac or smooth muscle cell contraction gene such as SM α-actin,Mhc11,SM22 and Calponin,thereby increasing the contractility of VSMCs.P49/STRAP(serum response factor binding protein 1,SRFBP1)is a cofactor for SRF and can bind to SRF.Endogenous P49/STRAP protein is mainly located in the nucleus and can directly interact with the transcriptional activation domain of SRF to promote the regulation of the target genes in the heart.However,there is no relevant study shows that P49/STRAP participates in the phenotype conversion of VSMCs.Previous studies in our laboratory found that P49/STRAP was significantly downregulated during the conversion of VSMCs from contractile to synthetic,and P49/STRAP positively regulated the transcription of contractile genes in VSMCs.In the early stage,we predicted that P49/STRAP may interact with MYOCD directly by bioinformatics.It is speculated that P49/STRAP synergistically promotes that SRF actives the transcription of contractile gene through interaction with MYOCD.Therefore,this paper intends to elucidate the molecular mechanism which P49/STRAP regulates the phenotypic transformation of VSMCs.In order to elucidate the molecular mechanism which P49/STRAP regulates the phenotypic transformation of VSMCs,this paper carried out the following research:1.The pEBG-P49/STRAP eukaryotic expression plasmid,pCMV-HA-MYOCD eukaryotic expression plasmid,ACTG2-pGL3 promoter reporter plasmid,P49/STRAP full-length eukaryotic expression plasmid and a series of truncated plasmids were constructed.The pEBG-P49/STRAP plasmid,a series of truncated plasmids and pCMV-HA-MYOCD plasmid was co-transfected into 293 t cells with ACTG2-pGL3 and RL-TK luciferase reporter plasmids to detect luciferase activity,which is to investigate the activation of transcription of ACTG2 by P49/STRAP and MYOCD.2.The eukaryotic expression plasmid pEGFP-N1-MYOCD was constructed,and pEBG-P49/STRAP was co-transfected with pEGFP-N1-MYOCD or empty plasmid into 293 t cells.The interaction between MYOCD and P49/STRAP was confirmed by immunoprecipitation and western blotting.3.A series of GST-tagged truncated P49/STRAP protein expression plasmids pEBG-P49/STRAP(aa 1-145),pEBG-P49/STRAP(aa 146-349)and pEBGP49/STRAP(aa 350-429)were co-transfected with pEGFP-N1-MYOCD expression vector or empty into 293 t cells,which identified the key domains that MYOCD protein interacts with P49/STRAP protein by immunoprecipitation and western blotting.The results showed that:1.Digestion and sequencing results showed that various expression plasmids and luciferase reporter plasmids were successfully constructed.The dual luciferase reporter gene results showed that MYOCD can activate the transcription of the ACTG2 contractile gene and the transcription is enhanced by the increase of MYOCD plasmid transfection.P49/STRAP does not activate transcription of the ACTG2 contractile gene alone,but promotes MYOCD-mediated transcriptional activation.The P49/STRAP(aa 146-349)plasmid and the P49/STRAP(aa 350-429)plasmid promoted MYOCDmediated transcriptional activation.2.The results of restriction enzyme digestion and sequencing showed that the pEGFP-N1-MYOCD expression vector was successfully constructed.Immunoprecipitation and Western blotting results showed that the P49/STRAP protein can co-precipitate with the MYOCD protein.3.Immunoprecipitation and Western blotting results showed that pEBGP49/STRAP(aa 1-146),pEBG-P49/STRAP(aa 146-349)and pEBG-P49/STRAP(aa 350-429)truncated proteins could co-precipitate with MYOCD.According to the results of dual luciferase activities,pEBG-P49/STRAP(aa 146-429)might interact with MYOCD.P49/STRAP does not have transcriptional activity,but it may interact with MYOCD via the aa 146-429 domain to promote transcription of the VSMCs contraction gene ACTG2 synergistically.