MG132 Induces Expression Of Monocyte Chemotactic Proteininduced Protein 1 In Vascular Smooth Muscle Cells

Objective: Human vascular smooth muscle cells(HVSMCs) was chosen as a model to identify the effect of MG132 on MCPIP1 expression, proliferation and pro-apoptotic in VSMCs, and sequentially elucidate the related molecular mechanisms by preforming modern molecular biology, such as Western blot, TUNEL assay, Brd U and so on.Methods: The DMEM/F-12 completed culture medium containing 10% FBS was used for culture HVSMCs in vitro.The effect of MG132-induced MCPIP1 production in HVSMCs:Experiment 1: VSMCs were divided into four groups: l) control group, 2) MG132(0.1 ?M) group, 3) MG132(1 ?M) group, MG132(10 ?M) group, cells were treated for 24 h; Western Blot was performed to determine the MCPIP1 expression in cell supernatants.Experiment 2: VSMCs were divided into two groups: 1) 10 ?M MG132 treatment in VSMCs were divided into various indicated time groups(0?1?2?4?8?12?24 h). 2) 10 ?M DMSO treatment in VSMCs were divided into various indicated time groups(0?1?2?4?8?12?24 h). Western Blot was performed to determine the MCPIP1 expression in cell supernatants.The effect of MG132-induced MCPIP1 production stability:VSMCs were divided into five groups: 1) control group, 2) DMSO control group, 3) MG132(10 ?M), 4) 10?M MG132 + 5?g/m L CHX + 10?M MG132(0?0.5?1?2 h), 5) 10?M MG132 + 5?g/m L CHX + 10?M DMSO(0?0.5?1?2 h). Western Blot was performed to determine the MCPIP1 expression in cell supernatants.The effect of different signaling pathway inhibitor(p38, AKT), gene transcript inhibitor and protein synthesis inhibitor on MG132-induced MCPIP1 production: VSMCs were divided into seven groups: 1) control group, 2) DMSO(10?M) group, 3) MG132(10?M), 4) MG132(10?M) + AMA(gene transcript inhibitor) group, 5) MG132(10?M) + CHX(protein synthesis inhibitor), 6) MG132(10?M)+ MK(AKT inhibitor), 7) MG132(10?M)+ SB(p38 inhibitor). Western Blot was performed to determine the MCPIP1 expression in cell supernatants.The effect of MCPIP1 induced the apoptosis in HVSMCs:Experiment 1: VSMCs were divided into three groups: l) control group, 2) GFP(Green Fluorescent Protein virus) overexpression group, 3) MCPIP1(MCPIP1 virus) overexpression group, Western Blot was performed to determine the MCPIP1 expression in cell supernatants.Experiment 2: VSMCs were divided into six groups: l) negative control group, 2) positive control group, 3) control group, 4) GFP(Green Fluorescent Protein virus) overexpression group, 5) MCPIP1(MCPIP1 virus) overexpression group, 6) MG132(10?M). The apoptosis and death of HVSMCs were determined by laser scanning cytometer(LSC) with terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling(TUNEL) assay.MCPIP1 inhibits HVSMC proliferation: VSMCs were divided into four groups: l) control group, 2) GFP(Green Fluorescent Protein virus) overexpression group, 3) MCPIP1(MCPIP1 virus) overexpression group, 4) MG132(10?M), VSMCs independently cultured in 0% FBS and 10% FBS condition. The proliferation of HVSMCs was determined by LSC with bromodeoxyuridine(Brd U) incorporation assay.Results:Treatment with MG132 was found to elevate MCPIP1 protein levels. The level of MCPIP1 protein was increased time-dependently and dose- dependently. It was through stimulation of the gene transcription, which was inhibited by ?-amanitin inhibition of gene transcription, without effects on MCPIP1 protein stability. Our further studies showed that MCPIP1 expression induced by MG132 was inhibited by the inhibitors of AKT and p38 kinase, suggesting a role of the AKTp38 pathway in MG132 effects. We also found that treatment with MG132 induces apoptosis, but overexpression of MCPIP1 inhibited bromodeoxyuridine(Brd U) incorporation in human VSMCs without significant apoptosis.Conclusions:1. MCPIP1 expression is induced by MG132 potentially through activation of the AKT-p38 pathway.2. MCPIP1 inhibits VSMC proliferation without induction of apoptosis.