The Pka Binding Site Of MLCK Play A Role In The Proliferation Migration Of VSMCs Induced By IL-8

Background:According to the World Health Organization' s report in 2015,the numbers of people death from cardiovascular disease are more than any other causes of death.More than three-quarters of the cardiovascular disease deaths occur in low and middle income countries.People die from this disease account for 51%of the total annual death caused in China.Therefore,it is very urgent to strengthen our country's ability in the study of cardiovascular disease.Cardiovascular disease is a series of disease usually associated with atherosclerosis,and the development of atherosclerosis is closely connected with some inflammatory cytokines.Interleukin-8(IL-8)is a kind of chemotactic cytokines.IL-8 has the effect of promoting mitosis,so it can directly cause cell proliferation.The study found that IL-8 can promote smooth muscle cells proliferation and migration,and plays an important role in the process of atherosclerosis,but the mechanism remains to be further studied.Myosin light chain kinase(MLCK)is one of the key enzymes related to cell contraction force.MLCK has a lot of functional domains,including myosin-binding domains,actin-binging domains,ATP activity domains,calmodulin-binding domains.MLCK can activate its downstream protein myosin light chain(MLC)and cause cell shrinkage.At the same time,MLCK can also be phosphorylated by variety of other kinases such as protein kinase A(PKA),protein kinase C(PKC),which ultimately affect cell function.Different binding sites of MLCK play a different role in regulating the cell function.In recent years,there are some reports that MLCK is closely related with the formation of atherosclerosis,but wether the binding sites of MLCK play a role in the development of atherosclerosis has not been reported.Objective:We overexpressed the full length of MLCK in GbaSM-4 cells in order to explore the role of MLCK in the smooth muscle cell proliferation and migration induced by IL-8.By PKA binding sites of MLCK dephosphorylated,we build a MLCK mutants to explore if the PKA binding site dephosphorylation play a role in regulate the proliferation and migration of VSMCs.Methods:(1)Cells:the cells of GbaSM-4 and MLCK-/VGba were gifts from professor Kohama.(2)Vector:MLCK eukaryotic expression vector pCDNA3.1+-Flag-MLCK(pCDNA3.1+-MLCK),and the PKA binding site phosphorylation of MLCK mutant vector pCDNA3.1+-Flag-MLCK-S993D(MLCK-S993D)were early build in our laboratory.The PKA binding site dephosphorylation of MLCK mutant vector pCDNA3.1+-Flag-MLCK-S993 A(MLCK-S993A)was constructed by this experiment.(3)The vector of pCDNA3.1+-MLCK transfected into GbaSM-4 cells,the cells overexpressed of MLCK(MLCK+/GbaSM-4),using the same method to transfect MLCK-S993D or MLCK-S993A into MLCK-/Gba cells,which can respectively express mutant protein of MLCK.(4)The CCK8 method was used to detect cells proliferation.(5)The Boyden Chamber method was used to detect cells migration.(6)RT-PCR method was used to detect the gene expression of cell proliferation and migration related cytokines,such as PCNA,CyclinDl and a-SMA.(7)Western blot method was used to detect the expression of MLCK,PCNA,a-SMA and p-ERK.(8)Gelatin zymography was used to detect the active of MMP2.(9)The SPSS 16.0 statistical software was used to analysis the experimental data,the difference was statistically significant if P<0.05.Results:(1)Stimulated with 20ng/mL IL-8 for 24h,can promote GbaSM-4 cells proliferation and migration.Overexpression of MLCK increased cells ability of proliferation and migration.(2)The PKA binding site dephosphorylation of MLCK mutant vector pCDNA3.1+-Flag-MLCK-S993 A was constructed successfully.pCDNA3.1+-MLCK,MLCK-S993D were early build in our laboratory.(3)Phosphorylated the PKA binding site of MLCK could promot the migration of GbaSM-4 cells induced by IL-8,while dephosphorylated the PKA binding site of MLCK,can inhibit the promoting effect.(4)Dephosphorylated the PKA binding site of MLCK,the expression of p-ERK was decreased.After pretreatment cells with U0126 for one hour,using 20 ng/mL IL-8 stimulate cells,the results show that dephosphorylation of MLCK's PKA binding sites could remedy the U0126 inhibitory effect on GbaSM-4 cell migration.Conclusions:(1)20ng/mL IL-8 promotes GbaSM-4 cell proliferation and migration,this effect may be related to increase MLCK expression.(2)Phosphorylation PKA binding site of MLCK is not associated with GbaSM-4 cell proliferation in vitro induced by IL-8.(3)The PKA binding site of MLCK played a role in the migration of GbaSM-4 cells induced by 20ng/mL IL-8.And the effect was relatived with MAPK/ERK signaling pathways.