IL-17 Stimulates The Expression Of MCP-1 In Cardiac Myocytes Via Act1/TRAF6/MAPK-dependent AP-1 Activation

IL-17 is an inflammatory factor which exists in the development of autoimmune diseases and it could promote the expression of chemokines.Monocyte chemoattractant protein(MCP-1)is a common chemokine and plays an important role in the infiltration of mononuclear cells in myocardial tissue of viral myocarditis(VMC).Our previous studies had found that MCP-1could mediate myocardial injury by its receptor CCR2,and IL-17 could aggravate myocardial injury by upregulating the expression of MCP-1.Therefore,it is meaningful to relieve the development of VMC by reducing the expression of MCP-1.However,the mechanism of the expression of MCP-1 induced by IL-17 in cardiac myocytes is still not explicit.Activate protein-1(AP-1)is a key nuclear transcription factors,related to the inflammation,which exists in the promoter sequence of genes,and it plays a vital role in many signal transduction pathways.The researchers paid more and more attention to AP-1 in recent years.The studies had found that IL-17 could activate the expression of AP-1 by combining the receptor of IL-17(IL-17R)on the variety of cell surfaces.Meanwhile,the activation of AP-1 could upregulate the expression of MCP-1 in some cells.However,it is not clear whether AP-1 was involved in the expression of MCP-1 induced by IL-17 in cardiac myocytes.IL-17 could trigger the downstream signal transduction pathways IL-17R-Act1-TRAF6 by combined theIL-17 R.There are studies showed that IL-17 could stimulate the expression of MCP-1 by mediating AP-1 in some cells,which was activated by MAPK signal transduction pathway.But how the signal transduction pathway acts on the expression of MCP-1 induced by IL-17 in myocardial cells is unknown.Using the predicted software,we found that the transcription factor binding sites were existed in the promoter of MCP-1,such as AP-1 binding site,NF-?B binding site and SP-1 binding site.Therefore,we conjecture that IL-17 combined with IL-17 R may stimulate the expression of MCP-1 in myocardial cells via Act1/TRAF6/MAPK-dependent AP-1activation,which could mediate the progress of VMC and provide a new target for the treatment of viral myocarditis.ObjectiveTo investigate the mechanism of IL-17 stimulates the expression of MCP-1 in cardiac myocytes via Act1/TRAF6/MAPK-dependent AP-1 activation.Methods1.The cardiac myocytes of BALB/c were isolated by differential attachment technique,and the myocardial cells were cultured.2.The specific primers were designed to amplify the promoter sequence of MCP-1,which contains AP-1 binding site.Then the promoter of MCP-1 sequence was combined with luciferase reporter gene vector pGL3-basic to construct the luciferase reporter gene vector pGL3-MCP-1-Luc.3.The relative fluorescence activity of pGL3-MCP-1-Luc induced by IL-17,KTCD1,Act1 siRNA,TRAF6 inhibitor,LY294002,or SB203580 was detected by the dual luciferase reporter gene system.4.Western Blot was used to detect the expression of Act1,TRAF6,MAPK and AP-1 induced by IL-17 at the different time.5.Western Blot and ELISA were used to detect the expression of MCP-1 and AP-1 in cardiac myocytes,which were stimulated by the signal transduction pathway small molecule inhibitor or siRNA.6.SPSS 17.0 software was used to establish the database and analyze the results.The comparison among the groups were used one-way ANOVA,and the comparison among the experimental group were used the LSD-T t test.P<0.05 was considered as a statistically significant mark.Results1.The identification of luciferase reporter gene vector pGL3-MCP-1-Luc containining MCP-1 promoter.It was clarified by enzyme digestion and DNA sequencing technology.Meanwhile,the activity of pGL3-MCP-1-Luc,which transfected into cardiac myocytes,was stimulated with different concentration of IL-17(0,5,10,50,100ng/mL),the fluorescence activity of MCP-1 promoter were increased with the increase of IL-17(P<0.01 vs.negative group).2.The screen of signal transduction pathway induced by IL-17 in cardiac myocytes.The fluorescence activity of the grouop of IL-17+Act1 siRNA,IL-17+TRAF6 inhibitor or IL-17+SB203580 was lower than the grouop of IL-17(P<0.05).The fluorescence activity of the group of IL-17+KCTD1 or IL-17+LY294002 was not significantly changed(P>0.05 vs.the saline group).3.The expression of Act1?TRAF6?MAPK?p-c-jun induced by IL-17 in cardiac myocytes.Cardiac myocytes were stimulated by IL-17 at 5min,15 min and 30 min,the results showed that the expression of Act1?TRAF6?MAPK?p-c-jun were increased as time flows(P<0.05).4.The effect of Act1/TRAF6/p38 MAPK and AP-1 on the expression of MCP-1induced by IL-17 in cardiac myocytes.Compared with the group of IL-17+Act1control or the group of IL-17,the expression of MCP-1 induced by IL-17+Act1siRNA was decreased(P<0.05).Compared with the group of IL-17+TRAF6 control or the grouop of IL-17,the expression of MCP-1 induced by IL-17 and TRAF6 inhibitor was decreased(P<0.05).Compared with the group of IL-17 and p38 control or the group of IL-17,the expression of MCP-1 induced by IL-17+SB203580 was decreased(P<0.05).The expression of MCP-1 induced by IL-17 was higher than the control group,and compared with IL-17,the expression of MCP-1 stimulated byIL-17+Curcuminn was lower(P<0.05).5.The relationship of the phosphorylation of c-jun and Act1/TRAF6/p38 MAPK on the expression of MCP-1 induced by IL-17 in cardiac myocytes.Compared with the negative control group,the phosphorylation of c-jun induced by IL-17 was increased.However,compared the group of IL-17+negative siRNA,the phosphorylation of c-jun of IL-17+Act1 siRNA was decreased.The phosphorylation of c-jun of the grouop of IL-17+TRAF6 inhibitor or IL-17+SB203580 was lower than the group induced by IL-17(P<0.05).Conclusions1.IL-17 could upregulate the expression of MCP-1 in cardiac myocytes by activating the phosphorylation of c-jun.2.IL-17 could upregulate the expression of MCP-1 in cardiac myocytes via the signal transduction pathway of Act1/TRAF6/p38MAPK3.IL-17 stimulates the expression of MCP-1 in cardiac myocytes via Act1/TRAF6/MAPK-dependent AP-1 activation.