CD137 Signaling May Affect The Expression Of NFATc1 By Activating TRAF6/JNK/AP-1 Pathway

ObjectiveTo investigate whether CD137-CD137 L signal through tumor necrosis factor receptor-associated factor-6/c-Jun amino-terminal kinase/activated protein-1(TRAF6/JNK/AP-1)pathway to regulate the expression of nuclear factor of activated T cell c1(NFATc1)in mouse vascular smooth muscle cell(VSMC).MethodsMouse primary VSMCs were cultured from aortic tissue block;and the forth-generation smooth muscle cells were used for the experiment.The whole experiment was divided into 6 groups: control group,CD137 stimulation group,CD137 inhibition group,siTRAF6 intervention group,siJNK intervention group and siAP-1 intervention group.The expression of TRAF6,JNK,AP-1 and NFATc1 mRNA were measured by real-time quantitative PCR(RT-PCR).The Apoptosis and proliferation of six group cells were detected by Flow Cytometry(FCM)and Cell Counting kit-8(CCK-8).The expression levels of TRAF6,phosphorylated JNK(p-JNK),phosphorylated AP-1(p-AP-1)and NFATc1 protein were detected by Western blot in each group.The fluorescence expression of TRAF6,p-JNK,p-AP-1 and NFATc1 were detected by immunofluorescence assay in each group.ResultsWhen Agonist CD137 mAb was used to treat with the VSMC,the expression levels of JNK,AP-1 and NFATc1 were increased obviously comparing with control group.(2)When Anti CD137 mAb was applied to treat with the VSMC,the expression levels of JNK,AP-1 and NFATc1 were decreased obviously comparing with control group.(3)When the expression of TRAF6 was blocked by TRAF6 si RNA,expression levels of JNK,AP-1 and NFATc1 were decreased after stimulated by agonist CD137 mAb comparing with the CD137 activated group.(4)When the expression of JNK was blocked by JNK siRNA,the expression levels of JNK,AP-1 and NFATc1 were decreased after stimulated by agonist CD137 mAb comparing with the CD137 activated group.(5)When the expression of AP-1 was blocked by AP-1 siRNA,the expression levels of AP-1 and NFATc1 were decreased after stimulated by agonist CD137 mAb comparing with the CD137 activated group.After the activation of CD137-CD137 L signal,the vascular smooth muscle cells could proliferate obviously.After the activation of CD137-CD137 L signal,the apoptosis of vascular smooth muscle cells can be significantly reduced.ConclusionsCD137 signaling may increase the expression of NFATc1 by activating TRAF6 / JNK /AP-1 pathway.CD137-CD137 L signal changes can affect the proliferation of mouse smooth muscle cells.