GM-CSF Induces MMP-2/-9 Expression In Fibroblasts And The Underlying Mechanism

Objective To investigate the regulation of MMP-2/9 by GM-CSF in fibroblasts and the molecular mechanism of GM-CSF underlying promoting wound healing.Methods1) ELISA, Gelatin zymography and Western Blot were applied to detect the concentration and activity of MMP-2/-9 in the supernatant of cultured fibroblasts induced by GM-CSF(0, 10, 50, 100, 500ng/ml).2) The phosphorylation of JNK and c-Jun in fibroblasts in GM-CSF treated group and SP600125-pretreated group were measured by Immunofluorescence and Western Blot; Real-time PCR was used to analyze the m RNA levels of MMP-2/-9 in fibroblasts in GM-CSF treated group and SP600125-pretreated group. The concentration of MMP-2/MMP-9 in fibroblasts in GM-CSF treated group and SP600125-pretreated group(at 6, 12, 24, 48h) were tested by ELISA.3) The proliferation of fibroblasts in GM-CSF treated group and SP600125-pretreated group was detected by cck-8 kit, the migration of fibroblasts in GM-CSF treated group and SP600125-pretreated group was measured by cell wound healing assay.Results1) The MMP-2/-9 expression in GM-CSF treated fibroblasts weresignificantly higher than that in control group(P<0.05), and the variation of MMP-2/9 induced by GM-CSF was in concentration- dependent manner.2) Compared to control group, the phosphorylation of JNK and c-Jun and the m RNA levels of MMP-2/9 in fibroblasts were highly enhanced in GM-CSF treated group(P<0.05). Pretreated by SP600125, the phosphorylation of c-Jun and the m RNA levels of MMP-2/-9 were dramatically inhibited compared to GM-CSF treated group. The levels of MMP-2/-9 in fibroblasts reached to climax at 24 hours after treatment by GM-CSF and gradually decreased from 24 to 48 hours. The level of MMP-2 in SP600125+GM-CSF group was significantly lower than control group(P<0.05), in the contrast, MMP-9 was higher than control group(P<0.05).3) GM-CSF had no effect on the proliferation of fibroblasts, but promoted fibroblasts migration which could be inhibited by SP600125.Conclusion: GM-CSF induced the synthesis, expression and secretion of MMP-2 and MMP-9 in fibroblasts through activation of JNK/c-Jun signaling pathway.