The Effects of Amphiregulin Induced MMP-13 Production in Human Osteoarthritis Synovial Fibroblast
Figure 3
PI3K/Akt is involved in AREG-mediated MMP-13 production in synovial fibroblasts. (a) OASFs were pretreated for 30 min with PI3K inhibitors (LY294002 (5 μM) and Wortmannin (0.5 μM)) and Akt inhibitor (Akti (5 μM)) for 30 min followed by stimulation with AREG for 24 h, and MMP-13 expression was examined by qPCR. ((b) and (c)) OASFs were pretreated for 30 min with PI3K inhibitors (LY294002 (5 μM) and Wortmannin (0.5 μM)) and Akt inhibitor (Akti (5 μM)) for 30 min followed by stimulation with AREG for 24 h; MMP-13 protein levels in cell lysates were determined by Western blot analysis. MMP-13 enzymatic activity in cell lysates and supernatants was measured using zymography. (d) OASFs were incubated with AREG for indicated time intervals, and p85 and Akt phosphorylation was examined by Western blot. (e) OASFs were transfected for 24 h with PI3K and Akt mutant followed by stimulation with AREG for 24 h, and MMP-13 expression was examined by qPCR. (f) OASFs were pretreated for 30 min with PD158780 (1 μM) and BIBX1382 (1 μM) followed by stimulation with AREG for 15 min; MMP-13 protein levels in cell lysates were determined by Western blot analysis. Results are expressed as the mean ± SEM . compared with control; compared with AREG-treated group.
