Figure 2

TNF-α induces MMP-9 expression via TNFR1-dependent c-Src cascade. (A) Cells were pretreated with various concentrations of TNF-α receptor antibody (TNFR Ab) for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) MC3T3-E1 cells were incubated with TNF-α (15 ng/ml) for the indicated time intervals, and the protein-protein interaction was determined by immunoprecipitation (IP) and Western blot using the antibodies as indicated. (C,D) Cells were treated with TNF-α (15 ng/ml) for (C) 24 h or (D) 6 h in the absence or presence of PP1. (D) The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared to the cells incubated with TNF-α alone. (E,G) Cells were pretreated with or without PP1 (30 μM) for 1 h or transfected with TRAF2 siRNA and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of c-Src was determined by Western blot using an anti-phospho-c-Src antibody. (F,G) Cells were transfected with c-Src siRNA or TRAF2 siRNA for 24 h and then incubated with TNF-α (15 ng/ml) for 24 h. (A,C,F,G) MMP-9 expression was determined as described in Figure 1. The cell lysates were determined by Western blot using an anti-c-Src, anti-TRAF2 or anti-GAPDH antibody.