Figure 8

TNF-α-induced MMP-9 expression is mediated through the NF-κB element in MMP-9 promoter leading to soluble ICAM-1 release. (A) Cells were transiently transfected with a wild-type MMP-9 promoter-luciferase reporter construct (WT-MMP9), and then incubated with TNF-α for the indicated time intervals. (B) The WT-MMP9 transfected cells were pretreated with anti-TNFR1 neutralizing antibody (TNFR-Ab, 10 μg/ml), PP1 (3 μM), U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), and Bay11-7082 (10 μM) for 1 h and then incubated with 15 ng/ml TNF-α for 6 h. (C) Cells were transfected with WT-MMP9 or mt-κB-MMP9 for 24 h and then incubated with TNF-α (15 ng/ml) for 6 h. The cell lysates were collected and determined the luciferase activity. (D,E) Cells were pretreated with GM6001 or MMP2/9i for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (D, upper panel) Conditioned media were collected and analyzed by gelatin zymography to determine the MMP-9 expression. The conditioned media were analyzed by trichloroacetic acid-protein precipitation and Western blot using an anti-sICAM-1 antibody. (E) The cell lysates were analyzed by Western blot to determine the expression of ICAM-1. (D, lower panel) Cells were pretreated with MMP-2/9i (10 μM), PP1 (10 μM), U0126 (10 μM), SB202190 (10 μM), SP600125 (10 μM), or Bay11-7082 (10 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for the indicated time intervals. The levels of sICAM-1 were determined in conditioned media using an sICAM-1 ELISA kit. Data are expressed as mean±SEM of three independent experiments. ^#P < 0.01, as compare to the cells exposed to vehicle (A,D) or TNF-α alone (B-D). (F) Schematic representation of signaling pathways involved in TNF-α-induced MMP-9 expression and sICAM-1 release in MC3T3-E1 cells. TNF-α stimulates two independent pathways through TNFR1/TRAF2 activates both c-Src-dependent MAPKs and c-Src-independent IKK/NF-κB pathways.