Figure 6
AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 expression through a Ca2+/PKC-α/Nox2/ROS cascade. (A) Cells were pretreated without or with tanshinone IIA (TSIIA, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. (B) Cells were pretreated without or with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h before exposure to 10 nM BK for 30 min. The nuclear fraction was collected and analyzed by Western blotting using an anti-c-Fos, phospho-c-Jun, or c-Jun antibody. (C) Cells were transiently cotransfected with pAP1-Luc and pGal for 24 h, pretreated with BAPTA (BAP), TG, Gö, Apo, DPI, or NAC for 1 h and then incubated with BK for 6 h. The AP-1 promoter activity in the cell lysates was determined. (D) Cells were transfected with scramble (scra) or c-Fos/c-Jun siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; #P < 0.01, as compared with the respective values of cells stimulated with BK only. The figure represents one of three similar experiments.