Figure 7

BK induces astrocytic migration through Nox/ROS-dependent AP-1 increasing MMP-9 gene expression. (A) Cells were incubated with BK (10 nM) for the indicated times (upper part), or pretreated with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h and then incubated with 10 nM BK for 30 min. The c-Fos and c-Jun AP-1 binding activity were analyzed by chromatin-IP (ChIP)-PCR assay. (B) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal for 24 h, pretreated with BAPTA (BAP), Gö, Apo, DPI, or transfection with c-Fos or c-Jun siRNA for 24 h and then incubated with BK for 16 h. (C) Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the AP-1 binding site (upper part). After cotransfection, luciferase activities of different MMP-9-promoter constructs after stimulation with or without BK (10 nM) for 16 h, were measured as relative MMP-9 promoter activity to that of β-galactosidase. (D) Cells were plated on 6-well culture plates, grew to confluence and starved with serum-free DMEM/F-12 medium for 24 h. Cells were pretreated with TG, Gö, Apo, DPI, NAC, TSIIA, or MMP-9i (9i) for 1 h and the monolayer cells were manually scratched with a blue tip as described in Methods, and then incubated with BK (10 nM) for 48 h. Phase contrast images of cells were taken at 48 h and the number of cell migration was counted as described in Methods. (E) Rat primary cultured astrocytes were pretreated with BAPTA (BAP), Gö, Apo, DPI, NAC, or TSIIA before exposure to BK for 16 h. The conditioned media and total RNA were collected and analyzed by zymography and RT-PCR. Data are expressed as the mean ± SEM (n = 3). ^#P < 0.01, as compared with the respective values of cells stimulated with BK only.