Figure 7

c-Jun/AP-1 binding element is essential for LTA-induced MMP-9 expression through a Ca²⁺/CaMKII/PDGFR/PI3K/JNK pathway in RBA-1 cells. (A) Time dependence of LTA-stimulated c-Jun/AP-1 binding activity. RBA-1 cells were incubated with 50 mg/ml LTA for the indicated time intervals, or cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with 50 mg/ml LTA for 30 min. c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. (B) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase for 24 h. The pGL-MMP-9-Luc-transfected cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with LTA for 16 h. (C) Activation of wild-type (WT) and AP-1-mutated (mt-AP1) MMP-9 promoter constructs by LTA. Cells were cotransfected with respective promoter constructs for 24 h and then incubated with 50 μg/ml LTA for 16 h. The values for beetle luciferase were normalized to that of b-galactosidase activity. (D) Cells were pretreated with CaMI, KN-62, BAPTA, or TG for 1 h and then incubated with LTA for 16 h. Total RNA were extracted and analyzed by RT-PCR. Data are expressed as mean ± SEM (B, C) or mean (A, D) of three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to LTA alone. The figure represents one of at least three individual experiments.