Figure 5.

Regulation of MMP-7 transcription is dependent on JNK/c-Jun and ERK/c-Fos activities in H₂O₂-treated SW620 cells. A, effects of DMA and MAPK inhibitors on H₂O₂-induced c-Jun and c-Fos translocation. Nucleoprotein was isolated from SW620 cells treated with DMA (100 μM), SP600125 (50 μM), or PD98059 (50 μM) for 30 min prior to H₂O₂ (5 μM) induction for 4 h. Afterward, Western blotting analysis was performed using specific antibodies against c-Jun and c-Fos. Expression of lamin B1 was examined in parallel to confirm that equal amounts of nucleoprotein were being analyzed for each condition. The right upper panel showed that the JNK inhibitor SP600125 and DMA effectively inhibited H₂O₂-induced c-Jun translocation. The right lower panel showed that the ERK inhibitor PD98059 and DMA effectively inhibited H₂O₂-induced c-Fos translocation. B, DMA suppressed H₂O₂-induced AP-1 transcriptional activity. AP-1 activation was induced by H₂O₂ treatment via the JNK/cJun and ERK/c-Fos signaling pathways. SW620 cells were transiently transfected with AP-1 luciferase reporter (pAP1-luc) and pRL-TK reference vector for 24 h and were co-cultured with DMA or MAPK inhibitors in the absence (□) or presence (■) of H₂O₂ (5 μM). Relative luciferase activities were measured by calculating the light emitted and were normalized by coexpression of pRL-TK Renilla luciferase (ratio multiplied by an arbitrary factor to set the control of H₂O₂ induction only to 100). The relative luciferase activities are presented as means ± SD. C, H₂O₂-stimulated MMP-7 transcription depends on both JNK and ERK signaling activation. Pretreatment with DMA (100 μM), SP600125 (50 μM), or PD98059 (50 μM) for 30 min prior to H₂O₂ (5 μM) induction for 4 h in SW620 cells significantly reduced the expression of MMP-7 mRNA compared with H₂O₂-only treatment (*p < 0.05). The bars in the lower panel denote means ± SD of three experiments for each condition determined from densitometry relative to GAPDH mRNA.