The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in tumor invasion and metastasis. In this study, the factors and signaling pathways that are involved in the regulation of the MMP-9 expression were examined in urinary bladder cancer HT1376 cells. Tumor necrosis factor-α (TNF-α) stimulated the secretion of MMP-9 in HT1376 cells, as shown by zymography and immunoblot analysis. At the level of transcription, TNF-α also stimulated 5'-flanking promoter activity of MMP-9. Transcription factor NF-κB, AP-1 and Sp-1 binding sites were identified by a gel shift assay to be cis-elements for TNF-α activation of the MMP-9 promoter. TNF-α activates multiple signaling pathways in HT1376 cells, including the extracellular signal-regulated kinase (ERK1/2), p38 MAP kinase and JNK pathways. Chemical inhibitors, which specifically inhibit each of these TNF-α-activated pathways, were used to examine the signaling pathways involved in TNF-α-mediated MMP-9 expression. The ERK1/2 inhibitor, U0126 and the p38 MAP kinase inhibitor, SB203580, significantly down-regulated TNF-α-induced MMP-9 expression and promoter activity. The transactivation of TNF-α-stimulated NF-κB, AP-1 and Sp-1 were inhibited by U0126 and SB203580 treatment. In conclusion, the findings of the present study indicate that TNF-α induces MMP-9 expression in HT1376 cells by activating transcription factors, which are involved in the ERK1/2- and p38 MAP kinase-mediated control of MMP-9 regulation, namely, NF-κB, AP-1 and Sp-1.

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