ERK1/2 Mediates TNF-α-Induced Matrix Metalloproteinase-9 Expression in Human Vascular Smooth Muscle Cells Via the Regulation of NF-κB and AP-1: Involvement of the Ras Dependent Pathway

The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in progression of atherosclerotic lesions. The role and importance of the signaling pathway in the transcriptional regulation of MMP-9 in human aortic smooth muscle cells (HASMC) was examined. Tumor necrosis factor-α (TNF-α) stimulated the secretion of MMP-9 in HASMC, as shown by zymography and immunoblot analysis. At the transcriptional levels, TNF-α also stimulated the 5′-flanking 710-bp promoter activity of MMP-9. Transcription factors NF-κB binding site (-601) and AP-1 binding site (-82) were identified as the cis-elements for TNF-α activation, as determined by gel shift assay and mutation analysis. Treatment with U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly down-regulated TNF-α-induced MMP-9 expression and promoter activity, whereas the inactive analog U0124 had no effect. Furthermore, the transactivation of TNF-α-stimulated NF-κB and AP-1 was inhibited by U0126 treatment. Finally, the transient transfection of HASMC with dominant negative Ras (RasN17) suppressed TNF-α-induced ERK activity, MMP-9 production, and promoter activity. Overexpression of RasN17 also abolished the TNF-α-stimulated NF-κB and AP-1 activity. In conclusion, the findings herein indicate the activation of the Ras/ERK pathway contributes to the induction of MMP-9 expression in HASMC. In addition, the transcription factors NF-κB and AP-1 that are involved in the Ras/ERK-mediated control of MMP-9 regulation on HASMC in response to TNF-α have now been identified.