TNF-α stimulates activation of pro-MMP2 in human skin through NF-κB mediated induction of MT1-MMP

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator during the inflammatory phase of wound healing, Excessive amounts of pro-inflammatory cytokines such as TNF-alpha are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during mound healing, angiogenesis and tumor metastasis. As with proinflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds, In this report we relate these two phenomena, TNF-alpha stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin, This suggests a mechanism whereby excess inflammation affects normal wound healing. To investigate this observation at the cellular and molecular levels, we examined TNF-alpha mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-alpha substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen, In marked contrast, collagen or TNF-alpha individually had little influence on the fibroblast-mediated pro-MMP-2 activation, One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-alpha significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen, Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-alpha and collagen activate the NF-kappaB pathway, In this report we provide three sets of evidence to support a hypothesis that activation of NF-kappaB is essential to induce MT1-MMP expression in fibroblasts after TNF-alpha exposure, First, SN50, a peptide inhibitor for NF-kappaB nuclear translocation, simultaneously blocked the TNF-alpha and collagen mediated MT1-MMP induction and pro-MMP-2 activation, Secondly, TNF-alpha induced I kappaB to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-kappaB activation, Lastly, a consensus binding site for p65 NF-kappaB (TGGAGCTTCC) was found in the 5'-flanking region of human mt1-mmp gene. Based on these results and previous reports, we propose a model to explain TNF-alpha activation of MMP-2 in human skin, Activation of NF-kappaB signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-alpha may affect matrix remodeling during wound healing and other physiological and pathological processes.