TNF-alpha induces matrix metalloproteinase-9 expression in A549 cells: role of TNFR1/TRAF2/PKCalpha-dependent signaling pathways

Abstract

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines, including TNF-alpha and contributes to airway inflammation. However, the mechanisms underlying TNF-alpha-induced MMP-9 expression in human A549 cells remain unclear. Here, we report that TNF-alpha-induced MMP-9 gene expression was mediated through the TNFR1/TRAF2/PKCalpha-dependent signaling pathways in A549 cells, determined by zymographic, RT-PCR, and Western blotting analyses. TNF-alpha-induced MMP-9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF-alpha-induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti-TNFR1 Ab followed by Western blot analysis against an anti-TRAF2 or anti-TNFR1 Ab. In addition, TNF-alpha-induced MMP-9 expression was also reduced by pretreatment with the inhibitor of PKCalpha (Gö6983), c-Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCalpha, Src, EGFR, Akt, p65, p300, and c-Jun. On the other hand, TNF-alpha stimulated the phosphorylation of c-Src, EGFR, Akt, JNK1/2, and c-Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF-alpha induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF-alpha-induced c-Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF-alpha induces MMP-9 expression via the TNFR1/TRAF2/PKCalpha-dependent JNK1/2/c-Jun and c-Src/EGFR/PI3K/Akt pathways.