Smad3, p38α and ERK1/2 cooperate in the induction of TIMP-3 gene expression in human fibroblasts.

<p>(<b>A</b>) Human gingival fibroblasts were serum starved for 18 h, and treated for 1 h with PD98059 (30 µM), or SB203580 (10 µM), specific chemical inhibitors for MEK1 or p38, respectively. Subsequently, TGF-β1 (5 ng/ml) was added, and the cultures incubated for 16 h. Total cellular RNAs were harvested and analyzed for the levels of TIMP-3, TIMP-1, PAI-1 and GAPDH mRNAs by Northern blot hybridizations. (<b>B</b>) Human gingival fibroblasts were transduced with recombinant adenoviruses for wild-type p38α (RAdp38α), constitutively active MKK3b (RAdMKK3bE), Smad3 (RAdSmad3), Smad4 (RAdSmad4), or with empty control virus (RAd66) at MOI 500, and incubated for 24 h. Total cellular RNA was analyzed with Northern blot hybridizations for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs. (<b>C</b>) Human gingival fibroblasts were transduced with recombinant adenoviruses for constitutively active MEK1 (RAdMEK1CA), constitutively active MKK3b (RAdMKK3bE), Smad3 (RAdSmad3) and control virus RAd66 as in (<b>B</b>). Total cellular RNA was analyzed with Northern blot hybridizations for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs.