Figure 1.
TGF-β induces TIMP-3 gene expression in a Smad-dependent manner in fibroblasts.
(A) Human gingival fibroblasts were treated with TGF-β1 (5 ng/ml) for 24 h. Thereafter, total cellular RNAs were harvested and analyzed for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs by Northern blotting. (B) EF7WT and EF7Smad4KO (Smad4 deficient) cells were treated with TGF-β1 (5 ng/ml) for 3 h and 12 h or left untreated (control). Total RNA was extracted and TIMP-3 and PAI-1 gene expression was determined by qRT-PCR. mRNA expression (mean+SD) is shown relative to 18S ribosomal RNA (n = 4). *p<0.05, **p<0.005 (t-test) for TGF-β vs. control cultures.
Figure 2.
Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4 null fibroblasts.
(A) EF7WT (wild-type) and EF7KO (Smad4 deficient) fibroblasts were transduced with recombinant adenovirus for HA-tagged Smad4 (RAdSmad4), or with empty control virus RAd66 at MOI 100 (EF7WT) or 300 (EF7KO). After 36 h incubation cell lysates were harvested and analyzed by Western blotting to detect the levels of endogenous and exogenous Smad4. Anti-HA antibody was used to detect adenovirally delivered Smad4 (upper panel) and anti-Smad4 to detect endogenous Smad4 (lower panel). (B) EF7KO fibroblasts were infected with adenoviruses RAdSmad4 or RAd66. After 36 h incubation the cells were stimulated with TGF-β1 (5 ng/ml) for different periods of time, as indicated. Total RNA was extracted and analyzed by qRT-PCR to determine TIMP-3 and PAI-1 mRNA levels. mRNA expression (mean ± SEM from two separate experiments, both run with duplicates) is shown relative to 18S ribosomal RNA. *p<0.05, t-test).
Figure 3.
Smad3 mediates TGF-β-elicited induction of TIMP-3 expression in human fibroblasts.
(A) Normal human gingival fibroblasts were transduced with recombinant adenoviruses for Smad2 (RAdSmad2), Smad3 (RAdSmad3), dominant negative Smad3 (RAdSmad3DN), or with empty control virus (RAd66) at MOI 500, and incubated for 18 h. Thereafter, the cells were treated with TGF-β1 for 24 h. The cell layers were harvested for RNA extraction and analyzed for the expression of TIMP-3 or GAPDH by Northern blot hybridizations. (B) Normal human gingival fibroblasts were infected with RAdSmad3, RAdSmad3DN, adenovirus for Smad7 (RAdSmad7), or with empty control virus (RAdpCA3) as in (A). Cells were treated with TGF-β1 for 24 h, the cell layers harvested and analyzed for the expression of TIMP-3 by Western blotting. Equal loading was confirmed by stripping and reprobing the same filter for β-actin.
Figure 4.
Smad3, p38α and ERK1/2 cooperate in the induction of TIMP-3 gene expression in human fibroblasts.
(A) 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) 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. (C) 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). Total cellular RNA was analyzed with Northern blot hybridizations for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs.
Figure 5.
A schematic representation of the complex interplay of TGF-β-signaling pathways regulating TIMP-3 expression in human fibroblasts.
Stimulation of human gingival fibroblasts with TGF-β results in activation of Smad3, ERK1/2 and p38 MAPK pathways. Activation of all three pathways is required for induction of TIMP-3 expression by TGF-β Smad3 associates with Smad4 and mediates induction of TIMP-3 expression by TGF-β. ERK1/2 and p38 MAPK pathways both co-operate with Smad3 in mediating the induction of TIMP-3 expression by TGF-β.