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TGF-β-elicited induction of tissue inhibitor of metalloproteinases (TIMP)-3 expression in fibroblasts involves complex interplay between Smad3, p38α, and ERK1/2.

Leivonen SK, Lazaridis K, Decock J, Chantry A, Edwards DR, Kähäri VM - PLoS ONE (2013)

Bottom Line: Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression.Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression.These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Turku, and Turku University Hospital, Turku, Finland.

ABSTRACT
Transforming growth factor-β (TGF-β) promotes extracellular matrix deposition by down-regulating the expression of matrix degrading proteinases and upregulating their inhibitors. Tissue inhibitor of metalloproteinases (TIMP)-3 is an ECM-associated specific inhibitor of matrix degrading metalloproteinases. Here, we have characterized the signaling pathways mediating TGF-β-induced expression of TIMP-3. Basal and TGF-β-induced TIMP-3 mRNA expression was abolished in Smad4-deficient mouse embryonic fibroblasts and restoring Smad4 expression rescued the response. Inhibition of Smad signaling by expression of Smad7 and dominant negative Smad3 completely abolished TGF-β-elicited expression of TIMP-3 in human fibroblasts, whereas overexpression of Smad3 enhanced it. Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression. Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression. These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.

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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.
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pone-0057474-g004: 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.

Mentions: Smad signaling is regulated through crosstalk with other signaling cascades, e.g. MAPK pathways p38, ERK1/2, and JNK, and Cam kinase II [20]. TGF-β activates ERK1/2 and p38 MAPK pathways in gingival fibroblasts [21]. In addition, our previous observations demonstrate, that Smad3 co-operates with p38 MAPK in regulating the expression of MMP-13 [25], and with ERK1/2 in the TGF-β-elicited expression of connective tissue growth factor (CTGF) in human gingival fibroblasts [26]. Therefore, we first examined whether ERK1/2 and p38 MAPK pathways play a role in mediating the effect of TGF-β on TIMP-3 gene expression in human gingival fibroblasts. We used PD98059 (30 µM), an inhibitor for MEK1, the upstream activator of ERK1/2, and SB203580 (10 µM), a specific chemical inhibitor for p38 MAPK. Interestingly, PD98059 and SB203580 potently down-regulated TGF-β-induced TIMP-3 mRNA expression (Figure 4A), indicating that both p38 and ERK1/2 MAPKs are crucial for mediating the effects of TGF-β on TIMP-3 expression. Treatment with PD98059 suppressed also TGF-β-induced PAI-1 expression, whereas SB20358 had a weaker inhibitory effect. In comparison, PD98059 and SB203580 had modest effects on the TGF-β-induced levels of TIMP-1 (Figure 4A).


TGF-β-elicited induction of tissue inhibitor of metalloproteinases (TIMP)-3 expression in fibroblasts involves complex interplay between Smad3, p38α, and ERK1/2.

Leivonen SK, Lazaridis K, Decock J, Chantry A, Edwards DR, Kähäri VM - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585359&req=5

pone-0057474-g004: 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.
Mentions: Smad signaling is regulated through crosstalk with other signaling cascades, e.g. MAPK pathways p38, ERK1/2, and JNK, and Cam kinase II [20]. TGF-β activates ERK1/2 and p38 MAPK pathways in gingival fibroblasts [21]. In addition, our previous observations demonstrate, that Smad3 co-operates with p38 MAPK in regulating the expression of MMP-13 [25], and with ERK1/2 in the TGF-β-elicited expression of connective tissue growth factor (CTGF) in human gingival fibroblasts [26]. Therefore, we first examined whether ERK1/2 and p38 MAPK pathways play a role in mediating the effect of TGF-β on TIMP-3 gene expression in human gingival fibroblasts. We used PD98059 (30 µM), an inhibitor for MEK1, the upstream activator of ERK1/2, and SB203580 (10 µM), a specific chemical inhibitor for p38 MAPK. Interestingly, PD98059 and SB203580 potently down-regulated TGF-β-induced TIMP-3 mRNA expression (Figure 4A), indicating that both p38 and ERK1/2 MAPKs are crucial for mediating the effects of TGF-β on TIMP-3 expression. Treatment with PD98059 suppressed also TGF-β-induced PAI-1 expression, whereas SB20358 had a weaker inhibitory effect. In comparison, PD98059 and SB203580 had modest effects on the TGF-β-induced levels of TIMP-1 (Figure 4A).

Bottom Line: Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression.Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression.These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Turku, and Turku University Hospital, Turku, Finland.

ABSTRACT
Transforming growth factor-β (TGF-β) promotes extracellular matrix deposition by down-regulating the expression of matrix degrading proteinases and upregulating their inhibitors. Tissue inhibitor of metalloproteinases (TIMP)-3 is an ECM-associated specific inhibitor of matrix degrading metalloproteinases. Here, we have characterized the signaling pathways mediating TGF-β-induced expression of TIMP-3. Basal and TGF-β-induced TIMP-3 mRNA expression was abolished in Smad4-deficient mouse embryonic fibroblasts and restoring Smad4 expression rescued the response. Inhibition of Smad signaling by expression of Smad7 and dominant negative Smad3 completely abolished TGF-β-elicited expression of TIMP-3 in human fibroblasts, whereas overexpression of Smad3 enhanced it. Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression. Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression. These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.

Show MeSH
Related in: MedlinePlus