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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 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.
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pone-0057474-g003: 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.

Mentions: As the results above with Smad4 deficient fibroblasts demonstrate that the TGF-β-induced expression of TIMP-3 is Smad-dependent in mouse fibroblasts, we studied this in detail also in human fibroblasts. Human gingival fibroblasts were transduced with recombinant adenoviruses for Smad2, Smad3, dominant negative Smad3 (RAdSmad2, RAdSmad3, and RAdSmad3DN, respectively), and with empty control adenovirus RAd66, and incubated for 18 h. Thereafter, the cells were treated with TGF-β for 24 h, as indicated. As shown in Figure 3A, a 24 h TGF-β stimulation of control virus RAd66 transduced cells resulted in the induction of TIMP-3 mRNA expression, as compared to untreated control cells. Interestingly, adenoviral overexpression of Smad3 markedly enhanced the TGF-β-elicited expression of TIMP-3 mRNAs (Figure 3A), whereas overexpression of Smad2 had no effect on the TGF-β-induced levels of TIMP-3 mRNA. In addition, adenoviral delivery of Smad3DN potently inhibited the up-regulatory effect of TGF-β on TIMP-3 expression (Figure 3A).


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585359&req=5

pone-0057474-g003: 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.
Mentions: As the results above with Smad4 deficient fibroblasts demonstrate that the TGF-β-induced expression of TIMP-3 is Smad-dependent in mouse fibroblasts, we studied this in detail also in human fibroblasts. Human gingival fibroblasts were transduced with recombinant adenoviruses for Smad2, Smad3, dominant negative Smad3 (RAdSmad2, RAdSmad3, and RAdSmad3DN, respectively), and with empty control adenovirus RAd66, and incubated for 18 h. Thereafter, the cells were treated with TGF-β for 24 h, as indicated. As shown in Figure 3A, a 24 h TGF-β stimulation of control virus RAd66 transduced cells resulted in the induction of TIMP-3 mRNA expression, as compared to untreated control cells. Interestingly, adenoviral overexpression of Smad3 markedly enhanced the TGF-β-elicited expression of TIMP-3 mRNAs (Figure 3A), whereas overexpression of Smad2 had no effect on the TGF-β-induced levels of TIMP-3 mRNA. In addition, adenoviral delivery of Smad3DN potently inhibited the up-regulatory effect of TGF-β on TIMP-3 expression (Figure 3A).

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