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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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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.
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pone-0057474-g001: 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.

Mentions: TGF-β has been previously shown to induce TIMP-1 and TIMP-3 gene expression in fibroblasts [12]. To confirm that TIMP-3 is a TGF-β responsive gene in human gingival fibroblasts, the cells were first treated with TGF-β1 for 24 h and analyzed for the expression of TIMP-3 mRNAs by Northern blot analysis. The TGF-β1 concentration used was 5 ng/ml, which has been shown to give maximal stimulation in fibroblasts [21], [24]–[26], [39], [40]. In addition, previous studies have shown that the response of fibroblasts to TGF-β1 in this concentration is comparable to TGF-β2 and TGF-β3 [40]–[42]. As shown in Figure 1A, TGF-β stimulation resulted in marked induction of TIMP-3, TIMP-1, and PAI-1 mRNA expression, as compared to untreated control cells. To study the Smad-dependence of the TGF-β-induced expression of TIMP-3, Smad4-deficient mouse embryonic fibroblasts, (EF7KO), and the corresponding wild type MEFs (EF7WT), were treated with TGF-β for 3 h and 12 h, as indicated. Thereafter, the expression of TIMP-3 and PAI-1 mRNAs were analyzed by qRT-PCR. As shown in Figure 1B, TGF-β stimulation of EF7WT fibroblasts resulted in potent induction of TIMP-3 and PAI-1 mRNA expression. However, in EF7KO cells the basal and TGF-β-induced expression of TIMP-3 mRNA was abolished, indicating that the Smad signaling pathway is essential for the expression of TIMP-3. In addition, the TGF-β-induced expression of PAI-1 mRNA was reduced, as compared to EF7WT control cells (Figure 1B). This is in accordance with previous observations showing that PAI-1 is a Smad-responsive gene [43], [44]. However, the TGF-β-induced PAI-1 mRNA expression was not completely abolished in EF7KO cells, indicating that other signaling pathways also participate in TGF-β induction of PAI-1 expression.


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)

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.
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pone-0057474-g001: 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.
Mentions: TGF-β has been previously shown to induce TIMP-1 and TIMP-3 gene expression in fibroblasts [12]. To confirm that TIMP-3 is a TGF-β responsive gene in human gingival fibroblasts, the cells were first treated with TGF-β1 for 24 h and analyzed for the expression of TIMP-3 mRNAs by Northern blot analysis. The TGF-β1 concentration used was 5 ng/ml, which has been shown to give maximal stimulation in fibroblasts [21], [24]–[26], [39], [40]. In addition, previous studies have shown that the response of fibroblasts to TGF-β1 in this concentration is comparable to TGF-β2 and TGF-β3 [40]–[42]. As shown in Figure 1A, TGF-β stimulation resulted in marked induction of TIMP-3, TIMP-1, and PAI-1 mRNA expression, as compared to untreated control cells. To study the Smad-dependence of the TGF-β-induced expression of TIMP-3, Smad4-deficient mouse embryonic fibroblasts, (EF7KO), and the corresponding wild type MEFs (EF7WT), were treated with TGF-β for 3 h and 12 h, as indicated. Thereafter, the expression of TIMP-3 and PAI-1 mRNAs were analyzed by qRT-PCR. As shown in Figure 1B, TGF-β stimulation of EF7WT fibroblasts resulted in potent induction of TIMP-3 and PAI-1 mRNA expression. However, in EF7KO cells the basal and TGF-β-induced expression of TIMP-3 mRNA was abolished, indicating that the Smad signaling pathway is essential for the expression of TIMP-3. In addition, the TGF-β-induced expression of PAI-1 mRNA was reduced, as compared to EF7WT control cells (Figure 1B). This is in accordance with previous observations showing that PAI-1 is a Smad-responsive gene [43], [44]. However, the TGF-β-induced PAI-1 mRNA expression was not completely abolished in EF7KO cells, indicating that other signaling pathways also participate in TGF-β induction of PAI-1 expression.

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