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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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Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4  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).
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pone-0057474-g002: Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4 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).

Mentions: To further elucidate the role of Smad signaling in TGF-β-induced TIMP-3 gene expression, we analyzed whether exogenous expression of Smad4 could rescue the TGF-β response of TIMP-3 expression in Smad4-deficient MEFs. We utilized adenoviral gene delivery of recombinant adenovirus expressing HA-tagged Smad4 (RAdSmad4). EF7WT and EF7KO cells were infected with an empty control virus RAd66 or RAdSmad4 and incubated for 36 h. Thereafter, the cells were treated with TGF-β1 for different periods of time up to 24 h, and the RNAs were subjected to qRT-PCR analysis. As shown in Figure 2A, uninfected or RAd66 infected EF7KO cells expressed no Smad4, as compared to EF7WT control cells. However, Smad4 expression was restored in RAdSmad4 infected EF7KO cells, as detected with Smad4 antibody or with HA-antibody, which detects only adenovirus-produced HA-Smad4 (Figure 2A). TGF-β was unable to induce TIMP-3 or PAI-1 expression in EF7KO cells transduced with control virus RAd66, whereas restoring Smad4 expression resulted in marked and significant upregulation of TIMP-3 expression in response to TGF-β (Figure 2B). Together, these results support the observation that TGF-β-induced TIMP-3 and PAI-1 gene expression is Smad-dependent.


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)

Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4  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).
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pone-0057474-g002: Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4 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).
Mentions: To further elucidate the role of Smad signaling in TGF-β-induced TIMP-3 gene expression, we analyzed whether exogenous expression of Smad4 could rescue the TGF-β response of TIMP-3 expression in Smad4-deficient MEFs. We utilized adenoviral gene delivery of recombinant adenovirus expressing HA-tagged Smad4 (RAdSmad4). EF7WT and EF7KO cells were infected with an empty control virus RAd66 or RAdSmad4 and incubated for 36 h. Thereafter, the cells were treated with TGF-β1 for different periods of time up to 24 h, and the RNAs were subjected to qRT-PCR analysis. As shown in Figure 2A, uninfected or RAd66 infected EF7KO cells expressed no Smad4, as compared to EF7WT control cells. However, Smad4 expression was restored in RAdSmad4 infected EF7KO cells, as detected with Smad4 antibody or with HA-antibody, which detects only adenovirus-produced HA-Smad4 (Figure 2A). TGF-β was unable to induce TIMP-3 or PAI-1 expression in EF7KO cells transduced with control virus RAd66, whereas restoring Smad4 expression resulted in marked and significant upregulation of TIMP-3 expression in response to TGF-β (Figure 2B). Together, these results support the observation that TGF-β-induced TIMP-3 and PAI-1 gene expression is Smad-dependent.

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