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Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes.

Wang X, McLennan SV, Allen TJ, Twigg SM - J Cell Commun Signal (2010)

Bottom Line: Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced.The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-beta neutralizing antibody and Alk 5 inhibitor (SB431542).Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-beta pathway signalling and partly through TrkA.

View Article: PubMed Central - PubMed

ABSTRACT
Connective tissue growth factor (CTGF), also known as CCN2, is implicated in fibrosis through both extracellular matrix (ECM) induction and inhibition of ECM degradation. The role of CTGF in inflammation in cardiomyocytes is unknown. In some mesenchymal cell systems, CTGF mediates effects through TGF-beta or tyrosine kinase cell surface receptor, TrkA, signalling. In this study, cellular mechanisms by which CTGF regulates pathways involved in fibrosis and inflammation were explored. Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced. CTGF treatment also increased pro-inflammatory cytokines TNF-alpha, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-beta1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-beta neutralizing antibody and Alk 5 inhibitor (SB431542). A specific blocker of TrkA activation, k252a, also abrogated CTGF-induced effects on fibrosis and gene expresison of MCP-1 and IL-8, but not TNF-alpha or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-beta pathway signalling and partly through TrkA.

No MeSH data available.


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CTGF phosphorylation of TrkA and upregulation of TGF-β1 mRNA. A, Addition of rhCTGF to H9c2 cells induced rapid phosphorylation of TrkA but did not increase total TrkA. B, rhCTGF application to cells induced TGF-β1 mRNA, which was inhibited by anti-TGF-β1 antibody or k252a. Data is mean±SD. *, P < 0.05, vs control. #P < 0.05, ##P < 0.01 vs CTGF treatment
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Fig3: CTGF phosphorylation of TrkA and upregulation of TGF-β1 mRNA. A, Addition of rhCTGF to H9c2 cells induced rapid phosphorylation of TrkA but did not increase total TrkA. B, rhCTGF application to cells induced TGF-β1 mRNA, which was inhibited by anti-TGF-β1 antibody or k252a. Data is mean±SD. *, P < 0.05, vs control. #P < 0.05, ##P < 0.01 vs CTGF treatment

Mentions: To confirm whether CTGF signals through TrkA, the H9c2 cells were treated with rhCTGF for up to 120 min and cell lysate was isolated and probed by Western immunoblot. Phosphorylation of TrkA was induced by added CTGF from 15 min (Fig. 3a). In contrast total TrkA was unchanged. These findings are consistent with those that we recently reported, in a less detailed time course (McLennan et al. 2004). As TGF-β1 gene expression is induced by TGF-β pathway activation, subsequent studies focussed on TGF-β1 mRNA as a read out at 24 h. These experiments showed that rhCTGF induced TGF-β1 mRNA levels, which was blocked by the TrkA activity inhibitor, k252a and also by anti-TGF-β antibody (Fig. 3b). Collectively, this data suggests that CTGF rapidly signals through TrkA activation and then through TGF-β protein dependent processes in H9c2 cells.Fig. 3


Regulation of pro-inflammatory and pro-fibrotic factors by CCN2/CTGF in H9c2 cardiomyocytes.

Wang X, McLennan SV, Allen TJ, Twigg SM - J Cell Commun Signal (2010)

CTGF phosphorylation of TrkA and upregulation of TGF-β1 mRNA. A, Addition of rhCTGF to H9c2 cells induced rapid phosphorylation of TrkA but did not increase total TrkA. B, rhCTGF application to cells induced TGF-β1 mRNA, which was inhibited by anti-TGF-β1 antibody or k252a. Data is mean±SD. *, P < 0.05, vs control. #P < 0.05, ##P < 0.01 vs CTGF treatment
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Related In: Results  -  Collection

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Fig3: CTGF phosphorylation of TrkA and upregulation of TGF-β1 mRNA. A, Addition of rhCTGF to H9c2 cells induced rapid phosphorylation of TrkA but did not increase total TrkA. B, rhCTGF application to cells induced TGF-β1 mRNA, which was inhibited by anti-TGF-β1 antibody or k252a. Data is mean±SD. *, P < 0.05, vs control. #P < 0.05, ##P < 0.01 vs CTGF treatment
Mentions: To confirm whether CTGF signals through TrkA, the H9c2 cells were treated with rhCTGF for up to 120 min and cell lysate was isolated and probed by Western immunoblot. Phosphorylation of TrkA was induced by added CTGF from 15 min (Fig. 3a). In contrast total TrkA was unchanged. These findings are consistent with those that we recently reported, in a less detailed time course (McLennan et al. 2004). As TGF-β1 gene expression is induced by TGF-β pathway activation, subsequent studies focussed on TGF-β1 mRNA as a read out at 24 h. These experiments showed that rhCTGF induced TGF-β1 mRNA levels, which was blocked by the TrkA activity inhibitor, k252a and also by anti-TGF-β antibody (Fig. 3b). Collectively, this data suggests that CTGF rapidly signals through TrkA activation and then through TGF-β protein dependent processes in H9c2 cells.Fig. 3

Bottom Line: Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced.The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-beta neutralizing antibody and Alk 5 inhibitor (SB431542).Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-beta pathway signalling and partly through TrkA.

View Article: PubMed Central - PubMed

ABSTRACT
Connective tissue growth factor (CTGF), also known as CCN2, is implicated in fibrosis through both extracellular matrix (ECM) induction and inhibition of ECM degradation. The role of CTGF in inflammation in cardiomyocytes is unknown. In some mesenchymal cell systems, CTGF mediates effects through TGF-beta or tyrosine kinase cell surface receptor, TrkA, signalling. In this study, cellular mechanisms by which CTGF regulates pathways involved in fibrosis and inflammation were explored. Murine H9c2 cardiomyocytes were treated with recombinant human (rh)CTGF and ECM formation gene expression: fibronectin, collagen type -I and -III and ECM degradation genes: TIMP-1, TIMP-2 and PAI-1 were found to be induced. CTGF treatment also increased pro-inflammatory cytokines TNF-alpha, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-beta1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF-beta neutralizing antibody and Alk 5 inhibitor (SB431542). A specific blocker of TrkA activation, k252a, also abrogated CTGF-induced effects on fibrosis and gene expresison of MCP-1 and IL-8, but not TNF-alpha or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF-beta pathway signalling and partly through TrkA.

No MeSH data available.


Related in: MedlinePlus