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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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Differential effects of TrkA on CTGF regulation of inflammatory gene expression. H9c2 cells were pretreated for 1 hr with K252a at 100 nM and 200 nM and then conditioned media with no treatment or CTGF (500 ng/ml) was added. The RNA was isolated at 24 h and mRNA levls of respective species was determine by RT-qPCR A, MCP-1 B, IL-8 C, TNF-α and D IL-6 mRNA. Data is mean±SD. *, P < 0.05, vs control respectively. #P < 0.05, ##P < 0.01, vs. CTGF treatment
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Fig4: Differential effects of TrkA on CTGF regulation of inflammatory gene expression. H9c2 cells were pretreated for 1 hr with K252a at 100 nM and 200 nM and then conditioned media with no treatment or CTGF (500 ng/ml) was added. The RNA was isolated at 24 h and mRNA levls of respective species was determine by RT-qPCR A, MCP-1 B, IL-8 C, TNF-α and D IL-6 mRNA. Data is mean±SD. *, P < 0.05, vs control respectively. #P < 0.05, ##P < 0.01, vs. CTGF treatment

Mentions: The ability of k252a to regulate other effects induced by rhCTGF was examined. As shown in Table 4, FN, Col-I and Col-III induction by CTGF treatment was found to be blocked by k252a. Similar profiles of results were obtained for TIMP-1 and TIMP-2 , while PAI-1 showed very strong inhibitory effects with k252a treatment with or without rhCTGF. For the pro- inflammatory cytokine gene expression studied, as shown in Fig. 4, two differential profiles were observed: MCP-1 and IL-8 showed the same trends as for the fibrotic markers (Fig. 4a–b ); in contrast, for TNF-α and IL-6 the k252a reagent added alone showed an increased trend compared with control, and ∼ additive effects with CTGF and k252a combined compared with CTGF addition alone (Fig. 4c–d). This data for TNF-α and IL-6 suggests that TrkA activity has a tonic inhibitory effect on steady state TNF-α and IL-6 and that upregulatory effects of rhCTGF on TNF-α and IL-6 (Fig. 1c) are not TrkA mediated and they persist when TrkA activity is blocked.Table 4


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)

Differential effects of TrkA on CTGF regulation of inflammatory gene expression. H9c2 cells were pretreated for 1 hr with K252a at 100 nM and 200 nM and then conditioned media with no treatment or CTGF (500 ng/ml) was added. The RNA was isolated at 24 h and mRNA levls of respective species was determine by RT-qPCR A, MCP-1 B, IL-8 C, TNF-α and D IL-6 mRNA. Data is mean±SD. *, P < 0.05, vs control respectively. #P < 0.05, ##P < 0.01, vs. CTGF treatment
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Related In: Results  -  Collection

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Fig4: Differential effects of TrkA on CTGF regulation of inflammatory gene expression. H9c2 cells were pretreated for 1 hr with K252a at 100 nM and 200 nM and then conditioned media with no treatment or CTGF (500 ng/ml) was added. The RNA was isolated at 24 h and mRNA levls of respective species was determine by RT-qPCR A, MCP-1 B, IL-8 C, TNF-α and D IL-6 mRNA. Data is mean±SD. *, P < 0.05, vs control respectively. #P < 0.05, ##P < 0.01, vs. CTGF treatment
Mentions: The ability of k252a to regulate other effects induced by rhCTGF was examined. As shown in Table 4, FN, Col-I and Col-III induction by CTGF treatment was found to be blocked by k252a. Similar profiles of results were obtained for TIMP-1 and TIMP-2 , while PAI-1 showed very strong inhibitory effects with k252a treatment with or without rhCTGF. For the pro- inflammatory cytokine gene expression studied, as shown in Fig. 4, two differential profiles were observed: MCP-1 and IL-8 showed the same trends as for the fibrotic markers (Fig. 4a–b ); in contrast, for TNF-α and IL-6 the k252a reagent added alone showed an increased trend compared with control, and ∼ additive effects with CTGF and k252a combined compared with CTGF addition alone (Fig. 4c–d). This data for TNF-α and IL-6 suggests that TrkA activity has a tonic inhibitory effect on steady state TNF-α and IL-6 and that upregulatory effects of rhCTGF on TNF-α and IL-6 (Fig. 1c) are not TrkA mediated and they persist when TrkA activity is blocked.Table 4

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