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

Xiaoyu Wang 0 Susan V. McLennan 0 Terri J. Allen 0 Stephen M. Twigg 0 0 T. J. Allen Baker Heart and Diabetes Research Institute , Melbourne, Australia 1 ) Discipline of Medicine, The University of Sydney , Sydney, Australia 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- 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 proinflammatory cytokines TNF-, IL-6, MCP-1 and IL-8. CTGF upregulated TGF-1 mRNA and rapidly induced phosphorylation of TrkA. The CTGF-induced pro-fibrotic and pro-inflammatory effects were blocked by anti-TGF- 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- or IL-6. Collectively, this data implicates CTGF in effects on pro-fibrotic genes and pro-inflammatory genes via TGF- pathway signalling and partly through TrkA. - The predominant cell type in the heart, the cardiomyocyte, is implicated in the pathologies of both fibrosis and inflammation that can occur in the myocardium. For example, the heart disease of diabetic cardiomyopathy (DCM) (Fang et al. 2004) involves inflammatory (Westermann et al. 2007; Wang & Cai 2006) and fibrotic changes (Samuel et al. 2008) including effects mediated by cardiomyocytes (Wang and Cai 2006; Wang et al. 2009). Pro-fibrotic factors such as collagen-I, collagen-III and fibronectin (Wang et al. 2009) and pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumour necrosis factor- (TNF-), are increased in DCM (Westermann et al. 2006). However the cellular mechanisms mediating these changes are still not completely understood. Also known as CCN2, the protein CTGF is considered to be a mediator involved in fibrotic processes (Leask and Abraham 2004) and it stimulates fibroblast growth and ECM protein up-regulation in vitro and in vivo. CTGF over-production has been proposed to play a major role in pathways that lead to fibrosis in many organs (Frazier et al. 1996; Igarashi et al. 1993). Increased CTGF expression has been described in diabetes including DCM (Wang et al. 2009; Way et al. 2002) and post-myocardial infarct (Dean et al. 2005). CTGF has been shown to induce the inhibitors of matrix metalloproteinases known as TIMPS, leading to ECM accumulation in renal cells (McLennan et al. 2004). Cellular mechanisms exploring how CTGF regulates ECM changes in cardiomyocytes remain minimally explored and whether CTGF has any effects on inflammatory processes in cardiomyocytes has not been reported. In some mesenchymal cell systems, CTGF appears to function by modulating bioactivity of other cytokines, especially TGF- (Abreu et al. 2002), indicating that TGF- protein is required for CTGF to exert its effect. In contrast, in a renal based cell system, CTGF was reported to function through the cell surface receptor, TrkA (tyrosine kinase A), leading to activation of TGF- inducible early gene (TIEG), and TGF- pathway signalling (Wahab et al. 2005). Thus in some cells both TrkA and TGF- based pathways have been implicated in CTGF effects. With this background, the aims of this study in cardiomyocytes were to test whether (a) CTGF regulates pro-fibrotic and pro-inflammatory gene expression in cardiomyocytes; (b) whether CTGF effects on fibrosis and inflammation are TGF- dependent; and/or through the cell surface receptor TrkA in cardiomyocytes. Materials and methods Recombinant human CTGF (rhCTGF) protein was produced using an adenoviral expression system, purified with heparin-Sepharose affinity chromatography using HiTrap Heparin HP columns (Amersham Biosciences, Piscataway, NJ, USA) and quantified as previously described (Tan et al. 2008). A chemical inhibitor of TrkA activity, k252a, purchased from Merck-Calbiochem, Melbourne, Australia was prepared by dissolving in DMSO, with final DMSO concentration in cell culture at 0.001%. Pan anti-TGF- neutralizing antibody was purchased from R&D systems Inc (Minneapolis, USA) and Alk 5 inhibitor (SB 431542) was purchased from Tocris Cookson Inc, Ellisville, Missouri USA. Rat nerve growth factor (NGF) was purchased from Sigma (St. Louis, Mo, USA). Antibodies for detection of phospho-TrkA (Tyr490) and total TrkA were purchased from Cell Signaling Technology, Danvers, MA, USA. The -tubulin antibody was purchased from Abcam Inc (Cambridge, MA, USA). Cell treatment The well studied murine H9c2 cells derived from embryonic rat heart myocardium were purchased from ATCC (Rockville, MD, USA). Cells were cultured in DMEM containing normal 5.5 mM glucose, 4.4 mM L-glutamine and 1.7 g/L bicarbonate supplemented with 10% FBS, 50 U/ml of penicillin and 50 g/ml of streptomycin. Each experiment was performed at least three times independently with in each experiment, triplicate wells of treatments. Cells were grown in 6-well plates until 80% confluent and then treated with reagents in serum-free fresh DMEM media containing 0.1% BSA (Sigma, St Louis, MO, USA). Cells were harvested at 24 h. Treatments included control media, rhCTGF, k252a (TrkA chemical blocker), Pan antiTGF- neutralizing antibody, SB 431542 (Alk 5 inhibitor) and combination treatments of rhCTGF with k252a or TGF- antibody or SB 431542. In the case of k252a or SB 431542 the highest relevant concentration of DMSO was used as control. DMSO alone was shown in preliminary studies not to affect mRNA levels measured compared with no DMSO addition (not shown). Total RNA isolation and analysis by quantitative real-time PCR Total RNA of each cell treatment was isolated from cells using the RNeasy Mini kit (Qiagen). RNA amount was quantitated spectrophometrically (Bio-Rad Laboratories Inc, CA, USA), and the purity was determined from the A260/A280 ratio (at or above 1.80, to 2.00). RNA was then reverse transcribed to cDNA using Random Hexamer Primer (Invitrogen, CA, USA) and SuperScript III Reverse Transcriptase (Invitrogen). The resulting cDNA was analyzed by quantitative realtime PCR using Rotor Gene 6000 (Corbett Research), using SYBR green as fluorescence dye, as described previously (Tan et al. 2008). Relative quantitation of mRNA expression in the gene of interest was calculated using the comparative threshold cycle number for each sample. In each case, the gene of interest expression level was normalized to 18s ribosomal RNA and related to the releva (...truncated)