Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling

The extracellular matrix (ECM) and transforming growth factor- (TGF)- are important in cardiac fibrosis, however, the effects of the ECM on TGF- signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF- signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF- signaling in HL-1 cells treated with and without (control) type I collagen (10 mu g/ml), which was co-administered with either an anti-1 integrin antibody (10 mu g/ml) or a stretch-activated channel inhibitor (gadolinium; 50 mu M). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen-activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 mu g/ml)-treated HL-1 cells were incubated with or without anti-1 integrin antibody (10 mu g/ml), gadolinium (50 mu M) or inhibitors of p38 (SB203580; 3 mu M), extracellular signal-regulated kinase (ERK; PD98059; 50 mu M) and c-Jun N-terminal kinase (JNK; SP600125; 50 mu M). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF- receptors (TGFRI and TGFRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFRII and phosphorylated FAK. Gadolinium down-regulated the expression levels of TGFRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen-induced activation of p38 and ERK1/2. In the presence of gadolinium and anti-1 integrin antibody, collagen regulated the expression levels of TGFRI, TGFRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen-induced cell proliferation was inhibited by the anti-1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFRI and TGFRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.