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-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF-β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF-β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF-β signaling in HL-1 cells treated with and without (control) type I collagen (10 μg/ml), which was co-administered with either an anti-β1 integrin antibody (10 μg/ml) or a stretch-activated channel inhibitor (gadolinium; 50 μM). 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 μg/ml)-treated HL-1 cells were incubated with or without anti-β1 integrin antibody (10 μg/ml), gadolinium (50 μM) or inhibitors of p38 (SB203580; 3 μM), extracellular signal-regulated kinase (ERK; PD98059; 50 μM) and c-Jun N-terminal kinase (JNK; SP600125; 50 μM). Compared with the control cells, the collagen-treated HL-1 cells had lower expression levels of type I and type II TGF-β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti-β1 integrin antibody reversed the collagen-induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI 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-β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII 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-β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin- or stretch-dependent and -independent signaling pathways.