Synergistic short-term and long-term effects of TGF-β1 and 3 on collagen production in differentiating myoblasts
Andi Shi, Michèle MG Hillege, Rob C. I. Wüst, Gang Wu, Richard T. Jaspers
Abstract
Skeletal muscle fibrosis and regeneration are modulated by transforming growth factor β (TGF-β) superfamily. Amongst them, TGF-β1 is a highly potent pro-fibrotic factor, while TGF-β3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. However, little is known about the individual and combined short- and long-term effects of TGF-β1 and TGF-β3 on collagen expression in myoblasts and myotubes. Here we show that in C2C12 myoblasts TGF-β1 and/or TGF-β3 increased mRNA expression of Ctgf and Fgf-2 persistently after 3 h and of Col1A1 after 24 h, while TGF-β1+TGF-β3 mitigated these effects after 48 h incubation. Gene expression of Tgf-β1 was enhanced by TGF-β1 and/or TGF-β3 after 24 h and 48 h. However, Tgfbr1 mRNA expression was reduced at 48 h. After 48 h incubation with TGF-β1 and/or TGF-β3, Col3A1 and Col4A1 mRNA expression levels were decreased. Myoblasts produced collagen after three days incubation with TGF-β1 and/or TGF-β3 in a dose independent manner. Collagen deposition was doubled when myoblasts differentiated into myotubes and TGF-β1 and/or TGF-β3 did not stimulate collagen production any further. TGF-β type I receptor (TGFBR1) inhibitor, LY364947, suppressed TGF-βs-induced collagen production. Collagen I expression was higher in myotubes than in myoblasts. TGF-β1 and/or TGF-β3 inhibited myotube differentiation which was antagonized by LY364947. These results indicate that both C2C12 myoblasts and myotubes produce collagen. Whereas TGF-β1 and TGF-β3 individually and simultaneously stimulate collagen production in C2C12 differentiating myoblasts, in myotubes these effects are less prominent. In muscle cells, TGF-β3 is ineffective to antagonize TGF-β1-induced collagen production.