Induction of alpha-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes

Abstract

The effects of serum, transforming growth factor (TGF) beta 1, bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myofibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.