Transforming growth factor(β)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly

Purpose. Recent studies indicate that transforming growth factor (TGF)(β) is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, α-actin (α-SMA). Although TGF(β) is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating α-SMA gene expression and ultimately myofibroblast development. Methods. Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGF(β) (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 μM), Gly-Arg-AL- Asp-Ser-Pro (GRADSP; 100 μM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for α-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit α-SMA. All experiments were repeated at least three times. Results. Keratocytes exposed to TGF(β) showed expression of α-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and α-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGF(β)-associated tyrosine phosphorylation of paxillin, pp125(tak), p130, PLCγ, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGF(β) showed a dose-dependent loss of α-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. Conclusions. The data suggest that TGF(β)-mediated α-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.