Recent studies of the inhibitory effects of ROD peptides on TGFβ induced keratocyte transformation and a-smooth muscle-specific actin (a-SM) expression suggest the presence of an outside-in signal transduction mechanism involving interactions between fibronectin (FN) and the FN receptors, a5βl integrin. The purpose of this study was to identify the role of integnn-dependent tyrosine phosphorylation in regulating a-SM gene expression and ultimately myofibroblast development. Methods: Since cell culture in serum containing media mimics myofibroblast transformation, all experiments were carried out on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGFR (1 ng/ml), GRGDdSP (50 uM), GRADSP (100 uM) or herbimycin A (0.1 to 10 nM) at 24 hr (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry, and proteins and mRNA collected for western and northern blotting using antibodies specific for a-SM actin, FN, focal adhesion proteins and phosphotyrosine (clones 4G10 and PY20); and probes specific for a 29 bp sequence of the 3 untranslated region of the rabbit a-SM, rabbit collagen a 1(1) chain (ARC 35), βactin, and G3PDH. All experiments were repeated at least twice. Results: Keratocytes exposed to TGFR for 72 hours showed de novo or enhanced expression of a-SM actin. FN, a-SM mRNA and collagen aim mRNA. Addition of GRGDdSP but not GRADSP (control) blocked expression of a-SM protein and mRNA but had no effect on TGFn enhanced collagen al(l) mRNA synthesis or FN expression. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGFR associated tyrosine phosphorylation ofpaxillin, pp!25FAK, p!30 PLCy and tensin which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGFR showed a dose dependent loss of a-SM actin protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of stress fibers and focal contacts but no effect on collagen a 1(1) Conclusions: The data indicate that TGFβ mediated a-SM gene expression leading to myofibroblast transformation is uniquely regulated by an outside-in phosphotyrosine signaling cascade most-likely associated with interactions between extracefluar FN and a5Sl inteorin.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - 1997|
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