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James V. Jester, Jiying Huang, Patricia A. Barry–Lane, Winston W-Y. Kao, W. Matthew Petroll, H. Dwight Cavanagh; Transforming Growth Factorβ–Mediated Corneal Myofibroblast Differentiation Requires Actin and Fibronectin Assembly. Invest. Ophthalmol. Vis. Sci. 1999;40(9):1959-1967.
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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 TGFβ 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 regulatingα
-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 TGFβ (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 TGFβ 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, andα
-SMA protein and mRNA. Immunoprecipitation of cell proteins with
4G10 or PY20 identified the TGFβ-associated tyrosine
phosphorylation of paxillin, pp125fak, p130, PLCγ, and
tensin, which was blocked by addition of GRGDdSP. Addition of
herbimycin A to keratocytes exposed to TGFβ showed a
dose-dependent loss of α-SMA protein and mRNA which correlated with
loss of tyrosine phosphorylation, absence of actin reorganization, and
conclusions. The data suggest that TGFβ-mediated α-SMA gene
expression leading to myofibroblast transformation may involve an
RGD-dependent phosphotyrosine signal transduction
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