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N. Sundarraj, E. Guerriero, J. Chen; Rho/ROCK Signaling in Regulation of TGFß1 Induced Phenotypic Changes Associated With Differentiation of Keratocytes to Myofibroblasts . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2322.
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Rho/ROCK signaling is involved in TGFß1 induced expression of α–smooth muscle actin (SMA) and assembly of actomyosin filament network in myofibroblasts. The purpose of the present studies was to determine whether Rho/ROCK signaling is involved in other phenotypic changes associated with keratocyte to myofibroblast differentiation.
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum free (SF) medium. They were treated with TGFß1 (1ng/ml) in the presence or absence of the ROCK inhibitor (Y27632). The RNA extracted from the cells was used for quantitative RT–PCR analyses of mRNAs encoding αSMA, ALDH1, lumican and keratocan and α3 (IV) collagen using SYBR Green RT–PCR reagents.
TGFß1 treatment of rabbit corneal keratocytes induced cell spreading and assembly of stress fibers and transcription of α–SMA mRNA and markedly decreased the expression of ALDH1, lumican, keratocan and α3(IV) collagen. Inhibition of ROCK activity with Y27632 in keratocytes did not affect the levels ALDH1, lumican, keratocan mRNA but induced a 60–70% decrease in α3(IV) collagen mRNA. Treatment of keratocytes with Y27632 for six hours prior to and for 48 hours during TGFß1 treatment resulted in an 80% decrease in the αSMA mRNA levels. However, ROCK inhibition did not prevent decreases in the expression of ALDHI, lumican, keratocan and α3(IV) collagen promoted by TGFß1.
The Rho/ROCK signaling pathway is involved in expression of α3(IV) collagen, and TGFß1 induced αSMA expression and actomyosin filament assembly. However, it does not regulate TGFß1 mediated decreases in the expression of KSPGs, ALDH1 or α3(IV) collagen. Thus, signaling pathway(s) for TGFß1 mediated decreases in the expression of these proteins is regulated independently of actomyosin filament assembly in myofibroblasts.
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