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Originally published In Press as doi:10.1167/iovs.08-3276 on March 25, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:3662-3670.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-3276
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Rho-Mediated Regulation of TGF-β1– and FGF-2–Induced Activation of Corneal Stromal Keratocytes

Jian Chen,1 Emily Guerriero,1 Yoshikazu Sado,2 and Nirmala SundarRaj1

1From the UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and the 2Division of Immunology, Shigei Medical Research Institute, Okayama, Japan.

PURPOSE. To investigate the role of Rho GTPase signaling in FGF-2– and TGF-β1–induced activation of corneal keratocytes.

METHODS. Keratocytes isolated from rabbit corneal stroma and plated in a serum-free medium were treated with FGF-2/heparin or TGF-β1 in the presence or absence of Rho inhibitor (C3 exoenzyme) or ROCK (Rho kinase) inhibitor (Y27632). Specific phenotypic changes were analyzed by immunocytochemistry and Western blot analysis, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR.

RESULTS. TGF-β1–induced expression of {alpha}-SMA and transcription of {alpha}-SMA mRNA in activated keratocytes were reduced by Rho or ROCK inhibition during the activation. In nonactivated keratocytes, the expression of {alpha}3(IV) collagen was downregulated by Rho-inhibition. TGF-β1– or FGF-2–induced downregulation of the expression of {alpha}3(IV) collagen and its mRNA was not significantly altered by Rho or ROCK inhibition. TGF-β1– and FGF-2–induced decreases in cell-associated and secreted KS, and lumican mRNA levels were prevented by Rho or ROCK inhibition. However, FGF-2–induced decreases in keratocan mRNA levels were prevented by Rho inhibition but not by ROCK inhibition. Whereas Rho inhibition downregulated both TGF-β1– and FGF-2–induced tenascin-C expression, ROCK inhibition was found to downregulate only TGF-β1–induced expression.

CONCLUSIONS. Rho signaling has a significant role in the activation of keratocytes. Rho, via ROCK-independent and/or -dependent pathways differentially regulates the TGF-β1–induced expression of {alpha}-SMA and TGF-β1– and FGF-2–induced de novo expression of tenascin-C and the downregulation of {alpha}3(IV) collagen and KSPGs, lumican and keratocan.






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