title: Rho-mediated regulation of TGF-beta1- and FGF-2-induced activation of corneal stromal keratocytes.
abstract: OBJECTIVE
  To investigate the role of Rho GTPase signaling in FGF-2- and TGF-beta1-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-beta1 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-beta1-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 alpha3(IV) collagen was downregulated by Rho-inhibition. TGF-beta1- or FGF-2-induced downregulation of the expression of alpha3(IV) collagen and its mRNA was not significantly altered by Rho or ROCK inhibition. TGF-beta1- 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-beta1- and FGF-2-induced tenascin-C expression, ROCK inhibition was found to downregulate only TGF-beta1-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-beta1-induced expression of alpha-SMA and TGF-beta1- and FGF-2-induced de novo expression of tenascin-C and the downregulation of alpha3(IV) collagen and KSPGs, lumican and keratocan.
authors.affiliation: UPMC Eye Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Emily
authors.initials: E
authors.lastname: Guerriero
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Sado
authors.forename: Nirmala
authors.initials: N
authors.lastname: SundarRaj