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N. Sundarraj, T. Leung, S. Anderson, L. DiCesare; Rho-Mediated Assembly of Stress Fibers is Differentially Regulated in Corneal Fibroblasts and Myofibroblasts . Invest. Ophthalmol. Vis. Sci. 2003;44(13):876.
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Purpose: During corneal stromal wound healing,the keratocytes are activated to fibroblasts and myofibroblasts. Myofibroblast transdifferentiation is accompanied by expression of α-smooth muscle actin (α-SMA), and modifications of focal adhesions (FAs). The assembly of FAs and stress fibers is regulated by signaling through the small GTPase Rho. In the present study, we evaluated the mechanisms of Rho signaling in the formation of stress fibers in corneal fibroblasts and myofibroblasts in vitro. Methods: Rabbit corneal stromal cells in culture were grown in a media containing bFGF2 and heparin, or TGFß1 to induce fibroblast or myofibroblast phenotypes, respectively. Rho activity in the cells was inhibited by microinjection of C3 transferase. Activities of the Rho effectors, ROK (Rho-associated kinase) and mDia1, were inhibited by microinjection of the cDNA constructs encoding dominant negative forms of these effectors. Double immunofluorescence technique was employed to identify the injected cells, the stress fiber organization and the distribution of specific proteins. Results: Inhibition of Rho activity with C3 exoenzyme resulted in the disassembly of stress fibers in both fibroblasts and myofibroblasts. Inhibition of ROK activity with a specific inhibitor, Y27632, or by overexpression of a dominant negative form of ROKα, resulted in stress fiber disassembly in the fibroblasts but did not affect a large portion of the α-SMA containing stress fibers in myofibroblasts. Inhibition of mDia1 activity by overexpression of a dominant negative form of mDia1 resulted in disassembly of the major portion of the stress fibers both in the fibroblasts and myofibroblasts and also the ROK inhibitor resistant stress fibers in myofibroblasts. Conclusions: The assembly of stress fibers is differentially regulated in fibroblasts and myofibroblasts by the Rho signaling pathways. Although mDia1 and ROK may have a cooperative role in the assembly of stress fibers in both cell types, mDia1 promotes assembly of distinct stress fibers, independently of ROK, in myofibroblasts
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