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Nirmala Sundarraj, Julie Wong Chong, Rajesh Bhattacharjee, Jian Chen; Rac GTPase Differentially Regulates TGF-bete1 and PDGF-BB-mediated Regulation of alpha-Smooth Muscle Actin Expression in Activated Corneal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2526.
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Cytokines and growth factors, released following an injury to corneal stroma, result in activation of specific signaling pathways which activate corneal keratocytes to fibrotic phenotypes (fibroblasts and myofibroblasts). The present study evaluated the role of Rac GTPase signaling pathway in the regulation of alpha-smooth muscle actin (SMA) expression in TGF-beta1 and PDGF-BB- activated corneal keratocytes.
Keratocytes isolated from rabbit corneal stroma, plated in a serum free (SF) medium, were treated with TGF-beta1 and/or PDGF-BB or fetal bovine serum (FBS) in the presence or absence of Rac inhibitor (NSC23766). In separate experiments keratocytes were infected with replication-defective inducible Tet-off adenoviral vectors encoding cDNAs for dominant negative (DN) or constitutively active (CA) HA-tagged Rac, and the expression of the recombinant proteins was induced during the activation of keratocytes. Alpha-SMA expression was analyzed immunocytochemically and by western blotting, and the relative abundance of alpha-SMA mRNAs was estimated by quantitative RT-PCR.
Immunocytochemical and western blot analyses indicated that Rac inhibition with NSC23766 or by overexpression of DN Rac during the activation of keratocytes with TGF-beta1 resulted in the inhibition of the expression of alpha-SMA and its mRNA in the resultant myofibroblasts. Overexpression of CA Rac upregulated the expression of alpha-SMA in the activated keratocytes. These findings indicated that Rac is involved in the activation of keratocytes to myofibroblasts. PDGF-BB or FCS decreased TGF-beta1-induced alpha-SMA expression. On the contrary to upregulation of TGF-beta1 induced alpha-SMA expression, Rac GTPase promoted PDGF- or FBS-mediated downregulation of alpha-SMA expression. Unlike in keratocytes, Rac-inhibition did not inhibit TGF-beta1 induced alpha-SMA expression in corneal fibroblasts but inhibited FBS-induced downregulation of alpha-SMA expression. Thus, Rac GTPase regulates alpha-SMA expression in TGF-beta1 activated keratocytes but not TGF-beta1 activated fibroblasts to myofibroblasts.
Rac GTPase differentially regulates downstream effects of TGF-beta1 and PDGF-BB signaling pathways leading to transdifferentiation of corneal keratocytes and fibroblasts to myofibroblasts. Therefore, Rac may function differentially at different stages of corneal wound healing process depending on specific growth factors present in the stroma.
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