Transgenic Expression of Rho GDP Dissociation Inhibitor (Rho GDI), an Endogenous Inhibitor of Rho Family of Proteins, in Mouse Lens Disrupts Lens Growth and Structure

Abstract

Abstract: : Purpose:To determine the significance of Rho GTPase (Rho, Rac and Cdc42) activity in lens epithelial cell proliferation and fiber cell differentiation, lens specific expression of Rho GDI, an endogenous GDP dissociation inhibitor for Rho GTPases was carried out using αA–crystallin promoter (αA–CP) alone and heterogenic αA–crystallin promoter/chick delta–crystallin enhancer (αA–CP/DE). Methods: Several lines of transgenic mice (Black 6 strain) expressing the bovine RhoGDIα in a lens fiber cell–specific, and lens epithelium– and fiber cell–specific manner have been developed using αA–CP alone and αA–CP/DE promoters, respectively. Lens histological changes were examined by light microscope with hematoxylin and eosin staining. Transgene expression and levels of Rho GDI protein were determined by RT–PCR and Western blot analysis, respectively. Results: The Rho GDI Transgenic F1 mice derived from several independent lines (>5) with the use of αA–CP/DE promoter exhibit ocular specific phenotypes including severe microphthalmia and cataract. The size and weights of these transgenic eyes were significantly reduced (>50%) compared to littermate controls. Histological analysis of day one and one month old mice revealed gross structural alterations in lens fiber integrity with abnormal fiber cell differentiation. Embryonic (E15) transgenic eyes showed no obvious abnormalities in lens size and retained normal primary and secondary fiber cell organization. Lens crystallin profiles assessed by the SDS–PAGE in 7 day old revealed alterations with obvious degradation. These transgenic lenses showed markedly increased levels of Rho GDI as compared to littermate controls. Transgenic lenses also demonstrated accumulation of Rho and Rac GTPases in the soluble lysates indicating their functional impairment by the over expression of Rho GDI. Several F0 transgenic mice (>8) derived from using αA–crystallin promoter alone also demonstrated ocular specific phenotypes including microphathalmia and cataract. Histological analysis of 3 F0 transgenic mice revealed abnormalities in lens fiber differentiation and organization. Conclusions: These preliminary data based on overexpression of Rho GDI in lens epithelium and in fiber demonstrate a critical role for the Rho family of proteins in lens fiber cell differentiation, lens growth and function.

Keywords: signal transduction • cytoskeleton • transgenics/knock-outs