Lens Specific Rlip76 Transgenic Mice Show Impaired Lens Development

Abstract

Purpose: : Lipid peroxidation (LPO) product, 4-hydroxynonenal (HNE) is involved in the etiology of oxidative stress induced cataractogenesis. We have earlier demonstrated that the over expression of RLIP76 (ral binding GTPase activating protein) provides resistance to ocular cells from oxidative stress induced injury through an accelerated disposition of HNE by the ATP-dependent transport of its glutathione conjugate. During present studies, we have developed RLIP76 (lens++++) Tg mice to investigate the effects of RLIP76 overexpression in lens.

Methods: : RalBP1 transgene was constructed which comprised of chick Δ1 crystallin enhancer-mouse α crystalline promoter-intron from rabbit β globin mouse RalBp1 cDNA, and a poly (A) signal from bovine growth hormone gene. A 5.3-kb transgene insert was released by Asp718-NotI digestion, and injected into pronuclei of B6C3F2 fertilized eggs. Five founders were identified using Southern blot analyses, and bred with C57BL/6J mice. Two of these lines had the highest numbers of transgene copies, and the mice hemizygous for the transgene exhibited smaller eyes with poorly developed lens compared to wild-type littermates. Expression of RLIP76 in the transgenic mice founders was analyzed by Southern blotting of the genomic DNA and PCR analysis of the RNA isolated from the lenses and was also confirmed by the Western blot and immunofluorescence analysis.

Results: : RLIP76 expression in Tg mice lenses was found to be high as compared to the WT lenses. Preliminary gross examination showed that the eyes of RLIP76 Tg mice were significantly smaller than those of the WT. The data on the size and weight of the eye balls also indicated that the eyes of the Tg mice were approximately 50% of the size of WT mice. Furthermore, the lens development was severely impaired in Tg mice. Histopathological examination confirmed a poor development of lenses in Tg mice. Since heat shock transcription factor 4 (HSF4) is known to be involved in the development of ocular lens, we compared the expression of HSF4 in WT and Tg lenses. Results of immunofluorescence and Western blot analyses indicated a remarkable suppression of HSF4 expression in Tg lens thereby suggesting that overexpression of RLIP76 inhibits HSF4- mediated differentiation of epithelial to fiber cells.

Conclusions: : These studies suggest a role of RLIP76 in the mechanisms of lens development and provide a suitable animal model for developmental studies (supported by EY04396).

Keywords: oxidation/oxidative or free radical damage • cataract • signal transduction