The long-range goal of this project is to elucidate the molecular mechanisms of expression and the role of the corneal Class 3 aldehyde dehydrogenase (ALDH3c; mouse AHD4). Studies in several mammalian species, including human, have revealed high AHD4 expression in cornea. Although this enzyme represents a major (10-40 percent) corneal soluble protein, its function and molecular mechanism(s) of expression in cornea remain unclear. It has been suggested that AHD4 might play a critical role in cornea by maintaining transparency and/or redox balance, facilitating absorption of UV light, and disposing of UV generated cytotoxic aldehydes. Genetic variants of AHD4 are known to exist in mice and humans. The SWR/J mouse strain, which exhibits a low AHD4 phenotype, shows extensive corneal clouding after UV exposure, compared with that in other mouse strains having high ocular AHD4 activity. They have cloned the AHD4 cDNA from SWR/J mouse cornea and found that this gene is indeed polymorphic. A correlation between the low AHD4 enzymatic activity phenotype and keratoconus (and other corneal pathology) has been reported in humans. They have previously cloned and sequenced the mouse Ahd4 gene. The principal investigator has recently identified the promoter and enhancer elements in a 3.2-kb 5' flanking region of this gene that are presumably involved in corneal expression. In order to elucidate the molecular mechanisms of the corneal AHD4 expression, as well as its protective role in the eye, they therefore propose to: 1) characterize cis-and/or trans-regulatory DNA elements responsible for cornea-specific Ahd4 gene expression. Ahd4 promoter/enhancer-luciferase DNA constructs will be used to transfect corneal epithelial cells lines available in their laboratory. in vitro footprinting and gel retardation assays will be used to characterize the cornea-specific DNA elements. 2) characterize the Ahd4 polymorphism in mice. To elucidate functional or structural differences in AHD4 protein variants, they will clone allelic cDNAs in expression vectors and express them in prokaryotic and human corneal cell lines. 3) develop a knockout mouse line having homozygous disruptions in the Ahd4 gene. These mouse lines will allow them to study the physiologic and protective role of corneal AHD4 against oxidative damage caused by UV irradiation. These studies will greatly enhance the understanding about the regulation and the role of the corneal Ahd4 gene. Because of conservation between human and mouse, and human polymorphisms in the ALDH3c gene are known to exist, these studies should help elucidate the Ahd4 gene as a genetic factor in the UVR-induced eye damage.
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