The overall aim of this application is to characterize the mechanism(s) regulating aldose reductase (AR2, EC1.1.1.21) gene expression in retinal pigment epithelial (RPE) cells in vitro, believed involved in the development of diabetic retinopathy in vivo. The rationale for this proposal rests on the postulated role of AR2 in the genesis of the chronic complications of diabetes, and the growing evidence that AR2 participates in, and is modulated by, physiological osmoregulation. The polyol hypothesis asserts that diabetic complications (such as retinopathy, neuropathy and nephropathy) result, in part, from the direct or indirect consequences of sorbitol production from glucose by AR2, a member of the monomeric, NADPH-dependent aldoketoreductase family. Despite its pivotal role in the polyol hypothesis, little is known about the regulation of AR2 gene expression. Preliminary studies from this laboratory have examined the change in AR2 mRNA in cultured RPE cells subjected to hyperosmotic media. Within 24 hours of exposure to 300 mM glucose, 3-O-methyl glucose or mannitol, AR2 mRNA increased 40 fold with a subsequent increase in sorbitol content. Of the four RPE cell lines studied extensively, three demonstrated this """"""""normal"""""""" AR2 induction in response to osmotic stress, but a fourth (RPE 91) exhibited constitutive high level expression of AR2 mRNA and accelerated and exaggerated accumulation of sorbitol. Aberrant expression of AR2 such as that exhibited by this fourth RPE cell line, if present in diabetic patients, could predispose to the development of diabetic complications. Based on these considerations, this application proposes four specific aims: 1) To quantitate the change in the steady-state AR2 mRNA level in RPE cells in response to exposure to glucose, 3- O-methylglucose and mannitol and to correlate the mRNA levels with changes in AR2 protein and sorbitol. 2) To determine whether alterations in AR2 gene transcription, and/or mRNA stability cause the changes in steady-state AR2 mRNA level. 3) To characterize the promoter of the AR2 gene, and identify the cis-acting regulatory sequences which regulate osmotic induction of transcription. 4) To determine the mechanism of aberrant AR2 expression in RPE 91.
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