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N Fatma, DP Singh, LT Chylack, T Shinohara; LEDGF Activates Alcohol and Aldehyde Dehydrogenase Genes: Detoxifies Ethanol, and Modifies the Metabolism of Vitamin A . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2343.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ethanol (EtOH) is a risk factor for the incidence and progression of age-related cataract. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) detoxify EtOH and convert vitamin A (retinol) into retinoic acid (RA) which is a key molecule for the normal growth and function of epithelial cells. Our preliminary DD-PCR and RT-PCR studies indicated that LEDGF 1) induced expression of ADH (classes 1,4) and ALDH-1,2 mRNAs in LECs and 2) bound to HSE (nGAAn) and STRE (A/TGGGGA/T) to activate these stress-genes. In our current project we have asked if LEDGF detoxifies EtOH and modifies retinol metabolism by modifying ADH and ALDH levels. Methods: RT-PCR and protein blots were performed to quantify the levels of LEDGF, ADH, and ALDH in LECs. The MTS assay was used to monitor LEC viability. ADH- and ALDH-CAT-constructs were used to monitor promoter activity. LEDGF binding to DNA was assessed with EMSA and super-shift assays. A pRARE-TA-SEAP reporter system (Clontech) was used to monitor retinoic acid levels in LECs. LEDGF-antisense was used to validate the results. Results: LEDGF protein and mRNA levels were elevated in LECs treated with EtOH. LEDGF bound to the HSE and STRE in promoters of ALD and ALDH. Cells over-expressing LEDGF activated expression of ALDH-1, 2 and two classes of ADH (I and IV). These cells showed significant resistance against EtOH stress. LECs and cos7 cells co-transfected with LEDGF cDNA and ADH or ALDH promoter constructs showed elevated CAT expression. EMSA studies with mutant probes confirmed that LEDGF bound specifically to the HSE and STRE. Levels of retinoic acid in LECs over-expressing LEDGF were higher than in cells transfected with vector. Conclusion: Ethanol stress increased the expression of LEDGF and elevated LEDGF in turn up-regulated the expression of ADH and ALDH genes. Thus, LEDGF may reduce the risk of cataract formation by protecting cells from alcohol toxicity and increasing the level of retinoic acid in LECs and appeared to contribute significantly to LEC vitality.
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