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Y. Takamura, N. Fatma, E. Kubo, T. Shinohara, D.P. Singh; Induction of Heavy Subunit Chain of –Glutamylcysteine Synthetase Expression by Tumor Necrosis Factor in Mice Lens Epithelial Cells: Role of LEDGF . Invest. Ophthalmol. Vis. Sci. 2004;45(13):392.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: LEDGF, a transcriptional anti–death factor, protects cells from various stresses by up–regulating stress associated genes. Tumor necrosis factor α (TNF α) is an inflammatory cytokine that generates oxidative stress and up regulates heavy subunit chain γ–glutamylcysteine synthetase (γ–GCS–HS). Since LEDGF is inducible to oxidative stress, we hypothesized that TNF α may regulate LEDGF and LEDGF may be one of the activators of γ–GCS–HS gene. Herein, we demonstrated that TNFα elevates LEDGF expression and that LEDGF consequently transactivates γ–GCS–HS gene by binding to heat shock (HSE; nGAAn) and stress response (STRE; A/TGGGGA/T) elements in the γ–GCS–HS gene. Methods: A DNA fragment of γ–GCS–HS (–819 to +525 nts) consisting of LEDGF binding sites was generated from a genomic human DNA using a PCR, and cloned into the pCAT Basic vector. Gel–mobility and supershift assays were conducted for LEDGF binding to HSE and STRE; CAT–ELISA was done to monitor promoter activity. Western analysis and real time quantitative PCR was used to assess levels of LEDGF and γ–GCS–HS mRNA and proteins in mLECs treated with TNF α at variable concentrations for variable time. The GSH level was quantified with a commercial kit (Oxis International Inc.). Results: Gel–mobility assay confirmed the binding of LEDGF to the HSE and STRE present in γ–GCS–HS promoter. CAT–ELISA showed activation of γ–GCS–HS promoter in cells overexpressing LEDGF. TNFα treatment of LECs for 3 to 24 hrs markedly increased LEDGF and γ–GCS–HS promoter activity with increase of abundance of LEDGF and γ–GCS–HS mRNA and proteins. Mutation of γ–GCS–HS promoters at HSE and STRE abolished the TNFα induced up–regulation of this gene promoter. Cells over expressing LEDGF or treated with TNF α revealed elevated GSH level (10–15%). Conclusions: TNFα regulation of LEDGF may be physiologically important, as elevated expression of LEDGF increases the expression of endogenous γ–GCS–HS gene, the catalytic subunit of the regulating enzyme in GSH biosynthesis, that may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.
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