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P Sharma, DP Shingh, N Fatma, N Chattopadhyay, LT Chylack, T Shinohara; Attenuation of LEDGF-DNA Binding Affinity and Down Regulation of LEDGF Promoter Activity in Human Lens Epithelial Cells Treated with TGFß1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2344.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:LEDGF, a transcription and survival factor, protects cells from environmental stress by activating cis-, stress response- (STRE), and heat shock (HSE) elements in stress genes. The LEDGF gene is inducible by oxidative stress. Transforming growth factor-ß (TGFß) has been implicated in cataractogenesis, terminal differentiation, and apoptosis. We investigated the expression and regulation of the LEDGF gene during TGFß-induced changes in human lens epithelial cells (hLECs). Methods: 3-5 x 105 hLECs were cultured in DMEM + 20% FBS in six-well plates for 24 h at 37°C in a CO2 incubator. The medium was replaced with 0.2% BSA-DMEM containing TGFß1 at varying concentrations, or TGFß1-free medium. Cultures were continued for varying time intervals. Nuclear extracts were prepared. Western blots and RT-PCR were used to monitor the expression of LEDGF. Gel-shift, super-shift assays were used to assess the LEDGF-DNA binding affinity, and a CAT assay was used to monitor LEDGF promoter activity. A specific p38 MAPK inhibitor, SB203580 (Calbiochem) and an antibody against phospho-p38 MAPK (Cell Signaling) were used to study the effects of MAPK on TGFß1 mediated signaling. Cell growth/inhibition and morphology were monitored with phase contrast and fluorescence microscopy. Trypan blue, the MTS assay, and DAPI staining were used to assess cell viability.Results: Human LECs treated with TGFß1 formed elongated fiber-like cells, multi-layered cellular aggregates, detached more frequently, and manifested increased inter-cellular spaces. These changes could be abolished by pretreating the cells with 5µM SB203580 o/n. Furthermore, 1ng/ml TGFß1 induced rapid phosphorylation of p38 MAPK. Moreover, TGFß1 treatment inactivated the LEDGF promoter, reduced the expression of LEDGF protein, and DNA binding affinity of LEDGF in nuclear extracts. Reduced binding of LEDGF to HSE (nGAAn), evident from gel-shift assay, was consistent with the noted attenuation of the protective effect of LEDGF. Conclusion:That the inactivation of LEDGF and the reduction of LEDGF-DNA-binding activity can be prevented by a MAPK inhibitor suggests that morphological changes induced by TGFß1, are mediated through the MAPK pathway. Inactivation of LEDGF may be a critical event in TGFß1 induced changes in hLECs.
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