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N. Fatma, E. Kubo, Y. Takamura, D.P. Singh; LEDGF and HSF1 Exclusively and Cooperatively Transactivate the Small Heat Shock Protein (Hsp) Genes and Provide Cellular Protection From Stress . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3852.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lens epithelium–derived growth factor (LEDGF) is a survival factor and acts as a transcription factor. At normal physiological condition, it binds to stress response (STRE;A/TGGGGA/T) and heat shock (HSE; nGAAn) elements in the stress genes and activates their transcription, thereby maintaining growth and survival of cells and also protecting cells from stress. Under stress condition, the regulatory HSF1 trimerizes and binds to HSE and stimulates the expression of Hsps and maximizes the stress response of cells. Herein, we explored the functional significance of the interaction between HSE–HSF1 and HSE–LEDGF in normal (unstressed) cells and in the stress– activation of the hsp27, and αB–crystallin genes. Methods: Mouse lens epithelial cells (mLECs) were generated from the lenses isolated from hsf1–/– and hsf1+/+ mice. Mouse embryonic cells (MEC) were also used in the study. Cells were cultured in DMEM with 10% FCS. Constructs of LEDGF in prokaryotic (pGEX2T–LEDGF) and eukaryotic (pEGFP–LEDGF) vectors, were prepared. MTS assay and Trypan blue exclusion tests were done to assess cell viability. TUNEL and DAPI staining were used to define cell death. Transactivation–efficiency of Hsp27 or αB–crystallin–CAT or their HSE mutants were monitored using CAT–ELISA at 37oC and 43oC and compared. Nuclear extracts isolated from heat stressed and unstressed cells were used for EMSA to assess the relative DNA binding affinity of LEDGF and HSF1 to HSE. Results: HSF1 depleted cells showed a low growth potential and underwent apoptosis following heat stress. RT–PCR and Western analysis revealed an elevated level of LEDGF in hsf1–/– cells. Transfection experiments disclosed the transactivation of HSP27– or αB–crystallin–CAT promoter in both hsf1–/– and hsf1+/+ cells at 37oC. However, increased promoter activity at 43oC suggests the cooperative role of HSF1 and LEDGF in transactivation. Down–regulation of transcriptional activity at 37oC and 43oC following co–transfection with a LEDGF–siRNA further validated the findings. EMSA revealed that LEDGF in nuclear extracts of heat stressed (43oC for 1h) and unstressed (37oC) hsf1–/– cells bound to nGAAn. Conclusions: Taken together our results suggest that LEDGF is an activator of heat shock genes for their constitutive expression at normal physiological condition necessary for cell survival, and cooperatively acts with HSF1 for rapid Hsps induction in cells during stress for providing cellular defense.
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