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N. Fatma, E. Kubo, K. Ishihara, Y. Takamura, D. P. Singh; LEDGF and HSF1 Interaction is Essential in Transactivation of Heat Shock Protein Genes for Lens Epithelial Cell Survival During Stress. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2613.
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© ARVO (1962-2015); The Authors (2016-present)
In normal physiological conditions, the nuclear protein LEDGF binds to stress response (STRE; A/TGGGGA/T) and heat shock (HSE; nGAAn) elements in the small heat shock protein (shsp) genes and protects cells by activating those genes. Under heat stress, LEDGF interacts with trimeric form of HSF1 and binds to HSE for rapid action of heat shock genes. We investigated the relative importance of bases in HSE, essential for synergetic binding of LEDGF and HSF1 to the same site, and explored the influence of cooperative binding of these factors in transactivating HSP27 and αB-crystallin genes and in protecting lens epithelial cells (LECs) facing stress.
HSF1-depleted (hsf1-/-), wild-type (hsf1+/+) mouse LECs, human LECs and LEDGF-siRNA interference experiments were used to ascertain the protective potential of LEDGF and HSF1. Cell-viability assay, TUNEL and DAPI staining were used to define cell death. Gel-mobility and super-shift assay were done to assess relative DNA binding affinity of LEDGF and HSF1 to HSE. Transcriptional activity of HSP27/αB-crystallin or their HSE mutants was monitored by CAT-ELISA at 37oC and 43oC. The interaction between LEDGF and HSF1 during heat stress was done by immunoprecipitation. An active form of HSF1 (pcDNA3/HSF1ΔRDT construct was used to validate the results. Levels of HSF1, LEDGF and shsp were monitored by Western analysis and RT-PCR.
Transfection experiments revealed that cells lacking LEDGF did not survive well and underwent apoptosis during stress, and showed reduced expression of HSP27 and αB-crystallin mRNA and protein. Transactivation experiments revealed significantly reduced promoter activity of HSP27/αB-crystallin. Cells lacking HSF1 could not upregulate shsp following heat stress and underwent apoptosis, suggesting LEDGF alone did not protect cells. Using a double-standard DNA oligomer with HSE element (nGAAn) and its repeats and their mutants in gel-shift assay coupled with transactivation assay, we found that inverted repeat was essential for cooperative binding of these molecules to HSE. Immunoprecipitation experiments showed that LEDGF interacts with trimeric HSF1 in cells.
Our results provide evidence that inverted repeats of nGAAn are essential for cooperative binding of LEDGF and HSF1 at HSE sites for transactivating heat shock genes and thereby protecting lens epithelial cells from heat stress.
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