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J.M. Kwong, N. Piri, M. Lalezary, J. Nguyen, A. Khattar, C. Yang, J. Caprioli; Expression of Heat Shock Transcription Factors 1 and 2 in Rat Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1568.
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We have previously demonstrated that the expression of the heat shock protein 72 (HSP72) is up–regulated in retinal ganglion cells (RGCs) in response to heat shock, excitotoxicity, hypoxia, and anoxia. In this study we investigate the expression of heat shock factors 1 (HSF1) and 2 (HSF2) in retina and their roles in transcriptional activation of HSP72.
Adult male Wistar rats weighing 300–350g were used to examine the expression of retinal HSF1 and HSF2 with immuno–histochemistry (n=6), Western blotting (n=4), and reverse transcription–PCR (RT–PCR; n=4) of HSF1 and HSF2. To confirm specificity to RGCs, adult rat retinal cells (n=8) were dissociated and RGCs were harvested with anti–Thy–1 (RGC marker) coated magnetic beads for protein analysis. Progressional deletion and mutational analysis of the HSP72 promoter region was carried out to determine the roles of the cis–acting elements, including HSE1 (binding site for HSF1) and HSE2 (binding site for HSF2), in HSP72 expression.
RT–PCR and Western blot analyses showed HSF1 and HSF2 expression at the mRNA and protein levels in adult rat retina. Positive immuno–reactivities corresponding to HSF1 and HSF2 were localized predominantly in the inner retina. The enriched RGC fractions were Thy–1, HSF1, HSF2 and HSP72 immuno–positive, but immuno–negative for glial fribillary acidic protein (a glial cell marker), indicating that HSF1 and HSF2 are expressed in the RGCs of the adult retina. Deletion of HSE2 from the HSP72 promoter led to an approximately 10–fold decrease in reporter gene expression level. Deletion of both HSE1 and HSE2 abolished gene expression of HSP72.
The observed results indicate that both HSF1 and HSF2 are expressed in RGCs and may be involved in the regulation of HSP72 expression in RGCs after stress.
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