Mechanism of heat shock signaling and glucocorticoid receptor cross -talk: Repression of HSF1 by GR

When activated by glucocorticoid hormone, glucocorticoid receptor (GR) functions as a transcription factor regulating expression of various genes. In intact cells, it forms a heterocomplex with heat shock proteins (HSPs). GR signaling pathway plays a crucial cytoprotective role in cells subjected to stress. Stress-induced synthesis of HSPs involves heat shock transcription factor (HSF1) activity. A cross talk between HSF1 and GR has thus been speculated. Earlier studies in our laboratory provided evidence for enhancement of GR-mediated gene expression due to HSF1 transactivity in heat shocked mouse fibroblasts (L929). We also made an interesting finding that heat shock signaling in turn, is inhibited by GR signaling. By using functional mutants of GR stably expressed in GR-negative CHO cells, and a GR antagonist RU486, we provided evidence for the inhibitiory effect to be GR specific. By using a constitutively-active mutant of HSF1, whose expression is controlled by doxycycline treatment, we characterized that heat shock response is inhibited at the level of HSF1-transactivity. EMSA data indicated that repression of HSF1 activity occurred at a stage distal to HSF1 DNA-binding. Requirement of intact DNA-binding domain of GR led us to speculate involvement of either GR-transactivation or transrepression. By CAT assay, we observed that GR-activity induced HSF1 repression as a very rapid effect. It suggested a possibility, that an interaction between transcription factors, led to inhibition of transactivity of HSF1. We also tested the possibility of transrepression induced by GR via plausible nGRE sequences in Hsp70 promoter. However, use of deletion constructs of Hsp70 promoter controlling CAT gene expression, provided us with data that strongly suggests a lack of any functional nGRE at heat shock protein promoters. With the knowledge of Dex-effect being rapid, we developed chromatin immunoprecipitation assay in order to assess the in vivo promoter occupancy of these factors (GR, HSF1) in intact cells. To our surprise, in contrast with our EMSA data, ChIP data indicated that HSF1 DNA-binding is abolished by GR. In the process of analyzing how GR signaling affects HSF1 Hsp70 promoter-binding, using ChIP assay, we detected a rapid release of DNA-bound HSF1 from Hsp70 promoter, in stressed cells, upon Dex treatment. Now that we know, that repression of HSF1 by GR occurs by inhibiting HSF1 DNA-binding, our studies will focus on understanding whether Dex induces removal of HSF1 from heat shock protein promoters, via other proteins such as C/EBPbeta and HSBP1 that are known to affect HSF1 DNA-binding. The study will enable us to further understand a mechanism by which GR ad HSF1 regulate each other and ensure recovery of cells from stress.