The glucocorticoid receptor (GR) is a ligand-activated transcription factor that acts to control specific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat shock proteins (hsp90, hsp70 and hsp56). A variety of in vitro approaches have suggested a role for these heat shock proteins (HSPs) as molecular chaperones controlling the assembly and maintenance of the hormone-competent GR heterocomplex. We and other laboratories have shown that the cellular heat shock response can cause a super activation of GR's ability to enhance transcription by a mechanism that likely involves the interaction of GR with the Pol II transcription complex. Thus, the cellular responses controlled by the GR may be stimulated not only by hormone but also by stress signals, and it is a goal of our laboratory to understand how the heat shock and GR signal mechanisms converge in cells subjected to both normal and stress conditions. As a further step in this direction, we propose to directly test the in vivo roles of hsp90, hsp70 and hsp56 in the GR signal mechanism by altering the levels of each HSP in cells through use of a tetracycline-inducible expression system (rTet). Both over- expression and down-regulation of HSPs will be achieved by inserting the cDNAs into the expression vectors in the sense and antisense orientations, and the effects of these changes on all stages of GR signaling will be measured. As the heat shock signal mechanism primarily involves the stress activation of the heat shock transcription factor (HSF) and its subsequent actions to increase synthesis of HSPs, we also propose to use the rTet expression system to test the involvement of HSF, hsp56, hsp70 and hsp90 in the stress potentiation of GR-mediated gene expression. In related projects, we will use DNA- and immuno-affinity purification protocols to detect proteins that interact with the GR of heat shocked cells, as well as differential display RT-PCR and RNA Northern blot techniques to identify GR-regulated gene products that may be involved in protection of cells from stress or in the heat shock potentiation mechanism itself. Understanding the intracellular mechanisms that modulate steroid receptor action is a central problem in eukaryotic gene expression and development. As steroids are also widely-used pharmacological agents for immunosuppression and cancer chemotherapy, the proposed study may provide the basis for augmentation of steroid-based therapies.
