Inhibition of heat shock transcription factor by GR

The GR is a hormone-activated transcription factor that acts to regulate sp ecific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat sh ock proteins. As heat shock proteins are produced by cells as an adaptive r esponse to stress, speculation has existed that communication between the h eat shock and glucocorticoid hormone signal pathways must exist. Only recen tly has evidence to support this hypothesis been reported. In almost all ca ses, the evidence has been of an ability of heat shock to cause a potentiat ion of the glucocorticoid hormone response. In this proposal, evidence is n ow presented that heat shock signaling can, in turn, be regulated by glucoc orticoids. In mouse L929 cells stably expressing a chloramphenicol acetyltr ansferase reporter controlled by the human heat shock protein70 promoter an d containing known binding sites for heat shock transcription factor 1 trea tment with glucocorticoid agonist (dexamethasone) results in a dose-depende nt decrease of stress-induced chloramphenicol acetyltransferase gene expres sion. In these cells, inhibition of heat shock protein70 promoter activity by dexamethasone was completely blocked by GR antagonist (RU486). Similar t reatment of L929 cells stably expressing a chloramphenicol acetyltransferas e reporter under the control of the constitutively active SV40 promoter sho wed no such inhibition by dexamethasone. More importantly, dexamethasone wa s also found to inhibit heat shock-induced expression of the major heat sho ck proteins-heat shock proteins70, 90, and 110. Thus, the inhibitory effect of dexamethasone appears to apply to most, if not all, heat shock transcri ption factor I-regulated genes. Although dexamethasone did not prevent the DNA-binding function of heat shock-activated heat shock transcription facto r 1, it did inhibit a constitutively active mutant of human heat shock tran scription factor 1 under nonstress conditions, suggesting that dexamethason e repression of heat shock transcription factor 1 was primarily through an inhibition of heat shock transcription factor 1 transcription enhancement a ctivity. To more accurately characterize the stage of GR signaling responsi ble for inhibition of heat shock transcription factor 1, a series of Chines e hamster ovary cells containing either no GR, wildtype mouse GR, or single -point mutations of GR were employed. Dexamethasone inhibition of heat shoc k-induced heat shock transcription factor 1 activity was observed in the pr esence of wild-type GR, but not in Chinese hamster ovary cells lacking GR, suggesting that signaling cascades other than GR were not involved in this effect of dexamethasone. Consistent with this conclusion was the observatio n that dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or c-jun N-terminal kinase), which are known to negatively regulate heat shoc k transcription factor 1. Dexamethasone inhibition of heat shock transcript ion factor 1 was not seen in Chinese hamster ovary cells expressing GR defe ctive for DNA-binding function. Moreover, dissociation of GR/Hsp90/Hsp70 co mplexes was observed in response to hormone for both the wild-type and DNA binding-defective forms of GR, demonstrating that release of Hsp90 or Hsp70 (both of which are known to keep heat shock transcription factor 1 in its inactive state) could be ruled out as a potential mechanism. Thus, it appea rs that GR-mediated transactivation or transrepression is required for the inhibitory effect of dexamethasone on heat shock transcription factor 1 act ivity. Taken as a whole, these results provide evidence for a novel mechanism of c ross-talk in which signaling by the GR can attenuate the heat shock respons e in cells through an inhibition of the transcription enhancement activity of HSF1.