Enhancement of glucocorticoid receptor-mediated gene expression by cellular stress: evidence for the involvement of a heat shock-initiated factor or process during recovery from stress

Recent reports have demonstrated the ability of cellular stress to cause a large increase in the maximal levels of steroid receptor-mediated gene expression, a process we refer to as the heat shock potentiation effect (HSPE). In the present work, we have analyzed the time of appearance of the HSPE on the glucocorticoid receptor (GR) of L929 cells stably-transfected with the MMTV-CAT reporter plasmid (LMCAT2 cells). In LMCAT2 cells exposed to heat shock (43°C, 2-h) before addition of 1µM dexamethasone, the first appearance of HSPE (CAT levels greater that hormonealone) occurred at 8 h of recovery and continued to increase by 24 h of recovery. Treatment of LMCAT2 cells with 1 µM dexamethasone for 2 h before heat or chemical shock (sodium arsenite) resulted in the same delayed onset pattern for the HSPE. Based on a [³⁵S]methionine assay and tests of L929 cells stably transfected with the constitutive pSV2-CAT reporter, evidence is provided that the delayed appearance of the HSPE is not due to the heat shock block of general protein synthesis or to specific repression of CAT mRNA expression or translation. By using short-term incubations (4 h) with dexamethasone during the recovery period, the peaks of HSPE expression during recovery were determined to be 12–16 h for CAT enzyme activities, and 4–8 h for CAT mRNA expression. Taken together, these results provide evidence that the timing of the HSPE is not dependent on the rate of GR activation, or on the type of stress, but rather on a factor or process that is either synthesized or activated during the recovery period following stress.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.