Heat and Chemical Shock Potentiation of Glucocorticoid Receptor Transactivation Requires Heat Shock Factor (HSF) Activity

Abstract

Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal pathways. An unresolved question concerning this cross-talk is whether heat shock factor (HSF1) activity is required for this response. We addressed this issue by modulating HSF1 activity with compounds acting by distinct mechanisms: sodium vanadate (SV), an inhibitor of protein phosphatases; and wortmannin, an inhibitor of DNA-dependent protein kinase. Using HSF1-and GR-responsive CAT reporters, we demonstrate that SV inhibits both HSF1 activity and the stress potentiation of GR, while having no effect on the hormone-free GR or HSF1. Paradoxically, SV increased hormone-induced GR activity in the absence of stress. In contrast, wortmannin increased HSF1 activity in stressed cells and had no effect on HSF1 in the absence of stress. Using the pMMTV-CAT reporter containing the negative regulatory element 1 site for DNA-dependent protein kinase, wortmannin was found to increase the GR response. However, in cells expressing a minimal promoter lacking negative regulatory element 1 sites, wortmannin had no effect on the GR in the absence of stress but increased the stress potentiation of GR. Our results show that the mechanism by which GR activity is increased in stressed cells requires intrinsic HSF1 activity. The glucocorticoid receptor (GR)1 is a member of the nuclear receptor family of proteins that act as hormone-activated transcription factors (1, 2). Since the GR is known to be involved in the organismal response to stress (3), and since the hormonefree GR is known to reside in the cytoplasm as a complex containing heat shock proteins (HSPs) (4), we and others have investigated the role of the heat shock response in controlling GR function. Early evidence to suggest a relationship between these responses includes the ability of heat shock or chemical stress (arsenite) to cause nuclear translocation of hormone-free GR in mouse L929 and Chinese hamster ovary cells (5, 6) and a partial increase in GR-mediated gene expression in Chinese hamster ovary cells subjected to heat or chemical shock in the absence of hormone (6). Heat shock-induced nuclear translocation of hormone-free GR has also been documented in the liver of rats subjected to whole-body hyperthermia (7, 8) as well as in COS cells expressing human GR (9). Evidence to suggest that heat shock-induced nuclear translocation of hormone-free GR can result in altered expression of endogenous genes has also been accumulating. For example, heat shock treatment of COS and Hela cells in the absence of hormone results in GR-mediated transrepression of the collagenase promoter (9), while heat shock treatment of murine macrophages results in a pattern of Fc receptor expression or repression that is identical to that observed in response to hormone (10). More recently, heat shock-induced translocation of hormone-free GR has been implicated in the process of stress-induced apoptosis in leukemic cells (11).In the presenc...

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

“…1A), with increased activity seen with vanadate ranging from 50 to 500 M. A potential explanation for this effect was that vanadate at these seemingly-high concentrations was actually a form of chemical shock, such as that seen with sodium arsenite, which we have previously shown will cause similar increases of GR activity in these same cells [21,22]. However, this mechanism was clearly not operating, as vanadate treatment (500 M) of L929 cells stably-transfected with an HSF1-responsive CAT reporter was found to inhibit heat and arsenite induction of HSF1 activity [15].…”

“…In previous work by our laboratory [15], studying the effects of heat shock on GR activity, we used sodium vanadate to inhibit HSF1 activity in stressed cells by a mechanism involving MAPK (ERK-1 and ERK-2)-mediated phosphorylation of HSF1 [19,20]. In those studies, the presumptive target of vanadate action was the dual-specificity MAPK phosphatase, MKP1, inhibition of which by vanadate has been shown to induce MAPK activity [17,18].…”

“…This was discovered as part of our efforts to use sodium vanadate to inhibit heat shock factor (HSF1) activity in stressed cells [15]. In that work, HSF1 inactivation was achieved through a mechanism that was consistent with the known ability of vanadate to act as an inhibitor of tyrosine phosphatases [16][17][18], resulting in subsequent inhibition of HSF1 via MAPK (ERK-2)-mediated phoshorylation [19,20].…”

Glucocorticoid-induced glucose release is abolished in trout hepatocytes with elevated hsp70 content

“…Since we used whole cell homogenates for our immunodetection, the observed levels correspond to the total GR pool (cytosolic and nuclear) and the lower levels correspond to GR downregulation and not nuclear translocation. Studies using transformed cells showed that HS and arsenite potentiated glucocorticoid-mediated gene expression (Li et al 2000, Wadekar et al 2001. This potentiation was shown to require HSF activity (Li et al 2000) suggesting a positive relationship between the cellular stress response and the glucocorticoid pathway at the transcriptional level.…”

“…Studies using transformed cells showed that HS and arsenite potentiated glucocorticoid-mediated gene expression (Li et al 2000, Wadekar et al 2001. This potentiation was shown to require HSF activity (Li et al 2000) suggesting a positive relationship between the cellular stress response and the glucocorticoid pathway at the transcriptional level. However, HS also decreased GR-binding activity and increased GR degradation in mouse AtT-20 cell lines (Ali & Vedeckis 1990-91) implying a decrease in cellular sensitivity to GR stimulation.…”