Enhancement of Glucocorticoid Receptor-Mediated Gene Expression by Constitutively Active Heat Shock Factor 1

Abstract

To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stress-activated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE(2)E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE(2)E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions.

Polymorphisms at the 5′ flanking region of the HSP90AA1 gene in native Turkish sheep breeds

“…In this respect, Wadekar et al (2001) showed that glucocorticoids can suppress the heat shock response in cells by inhibiting the actions of heat shock factor 1 (HSF1). In stressed cells, activation of GR signaling causes rapid inactivation of HSF1 by blocking its actions at the promoter of Hsp70 and probably other Hsp genes (Jones et al, 2004). Interestingly, the indel (AA) was completely linked to the C-G transversion at position À 660 reported by MarcosCarcavilla et al (2010b), which was also found in all breeds here analyzed.…”

Forkhead Box M1 Is Regulated by Heat Shock Factor 1 and Promotes Glioma Cells Survival under Heat Shock Stress

“…Overexpression of FoxM1 and Constitutively Active HSF1-Hs683 cells were transfected with FoxM1 expression plasmid pcDNA3-FoxM1, CMV-hHSF1 (ϩ) (constitutively active HSF1 plasmid), CMV-hHSF1 (Ϫ) (non-active HSF1 plasmid) (34), and control vector using Lipofectamine 2000. Expression of transfected genes was confirmed with Western blotting, and the FoxM1-transfected cells were treated with heat shock stress followed by cell cycle analysis.…”

Glucocorticoid receptor recruits to enhancers and drives activation by motif-directed binding

“…5F). Most of the other motifs positively predictive for enhancer activation were for factors that have been shown to participate with GR in genomic binding and/or transcriptional activation (e.g., HSF [Jones et al 2004]; CEBP [Steger et al 2010]; RAR/RXR [Tóth et al 2011]; VDR ; TEAD and FOX [Starick et al 2015]). Thus, these factors may act in concert with GR, given the widespread direct binding of GR in activated enhancers.…”