Potentiation of glucocorticoid receptor-mediated gene expression by heat and chemical shock.
We have examined the effects of heat shock on glucocorticoid receptor (GR)-mediated gene transcription in an L929 cell line derivative (LMCAT2) stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. Exposure of the LMCAT2 cells to heat or chemical shock resulted in a large increase in dexamethasone (Dex)-induced expression of CAT enzyme activity. This potentiation of hormone-induced MMTV-CAT expression was dependent on the magnitude of the stress event and on the Dex concentration, with maximal increases observed for 1 microM Dex after 2 h at 43 C or 2 h at 200 microM sodium arsenite. Heat shock potentiation of MMTV-CAT expression was not seen in an L929 cell derivative devoid of GR or in LMCAT2 cells treated with RU486 antagonist, suggesting that this effect of stress on CAT gene expression was mediated by the GR. Using a quantitative Western blot procedure, the amount of GR protein in the nucleus of cells subjected to combined heat shock and Dex treatment was no greater than the amount of nuclear GR in cells treated with hormone alone, indicating that the stress potentiation effect was not the result of increased nuclear translocation or retention by the GR. In addition, equally strong potentiations of MMTV-CAT expression were observed for cells subjected to heat shock either before or after Dex-mediated translocation of the GR to the nucleus. Thus, the major effect of stress on GR transcription enhancement activity appears to occur after the GR is bound to its high affinity nuclear acceptor sites. We have used a series of MMTV-CAT reporter constructs containing varying portions of the long terminal repeat regulatory region to show that a putative heat shock transcription factor-binding sequence at position -437 of the long terminal repeat is not required for this effect of heat shock on MMTV-CAT expression. A stress-induced increase in hormone-mediated CAT gene expression was observed for a minimal CAT reporter controlled by two synthetic glucocorticoid response elements and a TATA box sequence. Thus, it is unlikely that any DNA-binding transcription factor, other than GR, is required for this effect of stress on transcription by the hormone-bound GR. Based on these results, a model of heat shock enhancement of GR-mediated gene expression is developed in which stress acts on the DNA-bound GR, on a putative heat shock-activated adaptor, or on components of the RNA-polymerase-II complex.
Sanchez
E R
ER
Department of Pharmacology, Medical College of Ohio, Toledo 43699.
Hu
J L
JL
Zhong
S
S
Shen
P
P
Greene
M J
MJ
Housley
P R
PR
D000818:Animals; D018053:Arsenites / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006358:Hot Temperature; D007739:L Cells; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D017670:Sodium Compounds / Q000494:pharmacology*; D014162:Transfection
D018053:Arsenites; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D017670:Sodium Compounds; C028697:dexamethasone receptor; C017947:sodium arsenite; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase