Hormone-Independent Repression of AP-1-Inducible Collagenase Promoter Activity by Glucocorticoid Receptors

Abstract

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200-to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/ C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.The glucocorticoid receptor (GR) is a member of a family of ligand-dependent transcription factors capable of both positive and negative regulation of gene expression (4, 51, 69). In its unactivated form, the GR is part of a large heteromeric complex which includes hsp90 (18,35,45,61,67) and hsp56 (FKBP52) (42,57,61,63,81). Binding of the agonist stimulates receptor activation, dissociation from hsp90 (17, 66), and nuclear translocation, prerequisites for both transactivation and transrepression. For transactivation, specific in vitro binding of the activated GR to the glucocorticoid response element does not appear to be ligand dependent (22,79). However, in the absence of bound ligand, the nuclear form of the GR is a poor activator of target genes (52,65). This suggests that a ligandinduced conformational change, comparable to that inferred to be necessary for activation of the progesterone (2) and estrogen (5) receptors, is required for efficient activation (or derepression) of the transcriptional activating function present in the GR ligand-binding domain (34, 78) and/or for interaction of the receptor with other components of the transcription apparatus. Direct evidence for such a change has been provided by Simons et al., who demonstrated that affinity-labeled GR was less sensitive to proteolytic digestion than the unoccupied GR (74).Several mechanisms for GR-mediated transrepression ha...

Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells

“…In these model systems, it is necessary that at ®rst, ligand binds with its receptor to stimulate receptor activation, and next, the activated receptor complex be dissociated from hsp90, and then nuclear translocation be occurred. However, the recent report showed that GR-dependent transrepression of AP-1 was ligandindependent, which was observed in transfected cells after heat shock in the absence of ligand (Liu et al, 1995). In particular, the action mechanism of glucocorticoid hormone to inhibit the transcription of collagenases has been suggested by nuclear translocation of GR and its interfering the AP-1, the main After treatment with 10 mM of UA, 10 mM of Dexa or 10 mM of RU486 for 3 or 6 days, the cells were harvested and nuclear proteins (20 mg) were fractionated and transferred to Hybond-ECL.…”

“…Dexa activates and translocates GR from cytosol to nucleus by dissociating it from hsp90. The translocated GR inhibits the activity of AP-1 by directly binding to it (Jonat et al, 1990;Nicolson et al, 1990;SchuÈ le et al, 1990;Liu et al, 1995). MMP-9 has the AP-1 binding sequences on the promoter region .…”

Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells

“…In other GR heterozygous T-ALL systems, that is, CCRF-CEM cells with the L753F mutation that impairs ligand binding, 21 basal GR expression and/or auto-induction of the wild-type allele have been implicated in GC sensitivity. 4,22 To compare these parameters in the two human T-ALL systems, we first exploited 'real time' RT-PCR to quantitate GR expression prior to, and 12 h after, GC exposure at the mRNA level in GC-sensitive CCRF-CEM-C7H2 (phenotype GR L753F/wt ), 16 GC-resistant CEM-C1 (same phenotype GR L753F/wt ) 22 and Jurkat (phenotype GR R477H/wt ) T-ALL cells.…”

“…19 More specifically, CCRF-CEM cells, perhaps the best studied model for GC-induced apoptosis in human ALL, carry a GR gene mutation (L753F) on one allele that has already occurred in vivo 20 and markedly impairs ligand binding. 21 GC-resistant CCRF-CEM derivatives (mostly established by selection in GC-containing media) have either acquired loss-of-function mutations in the GR gene of the second allele, [13][14][15][16] have reduced basal GR levels 22 and/or fail to sufficiently auto-induce their functional GR gene upon GC exposure. 4 To gain further insights into the mechanism of GC resistance in leukemic cells, we investigated a different GCresistant T-ALL model, that is, the human T-ALL cell line Jurkat which is widely used in apoptosis research and has been exploited as a model for the molecular analysis of GR mutants.…”

The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K

“…The resultant RU486-liganded GR is efficiently imported into the nucleus but manifests a generalized defect in transcriptional activation. Nevertheless, this form of activated GR remains sufficient for transcriptional repression in certain cell systems and promoter contexts (45)(46)(47). Dexamethasone, a pure glucocorticoid agonist, inhibited IGF-I-stimulated activation of p70 S6K in a dose-related fashion within 4 h of exposure to the steroid (Fig.…”