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W V Vedeckis

Publications and source records attributed to W V Vedeckis.

At least 19 recordsLinked to original sources
PubMed2001

Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids.

A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

Alternative Splicing↗
PubMed1999

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

Apoptosis↗
PubMed1998

Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

Animals↗
PubMed1998

The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1.

Studies on the human glucocorticoid receptor (GR) promoter were carried out so as to understand the regulation of GR expression. A -2738 to +19 fragment of the human GR promoter was used to identify important regulatory elements involved in the control in GR expression in NIH 3T3 cells (mouse fibroblasts) and HeLa cells (human cervical carcinoma cells). Important regulatory domains in the distal region of the human GR promoter were identified by sequential 5' end deletion analysis. A region between -2490 and -2025 contributed 50% of the measured transcriptional activity to the promoter. Using DNase I footprint analysis, four sites in this region were identified: -2362 to -2339 (mouse footprint, mFP); -2301 to -2293 (distal YY1, dYY1); -2130 to -2122 (middle YY1, mYY1); and, -2086 to -2078 (proximal YY1, pYY1). Three sites contained an identical core sequence, CCAAGATGG and were identified as Yin Yang 1 (YY1) binding sites. The site located at -2362 to -2339 was footprinted in NIH 3T3 cells only. The sequence of this site is a direct repeat with a 2-nucleotide spacer region, and it does not share homology with any known transcription factor binding sites. Computer analysis of the entire promoter sequence revealed an additional YY1 site located at -260 to -249 (initiator YY1, iYY1) with the sequence CTCCTCCATTTTG. Electrophoretic mobility supershift assays, with an anti-YY1 antibody, were used to confirm YY1 binding to these four putative YY1 binding sites. Site-directed deletion of all three upstream YY1 sites but not the iYY1 site, or the iYY1 site alone, showed a approximately 60% decrease in transcriptional activity of the hGR promoter in HeLa cells but had no effect in NIH 3T3 cells. A similar (50%) decrease in the expression of a full-length hGR/luciferase reporter gene was obtained when HeLa cells were cotransfected with a full-length antisense YY1 expression plasmid. Additionally, a region between -1841 and -1689 contributed to hGR promoter activity in both cell types tested. An Sp1 binding site was identified in this region (-1748 to -1733) by DNase I footprint and mobility supershift analyses. The presence of four YY1 binding sites in the human GR promoter suggests that these sites play a critical role in GR gene regulation.

3T3 Cells↗
PubMed1998

Transrepression of c-jun gene expression by the glucocorticoid receptor requires both AP-1 sites in the c-jun promoter.

The c-jun protooncogene encodes a nuclear protein, cJun, which is a major component of the AP-1 transcription factor. AP-1 regulates various aspects of cell proliferation and differentiation. As an immediate early response gene, the expression of the c-jun gene is affected by various extracellular stimuli, such as serum, phorbol esters, and glucocorticoids. In mouse L929 fibroblasts, dexamethasone (DEX) treatment caused a 60% reduction of c-jun mRNA levels. Previous studies indicated that this reduction is due to the alteration of the transcription rate of the c-jun gene. To further investigate the molecular mechanisms of transcriptional repression of c-jun by DEX, a full-length human c-jun promoter, from -1780 to +731, was amplified from genomic DNA using PCR and then linked to the luciferase reporter gene. To identify the regulatory elements responsible for the down-regulation, nested deletions spanning the promoter were generated, and the promoter/luciferase constructs were transiently transfected into L929 cells. Upon hormone treatment, basal activity of the full-length c-jun promoter was reduced by approximately 40%, which accounts for two-thirds of the overall down-regulation observed at the mRNA level. This reduction of c-jun promoter activity was abolished after deletion of the region between -1780 to -63, where two AP-1 sites (-182 and -64) are located. Site-directed deletion of these AP-1 sites reduced the basal activity of the c-jun promoter and prevented repression by DEX. Repression of the c-jun gene is due to the transrepression activity of the glucocorticoid receptor (GR), as determined using GR mutants lacking this activity. Overexpression of cJun overcame the negative effect of DEX, suggesting that down-regulation of the c-jun gene by hormone is mediated by the interaction between the GR and the cJun protein. These studies are the first to show that glucocorticoids can repress c-jun promoter activity through the AP-1 sites in the c-jun promoter in mouse fibroblast cells. They also suggest that inhibition of cell proliferation by glucocorticoids may be due not only to the interference with AP-1 activity on other cellular genes, but also because of a direct transcriptional suppression of c-jun gene expression by the GR.

Animals↗
PubMed1997

Regulation of the glucocorticoid receptor gene by the AP-1 transcription factor.

The glucocorticoid receptor (GR) is a ligand-activated nuclear transcription factor, and AP- 1 (Fos/Jun or Jun/Jun) is a transcription factor whose components are nuclear proteins encoded by c-fos and c-jun protooncogenes. Serum stimulation of serum-starved NIH 3T3 cells resulted in an approx 188-fold induction of c-fos mRNA at 30 min and an approximately ninefold induction of c-jun mRNA at 1 h, followed by an increase in GR mRNA levels at 3-12 hour (twofold). Sequential induction of cFos, cJun, and GR protein levels also occurred. Overexpression of the cFos protein in NIH 3T3 cells (NIH 3T3 [cFos 3] and NIH 3T3 [cFos 10]) caused an increase in the endogenous GR protein. Previous and present studies showed that a putative AP-1 site within the GR promoter binds AP-1 proteins (both Jun and Fos family members). To address the molecular mechanism involved in transcriptional activation of the GR gene, we investigated the relevance of AP-1 binding complexes in this activation and in overall regulation of GR gene transcription. Transient transfection with a full length GR promoter linked to a luciferase gene into both NIH 3T3 (cFos 3) and NIH 3T3 (cFos 10) cells gave rise to an induction of luciferase activity. This induction was abolished following mutation or deletion of the GR AP-1 site from the promoter. These findings suggest that cFos is responsible for the induction of GR expression in serum-stimulated NIH 3T3 cells, and serum growth factors may stimulate GR transcription by a cFos-dependent mechanism at the putative AP-1 site. These studies support a role for the AP-1 transcription factor in regulating GR gene expression.

3T3 Cells↗
PubMed1996

Coordinate regulation of glucocorticoid receptor and c-jun gene expression is cell type-specific and exhibits differential hormonal sensitivity for down- and up-regulation.

We have previously proposed a novel mechanism for the coupled regulation of glucocorticoid receptor (GR) and c-jun transcription in triamcinolone acetonide (TA)-treated AtT-20 cells. This involved transcriptional interference of AP-1 (Fos/Jun)-driven gene transcription by the formation of inactive GR/Jun heterodimers. To further elucidate the molecular mechanism for GR autoregulation, the expression of GR and c-jun mRNA and protein levels were examined in both mouse L929 fibroblast cells and human CEM-C7 acute lymphoblastic leukemia cells. A rapid down-regulation of both GR and c-jun mRNA and protein levels occurs in TA-treated L929 cells. All-trans-retinoic acid (RA) treatment of Jun-deficient, mouse F9, teratocarcinoma cells causes the induction of c-jun expression. The increased expression of both c-jun mRNA and protein is accompanied by the induction of GR expression. These data further suggest that functional cJun is needed for the expression of the GR and c-jun genes in F9 cells. CEM-C7 cells undergo apoptosis after exposure to glucocorticoids. There is a parallel up-regulation of GR and c-jun mRNA levels in TA-treated CEM-C7 cells. This is accompanied by a concomitant increase in GR and cJun protein levels. Dose-response analyses reveal the expected coordinate regulation of both GR and c-jun mRNA and protein in L929 cells (decreasing) and in CEM-C7 cells (increasing). However, approximately 20-fold less TA is required for the inhibition of GR and c-jun expression as compared to that required for the stimulation of these two genes. These data demonstrate that the coordinate regulation of GR and c-jun gene expression is dose-dependent and cell type-specific. These results, along with previously reported data, suggest that GR complex formation with itself or with another transcription factor is important for the coordinate up- and down-regulation, respectively, of the GR and c-jun genes.

Animals↗
PubMed1996

Occupancy and composition of proteins bound to the AP-1 sites in the glucocorticoid receptor and c-jun promoters after glucocorticoid treatment and in different cell types.

The glucocorticoid receptor (GR) and c-jun promoters both contain activator protein-1 (AP-1) sites (GR AP-1 site and c-jun AP-1 site, respectively) that vary from the consensus AP-1 site. Electrophoretic mobility shift assays (EMSAs) were used to monitor GR AP-1 and c-jun AP-1 oligonucleotide binding by nuclear extracts from AtT-20 and L929 cells that were hormone- and vehicle-treated for 1, 6, or 24 h. Both AtT-20 and L929 cell nuclear extracts bound the c-jun AP-1 site somewhat better than the GRAP-1 site and, in the majority of cases, extracts from hormone-treated cells shifted both GRAP-1 and c-jun AP-1 oligonucleotides more than nontreated nuclear extracts. Supershift assays, using Jun and Fos family member-specific antibodies, showed that protein complexes formed by AtT-20 cell nuclear extracts bound to the c-jun AP-1 site were comprised of Jun family members, JunD, JunB, and cJun. No Fos family members were present. However, protein complexes from AtT-20 nuclear extracts that bound the GR AP-1 site were supershifted by JunD, JunB, cJun, and Fra-2 specific antibodies. In L929 cell nuclear extracts, the c-jun AP-1 site is bound by JunD and cJun. No clear association of Fos family members with the c-jun AP-1 site could be demonstrated. The GR AP-1 site bound protein complexes composed of JunD, JunB, Fra-2, and Fra-1 from L929 nuclear extracts. This demonstrates that the composition of the protein complexes that associate with the c-jun AP-1 site differs from those that bind the GR AP-1 site. These data also indicate that the protein complexes that bind the GR and c-jun AP-1 sites are cell-type-specific. Computer analysis also revealed five putative cyclic AMP response elements (CREs) in the GR promoter. Relative mobility shift and binding studies suggest that CRE binding protein (CREB), CREB modulator (CREM), or CREB/CREM may be associated with the c-jun AP-1 and/or GR AP-1 sites, but the association at these sites occurs at a lower binding affinity than for a consensus CRE. Nuclear extracts from AtT-20 and L929 cells were able to shift the CRE, and supershift analysis revealed that Jun family members are part of the protein complexes that bind the CRE. Pan Jun and pan Fos antibodies were able to supershift protein-CRE complexes formed using NIH 3T3 nuclear extracts. These data raise the possibility that the promiscuous binding of CREB and/or CREM to the AP-1 site, and AP-1 transcription factors to one or more CREs, in the GR promoter may contribute to the regulation of GR gene expression.

3T3 Cells↗
PubMed1995

Distribution of all-trans-retinoic acid in normal and vitamin A deficient mice: correlation to retinoic acid receptors in different tissues of normal mice.

The distribution of all-trans-retinoic acid (RA) was investigated using whole-body autoradiography of normal and partial vitamin A deficient (VAD) mice. Retinoic acid receptors (alpha, beta, and gamma) were also studied in normal mice using immunoblotting. Normal and VAD mice were injected with 5 muCi 14C RA. The distribution of RA was quantitatively studied using a computer-assisted image analysis system. 14C RA was incorporated 0.5 hr after RA administration in both normal and VAD mice, while the labelling peak was at 6 hr in most organs in normal and VAD mice. The most intense labeling was found in liver, kidney, intestine, lung, Harderian gland, and salivary gland at all time points. A band of M(r) 51K was found in all mouse tissues by immunoblotting using the polyclonal antibody RAR82 against total RARs or the RAR alpha-specific monoclonal antibody R alpha 13. In some tissue, an additional band of 55-58K was also found. Lung, large intestine, small intestine, testis, seminal vesicle, and spleen contained highest concentration of total RARs, while heart, lung, small intestine, spleen, salivary gland, and preputial gland had the highest concentration of RAR alpha. The uptake of labeled RA correlated well with RAR or RAR alpha concentration in the corresponding tissues.

Animals↗
PubMed1994

Porcine conceptus and endometrial retinoid-binding proteins.

Porcine conceptus secretory proteins were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialysed and concentrated by Amicon ultrafiltration for retinol and retinoic acid (RA) binding studies. Proteins in the 20-kDa range, conceptus-secreted retinol-binding protein (RBP), bound both [3H]retinol and [3H]RA specifically. Cross-competition experiments indicate that [3H]RA was completely displaced with excess cold retinol; however, excess cold RA did not completely displace [3H]retinol, suggesting that conceptus RBP has greater affinity for retinol than RA. Cellular RBP and retinoic acid receptor (RAR)-alpha and RAR-gamma mRNA transcripts (0.7 kb; 3.8 and 2.8 kb; 3.4 kb respectively) were detected in poly (A)+ RNA isolated from Day-15 conceptus, Day-15 pregnant endometrium, late pregnant myometrium and late pregnant fetal tissues of pigs by Northern blot analysis. RAR-alpha and RAR-gamma immunoreactive proteins were detected in extracts of Day-15 conceptus, Day-15 pregnant endometrium and late pregnant fetal tissues by Western blot analysis. Collectively, results indicate that biochemical molecules required for retinoid transport, metabolism and regulatory effects are present in porcine conceptus and endometrial tissues during early pregnancy in swine.

Animals↗
PubMed1994

Coordinate regulation of glucocorticoid receptor and c-jun mRNA levels: evidence for cross-talk between two signaling pathways' at the transcriptional level.

Autologous regulation of steroid receptors by their cognate ligands has been demonstrated for a number of nuclear receptor family members. To determine the molecular mechanism for glucocorticoid receptor (GR) autoregulation, the expression of glucocorticoid receptor mRNA and protein levels were examined in the mouse AtT-20 pituitary tumor cell line. The expression of c-jun and c-fos mRNA and protein was also examined in the same cell extracts. A rapid down-regulation of the GR protein was observed after treatment with the glucocorticoid analog, triamcinolone acetonide (TA). An oscillatory, parallel regulation of both GR and c-jun mRNA levels occurred. In contrast, POMC mRNA levels remained at a stable, low level during chronic TA treatment. Dose-response analyses also revealed a coordinate down-regulation of GR and c-jun (but not POMC or c-fos) mRNA levels. FOS protein levels were unaffected by TA treatment. Surprisingly, JUN protein levels were increased by TA, even when the c-jun mRNA levels were decreasing. Perhaps a derepression of c-jun mRNA translation occurs after TA treatment, and this may be due to GR/JUN heteromer formation interfering with JUN repression of c-jun mRNA translation. The effect of TA on GR and c-jun gene expression was a primary effect, as it occurred rapidly and was not inhibited by cycloheximide (CHX). Nuclear run-on transcription assays revealed a rapid (15 min) down-regulation in both GR and c-jun gene transcription rates, while POMC gene transcription was unaffected at this early time. Treatment of AtT-20 cells with all-trans retinoic acid gave different kinetics for GR and c-jun mRNA regulation than obtained with TA; however, the GR and c-jun mRNA levels were still coordinately regulated after retinoic acid treatment. Based upon these data, the promoter structures of the GR and c-jun genes, and previously published results, a novel mechanism for the coupled regulation of GR and c-jun transcription, via a direct transcriptional interference with AP-1 (FOS/JUN) activity, is proposed.

Animals↗
PubMed1992

Identification of human, mouse, and rat retinoic acid receptor alpha using monoclonal antibodies.

Monoclonal antibodies that recognize the human, mouse, and rat retinoic acid receptor alpha (RAR alpha) protein have been generated using synthetic peptides. Less well-characterized monoclonal antibodies were also generated against the RAR beta and RAR gamma proteins. Monoclonal antibodies of the IgG1 (R alpha 10) and IgG2a (R alpha 13) isotypes effectively and specifically recognize both the human and mouse RAR alpha protein. Preincubation of the antibodies with the synthetic RAR alpha peptide, but not with the RAR beta or RAR gamma peptides, blocked recognition of the approximately 55 kDa RAR alpha protein on western blots. These monoclonal antibodies also detected differing levels of RAR alpha in various rat tissues. These monoclonal antibodies will serve as powerful reagents to study the structure and regulation of the retinoic acid receptor protein.

Amino Acid Sequence↗
PubMed1991

Depletion of rat liver glucocorticoid receptor hormone-binding and its mRNA in sepsis.

Glucocorticoid receptor (GR) hormone-binding activity, its physical characteristics, and GR mRNA levels were studied in the liver, brain and muscle of normal (saline-injected) and hypermetabolic septic rats 24 h after the subcutaneous injections of E. coli. The GR levels (hormone-binding activity) declined by about 40%, 56%, and 40% in septic liver, brain, and muscle cytosol, respectively. The mechanism of the decrease in the GR levels in sepsis was studied in liver. The GR levels remained low (45% of control hormone-binding) even after 48 h of E. coli administration. The decrease in the liver GR occurred in the 9S untransformed GR. The 9S GR from septic liver transformed to the 4S form in proportions comparable to the control liver GR. In addition, the 4S GR from control and septic liver was capable of binding to DNA-cellulose to a similar extent. The GR mRNA level in septic liver declined by about 30%. Thus, a decrease in GR hormone-binding activity in sepsis appears to be due to a decline in the steady-state GR mRNA level and not from a change in the qualitative properties of the GR protein.

Animals↗
PubMed1991

Transfer RNA nucleoside composition in 13762 rat adenocarcinoma.

1. Abnormalities in patterns of tRNA methylation and in the activities of tRNA methyltransferases are well-documented phenomena. In this study, we focused our attention on tRNA from adenocarcinoma, a 9,10-dimethyl-1,2-benzanthracene-induced mammary tumor, because prior evidence has suggested the occurrence of an abnormal pattern of tRNA methylation. 2. Chemical postlabeling of tumor vs normal rat liver and mammary gland tRNAs revealed tumor specific differences in the modified nucleoside distribution, i.e., a 5.8-fold increase in tumor N-2-methylguanosine together with a 2.7-, 2.8-, 2.6- and 2.8-fold decrease in tumor 1-methyladenosine, dihydrouridine, pseudouridine and 5-methylcytidyne, respectively. 3. Class A tRNAs, a slower gel migrating group of tumor tRNAs, exhibited even lower 1-methyladenosine levels. Most of the remaining nucleosides in class A tRNAs showed molar ratios similar to those found in bulk tumor tRNA. However, N-2-methylguanosine levels in class A tRNA are intermediate between bulk tumor tRNA (2.8%) and mammary gland tRNA (0.49%). 4. The only qualitative difference found in tumor tRNA seems to be the absence of inosine usually present in tRNAs from liver and mammary tissues. 5. In spite of its abnormal methylation pattern adenocarcinoma tRNA binds to glucocorticoid receptor protein from mouse AtT-20 cells, generating a 6S tRNA-protein complex, in a fashion similar to that previously described for the endogenous tRNA isolated from the same cells.

Adenocarcinoma↗
PubMed1989

Regulation of glucocorticoid receptor protein and mRNA levels.

The level of steroid receptors in target cells affects the responsiveness of the cell to the hormone. In mouse AtT-20 cells, it has been shown that chronic glucocorticoid treatment causes a down-regulation of glucocorticoid receptor (GR) levels (F. Svec and M. Rudis, J. Biol. Chem., 256:5984-5987, 1981). The current study shows that chronic hormone treatment reduces the amount of GR mRNA to about 50% of that in untreated cells. A combined treatment of the cells with an inhibitor of RNA transcription and the glucocorticoid hormone causes a more rapid decrease in steady-state GR mRNA levels than either agent alone. This suggests that glucocorticoids regulate the expression of the GR gene posttranscriptionally, perhaps via destabilization of the GR mRNA. An additional transcriptional regulation by the steroid hormone is not ruled out by this observation. It was also found that heat shocking a variety of cell types at 42 degrees C not only causes an induction of heat shock proteins but also results in a dramatic decrease in the level of glucocorticoid-binding activity. GR labeled with a covalent ligand (dexamethasone 21-mesylate) was also reduced by heat shock, implying that heat shock caused an increased degradation in the GR protein itself. Finally, in vitro studies show that the GR is degraded in an ATP- and tRNA-dependent fashion in rabbit reticulocyte lysate. It therefore seems likely that the GR is degraded by the ubiquitin-dependent proteolytic pathway. Because ubiquitin is itself a heat shock protein, this may be the reason that the GR is rapidly degraded in heat-shocked cells. These studies point to possible mechanisms whereby the responsiveness of the cell to steroid hormones is altered by the regulation of the steroid receptor protein and mRNA levels.

Animals↗
PubMed1987

Interaction of RNA with transformed glucocorticoid receptor. I. Isolation and purification of the RNA.

The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.

Animals↗
PubMed1987

Interaction of RNA with transformed glucocorticoid receptor. II. Identification of the RNA as transfer RNA.

An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W. V. (1987) J. Biol. Chem., 262, 6771-6777). We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA. 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics. The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase. The purified [3H] aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form. The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides. The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the [3H]aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer. PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA. The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs. The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form. However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins. Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA). The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form. 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S. That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients. These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA.

Amino Acids↗