Abstract
The level of expression of the glucocorticoid receptor (GR) is the principal determinant of glucocorticoid sensitivity in most cells. GR levels are permanently ‘set’ in a tissue-specific manner in response to the perinatal environment, an effect we have previously shown to relate to differential expression of tissue-enriched alternative promoters/exons 1 of the GR gene. In adult animals, GR levels are dynamically regulated around the ‘set point’ by glucocorticoids themselves, with glucocorticoids down-regulating GR mRNA in most cells and tissues. Here we have examined whether autoregulation of GR mRNA by glucocorticoids involves differential promoter regulation. We show that, in contrast to tissue-specific programming of GR mRNA levels, autoregulation of GR mRNA in vivo does not involve differential regulation of variant exon 1-containing GR mRNAs in that the major variants are down-regulated to a similar extent by glucocorticoid treatment. Consistent with this, transfections of reporter constructs showed that the majority of GR promoters, which are contained within a 4.4 kb region upstream of exon 2, are similarly regulated by glucocorticoids, with two regions of the promoter redundantly required for glucocorticoid regulation. Thus transcriptional autoregulation can occur in adult tissues around the set point established by promoter selection in early life.
Introduction
Glucocorticoids exert effects on virtually all tissues, the majority of which are mediated by the type II glucocorticoid receptor (GR). Despite near ubiquitous expression, GR levels vary widely both between and within tissues (Reul et al. 1985, 1989). The level of GR expression is one of the principal determinants of glucocorticoid sensitivity (Vanderbilt et al. 1987) and both reduced and increased GR levels in transgenic mice alter glucocorticoid signalling with pathophysiological consequences (Pepin et al. 1992, King et al. 1995, Reichardt et al. 2000, Pazirandeh et al. 2002). Perinatally, GR levels are programmed to a ‘set point’ by environmental manipulations, including neonatal stress (de Kloet et al. 1990) or treatment with dexamethasone in utero (Nyirenda et al. 1998, Cleasby et al. 2003). However, in adults dynamic autoregulation by glucocorticoids around the ‘set point’ is perhaps the major short-term level of control over GR levels. In most cells and tissues, glucocorticoids decrease GR levels in a GR-dependent manner. However, there are important exceptions to this, notably some lymphoid cell lines in which glucocorticoids increase GR (Eisen et al. 1988). Furthermore, in vivo the effects of glucocorticoid manipulation are time- and tissue-dependent (see for example Sheppard et al. 1990, Holmes et al. 1995, 1997). Auto-regulation operates largely at a transcriptional level (Dong et al. 1988, Rosewicz et al. 1988, Burnstein et al. 1994), although post-translational mechanisms are also involved (Dong et al. 1988).
The GR gene is large, spanning over 100 kb. Transcription starts from a number of sites generating alternate exons 1, which are conserved between rats, mice and humans (Strähle et al. 1992, Gearing et al. 1993, Chen et al. 1999, McCormick et al. 2000, Breslin et al. 2001, Reynolds 2002). The 5′-heterogeneity of GR mRNA however, does not affect the encoded protein as the translation start lies within exon 2, common to all transcripts, and is preceded by an in-frame translation stop codon. We have previously shown that at least 11 potential exons 1 exist for the rat GR gene, six of which are present in GR mRNA in vivo (McCormick et al. 2000). The relative proportion of these variant GR mRNAs differs between different cell types (Strähle et al. 1992, McCormick et al. 2000) although in most rat tissues one variant (containing exon 110) predominates, being present in ≥50% GR mRNA (McCormick et al. 2000). Expression of some GR mRNA variants is restricted to certain tissues. Of the hippocampus, liver and thymus, exons 15 and 17 are specific to hippocampus, while exon 11 was found in thymus but not in the other tissues (McCormick et al. 2000). Interestingly, perinatal manipulations that permanently increase GR expression levels differentially regulate GR mRNA variants. Neonatal handling selectively increased hippocampal expression of GR mRNA containing the brain-specific exon 17, while prenatal glucocorticoid exposure decreased the proportion of the predominant GR mRNA variant containing exon 110 in liver, suggesting an increase in usage of a minor exon 1 variant (McCormick et al. 2000). Similar tissue-specific, alternately untranslated first exons occur in other nuclear receptor genes (Kastner et al. 1990, Leroy et al. 1991, Shi et al. 1992, Flouriot et al. 1998), including the closely related mineralocorticoid receptor (MR) (Kwak et al. 1993), with differential promoter regulation exerted by an autologous ligand (Leroy et al. 1991, Kwak et al. 1993, Zennaro et al. 1996, Flouriot et al. 1998).
Here, we address the dynamic regulation of GR mRNA levels by glucocorticoids and specifically investigate whether GR mRNA variants are differentially regulated in vivo in the hippocampus, liver and thymus and in vitro in transfected cells.
Materials and Methods
Experimental animals
Adult male Wistar rats (3 months, 200–250 g) were maintained under controlled lighting (lights on from 0700 to 1900 h) and temperature (22 °C) with water and food available ad libitum. To determine whether glucocorticoids differentially regulate GR mRNA variants, rats were adrenalectomised or sham operated under halothane (Fluothane, Merial, Harlow, UK) anaesthesia, followed by glucocorticoid replacement (or vehicle treatment) for 72 h. Doses of corticosterone acetate were chosen to approximate physiological or high physiological levels (0.2 mg/kg/day and 2 mg/kg/day, respectively) and were administered daily by subcutaneous injection at 1600 h. Sham operated controls were given vehicle injections daily. All animals were killed 72 h post-operatively between 0900 h–1000 h. Plasma corticosterone levels at time of sacrifice were: sham, 980 ± 260 nM; adrenalectomised with vehicle, 0.8 ± 0.5 nM; adrenalectomised with 0.2 mg/kg/day corticosterone acetate, 46 ± 10 nM; adrenalectomised with 2 mg/kg/day corticosterone acetate, 120 ± 35 nM.
All procedures were approved by the UK Home Office and were performed in strict accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
RNase protection assays
Total RNA was isolated from tissues by extraction with TRIzol (Invitrogen, Paisley, UK). Integrity was verified by electrophoresis on formaldehyde-agarose gels. For preparation of thymocytes and residual thymic epithelium, thymi were teased apart using syringe needles to release thymocytes, which were then cleared of contaminating erythrocytes using red blood cell lysis buffer (Sigma). Thymus tissue remaining was considered to be chiefly thymic epithelium. Exon 1-specific cRNA probes were synthesised as previously described (McCormick et al. 2000), and contain 186 nucleotides complementary to exon 2 (common to all GR mRNA variants) as well as exon 1. Assays were performed using a HybSpeed RPA Kit (Ambion Inc., Austin, TX, USA) as previously described (McCormick et al. 2000) and quantitated using a Fujifilm FLA-2000 phosphorimager and Aida v2.0 software (Raytek, Sheffield, UK). Actin cRNA was used as an internal control and was transcribed from pTRI-β-actin-125-Rat (Ambion Inc) using SP6 polymerase. The abundance of each exon 1 was expressed relative to total GR mRNA (calculated using the intensity of the exon 2-protected fragment), to assess any changes in the proportion of each variant in the total GR mRNA population between groups, or relative to actin, to assess any differences in the total amount of each variant between groups. For the latter, mean adx/veh values were arbitrarily set to 100% and other groups expressed relative to adx/veh.
In situ mRNA hybridisation
[35S]-UTP-labelled RNA probes were synthesised as previously described (Seckl et al. 1990). After DNase I treatment, unincorporated nucleotides were removed by passage over a Sephadex G-50 Nick column (Pharmacia Biotech, St Albans, UK). Exon 15-, 17- and exon 2-specific probes were generated from plasmids with subcloned exon 1-specific PCR products generated as described (McCormick et al. 2000). The template used to synthesise exon 11-specific cRNA was a subcloned RT-PCR product generated from mouse thymus total RNA using the following oligonucleotides: 5′ primer (5′-CCAA AGAGGAGTCACTGTA-3′), 3′ primer (5′-TCTGAA ACATCTTCCTGGCT-3′). The template used to synthesise exon 110-specific cRNA was a subcloned PCR product which was generated as previously described (McCormick et al. 2000). In situ mRNA hybridisation was performed as previously described (Seckl et al. 1990, Yau et al. 1997). Densitometry was performed using MCID-M4 version 3.0 (revision 1.5) software (Imaging Research Ltd, St Catherine’s, Ontario, Canada).
Cell culture and transfections
B103 (rat neuroblastoma) cells were maintained and transfected as previously described (McCormick et al. 2000). As B103 cells lack endogenous GR (unpublished observations), a GR expression plasmid, pRShGR (Giguere et al. 1986) was co-transfected. Following transfection, cells were treated with either vehicle or 10−6M dexamethasone. Plasmids P2, P16 and P17 have been previously described (McCormick et al. 2000). P2 contains a 4.4 kb fragment of the rat GR gene encoding −4438 to −9 (the ATG translation start is designated +1) which includes end of exon 2, fused to a luciferase exons 14–111 and the 5′ reporter gene. P16 and P17 encode fragments encoding −4438 to −3336 and −4438 to −2931 respectively, fused to luciferase. P18 and P19/10 are similar to P16, but contain fragments encoding −4438 to −2803 and −4438 to −2532 respectively, fused to luciferase. A 5′ deletion series of P2 was constructed by removal of appropriate restriction fragments to give P2(Δ− 3911), P2(Δ − 2808), P2(Δ − 2537) and P2(Δ − 1772).
Statistical analysis
Statistical analysis was performed by ANOVA followed by Fisher’s LSD Test. Significance was taken as P < 0.05.
Results
Glucocorticoids decrease variant exon 1-containing GR mRNAs to the same extent as total GR mRNA in vivo
Liver showed clear zonal expression of total GR mRNA, with higher expression periportally in all groups (Fig. 1A). Glucocorticoid treatment in adrenalectomised animals reduced GR mRNA to the same extent in periportal and perivenous regions compared with animals receiving vehicle, whereas adrenalectomy itself significantly increased GR mRNA levels only in periportal regions compared with sham-operated controls (Fig. 1B). As the largest difference in GR mRNA levels was seen between adrenalectomised animals receiving vehicle or physiological corticosterone, these groups were analysed for changes in variant GR mRNAs. GR mRNA variants containing exon 110 and exon 16 predominate in liver (McCormick et al. 2000). Both mRNA variants showed the same pattern of regulation as total GR mRNA (Fig. 1C and data not shown), with glucocorticoid replacement reducing expression of both 110- and 16-containing mRNAs by approximately 50%, compared with vehicle-treated control animals (Fig. 1C). In situ mRNA hybridisation experiments did not reveal any differential regulation of GR mRNA variants between periportal and perivenous areas (data not shown). Furthermore, the proportion of GR mRNA containing the predominant exon 110 (~ 65%) was unaffected by glucocorticoid manipulation (Fig. 1D), suggesting that differential promoter regulation of any of the other alternate exons 1 could only play a minor role in regulation of the total hepatic pool of GR mRNA. Indeed, although glucocorticoid treatment caused a small but significant decrease in the proportion of GR mRNA containing exon 16 (from 16% to 13% of the total) this effect could only account for a very small part of the overall decrease in total GR mRNA concentration and any biological significance is moot. Levels of GR mRNA containing exons 15 and 111 were below the level of detection in total liver RNA from any experimental group (data not shown).
Glucocorticoid replacement in adrenalectomised animals caused a decrease in total GR mRNA in both thymic cortex and medulla compared with vehicle treated control animals (Fig. 2A, B). RNase protection assays performed on total thymus RNA showed that the major GR mRNA variants expressed in thymus (containing exons 11, 16 and 110) showed a similar pattern of regulation by glucocorticoids to total GR mRNA between groups (Fig. 2C and data not shown). Glucocorticoid replacement decreased exon 11-containing GR mRNA by~ 50% and 110-containing GR mRNA by ~ 40%. Although the decrease in exon 16-containing GR mRNA did not quite reach statistical significance (P=0.07), there was a clear trend for glucocorticoid replacement to decrease levels to a similar extent to the other exons 1. Importantly, glucocorticoid treatment did not significantly alter the proportion of GR mRNA transcripts containing any of the variant exons 1 in the thymus (Fig. 2D), consistent with a similar regulatory effect of glucocorticoids on all variants. In addition, it is unlikely that there is differential regulation of exon 11 in a small subset of expressing cells as in untreated rats the proportion of GR mRNA that contains exon 11 (expressed as mean % of total GR mRNA± s.e.m., n=2–5) does not significantly differ between thymus (23 ± 10%), spleen (20 ± 10%), purified thymocytes (25 ± 3%) and thymic epithelium depleted of thymocytes (20 ± 9%). In support of this, no differential regulation of exon 1 variant mRNAs was seen using in situ mRNA hybridisation (data not shown).
In hippocampus, adrenalectomy increased total GR mRNA across all subfields, while low-dose corticosterone treatment to ‘replacement’ levels restored GR mRNA to levels similar to sham-operated controls (Fig. 3A). Administration of high dose corticosterone resulted in higher GR mRNA levels compared with replacement with more physiological levels (Fig. 3A). The overall pattern of hippocampal expression of GR mRNA variants containing exons 15, 17, 110 or 111 between groups was very similar and closely resembled that of total GR mRNA (Fig. 3B and data not shown), suggesting that none of the variants is differentially regulated. However, erences between groups did apart from 111 in CA1, the did not quite achieve statistical significance (for example, P=0.06 for in 17 CA3 and DG and for 111 in CA3), probably due to the low levels of expression of individual GR mRNA variants and lower signal/noise ratio for these short cRNA probes in the in situ mRNA hybridisation experiments. Exon 11-containing GR mRNA was not detectable in hippocampus by in situ mRNA hybridisation (data not shown), consistent with previous data from RNase protection assays (McCormick et al. 2000).
Alternate glucocorticoid receptor promoters are similarly regulated by glucocorticoids
The above results show that GR mRNA variants are similarly regulated by glucocorticoids in vivo. With the exception of the promoter that drives expression of the 11 variant, transcription of all of the other major variants initiates within a large CpG island located close to exon 2 (McCormick et al. 2000). To determine whether the CpG island promoters are glucocorticoid regulated, we transiently transfected B103 rat neuroblastoma cells (Schubert et al. 1974) with reporter construct P2, in which a 4.4 kb region encompassing the CpG island (including exons 14–111) is fused to luciferase within exon 2, just prior to the translation start of GR (McCormick et al. 2000) (Fig. 4A). Dexamethasone caused a significant decrease in P2 activity (Fig. 4B) that was dependent upon co-transfected GR expression plasmid (data not shown).
To determine whether all promoters were similarly affected by glucocorticoid, a 3′ deletion series of P2 was co-transfected into B103 cells with pRShGR. This series, in which each of the alternate exons 1 is fused directly to luciferase, has been previously described (McCormick et al. 2000) (and see Fig. 4A). Luciferase activity arises from chimaeric RNA transcripts encoding part of an alternate exon 1 of the GR gene at the 5′ end and represents the activity of the promoter which directs transcription through that individual exon 1. Although transcription may additionally initiate from alternate promoters present on the same genomic DNA fragment, these transcripts are not transcriptional fusions to luciferase and do not give rise to luciferase activity (there is no splice acceptor site upstream of the luciferase gene in these constructs).
The activity of all constructs in the 3′ deletion series was similarly decreased by dexamethasone (Fig. 4B). Thus, a region between −4438 and −3336, present in all constructs, appears to be dexamethasone-sensitive. To further delineate the dexamethasone-sensitive region(s) a 5′ deletion series of P2 was transiently co-transfected into B103 cells with pRShGR. Dexamethasone significantly decreased the activity of P2(Δ −3911) and P2(Δ −2808). However, glucocorticoid repression was lost with further deletion to −2537; neither P2(Δ −2537) nor P2(Δ −1772) were responsive to dexamethasone. These data therefore show the presence of two glucocorticoid-sensitive elements within the GR promoter; one between −4438 and −3336 and a second between −2808 and −2537.
Discussion
Here, we show that in contrast to MR (Kwak et al. 1993, Zennaro et al. 1996), GR mRNA variants, arising from differential promoter usage, are not differentially regulated by glucocorticoids in vivo, nor are the promoters that direct transcription of the majority of mRNA variants differentially regulated in vitro. We find that two regions of the rat GR CpG island promoter region contribute to glucocorticoid-sensitivity of the rat GR gene; the upstream region appearing to confer similar glucocorticoid repression upon transcription initiating from all downstream start sites.
Consistent with previous data (Kalinyak et al. 1987, Reul et al. 1989, Spencer et al. 1991), total GR mRNA levels in hippocampus, liver and thymus were decreased by 72 h glucocorticoid replacement in adrenalectomised rats, with close to a two-fold reduction in all three tissues. For each tissue, the pattern of glucocorticoid regulation of GR mRNA variants between the groups was similar, with no evidence for a striking differential decrease in one (or more) variants that could account for the down-regulation of total GR mRNA. In support of this, the proportion of each mRNA variant within the total pool of GR mRNA was either unchanged, or changed very little, by glucocorticoid treatment. In addition, we found no evidence for region-specific differential regulation of mRNA variants in any of the tissues examined; within subfields of hippocampus, between periportal and perivenous regions of liver, or between thymic cortex and medulla. These data rule out the possibility that one or more of the mRNA variants reflect constitutive transcription initiating at a subset of the GR transcription start sites within the CpG island, with others being hormonally regulated.
Mouse and human T-cell lines, which are sensitive to the pro-apoptotic effects of glucocorticoids, up-regulate GR mRNA levels in response to glucocorticoids (Eisen et al. 1988, Tonko et al. 2001). In the case of human CEM-7 cells this has recently been shown to involve increased 1A (equivalent to 11 in rat) promoter activity (Breslin et al. 2001). However, with 72 h glucocorticoid replacement in adrenalectomised rats, we found the proportion of GR mRNA containing exon 11 was unchanged in thymus, ruling out a discordant effect on exon 11 and the downstream promoter region encompassing exons 14–111. There are a number of possible explanations for this finding. First, the up-regulation of promoter 1A/11 by glucocorticoids may be restricted to a specific minor subset of thymocytes, masked in our experiments. However, GR mRNA containing exons 11 and 110 are expressed at similar levels (≈20–25% and 50–70% of total GR mRNA respectively) in whole thymus, thymocytes, thymic epithelium and spleen, and both mRNA variants showed the same distribution as total GR mRNA in the in situ mRNA hybridisation after glucocorticoid manipulations. Second, it is possible that apoptosis was rapidly induced in thymocytes in which the 11 variant was up-regulated; thus these cells would have already been eliminated leaving only cells in which GR mRNA was down-regulated by glucocorticoids. Although glucocorticoid-induced apoptosis occurs over ~ 48 h in T-cell lines, evidence suggests that it occurs much more rapidly (over 24 h) in thymocytes (reviewed in (Thompson 1999)). However, if this was the case, we would expect that the proportion of GR mRNA containing exon 11 would be decreased by glucocorticoids, and this was not apparent even though effects of glucocorticoids upon thymus size and cellularity are significant at 72 h. Third, and most likely, all the mRNA variants may be similarly regulated in all cells. Pedersen and Vedeckis have recently shown that although the magnitude of glucocorticoid regulation of mouse GR mRNA variants (including 1A) differs between variants, the overall direction of regulation (up or down) is dependent on cell type and does not differ between mRNA variants (Pedersen et al. 2003). Thus, in vivo, although we cannot rule out differential regulation of promoter activity over the short term (24 h), there is no differential regulation apparent over 72 h.
Since the changes in total GR mRNA expression with glucocorticoid manipulations are not accounted for by changes in expression of a specific exon 1, this suggests that the mechanism by which glucocorticoids regulate GR expression is by uniformly altering activity of all the promoters of the GR gene, rather than by inducing changes in the activity of specific alternate promoters in different tissues. This was supported by the transfection experiments in which all constructs in the 3′ deletion series were down-regulated by dexamethasone. These constructs share a common region between −4438 and −3336, suggesting that this area contains elements associated with dexamethasone sensitivity which must therefore exert a repressive effect on transcription from downstream initiation sites in response to dexamethasone. An additional region involved in glucocorticoid autoregulation of GR between −2808 and −2537 was identified using a 5′ deletion series. The effect of the two glucocorticoid-responsive regions was not additive. Previous work using constructs in which a region of the human GR promoter was fused directly (within exon 1C, equivalent to rat exon 110) to a CAT reporter gene showed a similar level of dexamethasone-associated repression when transfected into CV-1 cells (Leclerc et al. 1991). Repression was lost with deletion to −470, relative to the transcription start of exon 1C, which corresponds to −2763 on the rat GR promoter. Furthermore, a 500 bp region between −750 and −250 relative to the transcription start of exon 1C (corresponding to the region between −3156 and −2656 on the rat GR promoter) conferred orientation-dependent repression upon a heterologous promoter (Leclerc et al. 1991). This region overlaps with the dexamethasone-sensitive element between −2808 and −2537, localised during this investigation (Fig. 5), and suggests that the glucocorticoid-responsive region may lie towards the 5′ end of this region. The previously noted orientation-dependence of the repression is intriguing, as the 500 bp region of the human promoter contains the homologous sequence to exons 17, 18 and the 5′ part of 19, all of which can be detected by RT-PCR in human GR mRNA (Reynolds 2002), suggesting that the repression may be mediated directly upon the promoters within that region. In our experiments, we found that promoter fusions of P17, 18 and 19/10 were all glucocorticoid repressed. It is unclear at present how the autoregulation of GR gene transcription is effected. GR negatively regulates transcription through several different mechanisms, including DNA-independent (for example, by interfering with AP-1- or NF-kB- mediated transcriptional activation; reviewed by de Bosscher et al. (2003)) and DNA-dependent means (for example, by binding to negative glucocorticoid response elements, nGREs (de Bosscher et al. 2003)). However, sequence analysis of the two glucocorticoid-sensitive regions did not reveal the presence of any sequences likely to act as nGREs, nor were there any sequences showing a close resemblance to AP-1, or other known cis-acting sites within the two regions.
Thus, although perinatal programming of GR expression is associated with permanent changes in activity of specific promoters, which subsequently determines the ‘set-point’ of GR expression in different tissues (McCormick et al. 2000), differential promoter usage plays little, if any, role in the dynamic autoregulation of GR expression by glucocorticoids. The molecular mechanisms that determine GR mRNA levels in early life, events which have been postulated as central to the ‘foetal origins of disease’ (Seckl et al. 2000), may involve epigenetic changes to the GR gene chromatin structure which are subsequently maintained throughout the lifetime of the animal, providing a persistent adaption to the prevailing environment at the time of birth. In contrast, the dynamic autoregulation of GR expression will provide short-term adaptions to stressful events, occurring during adult life.
GR mRNA is expressed in a clear zonal manner in liver and glucocorticoids down-regulate expression of GR mRNA containing variant exons 16 and 110. (A) Representative autoradiographs of in situ mRNA hybridisation showing higher expression of total GR mRNA in periportal (pp) than in perivenous (pv) regions of liver from adrenalectomised animals receiving receiving vehicle (adx/veh), 0.2 mg/kg/day corticosterone (adx/phys), or sham operated controls (sham). ‘Sense’ indicates background levels of hybridisation to the corresponding sense control RNA probe. (B) In situ mRNA hybridisation shows similar down-regulation of GR mRNA levels in both periportal and perivenous regions of liver. All values are based on silver grain counts and are expressed relative to GR mRNA levels in the corresponding region of adrenalectomised animals receiving vehicle (adx/veh), arbitrarily set to 100%. Glucocorticoid treated animals received 0.2 mg/kg/day corticosterone (adx/phys) or 2 mg/kg/day corticosterone (adx/high). (C) Data from RNase protection assays showing decreased levels of GR mRNA variants, relative to actin mRNA, in livers of adrenalectomised animals receiving 0.2 mg/kg/day corticosterone compared with those receiving vehicle, arbitrarily set to 100%. (D) Glucocorticoid treatment has little or no effect on the proportion of total GR mRNA containing exon 16 or exon 110. Shaded bars, adx/veh; solid bars, adx/phys. Data are mean ± s.e.m., n = 5–8 per group. *, P<0.05 (compared with adx/veh).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05773
GR mRNA variants are glucocorticoid-regulated in thymus, in a similar manner to total GR mRNA. (A) Representative autoradiographs of in situ mRNA hybridisation showing higher levels of total GR mRNA in thymic cortex (c) than medulla (m) of adrenalectomised animals that received vehicle (adx/veh), 0.2 mg/kg/day corticosterone (adx/phys), or that were sham-operated (sham). ‘Sense’ indicates background levels of hybridisation to the corresponding sense control RNA probe. (B) In situ mRNA hybridisation shows similar glucocorticoid down-regulation of GR mRNA levels in both thymic cortex and medulla. Glucocorticoid-treated animals received 0.2 mg/kg/day corticosterone (adx/phys) or 2 mg/kg/day corticosterone (adx/high). Values represent optical density measurements and are expressed relative to GR mRNA levels in the corresponding region of adrenalectomised animals receiving vehicle (adx/veh), arbitrarily set to 100%. (C) Data from RNase protection assays showing levels of GR mRNA variants, relative to actin mRNA, in livers of adrenalectomised animals receiving 0.2 mg/kg/day corticosterone (adx/phys) compared with those receiving vehicle (adx/veh), arbitrarily set to 100%. (D) Glucocorticoid treatment has no effect on the proportion of total GR mRNA containing alternate exons 1. Shaded bars, adx/veh; solid bars, adx/phys. Data are mean ± s.e.m., n = 5–8 per group.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05773
Total GR mRNA levels in hippocampus are decreased by glucocorticoid treatment, without differential regulation of variant exon 1-containing mRNAs. Levels of GR mRNA levels were measured by in situ mRNA hybridisation and analysed by densitometry. (A) Levels of total GR mRNA in CA1, CA3 and CA4 subfields of hippocampus and dentate gyrus (DG) in all groups, sham adrenalectomised animals (sham) and adrenalectomised animals receiving vehicle (adx/veh), 0.2 mg/kg/day corticosterone (adx/phys) or 2 mg/kg/day (adx/high). GR mRNA levels in CA1 of sham animals were arbitrarily set to 100. (B)–(E) Changes in levels of GR mRNA variants containing alternate exons 1 in adrenalectomised animals receiving 0.2 mg/kg/day corticosterone (adx/phys, solid bars) relative to those receiving vehicle (adx/veh, shaded bars), arbitrarily set to 100%. Data are mean ± s.e.m., n = 5–8 per group. *, P<0.05 (compared with adx/veh).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05773
Dexamethasone decreases GR promoter activity. (A) Diagrammatic representation of constructs used in transfection assays. Restriction fragments containing regions of the rat GR gene were fused to a luciferase reporter gene. Constructs in the 3′ deletion series (P19/10, P18, P17 and P16) are fused within individual exons 1 and do not contain a splice acceptor site; constructs in the 5′ deletion series (P2Δ −3912, P2Δ −2806, P2Δ −2535 and P2Δ −1770) all contain the splice acceptor site at the beginning of exon 2. (B) and (C) Dexamethasone repression of promoter activity in B103 neuroblastoma cells co-transfected with pRShGR and the 3′ deletion series (B) or the 5′ deletion series (C) of P2. Activity of P2 in the absence of dexamethasone was nominally set to 100%, and activity of all other constructs expressed relative to that value. Values represent means ± s.e.m. of at least 6 independent transfections. *, P<0.05 (compared with vehicle-treated control).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05773
The sequence of the glucocorticoid-responsive region of the rat GR promoter region between −2808 and −2537. The rat GR promoter region between −2808 and −2537 is shown, aligned with the corresponding region of the human GR gene promoter. Relevant restriction sites used to generate reporter constructs are indicated with a solid underline. A deletion of the human GR gene promoter, which results in loss of glucocorticoid repression, overlaps with the rat GR gene promoter and the overlapping region is indicated with a dashed underline.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05773
(Alistair I Freeman and Helen L Munn contributed equally to this work)
We are very grateful to June Noble for help with animal surgery and to Clare Blackburn for advice on thymus histology. We thank members of the Molecular Endocrinology Group, especially Chris Kenyon, Megan Holmes, Mark Cleasby, Brian Walker and Joyce Yau for many helpful discussions and for comments on the manuscript.
Funding
This work was supported by a Wellcome Trust Programme Grant and project grants from the Wellcome Trust. AIF was supported by a 5 year veterinary PhD award from the Wellcome Trust, HLM was supported by a 4 year PhD award from the Wellcome Trust. The authors are not aware of any conflicts of interest that would prejudice the impartiality of this work.
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