00l3-7227/91/1294-2166$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 4 Printed in U.S.A.
Hormonal Regulation of Type II Glucocorticoid Receptor Messenger Ribonucleic Acid in Rat Brain* ANDY PEIFFER, BENOIT LAPOINTE, AND NICHOLAS BARDEN Laboratory of Molecular Psychogenetics, CHUL Research Centre and Laval University, Ste-Foy, Quebec, Canada GlV 4G2
tomy only in amygdala. Corticosterone increased amygdala transcript levels in OVX and ADX/OVX animals. Estradiol administration to intact animals raised the GR mRNA content of amygdala, while progesterone treatment had no effect on any of the brain regions studied. We conclude that there exists heterogeneity with respect to type II GR mRNA regulation by corticosterone and dexamethasone in brain regions of ADX female rats, and that certain limbic structures show greater sensitivity to these hormonal manipulations, suggesting a more prominent role in the regulation of the hypothalamic-pituitary-adrenal axis. Our results also suggest that circulating estrogens can influence the sensitivity of brain structures (i.e. hypothalamus and amygdala) to glucocorticoids by altering GR mRNA levels. These regions may represent integration sites at which gonadal steroids are able to alter stress hormone secretion. {Endocrinology 129: 2166-2174, 1991)
ABSTRACT. Differences in the regulation of type II glucocorticoid receptor (GR) mRNA levels in female rat brain regions involved in the control of the hypothalamic-pituitary-adrenal axis were studied by Northern blot analysis after chronic administration of corticosterone or dexamethasone to adrenalectomized (ADX), ovariectomized (OVX), and ADX/OVX animals. The effect of chronic estradiol or progesterone treatment of intact animals was also studied. Our results show that type II GR mRNA levels of ADX animals were significantly increased above control values in amygdala (140%) and hippocampus (196%), but not in hypothalamus. These increased transcript levels were down-regulated by corticosterone or dexamethasone, with the exception of those in the amygdala, where corticosterone had no effect. Ovariectomy significantly increased hypothalamic GR mRNA content (174%) over control values, and this increase was sensitive to dexamethasone. The combined effect of adrenalectomy/ovariectomy on GR mRNA levels was greater than that of adrenalec-
I
N THE central nervous system glucocorticoids influence neuronal excitability and neurotransmitter metabolism, alter protein synthesis, and regulate hypothalamic-pituitary-adrenal (HPA) axis activity (1, 2). Intracellular glucocorticoid receptors (GR) confer tissue responsiveness to circulating glucocorticoids and mediate their genomic and posttranslational effects (3). GR in brain structures were first reported by McEwen et al. (4), and the presence of two distinct GR systems in rat brain has since been established. The type I GR has high affinity for corticosterone (5), the endogenous glucocorticoid in rat, and resembles the rat kidney mineralocorticoid receptor with respect to biochemical and structural properties (6-8). The type II GR, found in nearly all peripheral tissues, binds corticosterone with lower affinity, but has high affinity for the synthetic ligand dexamethasone (9). Each receptor has a distinct localization pattern in rat Received April 29, 1991. Address all correspondence and requests for reprints to: Dr. Nicholas Barden, Laboratory of Molecular Psychogenetics, CHUL Research Centre, 2705 boulevard Laurier, Ste Foy, Quebec, Canada GlV 4G2. * This work was supported by a grant from the Medical Research Council of Canada (to N.B.).
brain, with high concentrations of type I receptor binding in lateral septum, subiculum, and hippocampus (10). The type II receptor has a much wider distribution in rat brain, with particularly high density in the lateral septum, the CAl and CA2 hippocampal fields, certain brainstem nuclei, and the hypothalamic paraventricular nuclei (11). Colocalization of both GR subtypes to certain brain regions (e.g. hippocampus) has evoked the possibility that these structures are able to respond in a binary manner to circulating glucocorticoids (12, 13). Thus, occupation of the high affinity, low capacity type I sites may vary throughout the circadian rhythm and be complete at basal corticosterone levels, while the lower affinity type II sites may be fully occupied during stress when corticosterone levels are high. Binding studies have shown that circulating glucocorticoids regulate the tissue GR receptor concentration in in vitro systems (14), peripheral tissues (15), and neural structures, where differential down-regulatory responses to glucocorticoids have been shown depending upon brain region (16, 17). Heterologous regulation of GR levels in 1 brain by hormones other than adrenal steroids, such as insulin, thyroid hormone, vasopressin, and ACTH, has
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BRAIN GR mRNA REGULATION
also been described (18-20). Sex differences in rat brain glucocorticoid binding, which are further amplified by ovariectomy (OVX), have been reported by Turner and Weaver (21). Furthermore, brain GR concentrations have been shown to vary as a result of developmental stage, neonatal handling, early corticosterone exposure, and aging (18, 22, 23). With the recent cloning of rat, human, and mouse GR cDNAs (24-26), it has become possible to study the distribution and regulation of brain GR mRNA. Homologous down-regulation of GR has been shown to involve changes in GR mRNA levels, and glucocorticoids exert this control by influencing GR gene transcription (27, 28). In situ hybridization studies have mapped out GR mRNA distribution in rat brain (29) and have shown that transcript levels vary within different neuronal subfields of hippocampus (30). Recently, Herman et al. (31) have demonstrated that the sensitivity of type I and type II GR mRNA levels to regulation by steroids differs depending upon localization within the hippocampus. Differences in steroid hormone regulation of GR mRNA transcripts is also tissue specific (32), a phenomenon which is present early in development (33). Age-related changes in brain type II receptor mRNA levels occur, and these are most pronounced in hippocampus (34). The purpose of this study was 3-fold. The first objective was to study differences in the regulation of type II GR mRNA levels in brain regions of adrenalectomized (ADX) female rats after chronic administration of dexamethasone or corticosterone. Since type II receptors have differing sensitivities to these two steroids and show tissue-specific distribution in brain, it is possible that regulation of type II GR mRNA levels by glucocorticoids differs depending upon the brain region. Our results broaden the conclusion of previous reports by showing response heterogeneity of type II GR mRNA regulation by corticosterone and dexamethasone in the hypothalamus, amygdala, and hippocampus. Secondly, we wanted to determine whether sex steroids influence type II GR mRNA levels in these three brain regions, since we have previously shown that estrogens down-regulate rat pituitary GR mRNA levels (35, 36). The data presented in this report demonstrate that estrogens influence GR mRNA levels in female rat brain, although regional differences exist with respect to this regulation. Finally, we used an ADX/ovariectomized (ADX/OVX) rat model and glucocorticoid administration studies to OVX rats to determine whether glucocorticoids and female sex steroids act independently to influence brain GR mRNA levels. This has been shown to be the case in the anterior pituitary (36).
Materials and Methods Animals Female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA; 175-250 g BW) were maintained on a 12-h
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light, 12-h dark cycle, with access to food and water ad libitum. On the first day of the experiments, animals were ADX and/or OVX (control animals were sham-operated) under anesthesia. After surgery, animals were minimally handled except for the once daily ip hormone injections at approximately 1600 h. Corticosterone and dexamethasone were given at doses reported to return adrenalectomy (ADX)-induced ACTH plasma levels to within the normal range (37). Estrogen and progesterone therapies were given at pharmacological doses. Dosage regimens of the hormone treatments are listed in Table 1. All hormone preparations were suspended in vegetable oil, and control animals received injections of vehicle alone. On the morning after the last injection animals were killed by decapitation, their brains were quickly removed, and the selected brain regions were dissected. Samples were immediately frozen in liquid nitrogen and stored at -80 C before RNA extraction. Preparation of RNA Total RNA was isolated from brain samples by a modification of the method described by Chirgwin et al. (38). Frozen tissue was homogenized in 0.66 ml 4 M guanidium isothiocyanate, 50 mM sodium citrate (pH 7.0), 0.1 M /?-mercaptoethanol, 0.5% sodium lauryl sarcosine, and a trace of Antifoam-A (Sigma, St. Louis, MO), with 10 strokes of a 2.0-ml PotterElvejeim apparatus. After the addition of 0.33 ml 80% (wt/vol) cesium chloride (CsCl) in homogenization buffer to the homogenate, the mixture was carefully layered on 0.3 ml of a 5.7 M CsCl-100 mM EDTA, pH 7.5, cushion in a 1.5-ml Beckman polycarbonate tube (Palo Alto, CA) and centrifuged in a Beckman TL-100 centrifuge at 70,000 rpm for 4 h at 20 C. Pellets were resuspended in TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA) containing 1% sodium dodecyl sulfate (SDS). RNA was twice precipitated from 0.3 M sodium acetate by the addition of 2 vol ethanol at -80 C. RNA was collected by centrifugation at 15,000 x g at 4 C for 20 min. The final pellet was dried under vacuum and resuspended in 150 n\ 1 x TE. Aliquots were taken for estimation of purity and RNA content by spectrophotometric absorbance at 260/280 nm. Nylon filter hybridization Northern blot filters were prepared by electrophoresis of 20 Mg RNA on a denaturing 1% agarose gel containing formaldehyde at 30 mamp overnight, with subsequent transfer of the separated RNA to a nylon filter (Hybond-N, Amersham, Arlington Heights, IL) by passive diffusion under high salt concentrations (39). GR mRNA affixed to nylon filters was hybridized to a 2.2kilobase (kb) GR cRNA radiolabeled probe. Probe copies were generated from a GR cDNA fragment [kindly provided by Dr. TABLE
1. Hormone dosage regimen of rats Hormone
Dose
Duration
(per 100 g BW/day)
(days)
Corticosterone Dexamethasone 17/3-Estradiol Progesterone
2.5 mg 0.1 mg 3.0 Mg 1.0 mg
14 14 14
Hormones were administered by once daily ip injections.
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BRAIN GR mRNA REGULATION
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R. Miesfeld (24)] inserted into the pGEM-1 Riboprobe system vector (Promega Biotech, Madison, WI). Incubation of the linearized vector with a-32P-labeled ribonucleotides and bacteriophage T7 RNA polymerase produced GR cRNA antisense strand copies. Simultaneous measurement of /3-actin mRNA content by hybridization of filters with a jS-actin cRNA probe produced from a 1500-basepair /3-actin Pstl fragment inserted in pGEM-1 was used to control for any variations in total RNA amounts between samples. Nylon filters were prehybridized for 4 h at 42 C in 50% formamide, 5 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 8 x Denhardt's (50 X Denhardt's = 5% Ficoll, 5% polyvinylpyrrolidone, and 5% BSA), 50 mM sodium phosphate (pH 6.5), 250 Mg/ml denatured salmon sperm DNA (ssDNA), 250 ^g/ml tRNA, and 0.1% SDS. The filters were simultaneously hybridized with the GR probe and the /3-actin probe at 60 C for 18-24 h in the same buffer containing 1.5 x 106 cpm 32P-labeled GR cRNA and 1.0 x 106 cpm 32P-labelled /3-actin cRNA/ml buffer. At the end of the incubation, filters were washed in 1 X SSC-0.1% SDS twice for 30 min each time at room temperature and then in 0.1 x SSC0.1% SDS twice for 1 h each time at 65 C. To completely remove nonspecifically bound probe, filters were incubated with ribonuclease-A (1 ^ig/mL) in 2 X SSC for 3 min and washed twice for 15 min each time in 2 X SSC. Filters were air dried and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) with a Cronex intensifying screen (DuPont, Wilmington, DE) at —80 C in a light-proof film cassette. The resulting autoradiograms were analyzed by measurements of mean gray tone with a Ras image analysis system (Amersham). All comparisons of mRNA levels on autoradiograms were made from RNA samples hybridized on the same filter. Statistical analysis The statistical significance of any difference between groups was analyzed with the Duncan-Kramer multiple range test (40) after analysis of variance (ANOVA).
Results The presence of type II GR mRNA was confirmed in the three brain regions studied, and Fig. 1 shows that the signal obtained with the 32P-labeled GR probe in hypothalamus corresponds to the ~7.0-kb GR mRNA transcript previously described in rat hepatoma cells (24), rat brain (27, 29), and pituitary gland (35, 36). Measurements of the relative abundance of transcript levels in the brain regions studied suggest that in female rats, type II GR mRNA levels are slightly higher in hypothalamus than in hippocampus (Fig. 2). Transcript levels in the amygdala (not shown) of female rats are approximately 50% of the amounts found in hypothalamus. We also compared transcript levels in male Sprague-Dawley rats and found that the hypothalamic GR mRNA content of these animals is approximately 60% higher than that in hippocampus. Comparison between sexes showed that type II mRNA levels are slightly
Endo • 1991 Vol 129 • No 4
-«-6.5-7.0 Kb -»-28SrRNA -«-2.0-2.2 Kb
B FIG. 1. Double hybridization technique used on a Northern blot of total RNA in female rat hypothalamus. After electrophoresis of 20 fig total RNA extracted from female rat hypothalamus (see Materials and Methods), three identical blots were hybridized with /3-actin cRNA probe (A), GR cRNA probe (B), or simultaneously with both probes (C). A ~7.0-kb autoradiographic signal of the GR mRNA transcript was obtained, similar to the signal previously reported in female rat anterior pituitary (65). The 2.2-kb band corresponds to /3-actin mRNA. The simultaneous hybridization technique, which produced the same bands, was used in subsequent experiments.
higher in male us. female hypothalamus, but the GR mRNA content in the hippocampus is essentially the same. ADX experiments Type II GR mRNA levels in ADX animals were significantly increased above control values in amygdala and hippocampus, but not in hypothalamus (Fig. 3). GR mRNA concentrations in amygdala increased by 40% and were almost doubled in hippocampus. Corticosterone or dexamethasone replacement in these animals had no significant effect on GR mRNA levels in hypothalamus. In amygdala only dexamethasone treatment significantly reduced ADX-induced GR mRNA levels to a value not significantly different from that in controls. In hippocampus, both corticosterone and dexamethasone reversed the ADX-induced increase in GR mRNA levels. OVX experiments The effect of OVX on type II mRNA levels was significant only in hypothalamus, where the GR mRNA levels of OVX animals rose to 174% of control values (Fig. 4). Treatment of animals with pharmacological doses of estradiol had the opposite effect of OVX on transcript levels in hypothalamus, where transcript levels were decreased to 65% of control values, although this drop was not significant. In amygdala, GR mRNA content was significantly increased to 188% of control values after estradiol treatment. No detectable effect was seen in hippocampus, and progesterone did not significantly affect GR mRNA content in any of the brain regions studied. Glucocorticoid treatment of OVX animals had the following effects. Corticosterone treatment did not significantly affect hypothalamic GR mRNA levels, while
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BRAIN GR mRNA REGULATION
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FlG. 2. Relative comparison of GR mRNA transcript levels in female and male rat brains. Total RNA was extracted from hypothalamus and hippocampus of adult Sprague-Dawley rats (150-200 g BW) and hybridized on Northern blots. Results are the mean ± SEM for either brain region of male or female rats (n = 5), expressed in arbitrary units of the GR mRNA/(8-actin mRNA ratio. Thus, the ratio of hypothalamic GR mRNA content in males was 0.9 ±0.1 vs. 0.7 ± 0.08 in females, while the results in hippocampus were 0.55 ± 0.08 in males and 0.52 ± 0.07 in females. ANOVA by the Duncan-Kramer test showed that these results were not significant.
Male Female
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HYPOTHALAMUS dexamethasone treatment lowered transcript levels to 58% of control values. Thus, OVX renders hypothalamic GR mRNA sensitive to regulation by dexamethasone, in contrast to the absence of any effects of dexamethasone in ADX animals. Corticosterone treatment of OVX animals caused a significant increase in GR mRNA to 233% of control values in amygdala. This contrasts with the absence of any significant effect of corticosterone administration on GR mRNA content in the amygdala of intact (not shown) and ADX animals. Dexamethasone treatment had no effect in amygdala, and neither corticosterone nor dexamethasone altered GR mRNA content in hippocampus in OVX animals (Fig. 4). Neither corticosterone nor dexamethasone had any significant effect on GR mRNA in amygdala or hippocampus when administered to intact controls (not shown). ADX/OVX experiments The combined effect of ADX/OVX on GR mRNA levels in hypothalamus was no greater than the effect of ADX or OVX alone (Fig. 5). As in ADX or OVX animals, corticosterone replacement did not significantly affect hypothalamic transcript levels, but dexamethasone treatment lowered GR mRNA concentyrations to 68% of control values. This was similar to its effect in OVX animals. The amygdala GR mRNA content in ADX/OVX animals was slightly but significantly greater than the increase induced by ADX alone. This augmented effect of ADX/OVX on GR mRNA levels contrasts with the
HIPPOCAMPUS
absence of any influence of OVX alone. Corticosterone replacement in ADX/OVX animals increased amygdala GR transcripts to over 250% of control levels, an effect similar to that seen in OVX animals. As was seen in the ADX animals, dexamethasone treatment of ADX/OVX animals significantly reduced transcript levels in the amygdala. The influence of ADX/OVX on hippocampal GR mRNA content was no greater than the increase induced by ADX alone. Corticosterone and dexamethasone treatment, either of which significantly reduced the ADXinduced increase in hippocampal GR mRNA content, did not significantly influence the increased GR mRNA levels in ADX/OVX animals, although dexamethasone treatment resulted in a partial reversal. Discussion The present study shows that regional differences with respect to hormonal regulation of type II GR mRNA exist in hypothalamus, amygdala, and hippocampus. Cellular GR in these regions are thought to mediate HPA axis activity by altering CRF secretion from the hypothalamic paraventricular nucleus (41). Homologous regulation of the GR concentration, which alters the sensitivity of brain structures to circulating glucocorticoids, has been shown to involve changes in GR mRNA levels (27). Studies using nuclear run-on assays have shown that glucocorticoids down-regulate GR mRNA levels by influencing GR gene transcription and not mRNA half-
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BRAIN GR mRNA REGULATION
2170 200 r
HYPOTHALAMUS
AMYGDALA
too
100 -
18 ADX
10
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ADX+Cort
ADX+Dex
FIG. 3. Effect of ADX and glucocorticoid replacement on GR mRNA content in hypothalamus, hippocampus, and amygdala. After 14 days of corticosterone (Cort) or dexamethasone (Dex) administration to ADX rats, total RNA of the corresponding brain region from each animal was extracted and hybridized on Northern blots. Control animals and one ADX group received injections of vegetable oil. Results are the mean ± SEM for each group of rats (the number of animals is indicated in the bar) expressed as percentages of GR mRNA//3-actin mRNA ratios relative to control values normalized to 100%. Variation within control groups, which also applies to subsequent figures, were: hypothalamus, 102.4 ± 7.0; hippocampus, 100.5 ± 10.5; and amygdala, 99.9 ± 4.3. ANOVA shows that ADX significantly increased GR mRNA content over control values [*, P < 0.05; • * , P < 0.01 (by DuncanKramer test)] in hippocampus and amygdala. ADX and administration of corticosterone or dexamethasone to ADX rats did not significantly change GR mRNA levels with respect to control values in hypothalamus. In hippocampus, both corticosterone and dexamethasone caused decreases that were significantly lower than the ADX values (*, P < 0.05; **, P < 0.01), but were not significantly different with respect to control values. In amygdala, only dexamethasone decreased transcript levels below ADX values, while corticosterone treatment did not significantly alter the ADX-induced increase in GR mRNA content.
life (28), although several investigators have proposed that glucocorticoids may regulate GR mRNA levels through posttranscriptional mechanisms as well (42). GR binding studies have found high concentrations of type II GR in hypothalamus, amygdala, and hippocampus (12) and have shown that receptor concentrations
Endo'1991 Vol 129 • No 4
increase after ADX (23). The extent of the increase depended not only upon the region studied, but also upon whether [3H]corticosterone or [3H]dexamethasone was used as the ligand. Sapolsky and McEwen (17) have shown that the extent of down-regulation by corticosterone and dexamethasone varies with anatomical site in rat brain. In the first part of our study we demonstrate that removal of the endogenous ligand, corticosterone, by ADX of female rats caused type II GR mRNA transcript levels to increase significantly in hippocampus and amygdala, but not in hypothalamus. Replacement studies in ADX animals with corticosterone or dexamethasone demonstrate that these two steroids have different effects on GR mRNA levels depending upon the brain region. While corticosterone was able to reduce the ADX-induced increase in hipppocampus, no significant effect was seen in amygdala or hypothalamus. Dexamethasone administration lowered GR mRNA levels in hippocampus and amygdala of ADX, but not intact, animals. Thus, regulation of GR mRNA concentrations by glucocorticoids in female rat brain varies depending upon the region studied and the ligand used. Our results are generally in agreement with those of previous studies, which have shown that hippocampal type II GR transcript levels increase after ADX and are differentially regulated by glucocorticoids. Reul et al (43) concluded that in male rats the ADX-induced increase in hippocampal transcript levels was transient (4-8 days), but was prevented by dexamethasone administration. Sheppard et al. (32), using female rats, showed that the ADXinduced increase in hippocampal GR mRNA levels was suppressed by corticosterone administration, but not by dexamethasone, in contrast with our results. Plasma dexamethasone levels were not measured in either study, so our results with dexamethasone could be attributed to the different dexamethasone dosages or routes of administration, since the aforementioned investigators administered dexamethasone (0.25 /ug/ml) in the rats' drinking solution for 5 days during their replacement studies, while we used once-daily ip injections. In agreement with our results, Herman et al. (31), studying 8-day ADX male rats, have shown, using semiquantitative in situ hybridization analysis, that type II GR mRNA levels, which are particularly increased in hippocampal subfields CAl2 and the dentate gyrus, are down-regulated by high dose dexamethasone administration. In male rats, Chao et al. (44), measuring hippocampal type II mRNA using the RNAse T2 protection assay, did not detect any significant effects of ADX or corticosterone replacement (50 mg/kg, once daily, sc) on transcript levels. The differences in tissue response to corticosterone or dexamethasone seen in our study and others have been attributed to several factors that might influence effective local concentrations of these two hormones. The
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BRAIN GR mRNA REGULATION HYPOTHALAMUS
200
100
10
10
28
10
10
HIPPOCAMPUS 200
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FlG. 4. OVX or administration of sex steroids alters rat brain GR mRNA content. The effects of OVX or the chronic administration of either estradiol or progesterone on GR mRNA content were studied in hypothalamus, hippocampus, and amygdala. Results of the regulation of transcript levels after 14 days of corticosterone (Cort) or dexamethasone (Dex) injections to OVX rats are also shown. Hormone dosage regimens are shown in Table 1. Control animals and one OVX group received injections of vegetable oil. Results are expressed as percentages of GR mRNA/j8-actin mRNA ratios relative to control values normalized to 100%, with the panels representing the mean ± SEM for each group of rats (the number of animals is indicated in the panel). Oneway ANOVA shows that estradiol administration to intact rats significantly increased GR mRNA content only in amygdala [*, P < 0.05; • * , P < 0.01 (by Duncan-Kramer test)], while progesterone had no significant effect in any of the brain regions studied. A significant increase in GR transcript levels was seen only in the hypothalamus of 14-day OVX rats. Dexamethasone (and not corticosterone) treatment of OVX animals altered the OVX-induced increase in hypothalamic GR mRNA content. Thus, dexamethasone-treated rats had hypothalamic GR transcript levels that were not significantly different from control values. Although OVX had no significant effect in amygdala,
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presence of proteins in the brain that act similarly to corticosteroid-binding globulin, previously identified in rat plasma and pituitary (45), and bind corticosterone to decrease its concentration locally could explain the noneffectiveness of corticosterone at suppressing GR mRNA in certain brain regions. The metabolism of corticosterone, but not dexamethasone, by modifying enzymes such as 11/3-hydroxydehydrogenase and the shorter half-life of corticosterone could also influence corticosterone's effect on GR mRNA regulation in the brain. The presence of two GR systems which are differentially expressed throughout the brain may also account in part for the regional specificity of type II GR mRNA regulation seen in our experiments. The type I GR has at least 1 order of magnitude higher affinity for corticosterone over type II GR, which only becomes fully occupied when endogenous corticosterone attains stress levels (13). Current thought holds that both GR subtypes are involved in controlling ACTH secretion and that the lower affinity type II receptors act principally to prevent overactivity of the HPA axis in response to stress. At the doses of corticosterone used in our experiments, both receptor systems would be occupied (12). Since dexamethasone would be thought to activate predominantly, but not exclusively, the type II GR system, the differing effects of these two glucocorticoids probably reflect 1) the level of activation of each receptor system and 2) their respective roles in type II GR mRNA regulation within a given brain region. Occupied type I and type II receptors might both be able to interact with the glucocorticoid-responsive elements of the type II GR gene [which have been identified by Okret et al. (46)] to regulate transcript levels. The effects of reproductive steroids on brain GR mRNA levels were also studied, since estrogens have been shown to affect certain parameters of the stress hormone axis. Sexual dimorphism in the rat has been reported for cortisol secretion, plasma corticosteroidbinding globulin, and the glucocorticoid secretory response to stress (47, 48). Turner and Weaver (21) have reported sex differences in rat brain glucocorticoid binding which are further amplified by OVX, and female rats have been shown to adapt differently to stress in an animal model of depression (49). We have previously shown that OVX increases type II GR mRNA levels in both the anterior pituitary gland and neurointermediate lobe of rats (35, 36), and this has recently been confirmed by ligand binding studies (50). The results of the present study show that OVX also administration of corticosterone (and not dexamethasone) to OVX rats significantly increased GR mRNA levels in amygdala over control values.
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BRAIN GR mRNA REGULATION
2172 *
200 -
HYPOTHALAMUS
100 -
8
10
9
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OC
300
HIPPOCAMPUS
r
E O "O CT5 i =
200
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E DC CD AMYGDALA 300
200
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ADX/OVX+Cort
ADX/OVX+Dex
FIG. 5. Combined effect of ADX and OVX on GR mRNA content in rat hypothalamus, hippocampus, and amygdala. After 14 days of corticosterone (Cort) or dexamethasone (Dex) administration to ADX/ OVX rats, total RNA of the corresponding brain region from each individual animal was extracted and hybridized on Northern blots. Control animals and one ADX/OVX group received injections of vegetable oil. Results are the mean ± SEM for each group of rats (the number of animals is indicated in the panels), expressed as percentages of GR mRNA//3-actin mRNA ratios relative to control values normalized to 100%. ANOVA shows that ADX/OVX animals had GR transcript levels that were significantly increased above control values in all three brain regions studied [*, P < 0.05; • • , P < 0.01 (by DuncanKramer test)]. In amygdala, GR mRNA content was slightly but significantly greater than the increase induced by ADX alone (P < 0.05). The only effect of corticosterone treatment was to cause a significant increase in amygdala GR mRNA content with respect to ADX/OVX values (P < 0.01). In the hypothalamus and amygdala of ADX/OVX rats, dexamethasone decreased GR transcript levels to values not significantly different from controls.
significantly increased type II GR mRNA levels in the hypothalamus, but not in the other two brain areas studied. Estrogen administration to intact animals re-
E n d o • 1991 Vol 129 • No 4
sulted in a downward trend of GR mRNA levels in the hypothalamus and a significant increase in the amygdala. Neither treatment significantly affected hippocampal transcript levels. Thus, estrogenic influence on type II GR mRNA levels, which may fluctuate with the estrous cycle, varies depending upon the brain region studied. These effects may be mediated by estrogen receptors, which have been localized to hypothalamus, hippocampus, and other regions in rat brain (51). The level of estrogen receptors is particularly high in anterior hypothalamus, while the only significant binding in hippocampus occurs over interneurons of the dentate gyrus. The fact that OVX significantly increased hypothalamic GR mRNA levels, but had little effect in hippocampus, could be due at least partially to this difference in regional estrogen receptor distribution. Estrogens have been shown to regulate mRNA levels of cellular oncogenes such as c-fos (52) and erb-B (53). This transcriptional regulation occurs via estrogen-responsive elements, which have been shown to mediate estrogen regulation of the vitellogenin gene (54) and are similar to but distinct from glucocorticoid-responsive elements (55). The fact that estrogen increases GR mRNA in certain brain regions may be a protective effect against cytoplasmic degradation, since estrogens have been shown to stabilize mRNA in other systems (56) In OVX animals (as opposed to ADX animals), hypothalamic GR mRNA levels were rendered sensitive to regulation by dexamethasone. This phenomenon, which could not be explained by effects that estrogens are known to have on corticosteroid-binding globulin levels, since dexamethasone does not bind to corticosteroidbinding globulin, may reflect an induction of functional GR in OVX animals. The dexamethasone sensitivity of hypothalamic GR mRNA is maintained in ADX/OVX animals. OVX also caused corticosterone administration to greatly increase amygdala GR mRNA levels, in contrast with ADX animals, in which corticosterone administration did not significantly affect GR mRNA values. Since estrogen administration to rats has been shown to increase plasma corticosteroid-binding globulin levels (47), it is possible that estrogen removal may reduce local concentrations of similarly acting proteins in brain and account for the up-regulation of GR mRNA seen in amygdala upon corticosterone administration. Up-regulation of transcript levels in the amygdala induced by corticosterone was maintained in ADX/OVX animals, in which the combined effect of castration and adrenal ablation was to raise GR mRNA significantly above control values, in contrast to OVX or ADX alone. Dexamethasone did not alter amygdala GR mRNA concentrations in intact or OVX animals, but reversed the increased levels found subsequent to ADX or ADX/ OVX. ADX/OVX animals did not have hippocampal GR
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BRAIN GR mRNA REGULATION
mRNA levels that differed significantly from those of ADX animals, although regulation of transcript levels was rendered less sensitive to corticosterone or dexamethasone. This may point to a possible modulatory role in glucocorticoid feedback regulation for the specific subpopulations of hippocampal neurons to which estrogen receptors have been localized (57). Progesterone did not have any significant effect on GR mRNA levels in any of the regions studied, although progesterone receptors have been localized to rat brain hypothalamus, amgydaloid nuclei, hippocampal subfields, and limbic nuclei (58). This result suggests that the observed effects of OVX on GR mRNA levels in brain regions is not caused by the concomitant removal of circulating progesterone. Progesterone, a known GR antagonist, has been shown to enhance dissociation of the ligand-GR complex in vitro (59), while physiological studies have shown that progesterone's effects on hypothalamic gonadotropin release are abolished in ADX rats (60). In conclusion, this study provides further evidence that central structures involved in the physiological control of HPA axis activity respond differentially to alterations in plasma glucocorticoid levels. Despite the widespread distribution of the low affinity type II GR throughout the brain, certain structures (notably the hippocampus) show greater changes in GR mRNA content after ADX or glucocorticoid replacement. This phenomenon may suggest a more prominent role for such limbic structures in the regulation of CRF neuronal activity and ACTH pituitary secretion. Our results also suggest that circulating estrogens are able to influence the sensitivity of brain structures to glucocorticoids by altering GR mRNA levels, although we do not know whether these changes lead to corresponding changes in receptor protein levels. Interestingly, estrogens may control the sensitivity of brain structures without altering GR transcript levels. This possibility is strengthened by our observations in the amygdala, where corticosterone treatment of OVX animals increased GR mRNA content, although OVX alone had no significant effect. The local influence of estrogens may also account for sex differences in central GR mRNA levels. The effects of OVX were most pronounced in hypothalamus, where type II immunoreactivity is known to be highest in the paraventricular nuclei. Thus, the hypothalamus may be an important integration site at which gonadal steroids are able to alter stress hormone secretion.
References 1. McEwen BS, Davis PG, Parsons B, Pfaff DW 1979 The brain as a target for hormone action. In: Cowan WM, Hall ZW, Kandel ER (eds) Annual Review of Neuroscience. Annual Reviews, Palo Alto, vol 2:65-112 2. Keller-Wood M, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1-24
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3. Grove JR, Dieckmann BS, Schroer TA, Ringold GM 1980 Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescenceactivated cell sorting. Cell 21:47-56 4. McEwen BS, Weiss JM, Schwartz LS 1968 Selective retention of corticosterone by limbic structures in rat brain. Nature 220:911912 5. de Kloet ER, Veldhuis HD, Wagenaars JL, Bergink EW 1984 Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus. J Steroid Biochem 21:173-178 6. Krozowski ZS, Funder JW 1983 Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc Natl Acad Sci USA 80:6056-6060 7. Arriza JL, Simerly RB, Swanson LW, Evans RM 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1:887-900 8. Patel PD, Sherman TG, Goldman DJ, Watson SJ 1989 Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus. Mol Endocrinol 3:1877-1885 9. Funder JW, Feldman D, Edelman IS 1973 Glucocorticoid receptors in rat kidney: the binding of tritiated dexamethasone. Endocrinology 92:1005-1013 10. Warembourg M 1975 Radioautographic study of the rat brain and pituitary after injection of 1-2 3H-corticosterone. Brain Res 89:6170 11. Warembourg M 1975 Radioautographic study of the rat brain and pituitary after injection of 3H-dexamethasone. Cell Tissue Res 161:183-191 12. Reul JMHM, de Kloet ER 1985 Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505-2511 13. Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobsen L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. In: Clark JH (ed) Recent Progress in Hormone Research. Academic Press, New York, vol 43:113-173 14. Svec F, Rudis M 1981 Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell. J Biol Chem 256:5984-5987 15. Mayer M, Rosen F 1978 Effect of endocrine manipulations on glucocorticoid binding capacity in rat skeletal muscle. Acta Endocrinol (Copenh) 88:199-208 16. Sapolsky RM, Krey LC, McEwen BS 1984 Stress down-regulates corticosterone receptors in a site-specific manner in the brain. Endocrinology 114:287-292 17. Sapolsky RM, McEwen BS 1985 Downregulation of neural corticosterone receptors by corticosterone and dexamethasone. Brain Res 3:161-165 18. Meaney MJ, Aitken DH, Sapolsky RM 1987 Thyroid hormones influence the development of hippocampal glucocorticoid receptors in the rat: a mechanism for the effects of postnatal handling on the development of the adrenocortical stress response. Neuroendocrinology 45:278-283 19. Veldhuis HD, de Kloet ER 1982 Vasopressin-related peptides increase the hippocampal corticosterone receptor capacity of diabetes insipidus (Brattleboro) rats. Endocrinology 110:153-157 20. Veldhuis HD, de Kloet ER 1982 Significance of ACTH4-10 in the control of hippocampal corticosterone receptor capacity of hypophysectomized rats. Neuroendocrinology 34:374-380 21. Turner BB, Weaver DA 1985 Sexual dimorphism of glucocorticoid binding in rat brain. Brain Res 343:16-23 22. Sapolsky RM, Krey LC, McEwen BS 1983 Corticosterone receptors decline in a site-specific manner in the aged rat. Brain Res 289:235240 23. Olpe H-R, McEwen BS 1976 Glucocorticoid binding to receptorlike proteins in rat brain and pituitary: ontogenetic and experimentally induced changes. Brain Res 105:121-128 24. Miesfeld R, Okret S, Wikstrom A-C, Wrange O, Gustafsson J-A, Yamamoto KR 1984 Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312:779781 25. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfels MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635-641
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26. Danielsen M, Northrop JP, Ringold GM 1986 The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2522 27. Pepin M-C, Beaulieu S, Barden N 1990 Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex. Cell Mol Neurobiol 10:227-235 28. Rosewicz S, McDonald AR, Maddux BA, Goldfine ID, Miesfeld RL, Logsdon CD 1988 Mechanism of glucocorticoid receptor downregulation by glucocorticoids. J Biol Chem 263:2581-2584 29. Aronsson M, Fuxe K, Dong Y, Agnati LF, Okret S, Gustafsson JA 1988 Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization. Proc Natl Acad Sci USA 85:93319335 30. Yang G, Matocha MF, Rapoport SI 1988 Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization. Mol Endocrinol 2:682-685 31. Herman JP, Patel PD, Akil H, Watson SJ 1989 Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat. Mol Endocrinol 3:1886-1894 32. Sheppard KE, Roberts JL, Blum M 1990 Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus. Endocrinology 127:431-439 33. Kalinyak JE, Griffin CA, Hamilton RW, Bradshaw JG, Perlman AJ, Hoffman AR 1989 Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat. J Clin Invest 84:1843-1848 34. Peiffer A, Barden N, Meaney MJ, Age-related changes in glucocorticoid receptor binding and mRNA levels in the rat brain and pituitary. Neurobiol Aging, in press 35. Peiffer A, Barden N 1988 Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy. Mol Cell Endocrinol 55:115-120 36. Peiffer A, Barden N 1987 Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland. Mol Endocrinol 1:435-440 37. Beaulieu S, Gagne B, Barden N 1988 Glucocorticoid regulation of proopiomelanocortin messenger ribonucleic acid content of rat hypothalamus. Mol Endocrinol 2:727-731 38. Chirgwin JM, Pryzbyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 39. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 40. Kramer CY 1956 Extension of multiple range tests to group means with unequal number of replications. Biometrics 12:307-310 41. Paull WK, Gibbs FP1983 The corticotropin-releasing factor (CRF) neurosecretory system in intact, adrenalectomized, and adrenalectomized-dexamethasone treated rats. An immunocytochemical analysis. Histochemistry 78:303-316 42. Dong Y, Poellinger L, Gustafsson J-A, Okret S 1988 Regulation of glucocorticoid receptor expression: evidence for transcriptional and
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posttranslational mechanisms. Mol Endocrinol 2:1256-1264 43. Reul JMHM, Pearce PT, Funder JW, Krozowski ZS 1989 Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration. Mol Endocrinol 3:1674-1680 44. Chao HM, Choo PH, McEwen BS 1989 Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain. Neuroendocrinology 50:365-371 45. de Kloet ER, Burbach P, Mulder GH 1977 Localization and role of transcortin-like molecules in the anterior pituitary. Mol Cell Endocrinol 7:261-273 46. Okret S, Poellinger L, Dong Y, Gustafsson J-A 1986 Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. Proc Natl Acad Sci USA 83:5899-5903 47. Kitay JI 1961 Sex differences in adrenal cortical secretion in the rat. Endocrinology 68:818-824 48. Kant GJ, Lenox RH, Bunnell BN, Mougey EH, Pennington LL, Meyerhoff JL 1983 Comparison of stress response in male and female rats: pituitary cyclic AMP and plasma prolactin, growth hormone and corticosterone. Psychoneuroendocrinology 8:421-428 49. Kennett GA, Chaouloff F, Marcou M, Curzon G 1986 Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonin. Brain Res 382:416-421 50. Turner BB 1990 Sex difference in glucocorticoid binding in rat pituitary is estrogen dependent. Life Sci 46:1399-1406 51. Rainbow TC, Parsons B, MacLusky NJ, McEwen BS 1982 Estradiol receptor levels in rat hypothalamic and limbic nuclei. J Neurosci 2:1439-1445 52. Loose-Mitchell DS, Chiapetta C, Stancel GM 1988 Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2:946951 53. Lingham R, Stancel GM, Loose-Mitchell DS 1988 Estrogen induction of the epidermal growth factor receptor mRNA. Mol Endocrinol 2:230-235 54. Klein-Hitpa/3 L, Schorpp M, Wagner U, Ryffel GU 1986 An estrogen-responsive element derived from the 5' flanking region of Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46:1053-1061 55. Klock G, Strahle U, Schiitz G 1987 Oestrogen and glucocorticoid responsive elements are closely related but distinct. Nature 329:734-736 56. Brock ML, Shapiro DJ 1984 Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34:207-214 57. Loy R, Gerlach JL, McEwen BS 1988 Autoradiographic localization of estrogen-binding neurons in rat hippocampus and entorhinal cortex. Dev Brain Res 39:245-251 58. Parsons B, Rainbow TC, MacLusky NJ, McEwen BS 1982 Progestin receptor levels in rat hypothalamic and limbic nuclei. J Neurosci 2:1446-1452 59. Svec F, Yeakley J, Harrison RW 1980 Progesterone enhances glucocorticoid dissociation from the AtT-20 cell glucocorticoid receptor. Endocrinology 107:566-572 60. Sarkar DK, Fink G 1979 Effects of gonadal steroids on output of luteinizing hormone releasing factor into hypophysial portal blood in the female rat. J Endocrinol 80:303-313
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Vol. 129, No. 4 Printed in U.S.A.
Hormonal Regulation of Type II Glucocorticoid Receptor Messenger Ribonucleic Acid in Rat Brain* ANDY PEIFFER, BENOIT LAPOINTE, AND NICHOLAS BARDEN Laboratory of Molecular Psychogenetics, CHUL Research Centre and Laval University, Ste-Foy, Quebec, Canada GlV 4G2
tomy only in amygdala. Corticosterone increased amygdala transcript levels in OVX and ADX/OVX animals. Estradiol administration to intact animals raised the GR mRNA content of amygdala, while progesterone treatment had no effect on any of the brain regions studied. We conclude that there exists heterogeneity with respect to type II GR mRNA regulation by corticosterone and dexamethasone in brain regions of ADX female rats, and that certain limbic structures show greater sensitivity to these hormonal manipulations, suggesting a more prominent role in the regulation of the hypothalamic-pituitary-adrenal axis. Our results also suggest that circulating estrogens can influence the sensitivity of brain structures (i.e. hypothalamus and amygdala) to glucocorticoids by altering GR mRNA levels. These regions may represent integration sites at which gonadal steroids are able to alter stress hormone secretion. {Endocrinology 129: 2166-2174, 1991)
ABSTRACT. Differences in the regulation of type II glucocorticoid receptor (GR) mRNA levels in female rat brain regions involved in the control of the hypothalamic-pituitary-adrenal axis were studied by Northern blot analysis after chronic administration of corticosterone or dexamethasone to adrenalectomized (ADX), ovariectomized (OVX), and ADX/OVX animals. The effect of chronic estradiol or progesterone treatment of intact animals was also studied. Our results show that type II GR mRNA levels of ADX animals were significantly increased above control values in amygdala (140%) and hippocampus (196%), but not in hypothalamus. These increased transcript levels were down-regulated by corticosterone or dexamethasone, with the exception of those in the amygdala, where corticosterone had no effect. Ovariectomy significantly increased hypothalamic GR mRNA content (174%) over control values, and this increase was sensitive to dexamethasone. The combined effect of adrenalectomy/ovariectomy on GR mRNA levels was greater than that of adrenalec-
I
N THE central nervous system glucocorticoids influence neuronal excitability and neurotransmitter metabolism, alter protein synthesis, and regulate hypothalamic-pituitary-adrenal (HPA) axis activity (1, 2). Intracellular glucocorticoid receptors (GR) confer tissue responsiveness to circulating glucocorticoids and mediate their genomic and posttranslational effects (3). GR in brain structures were first reported by McEwen et al. (4), and the presence of two distinct GR systems in rat brain has since been established. The type I GR has high affinity for corticosterone (5), the endogenous glucocorticoid in rat, and resembles the rat kidney mineralocorticoid receptor with respect to biochemical and structural properties (6-8). The type II GR, found in nearly all peripheral tissues, binds corticosterone with lower affinity, but has high affinity for the synthetic ligand dexamethasone (9). Each receptor has a distinct localization pattern in rat Received April 29, 1991. Address all correspondence and requests for reprints to: Dr. Nicholas Barden, Laboratory of Molecular Psychogenetics, CHUL Research Centre, 2705 boulevard Laurier, Ste Foy, Quebec, Canada GlV 4G2. * This work was supported by a grant from the Medical Research Council of Canada (to N.B.).
brain, with high concentrations of type I receptor binding in lateral septum, subiculum, and hippocampus (10). The type II receptor has a much wider distribution in rat brain, with particularly high density in the lateral septum, the CAl and CA2 hippocampal fields, certain brainstem nuclei, and the hypothalamic paraventricular nuclei (11). Colocalization of both GR subtypes to certain brain regions (e.g. hippocampus) has evoked the possibility that these structures are able to respond in a binary manner to circulating glucocorticoids (12, 13). Thus, occupation of the high affinity, low capacity type I sites may vary throughout the circadian rhythm and be complete at basal corticosterone levels, while the lower affinity type II sites may be fully occupied during stress when corticosterone levels are high. Binding studies have shown that circulating glucocorticoids regulate the tissue GR receptor concentration in in vitro systems (14), peripheral tissues (15), and neural structures, where differential down-regulatory responses to glucocorticoids have been shown depending upon brain region (16, 17). Heterologous regulation of GR levels in 1 brain by hormones other than adrenal steroids, such as insulin, thyroid hormone, vasopressin, and ACTH, has
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BRAIN GR mRNA REGULATION
also been described (18-20). Sex differences in rat brain glucocorticoid binding, which are further amplified by ovariectomy (OVX), have been reported by Turner and Weaver (21). Furthermore, brain GR concentrations have been shown to vary as a result of developmental stage, neonatal handling, early corticosterone exposure, and aging (18, 22, 23). With the recent cloning of rat, human, and mouse GR cDNAs (24-26), it has become possible to study the distribution and regulation of brain GR mRNA. Homologous down-regulation of GR has been shown to involve changes in GR mRNA levels, and glucocorticoids exert this control by influencing GR gene transcription (27, 28). In situ hybridization studies have mapped out GR mRNA distribution in rat brain (29) and have shown that transcript levels vary within different neuronal subfields of hippocampus (30). Recently, Herman et al. (31) have demonstrated that the sensitivity of type I and type II GR mRNA levels to regulation by steroids differs depending upon localization within the hippocampus. Differences in steroid hormone regulation of GR mRNA transcripts is also tissue specific (32), a phenomenon which is present early in development (33). Age-related changes in brain type II receptor mRNA levels occur, and these are most pronounced in hippocampus (34). The purpose of this study was 3-fold. The first objective was to study differences in the regulation of type II GR mRNA levels in brain regions of adrenalectomized (ADX) female rats after chronic administration of dexamethasone or corticosterone. Since type II receptors have differing sensitivities to these two steroids and show tissue-specific distribution in brain, it is possible that regulation of type II GR mRNA levels by glucocorticoids differs depending upon the brain region. Our results broaden the conclusion of previous reports by showing response heterogeneity of type II GR mRNA regulation by corticosterone and dexamethasone in the hypothalamus, amygdala, and hippocampus. Secondly, we wanted to determine whether sex steroids influence type II GR mRNA levels in these three brain regions, since we have previously shown that estrogens down-regulate rat pituitary GR mRNA levels (35, 36). The data presented in this report demonstrate that estrogens influence GR mRNA levels in female rat brain, although regional differences exist with respect to this regulation. Finally, we used an ADX/ovariectomized (ADX/OVX) rat model and glucocorticoid administration studies to OVX rats to determine whether glucocorticoids and female sex steroids act independently to influence brain GR mRNA levels. This has been shown to be the case in the anterior pituitary (36).
Materials and Methods Animals Female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA; 175-250 g BW) were maintained on a 12-h
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light, 12-h dark cycle, with access to food and water ad libitum. On the first day of the experiments, animals were ADX and/or OVX (control animals were sham-operated) under anesthesia. After surgery, animals were minimally handled except for the once daily ip hormone injections at approximately 1600 h. Corticosterone and dexamethasone were given at doses reported to return adrenalectomy (ADX)-induced ACTH plasma levels to within the normal range (37). Estrogen and progesterone therapies were given at pharmacological doses. Dosage regimens of the hormone treatments are listed in Table 1. All hormone preparations were suspended in vegetable oil, and control animals received injections of vehicle alone. On the morning after the last injection animals were killed by decapitation, their brains were quickly removed, and the selected brain regions were dissected. Samples were immediately frozen in liquid nitrogen and stored at -80 C before RNA extraction. Preparation of RNA Total RNA was isolated from brain samples by a modification of the method described by Chirgwin et al. (38). Frozen tissue was homogenized in 0.66 ml 4 M guanidium isothiocyanate, 50 mM sodium citrate (pH 7.0), 0.1 M /?-mercaptoethanol, 0.5% sodium lauryl sarcosine, and a trace of Antifoam-A (Sigma, St. Louis, MO), with 10 strokes of a 2.0-ml PotterElvejeim apparatus. After the addition of 0.33 ml 80% (wt/vol) cesium chloride (CsCl) in homogenization buffer to the homogenate, the mixture was carefully layered on 0.3 ml of a 5.7 M CsCl-100 mM EDTA, pH 7.5, cushion in a 1.5-ml Beckman polycarbonate tube (Palo Alto, CA) and centrifuged in a Beckman TL-100 centrifuge at 70,000 rpm for 4 h at 20 C. Pellets were resuspended in TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA) containing 1% sodium dodecyl sulfate (SDS). RNA was twice precipitated from 0.3 M sodium acetate by the addition of 2 vol ethanol at -80 C. RNA was collected by centrifugation at 15,000 x g at 4 C for 20 min. The final pellet was dried under vacuum and resuspended in 150 n\ 1 x TE. Aliquots were taken for estimation of purity and RNA content by spectrophotometric absorbance at 260/280 nm. Nylon filter hybridization Northern blot filters were prepared by electrophoresis of 20 Mg RNA on a denaturing 1% agarose gel containing formaldehyde at 30 mamp overnight, with subsequent transfer of the separated RNA to a nylon filter (Hybond-N, Amersham, Arlington Heights, IL) by passive diffusion under high salt concentrations (39). GR mRNA affixed to nylon filters was hybridized to a 2.2kilobase (kb) GR cRNA radiolabeled probe. Probe copies were generated from a GR cDNA fragment [kindly provided by Dr. TABLE
1. Hormone dosage regimen of rats Hormone
Dose
Duration
(per 100 g BW/day)
(days)
Corticosterone Dexamethasone 17/3-Estradiol Progesterone
2.5 mg 0.1 mg 3.0 Mg 1.0 mg
14 14 14
Hormones were administered by once daily ip injections.
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BRAIN GR mRNA REGULATION
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R. Miesfeld (24)] inserted into the pGEM-1 Riboprobe system vector (Promega Biotech, Madison, WI). Incubation of the linearized vector with a-32P-labeled ribonucleotides and bacteriophage T7 RNA polymerase produced GR cRNA antisense strand copies. Simultaneous measurement of /3-actin mRNA content by hybridization of filters with a jS-actin cRNA probe produced from a 1500-basepair /3-actin Pstl fragment inserted in pGEM-1 was used to control for any variations in total RNA amounts between samples. Nylon filters were prehybridized for 4 h at 42 C in 50% formamide, 5 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 8 x Denhardt's (50 X Denhardt's = 5% Ficoll, 5% polyvinylpyrrolidone, and 5% BSA), 50 mM sodium phosphate (pH 6.5), 250 Mg/ml denatured salmon sperm DNA (ssDNA), 250 ^g/ml tRNA, and 0.1% SDS. The filters were simultaneously hybridized with the GR probe and the /3-actin probe at 60 C for 18-24 h in the same buffer containing 1.5 x 106 cpm 32P-labeled GR cRNA and 1.0 x 106 cpm 32P-labelled /3-actin cRNA/ml buffer. At the end of the incubation, filters were washed in 1 X SSC-0.1% SDS twice for 30 min each time at room temperature and then in 0.1 x SSC0.1% SDS twice for 1 h each time at 65 C. To completely remove nonspecifically bound probe, filters were incubated with ribonuclease-A (1 ^ig/mL) in 2 X SSC for 3 min and washed twice for 15 min each time in 2 X SSC. Filters were air dried and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) with a Cronex intensifying screen (DuPont, Wilmington, DE) at —80 C in a light-proof film cassette. The resulting autoradiograms were analyzed by measurements of mean gray tone with a Ras image analysis system (Amersham). All comparisons of mRNA levels on autoradiograms were made from RNA samples hybridized on the same filter. Statistical analysis The statistical significance of any difference between groups was analyzed with the Duncan-Kramer multiple range test (40) after analysis of variance (ANOVA).
Results The presence of type II GR mRNA was confirmed in the three brain regions studied, and Fig. 1 shows that the signal obtained with the 32P-labeled GR probe in hypothalamus corresponds to the ~7.0-kb GR mRNA transcript previously described in rat hepatoma cells (24), rat brain (27, 29), and pituitary gland (35, 36). Measurements of the relative abundance of transcript levels in the brain regions studied suggest that in female rats, type II GR mRNA levels are slightly higher in hypothalamus than in hippocampus (Fig. 2). Transcript levels in the amygdala (not shown) of female rats are approximately 50% of the amounts found in hypothalamus. We also compared transcript levels in male Sprague-Dawley rats and found that the hypothalamic GR mRNA content of these animals is approximately 60% higher than that in hippocampus. Comparison between sexes showed that type II mRNA levels are slightly
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-«-6.5-7.0 Kb -»-28SrRNA -«-2.0-2.2 Kb
B FIG. 1. Double hybridization technique used on a Northern blot of total RNA in female rat hypothalamus. After electrophoresis of 20 fig total RNA extracted from female rat hypothalamus (see Materials and Methods), three identical blots were hybridized with /3-actin cRNA probe (A), GR cRNA probe (B), or simultaneously with both probes (C). A ~7.0-kb autoradiographic signal of the GR mRNA transcript was obtained, similar to the signal previously reported in female rat anterior pituitary (65). The 2.2-kb band corresponds to /3-actin mRNA. The simultaneous hybridization technique, which produced the same bands, was used in subsequent experiments.
higher in male us. female hypothalamus, but the GR mRNA content in the hippocampus is essentially the same. ADX experiments Type II GR mRNA levels in ADX animals were significantly increased above control values in amygdala and hippocampus, but not in hypothalamus (Fig. 3). GR mRNA concentrations in amygdala increased by 40% and were almost doubled in hippocampus. Corticosterone or dexamethasone replacement in these animals had no significant effect on GR mRNA levels in hypothalamus. In amygdala only dexamethasone treatment significantly reduced ADX-induced GR mRNA levels to a value not significantly different from that in controls. In hippocampus, both corticosterone and dexamethasone reversed the ADX-induced increase in GR mRNA levels. OVX experiments The effect of OVX on type II mRNA levels was significant only in hypothalamus, where the GR mRNA levels of OVX animals rose to 174% of control values (Fig. 4). Treatment of animals with pharmacological doses of estradiol had the opposite effect of OVX on transcript levels in hypothalamus, where transcript levels were decreased to 65% of control values, although this drop was not significant. In amygdala, GR mRNA content was significantly increased to 188% of control values after estradiol treatment. No detectable effect was seen in hippocampus, and progesterone did not significantly affect GR mRNA content in any of the brain regions studied. Glucocorticoid treatment of OVX animals had the following effects. Corticosterone treatment did not significantly affect hypothalamic GR mRNA levels, while
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BRAIN GR mRNA REGULATION
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1.00
• •
CL
i ratii
O
0.75
actin r
E *3* ce of fype I
FlG. 2. Relative comparison of GR mRNA transcript levels in female and male rat brains. Total RNA was extracted from hypothalamus and hippocampus of adult Sprague-Dawley rats (150-200 g BW) and hybridized on Northern blots. Results are the mean ± SEM for either brain region of male or female rats (n = 5), expressed in arbitrary units of the GR mRNA/(8-actin mRNA ratio. Thus, the ratio of hypothalamic GR mRNA content in males was 0.9 ±0.1 vs. 0.7 ± 0.08 in females, while the results in hippocampus were 0.55 ± 0.08 in males and 0.52 ± 0.07 in females. ANOVA by the Duncan-Kramer test showed that these results were not significant.
Male Female
0.50
03
03 £
?cr °-25 CD
DC 0.00
HYPOTHALAMUS dexamethasone treatment lowered transcript levels to 58% of control values. Thus, OVX renders hypothalamic GR mRNA sensitive to regulation by dexamethasone, in contrast to the absence of any effects of dexamethasone in ADX animals. Corticosterone treatment of OVX animals caused a significant increase in GR mRNA to 233% of control values in amygdala. This contrasts with the absence of any significant effect of corticosterone administration on GR mRNA content in the amygdala of intact (not shown) and ADX animals. Dexamethasone treatment had no effect in amygdala, and neither corticosterone nor dexamethasone altered GR mRNA content in hippocampus in OVX animals (Fig. 4). Neither corticosterone nor dexamethasone had any significant effect on GR mRNA in amygdala or hippocampus when administered to intact controls (not shown). ADX/OVX experiments The combined effect of ADX/OVX on GR mRNA levels in hypothalamus was no greater than the effect of ADX or OVX alone (Fig. 5). As in ADX or OVX animals, corticosterone replacement did not significantly affect hypothalamic transcript levels, but dexamethasone treatment lowered GR mRNA concentyrations to 68% of control values. This was similar to its effect in OVX animals. The amygdala GR mRNA content in ADX/OVX animals was slightly but significantly greater than the increase induced by ADX alone. This augmented effect of ADX/OVX on GR mRNA levels contrasts with the
HIPPOCAMPUS
absence of any influence of OVX alone. Corticosterone replacement in ADX/OVX animals increased amygdala GR transcripts to over 250% of control levels, an effect similar to that seen in OVX animals. As was seen in the ADX animals, dexamethasone treatment of ADX/OVX animals significantly reduced transcript levels in the amygdala. The influence of ADX/OVX on hippocampal GR mRNA content was no greater than the increase induced by ADX alone. Corticosterone and dexamethasone treatment, either of which significantly reduced the ADXinduced increase in hippocampal GR mRNA content, did not significantly influence the increased GR mRNA levels in ADX/OVX animals, although dexamethasone treatment resulted in a partial reversal. Discussion The present study shows that regional differences with respect to hormonal regulation of type II GR mRNA exist in hypothalamus, amygdala, and hippocampus. Cellular GR in these regions are thought to mediate HPA axis activity by altering CRF secretion from the hypothalamic paraventricular nucleus (41). Homologous regulation of the GR concentration, which alters the sensitivity of brain structures to circulating glucocorticoids, has been shown to involve changes in GR mRNA levels (27). Studies using nuclear run-on assays have shown that glucocorticoids down-regulate GR mRNA levels by influencing GR gene transcription and not mRNA half-
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BRAIN GR mRNA REGULATION
2170 200 r
HYPOTHALAMUS
AMYGDALA
too
100 -
18 ADX
10
8
ADX+Cort
ADX+Dex
FIG. 3. Effect of ADX and glucocorticoid replacement on GR mRNA content in hypothalamus, hippocampus, and amygdala. After 14 days of corticosterone (Cort) or dexamethasone (Dex) administration to ADX rats, total RNA of the corresponding brain region from each animal was extracted and hybridized on Northern blots. Control animals and one ADX group received injections of vegetable oil. Results are the mean ± SEM for each group of rats (the number of animals is indicated in the bar) expressed as percentages of GR mRNA//3-actin mRNA ratios relative to control values normalized to 100%. Variation within control groups, which also applies to subsequent figures, were: hypothalamus, 102.4 ± 7.0; hippocampus, 100.5 ± 10.5; and amygdala, 99.9 ± 4.3. ANOVA shows that ADX significantly increased GR mRNA content over control values [*, P < 0.05; • * , P < 0.01 (by DuncanKramer test)] in hippocampus and amygdala. ADX and administration of corticosterone or dexamethasone to ADX rats did not significantly change GR mRNA levels with respect to control values in hypothalamus. In hippocampus, both corticosterone and dexamethasone caused decreases that were significantly lower than the ADX values (*, P < 0.05; **, P < 0.01), but were not significantly different with respect to control values. In amygdala, only dexamethasone decreased transcript levels below ADX values, while corticosterone treatment did not significantly alter the ADX-induced increase in GR mRNA content.
life (28), although several investigators have proposed that glucocorticoids may regulate GR mRNA levels through posttranscriptional mechanisms as well (42). GR binding studies have found high concentrations of type II GR in hypothalamus, amygdala, and hippocampus (12) and have shown that receptor concentrations
Endo'1991 Vol 129 • No 4
increase after ADX (23). The extent of the increase depended not only upon the region studied, but also upon whether [3H]corticosterone or [3H]dexamethasone was used as the ligand. Sapolsky and McEwen (17) have shown that the extent of down-regulation by corticosterone and dexamethasone varies with anatomical site in rat brain. In the first part of our study we demonstrate that removal of the endogenous ligand, corticosterone, by ADX of female rats caused type II GR mRNA transcript levels to increase significantly in hippocampus and amygdala, but not in hypothalamus. Replacement studies in ADX animals with corticosterone or dexamethasone demonstrate that these two steroids have different effects on GR mRNA levels depending upon the brain region. While corticosterone was able to reduce the ADX-induced increase in hipppocampus, no significant effect was seen in amygdala or hypothalamus. Dexamethasone administration lowered GR mRNA levels in hippocampus and amygdala of ADX, but not intact, animals. Thus, regulation of GR mRNA concentrations by glucocorticoids in female rat brain varies depending upon the region studied and the ligand used. Our results are generally in agreement with those of previous studies, which have shown that hippocampal type II GR transcript levels increase after ADX and are differentially regulated by glucocorticoids. Reul et al (43) concluded that in male rats the ADX-induced increase in hippocampal transcript levels was transient (4-8 days), but was prevented by dexamethasone administration. Sheppard et al. (32), using female rats, showed that the ADXinduced increase in hippocampal GR mRNA levels was suppressed by corticosterone administration, but not by dexamethasone, in contrast with our results. Plasma dexamethasone levels were not measured in either study, so our results with dexamethasone could be attributed to the different dexamethasone dosages or routes of administration, since the aforementioned investigators administered dexamethasone (0.25 /ug/ml) in the rats' drinking solution for 5 days during their replacement studies, while we used once-daily ip injections. In agreement with our results, Herman et al. (31), studying 8-day ADX male rats, have shown, using semiquantitative in situ hybridization analysis, that type II GR mRNA levels, which are particularly increased in hippocampal subfields CAl2 and the dentate gyrus, are down-regulated by high dose dexamethasone administration. In male rats, Chao et al. (44), measuring hippocampal type II mRNA using the RNAse T2 protection assay, did not detect any significant effects of ADX or corticosterone replacement (50 mg/kg, once daily, sc) on transcript levels. The differences in tissue response to corticosterone or dexamethasone seen in our study and others have been attributed to several factors that might influence effective local concentrations of these two hormones. The
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BRAIN GR mRNA REGULATION HYPOTHALAMUS
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FlG. 4. OVX or administration of sex steroids alters rat brain GR mRNA content. The effects of OVX or the chronic administration of either estradiol or progesterone on GR mRNA content were studied in hypothalamus, hippocampus, and amygdala. Results of the regulation of transcript levels after 14 days of corticosterone (Cort) or dexamethasone (Dex) injections to OVX rats are also shown. Hormone dosage regimens are shown in Table 1. Control animals and one OVX group received injections of vegetable oil. Results are expressed as percentages of GR mRNA/j8-actin mRNA ratios relative to control values normalized to 100%, with the panels representing the mean ± SEM for each group of rats (the number of animals is indicated in the panel). Oneway ANOVA shows that estradiol administration to intact rats significantly increased GR mRNA content only in amygdala [*, P < 0.05; • * , P < 0.01 (by Duncan-Kramer test)], while progesterone had no significant effect in any of the brain regions studied. A significant increase in GR transcript levels was seen only in the hypothalamus of 14-day OVX rats. Dexamethasone (and not corticosterone) treatment of OVX animals altered the OVX-induced increase in hypothalamic GR mRNA content. Thus, dexamethasone-treated rats had hypothalamic GR transcript levels that were not significantly different from control values. Although OVX had no significant effect in amygdala,
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presence of proteins in the brain that act similarly to corticosteroid-binding globulin, previously identified in rat plasma and pituitary (45), and bind corticosterone to decrease its concentration locally could explain the noneffectiveness of corticosterone at suppressing GR mRNA in certain brain regions. The metabolism of corticosterone, but not dexamethasone, by modifying enzymes such as 11/3-hydroxydehydrogenase and the shorter half-life of corticosterone could also influence corticosterone's effect on GR mRNA regulation in the brain. The presence of two GR systems which are differentially expressed throughout the brain may also account in part for the regional specificity of type II GR mRNA regulation seen in our experiments. The type I GR has at least 1 order of magnitude higher affinity for corticosterone over type II GR, which only becomes fully occupied when endogenous corticosterone attains stress levels (13). Current thought holds that both GR subtypes are involved in controlling ACTH secretion and that the lower affinity type II receptors act principally to prevent overactivity of the HPA axis in response to stress. At the doses of corticosterone used in our experiments, both receptor systems would be occupied (12). Since dexamethasone would be thought to activate predominantly, but not exclusively, the type II GR system, the differing effects of these two glucocorticoids probably reflect 1) the level of activation of each receptor system and 2) their respective roles in type II GR mRNA regulation within a given brain region. Occupied type I and type II receptors might both be able to interact with the glucocorticoid-responsive elements of the type II GR gene [which have been identified by Okret et al. (46)] to regulate transcript levels. The effects of reproductive steroids on brain GR mRNA levels were also studied, since estrogens have been shown to affect certain parameters of the stress hormone axis. Sexual dimorphism in the rat has been reported for cortisol secretion, plasma corticosteroidbinding globulin, and the glucocorticoid secretory response to stress (47, 48). Turner and Weaver (21) have reported sex differences in rat brain glucocorticoid binding which are further amplified by OVX, and female rats have been shown to adapt differently to stress in an animal model of depression (49). We have previously shown that OVX increases type II GR mRNA levels in both the anterior pituitary gland and neurointermediate lobe of rats (35, 36), and this has recently been confirmed by ligand binding studies (50). The results of the present study show that OVX also administration of corticosterone (and not dexamethasone) to OVX rats significantly increased GR mRNA levels in amygdala over control values.
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BRAIN GR mRNA REGULATION
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FIG. 5. Combined effect of ADX and OVX on GR mRNA content in rat hypothalamus, hippocampus, and amygdala. After 14 days of corticosterone (Cort) or dexamethasone (Dex) administration to ADX/ OVX rats, total RNA of the corresponding brain region from each individual animal was extracted and hybridized on Northern blots. Control animals and one ADX/OVX group received injections of vegetable oil. Results are the mean ± SEM for each group of rats (the number of animals is indicated in the panels), expressed as percentages of GR mRNA//3-actin mRNA ratios relative to control values normalized to 100%. ANOVA shows that ADX/OVX animals had GR transcript levels that were significantly increased above control values in all three brain regions studied [*, P < 0.05; • • , P < 0.01 (by DuncanKramer test)]. In amygdala, GR mRNA content was slightly but significantly greater than the increase induced by ADX alone (P < 0.05). The only effect of corticosterone treatment was to cause a significant increase in amygdala GR mRNA content with respect to ADX/OVX values (P < 0.01). In the hypothalamus and amygdala of ADX/OVX rats, dexamethasone decreased GR transcript levels to values not significantly different from controls.
significantly increased type II GR mRNA levels in the hypothalamus, but not in the other two brain areas studied. Estrogen administration to intact animals re-
E n d o • 1991 Vol 129 • No 4
sulted in a downward trend of GR mRNA levels in the hypothalamus and a significant increase in the amygdala. Neither treatment significantly affected hippocampal transcript levels. Thus, estrogenic influence on type II GR mRNA levels, which may fluctuate with the estrous cycle, varies depending upon the brain region studied. These effects may be mediated by estrogen receptors, which have been localized to hypothalamus, hippocampus, and other regions in rat brain (51). The level of estrogen receptors is particularly high in anterior hypothalamus, while the only significant binding in hippocampus occurs over interneurons of the dentate gyrus. The fact that OVX significantly increased hypothalamic GR mRNA levels, but had little effect in hippocampus, could be due at least partially to this difference in regional estrogen receptor distribution. Estrogens have been shown to regulate mRNA levels of cellular oncogenes such as c-fos (52) and erb-B (53). This transcriptional regulation occurs via estrogen-responsive elements, which have been shown to mediate estrogen regulation of the vitellogenin gene (54) and are similar to but distinct from glucocorticoid-responsive elements (55). The fact that estrogen increases GR mRNA in certain brain regions may be a protective effect against cytoplasmic degradation, since estrogens have been shown to stabilize mRNA in other systems (56) In OVX animals (as opposed to ADX animals), hypothalamic GR mRNA levels were rendered sensitive to regulation by dexamethasone. This phenomenon, which could not be explained by effects that estrogens are known to have on corticosteroid-binding globulin levels, since dexamethasone does not bind to corticosteroidbinding globulin, may reflect an induction of functional GR in OVX animals. The dexamethasone sensitivity of hypothalamic GR mRNA is maintained in ADX/OVX animals. OVX also caused corticosterone administration to greatly increase amygdala GR mRNA levels, in contrast with ADX animals, in which corticosterone administration did not significantly affect GR mRNA values. Since estrogen administration to rats has been shown to increase plasma corticosteroid-binding globulin levels (47), it is possible that estrogen removal may reduce local concentrations of similarly acting proteins in brain and account for the up-regulation of GR mRNA seen in amygdala upon corticosterone administration. Up-regulation of transcript levels in the amygdala induced by corticosterone was maintained in ADX/OVX animals, in which the combined effect of castration and adrenal ablation was to raise GR mRNA significantly above control values, in contrast to OVX or ADX alone. Dexamethasone did not alter amygdala GR mRNA concentrations in intact or OVX animals, but reversed the increased levels found subsequent to ADX or ADX/ OVX. ADX/OVX animals did not have hippocampal GR
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BRAIN GR mRNA REGULATION
mRNA levels that differed significantly from those of ADX animals, although regulation of transcript levels was rendered less sensitive to corticosterone or dexamethasone. This may point to a possible modulatory role in glucocorticoid feedback regulation for the specific subpopulations of hippocampal neurons to which estrogen receptors have been localized (57). Progesterone did not have any significant effect on GR mRNA levels in any of the regions studied, although progesterone receptors have been localized to rat brain hypothalamus, amgydaloid nuclei, hippocampal subfields, and limbic nuclei (58). This result suggests that the observed effects of OVX on GR mRNA levels in brain regions is not caused by the concomitant removal of circulating progesterone. Progesterone, a known GR antagonist, has been shown to enhance dissociation of the ligand-GR complex in vitro (59), while physiological studies have shown that progesterone's effects on hypothalamic gonadotropin release are abolished in ADX rats (60). In conclusion, this study provides further evidence that central structures involved in the physiological control of HPA axis activity respond differentially to alterations in plasma glucocorticoid levels. Despite the widespread distribution of the low affinity type II GR throughout the brain, certain structures (notably the hippocampus) show greater changes in GR mRNA content after ADX or glucocorticoid replacement. This phenomenon may suggest a more prominent role for such limbic structures in the regulation of CRF neuronal activity and ACTH pituitary secretion. Our results also suggest that circulating estrogens are able to influence the sensitivity of brain structures to glucocorticoids by altering GR mRNA levels, although we do not know whether these changes lead to corresponding changes in receptor protein levels. Interestingly, estrogens may control the sensitivity of brain structures without altering GR transcript levels. This possibility is strengthened by our observations in the amygdala, where corticosterone treatment of OVX animals increased GR mRNA content, although OVX alone had no significant effect. The local influence of estrogens may also account for sex differences in central GR mRNA levels. The effects of OVX were most pronounced in hypothalamus, where type II immunoreactivity is known to be highest in the paraventricular nuclei. Thus, the hypothalamus may be an important integration site at which gonadal steroids are able to alter stress hormone secretion.
References 1. McEwen BS, Davis PG, Parsons B, Pfaff DW 1979 The brain as a target for hormone action. In: Cowan WM, Hall ZW, Kandel ER (eds) Annual Review of Neuroscience. Annual Reviews, Palo Alto, vol 2:65-112 2. Keller-Wood M, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1-24
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3. Grove JR, Dieckmann BS, Schroer TA, Ringold GM 1980 Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescenceactivated cell sorting. Cell 21:47-56 4. McEwen BS, Weiss JM, Schwartz LS 1968 Selective retention of corticosterone by limbic structures in rat brain. Nature 220:911912 5. de Kloet ER, Veldhuis HD, Wagenaars JL, Bergink EW 1984 Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus. J Steroid Biochem 21:173-178 6. Krozowski ZS, Funder JW 1983 Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc Natl Acad Sci USA 80:6056-6060 7. Arriza JL, Simerly RB, Swanson LW, Evans RM 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1:887-900 8. Patel PD, Sherman TG, Goldman DJ, Watson SJ 1989 Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus. Mol Endocrinol 3:1877-1885 9. Funder JW, Feldman D, Edelman IS 1973 Glucocorticoid receptors in rat kidney: the binding of tritiated dexamethasone. Endocrinology 92:1005-1013 10. Warembourg M 1975 Radioautographic study of the rat brain and pituitary after injection of 1-2 3H-corticosterone. Brain Res 89:6170 11. Warembourg M 1975 Radioautographic study of the rat brain and pituitary after injection of 3H-dexamethasone. Cell Tissue Res 161:183-191 12. Reul JMHM, de Kloet ER 1985 Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505-2511 13. Dallman MF, Akana SF, Cascio CS, Darlington DN, Jacobsen L, Levin N 1987 Regulation of ACTH secretion: variations on a theme of B. In: Clark JH (ed) Recent Progress in Hormone Research. Academic Press, New York, vol 43:113-173 14. Svec F, Rudis M 1981 Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell. J Biol Chem 256:5984-5987 15. Mayer M, Rosen F 1978 Effect of endocrine manipulations on glucocorticoid binding capacity in rat skeletal muscle. Acta Endocrinol (Copenh) 88:199-208 16. Sapolsky RM, Krey LC, McEwen BS 1984 Stress down-regulates corticosterone receptors in a site-specific manner in the brain. Endocrinology 114:287-292 17. Sapolsky RM, McEwen BS 1985 Downregulation of neural corticosterone receptors by corticosterone and dexamethasone. Brain Res 3:161-165 18. Meaney MJ, Aitken DH, Sapolsky RM 1987 Thyroid hormones influence the development of hippocampal glucocorticoid receptors in the rat: a mechanism for the effects of postnatal handling on the development of the adrenocortical stress response. Neuroendocrinology 45:278-283 19. Veldhuis HD, de Kloet ER 1982 Vasopressin-related peptides increase the hippocampal corticosterone receptor capacity of diabetes insipidus (Brattleboro) rats. Endocrinology 110:153-157 20. Veldhuis HD, de Kloet ER 1982 Significance of ACTH4-10 in the control of hippocampal corticosterone receptor capacity of hypophysectomized rats. Neuroendocrinology 34:374-380 21. Turner BB, Weaver DA 1985 Sexual dimorphism of glucocorticoid binding in rat brain. Brain Res 343:16-23 22. Sapolsky RM, Krey LC, McEwen BS 1983 Corticosterone receptors decline in a site-specific manner in the aged rat. Brain Res 289:235240 23. Olpe H-R, McEwen BS 1976 Glucocorticoid binding to receptorlike proteins in rat brain and pituitary: ontogenetic and experimentally induced changes. Brain Res 105:121-128 24. Miesfeld R, Okret S, Wikstrom A-C, Wrange O, Gustafsson J-A, Yamamoto KR 1984 Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312:779781 25. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfels MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635-641
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26. Danielsen M, Northrop JP, Ringold GM 1986 The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J 5:2513-2522 27. Pepin M-C, Beaulieu S, Barden N 1990 Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex. Cell Mol Neurobiol 10:227-235 28. Rosewicz S, McDonald AR, Maddux BA, Goldfine ID, Miesfeld RL, Logsdon CD 1988 Mechanism of glucocorticoid receptor downregulation by glucocorticoids. J Biol Chem 263:2581-2584 29. Aronsson M, Fuxe K, Dong Y, Agnati LF, Okret S, Gustafsson JA 1988 Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization. Proc Natl Acad Sci USA 85:93319335 30. Yang G, Matocha MF, Rapoport SI 1988 Localization of glucocorticoid receptor messenger ribonucleic acid in hippocampus of rat brain using in situ hybridization. Mol Endocrinol 2:682-685 31. Herman JP, Patel PD, Akil H, Watson SJ 1989 Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat. Mol Endocrinol 3:1886-1894 32. Sheppard KE, Roberts JL, Blum M 1990 Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus. Endocrinology 127:431-439 33. Kalinyak JE, Griffin CA, Hamilton RW, Bradshaw JG, Perlman AJ, Hoffman AR 1989 Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat. J Clin Invest 84:1843-1848 34. Peiffer A, Barden N, Meaney MJ, Age-related changes in glucocorticoid receptor binding and mRNA levels in the rat brain and pituitary. Neurobiol Aging, in press 35. Peiffer A, Barden N 1988 Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy. Mol Cell Endocrinol 55:115-120 36. Peiffer A, Barden N 1987 Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland. Mol Endocrinol 1:435-440 37. Beaulieu S, Gagne B, Barden N 1988 Glucocorticoid regulation of proopiomelanocortin messenger ribonucleic acid content of rat hypothalamus. Mol Endocrinol 2:727-731 38. Chirgwin JM, Pryzbyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 39. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 40. Kramer CY 1956 Extension of multiple range tests to group means with unequal number of replications. Biometrics 12:307-310 41. Paull WK, Gibbs FP1983 The corticotropin-releasing factor (CRF) neurosecretory system in intact, adrenalectomized, and adrenalectomized-dexamethasone treated rats. An immunocytochemical analysis. Histochemistry 78:303-316 42. Dong Y, Poellinger L, Gustafsson J-A, Okret S 1988 Regulation of glucocorticoid receptor expression: evidence for transcriptional and
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posttranslational mechanisms. Mol Endocrinol 2:1256-1264 43. Reul JMHM, Pearce PT, Funder JW, Krozowski ZS 1989 Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration. Mol Endocrinol 3:1674-1680 44. Chao HM, Choo PH, McEwen BS 1989 Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain. Neuroendocrinology 50:365-371 45. de Kloet ER, Burbach P, Mulder GH 1977 Localization and role of transcortin-like molecules in the anterior pituitary. Mol Cell Endocrinol 7:261-273 46. Okret S, Poellinger L, Dong Y, Gustafsson J-A 1986 Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. Proc Natl Acad Sci USA 83:5899-5903 47. Kitay JI 1961 Sex differences in adrenal cortical secretion in the rat. Endocrinology 68:818-824 48. Kant GJ, Lenox RH, Bunnell BN, Mougey EH, Pennington LL, Meyerhoff JL 1983 Comparison of stress response in male and female rats: pituitary cyclic AMP and plasma prolactin, growth hormone and corticosterone. Psychoneuroendocrinology 8:421-428 49. Kennett GA, Chaouloff F, Marcou M, Curzon G 1986 Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonin. Brain Res 382:416-421 50. Turner BB 1990 Sex difference in glucocorticoid binding in rat pituitary is estrogen dependent. Life Sci 46:1399-1406 51. Rainbow TC, Parsons B, MacLusky NJ, McEwen BS 1982 Estradiol receptor levels in rat hypothalamic and limbic nuclei. J Neurosci 2:1439-1445 52. Loose-Mitchell DS, Chiapetta C, Stancel GM 1988 Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2:946951 53. Lingham R, Stancel GM, Loose-Mitchell DS 1988 Estrogen induction of the epidermal growth factor receptor mRNA. Mol Endocrinol 2:230-235 54. Klein-Hitpa/3 L, Schorpp M, Wagner U, Ryffel GU 1986 An estrogen-responsive element derived from the 5' flanking region of Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46:1053-1061 55. Klock G, Strahle U, Schiitz G 1987 Oestrogen and glucocorticoid responsive elements are closely related but distinct. Nature 329:734-736 56. Brock ML, Shapiro DJ 1984 Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34:207-214 57. Loy R, Gerlach JL, McEwen BS 1988 Autoradiographic localization of estrogen-binding neurons in rat hippocampus and entorhinal cortex. Dev Brain Res 39:245-251 58. Parsons B, Rainbow TC, MacLusky NJ, McEwen BS 1982 Progestin receptor levels in rat hypothalamic and limbic nuclei. J Neurosci 2:1446-1452 59. Svec F, Yeakley J, Harrison RW 1980 Progesterone enhances glucocorticoid dissociation from the AtT-20 cell glucocorticoid receptor. Endocrinology 107:566-572 60. Sarkar DK, Fink G 1979 Effects of gonadal steroids on output of luteinizing hormone releasing factor into hypophysial portal blood in the female rat. J Endocrinol 80:303-313
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