Regulation of Glucocorticoid Receptor and Mineralocorticoid Receptor ...

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 4 Printed in U.S.A.

Regulation of Glucocorticoid Receptor and Mineralocorticoid Receptor Messenger Ribonucleic Acids by Selective Agonists in the Rat Hippocampus HELEN M. CHAO, LI YUN MA, BRUCE S. MCEWEN,

AND

RANDALL R. SAKAI

The Rockefeller University (H.M.C., B.S.M.), Laboratory of Neuroendocrinology, New York, New York 10021; University of Pennsylvania (L.Y.M., R.R.S.), Department of Animal Biology, Philadelphia, Pennsylvania 19104 ABSTRACT Adrenal steroids can have prodigious effects on the structure, function, and survival of hippocampal neurons. In the rat hippocampus, the actions of adrenal steroids are mediated by two receptor types, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Using in situ hybridization, we have examined the regulation of the messenger RNAs (mRNAs) encoding the glucocorticoid and mineralocorticoid receptors, by aldosterone, which acts selectively through MR, and by RU28362, which acts selectively through GR. Our results

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HE HIPPOCAMPUS is highly sensitive to glucocorticoid action by virtue of the prominence in this brain region of two distinct adrenal steroid receptor subtypes. The mineralocorticoid receptor (MR or Type I receptor) has a high affinity for corticosterone and aldosterone (1, 2). In the hippocampus, MR messenger RNA (mRNA) is most abundant in CA2 pyramidal cells and is expressed at moderate levels in CA1, CA3, and the granule cells of the dentate gyrus (3), a pattern that reflects that observed for receptor binding (4, 5). The glucocorticoid receptor (GR or Type II receptor), which is expressed in most brain regions, has a relatively low affinity for aldosterone, a high affinity for dexamethasone, and is selectively activated by the synthetic agonist RU28362 (6, 7). In the hippocampus, the highest level of GR mRNA expression is in CA1 and lowest in CA3, with an intermediate level expressed in the dentate gyrus (3), a distribution similar to that for hippocampal GR protein as observed by immunocytochemistry (5, 8). Receptor binding studies indicate that hippocampal GR and MR are sensitive to changes in the available levels of steroid ligand. Withdrawal of endogenous steroids via bilateral adrenalectomy results in an increase in the number of adrenal steroid receptor binding sites, which can be reversed or prevented by steroid treatment (6, 9, 10). These results suggest that adrenal steroids may regulate their target receptors at the level of gene expression. Our studies sought to investigate whether changes in circulating steroid levels would affect the expression of the mRNAs encoding GR and MR in the rat hippocampus. By using ligands that were specific for each receptor subtype in the replacement regiReceived September 9, 1997. Address all correspondence and requests for reprints to: Helen M. Chao, The Rockefeller University, 1230 York Avenue, Box 165, New York, New York 10021. E-mail: [email protected].

demonstrate that there is autoregulation of each receptor subtype, such that activation of GR regulates GR mRNA levels and MR activation regulates MR mRNA expression. In addition, there is evidence that aldosterone, acting through MR, can affect the expression of GR mRNA. The extent to which a specific agonist can produce a significant change in the expression of a particular steroid receptor mRNA varies between the different subfields of the hippocampus. (Endocrinology 139: 1810 –1814, 1998)

men, it was possible to distinguish between autoregulation and reciprocal regulation of GR and MR, in the different hippocampal subfields. Materials and Methods Experimental animals Adult male Sprague-Dawley rats (CD strain, Harlan, Indianapolis, IN) were maintained on a 12-h light, 12-h dark cycle and had access to both water and 0.5 m NaCl from 7 days before surgery, to the time when the rats were euthanized. Exp 1. Animals were 1) sham-operated and implanted with mock minipumps (Sham); 2) adrenalectomized and implanted with mock minipumps (ADX); 3) ADX and implanted with Alzet no. 2001 minipumps delivering aldosterone at 1 mg/h (ADX 1 Aldo); and 4) ADX and implanted with minipumps delivering corticosterone at 10 mg/h (ADX 1 CORT); n 5 5– 6 per treatment group. The aldosterone (Sigma Chemical Co., St. Louis, MO) and corticosterone (Steraloids, Witton, NH) were delivered in propylene glycol. Daily fluid intakes were monitored following surgery. Animals were euthanized 7 days after surgery, and brains and trunk blood were collected. Plasma corticosterone levels were assessed by RIA (11). Exp 2. Animals were 1) sham-operated and implanted with mock minipumps (Sham); 2) adrenalectomized and implanted with mock minipumps (ADX); 3) ADX and implanted with Alzet no. 2001 minipumps delivering aldosterone at 1 mg/h (ADX 1 Aldo); 4) ADX and implanted with minipumps delivering RU28362 at 10 mg/h (ADX 1 RU); and 5) ADX and implanted with minipumps delivering aldosterone at 1 mg/h and RU28362 at 10 mg/h (ADX 1 Aldo1 RU); n 5 5– 6 per treatment group. The aldosterone (Sigma) and RU28362 (Roussel-Uclaf, Romainville, France) were delivered in propylene glycol. Daily fluid intakes were monitored, and animals exhibiting aberrant intake levels were eliminated from the study. Seven days after surgery, body weights were recorded, animals were euthanized, and brains and trunk blood were collected. Plasma steroid levels were assessed by RIA (Diagnostic Products Corporation, Los Angeles, CA).

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In situ hybridization Brains were removed, immediately frozen, and stored at 270 C. Sixteen-micron sections were prepared on a cryostat microtome, collected on gelatin-coated slides, and stored frozen until hybridization. Before hybridization, sections were fixed in 4% formaldehyde in PBS, acetylated in a solution of 0.25% acetic anhydride in 0.1 m triethanolamine-HCl, pH 8.0, rinsed in 2 3 SSC, and allowed to air-dry. Antisense riboprobes radioactively labeled with 35S-UTP were transcribed from complementary DNA clones corresponding to the rat glucocorticoid and mineralocorticoid receptors as previously described (12). The hybridization mix (50% formamide; 10% dextran sulfate; 600 mm NaCl; 1 3 Denhardt’s solution; 10 mm Tris-HCl, pH 7.5; 1 mm EDTA, pH 8; 100 mg/ml denatured salmon testis DNA; 10 mm DTT; radiolabeled probe) was added at 0.2 ml per slide, the slides were coverslipped, and the sections were incubated overnight at 55 C. Following hybridization, the coverslips were floated off, and the sections were rinsed in 2 3 SSC. The sections were treated with 10 mg/ml RNAse A, washed in RNase A buffer and in 2 3 SSC at room temperature, followed by 0.5 3 SSC at 55 C. The sections were allowed to air dry and then were apposed to x-ray film for autoradiography. The optical densities of the autoradiographic images were determined on the Imaging Research (St. Catherines, Ontario, Canada) image analysis system. The value of the low hybridization signal in CA1 stratum radiatum was taken (by definition) as background and subtracted from the optical density values for the hippocampal cell layers. The data were expressed as optical density (means 6 sem). For each animal, the optical density value represented the average of measurements from two to three brain sections per slide and two to three slides per animal. Statistical analysis was by one-way ANOVA followed by Tukey’s posthoc test, with P # 0.05 as the criterion for statistical significance.

Results Exp 1

Steroid replacement of adrenalectomized animals with aldosterone or corticosterone. Daily saline and water intake (Fig. 1) and plasma corticosterone levels were measured as indicators of the efficacy of the bilateral adrenalectomy and of the steroid replacement. The replacement of aldosterone to ADX animals restored their saline intake to levels comparable with Sham animals, whereas corticosterone replacement did not appreciably alter saline intake compared with the ADX animals (Fig. 1A). Plasma corticosterone levels were measurable in the Sham group (7.48 6 0.71 mg%) and the ADX 1 CORT group (10.88 6 1.35 mg%), but for the ADX and ADX 1 Aldo groups these levels were below the limit of detection for this RIA. In the adrenalectomized animals, there was a significant induction in GR mRNA expression in all hippocampal subfields examined, relative to the Sham animals (Fig. 2). Replacement with aldosterone resulted in a significant decrease in GR mRNA levels compared with ADX animals. Replacement with corticosterone completely reversed the ADX effect in all hippocampal subfields. Adrenalectomy resulted in a significant increase in MR mRNA levels in pyramidal cells but not in the granule cells of the dentate gyrus (Fig. 3). In aldosterone-replaced animals, pyramidal cell MR levels were significantly decreased compared with ADX animals. Corticosterone replacement restored the MR mRNA expression in CA1, CA2, and CA3 to levels comparable with adrenal-intact animals. Exp 2

Steroid replacement of adrenalectomized animals with aldosterone and/or RU28362. Daily saline and water intake (Fig. 4) and

FIG. 1. Daily saline intake (A) and daily water intake (B) in Exp 1. Daily intake levels were monitored from the day of surgery (Day 1) until the day the rats were euthanized.

FIG. 2. Hippocampal GR mRNA expression in Exp 1. Levels of GR mRNA expression were assessed in the CA1 pyramidal cell layer (CA1), CA3 pyramidal cell layer (CA3), and granule cell layer of the dentate gyrus (DG). Statistical analysis indicated that the ADX animals were significantly different from the Sham animals in all regions examined and that the ADX 1 Aldo group was different from the Sham group in the CA3 subregion (*, P # 0.05).

plasma steroid levels and body weight (Fig. 5) were measured as indicators of the efficacy of the surgery and of the steroid replacement. Plasma corticosterone levels were measurable in the Sham group (30.37 6 4.7 mg%), whereas

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FIG. 3. Hippocampal MR mRNA expression in Exp 1. Levels of MR mRNA expression were assessed in the CA1 pyramidal cell layer (CA1), CA2 pyramidal cell layer (CA2), CA3 pyramidal cell layer (CA3), and granule cell layer of the dentate gyrus (DG). Statistical analysis indicated that the ADX animals were significantly different from the Sham animals in CA1, CA2, and CA3, and that the ADX 1 Aldo group was different from the Sham group in CA2 and CA3 (*, P # 0.05).

plasma aldosterone levels were detectable in the Sham group and in the ADX groups that received aldosterone (Fig. 5A). The profile of saline intake was comparable with that observed in Exp 1 for the Sham, ADX, and aldosterone-replaced animals; however, the ADX 1 RU group exhibited an anomalous pattern (Fig. 4A). Moreover, the ADX 1 RU animals showed the lowest body weight gain during the study (Fig. 5B). Adrenalectomy resulted in an induction of GR mRNA expression in CA1 and CA3, relative to Sham animals (Fig. 6). In CA1 pyramidal cells, the ADX-induced increase was reversed by RU28362, whereas in CA3 pyramidal cells replacement with aldosterone was more effective in decreasing GR mRNA levels. In the adrenalectomized animals, there was a significant increase in MR mRNA levels in hippocampal pyramidal cells but not in the granule cells of the dentate gyrus, compared with Sham animals (Fig. 7). The ADX-induced increase was reversed by aldosterone replacement, in the presence or absence of RU28362, in CA2 and CA3. Discussion

Glucocorticoids can cause dramatic changes in the morphology, electrophysiology, and survival of neurons in the hippocampus, which may underlie their role in modulating cognition. In many of these processes, there are markedly different consequences to activation of each receptor subtype. Steroid agonists specific for MR facilitate long-term potentiation and reduce calcium currents in hippocampal neurons, whereas GR-specific agonists have the opposite effect on these parameters of neuronal excitability. The survival of granule cells in the dentate gyrus is dependent upon activation of MR, whereas GR activation can potentiate neurodegenerative and neurotoxic damage to hippocampal pyramidal cells (13). Adrenal steroid receptors act as transcriptional regulators

FIG. 4. Daily saline intake (A) and daily water intake (B) in Exp 2. Intake levels were monitored from 2 days before surgery until the rats were euthanized, with the day of surgery designated as Day 1.

of a variety of gene products affecting hippocampal function including neurotransmitters, neurotransmitter receptors, and neurotrophic factors (13–16). In the classical model of genomic regulation by GR and MR, ligand binding causes a conformational change in the receptor that results in nuclear translocation and association as homodimers with response elements in the target gene promoters (17, 18). In vitro studies have demonstrated that GR and MR are also able to form a heterodimeric complex, with transcriptional activator properties distinct from the homodimers (19, 20). The coexpression of GR and MR in hippocampal neurons (21, 22) suggests that such heterodimers may form in vivo as well. Receptor binding studies have provided evidence that, in the hippocampus, the number of GR and MR binding sites is susceptible to regulation by adrenal steroid ligands (6, 9, 10). In situ hybridization provides the anatomical resolution required to examine the regional specificity of this regulatory mechanism within the hippocampus. In the pyramidal cell layers of the hippocampus, we have found the expression of GR and MR to be responsive to adrenal steroid regulation, whereas such regulation in the granule cells of the dentate gyrus was equivocal. Because adrenalectomy has been shown to result in granule cell death (23), the possibility remains that, in measurements of the entire granule cell layer, neuronal loss could be obscuring increases in steroid


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FIG. 7. Hippocampal MR mRNA expression in Exp 2. Levels of MR mRNA expression were assessed in the CA1 pyramidal cell layer (CA1), CA2 pyramidal cell layer (CA2), CA3 pyramidal cell layer (CA3), and granule cell layer of the dentate gyrus (DG). Statistical analysis indicated that the ADX animals were significantly different from the Sham animals in CA1, CA2, and CA3, and that the ADX 1 RU group was different from the Sham group in CA2 and CA3 (*, P # 0.05).

FIG. 5. Plasma aldosterone levels (A) and body weight measurements (B) in Exp 2. Plasma aldosterone levels in the ADX and ADX 1 RU animals were below the limit of detection by this RIA.

FIG. 6. Hippocampal GR mRNA expression in Exp 2. Levels of GR mRNA expression were assessed in the CA1 pyramidal cell layer (CA1), CA3 pyramidal cell layer (CA3), and granule cell layer of the dentate gyrus (DG). Statistical analysis indicated that the ADX animals were significantly different from the Sham animals in CA1 and CA3 and that the ADX1RU group was different from the Sham group in the CA3 region (*, P # 0.05).

receptor expression in the cells that survive, a question that might be resolved by single-cell analysis of receptor mRNA expression. Previous studies have demonstrated that RU28362 is se-

lective for GR, whereas aldosterone selectively activates MR (6, 7, 23). The results obtained through receptor activation with these specific agonists indicate that in the hippocampus there is autoregulation of each adrenal receptor subtype. In the CA1 hippocampal subfield where GR mRNA is most abundant, the level of GR expression is regulated by RU28362, the GR-specific ligand. Similarly, in CA2 where levels of MR mRNA are highest, MR expression is regulated by the MR-activating ligand aldosterone but is unaffected by activation of GR by RU28362. In addition, we have found evidence that activation of MR can affect expression of GR mRNA. In CA3 pyramidal cells (where levels of GR are relatively low), aldosterone can act through MR to reverse the ADX-induced increase of GR mRNA as well as MR mRNA. These studies provide evidence that steroid receptor activation can regulate the expression of the GR and MR genes. In hippocampal cells that coexpress GR and MR, changes in receptor levels will alter the ratio between GR homodimers, MR homodimers, and the putative GR/MR heterodimers, which will in turn determine the extent to which target genes are activated. Adrenal steroid regulation of neurotransmitters and neurotransmitter receptors can influence hippocampal activity, and changes in neurotrophin expression can affect the morphology and survival of hippocampal neurons. Future studies will seek to correlate target gene regulation by a specific ligand with the known differential effects of GR and MR activation on the structure and function of hippocampal neurons. Acknowledgment The authors would like to thank Dr. James P. Herman (University of Kentucky Medical Center) for providing the GR and MR complementary DNA clones and Roussel-Uclaf (Romainville, France) for providing RU28362.

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