0013-7227/90/1271-0431$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 1 Printed in U.S.A.
Differential Regulation of Type II Corticosteroid Receptor Messenger Ribonucleic Acid Expression in the Rat Anterior Pituitary and Hippocampus* KAREN E. SHEPPARD, JAMES L. ROBERTS, AND MARIANN BLUM Fishberg Research Center in Neurobiology, Mt. Sinai Medical Center, New York, New York 10029
ABSTRACT. The present study was designed to characterize the regulation of the type II corticosteroid receptor (GR) mRNA in two tissues involved in the control of the hypothalamicpituitary-adrenal axis. We have used a solution hybridization/ Si nuclease protection assay to quantitate GR mRNA levels in the rat hippocampus and anterior pituitary after CRF, dexamethasone (DEX), or corticosterone (CORT) treatment. In general, hippocampal GR mRNA levels increased after removal of endogenous corticosteroids by surgical adrenalectomy and decreased in response to glucocorticoid treatment. More specifically, in the hippocampus 1) GR mRNA expression was decreased when adrenalectomized (ADX) animals were replaced with a relatively low dose of CORT, but not with a low dose of DEX; 2) acutely, CRF was more effective than DEX in decreasing the levels of GR mRNA in intact animals; however, under the same paradigm in ADX animals, DEX decreased the level of GR mRNA, whereas CRF was ineffective; and 3) in contrast to the decrease in GR mRNA levels observed after acute and low doses of glucocorticoid treatment, chronic treatment with
decrease in GR mRNA expression after CRF treatment is probably via the release of glucocorticoids, and that this tissue is more sensitive to endogenous glucocorticoids than DEX. Anterior pituitary GR mRNA was differentially regulated compared with that in the hippocampus. In marked contrast to GR mRNA in the hippocampus, ADX did not alter anterior pituitary GR mRNA expression, and glucocorticoid treatment led to an increase in GR mRNA levels. In the anterior pituitary 1) glucocorticoid treatment led to an increase in GR mRNA expression, when replaced with a relatively low dose of DEX, but not when replaced with a low dose of CORT; 2) acutely, neither CRF nor DEX altered levels of GR mRNA in intact animals; however, under the same paradigm DEX increased levels in ADX animals; and 3) chronic DEX or CORT treatment of intact animals elevated levels of anterior pituitary GR mRNA. In summary, these data have demonstrated tissue-specific regulation of GR mRNA in the hippocampus and anterior pituitary, which is dependent on both the dose and length of treatment and, in addition, on the glucocorticoid itself. {Endocrinology 127: 431-
either DEX or CORT did not change the level of hippocampal
439, 1990)
GR mRNA. These results suggest that in the hippocampus the
T
WO TYPES of receptors for adrenal steroids have been identified in the brain (1). The type I corticosteroid receptor, also referred to as the corticosterone (CORT)-preferring site or mineralocorticoid receptor, has similar, if not equal, apparent affinity in vitro for both the endogenous mineralocorticoid (aldosterone) and glucocorticoid (CORT in rat and cortisol in man) (2-6). However, in vivo the apparent affinity of these steroids for the type I corticosteroid receptor differs depending on the presence of tissue-modifying factors (7-9). The type II corticosteroid receptor [GR; classical dexamethasone (DEX)-binding site] has higher affinity for glucocorticoids than mineralocorticoids (5, 10). The type II corticosteroid receptor is present in nearly all mammalian tissues and has a multitude of physiological funcReceived February 5, 1990. Address all correspondence and requests for reprints to: Karen E. Sheppard, Fishberg Research Center in Neurobiology, Box 1065, Mt. Sinai Medical Center, New York, New York 10029. * This work was supported in part by a Fulbright Postdoctoral Fellowship (to K.E.S.) and NIH Grant DK-27484 (to J.L.R. and M.B.).
tions. In both the brain (in particular the hippocampus) and the anterior pituitary, one of the many actions of this receptor is its involvement in the feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis (1114). The cellular response to glucocorticoids is dependent on both the availability of steroid and the presence and cellular concentration of functional receptor (15-18). Ligand-induced down-regulation of the type II corticosteroid receptor and its increase in response to the removal of glucocorticoids by surgical adrenalectomy (ADX) have been shown by steroid binding assays (1923). Studies on corticosteroid receptor regulation by binding assays requires either the removal of endogenous ligand, which in itself can alter receptor concentration, or the complete exchange of endogenous/administered ligand for radiolabeled ligand. The manipulations needed for these assays potentially alter receptor-ligand binding characteristics and make acute studies on the effects of glucocorticoid treatment on receptor levels difficult. An-
431
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RAT GR mRNA REGULATION
432
other approach to studying the regulation of corticosteroid receptors is by examining gene expression. By measuring corticosteroid receptor gene expression, the problems associated with the binding assays can potentially be avoided and, in addition, additional information can be provided regarding the level at which the regulation is occurring. Recently, there have been several studies examining the regulation of these receptors at the mRNA level. From the studies in rats (24-27) and cultured cell lines (28-30) it appears that the regulation of GR mRNA by glucocorticoids differs depending on the cell type and mode of glucocorticoid administration. In the following study we have used a specific type II corticosteroid receptor antisense RNA probe in conjunction with a solution hybridization/Si nuclease protection assay to quantitate levels of GR mRNA in rat hippocampus and anterior pituitary after the administration of physiological and pharmacological doses of both the endogenous glucocorticoid CORT and the synthetic glucocorticoid DEX. We report that in two tissues involved in the regulation of the HPA axis (hippocampus and anterior pituitary), GR mRNA is differentially regulated in terms of 1) the direction of the response to glucocorticoids (in the hippocampus glucocorticoids decrease GR mRNA, whereas in the anterior pituitary glucocorticoids increase GR mRNA); 2) the sensitivity to either CORT or DEX (hippocampus is more sensitive to CORT, whereas anterior pituitary is more sensitive to DEX); and 3) the length of treatment (hippocampus responds rapidly but after chronic treatment there is no change in GR mRNA, whereas in the anterior pituitary chronic treatment results in an increase in GR mRNA).
Materials and Methods Animals Adult female Sprague-Dawley rats, weighing 250-300 g, purchased from Blue Spruce Farms (Altamont, NY) were used in all experiments. Animals were maintained on a 12-h light-dark cycle and fed rat chow and water ad libitum. Sham and bilateral ADX were performed under light ether anaesthesia between 1000-1100 h. After ADX, animals were given 0.9% NaCl solution to drink. Acute studies Animals (five or six per group) were sc injected with 20 ng synthetic rat (r) CRF (gift of Drs. J. Rivier and W. Vale, Salk Institute, La Jolla, CA), 20 ng DEX (Sigma Chemical Co., St. Louis, MO), or CRF vehicle (0.1% BSA and 0.1% ascorbic acid in sterile saline) and killed by decapitation 4 h later. For acute studies in adrenalectomized (ADX) animals, rats were either sham or bilaterally ADX 20 h before treatment.
E n d o • 1990 Vol 127 • No 1
Chronic glucocorticoid studies In chronic studies, animals (five or six per group) received placebo, CORT, or DEX via slow release pellets (Innovative Research of America, Toledo, OH). Placebo pellet (cholesterol, methylcellulose, and lactose), two 100-mg CORT pellets (CORT mixed with cholesterol, methylcellulose, and lactose), or one 10-mg DEX pellet (DEX mixed with cholesterol, methylcellulose, and lactose) was implanted sc behind the shoulder while the animal was under light ether anesthesia. Steroid pellets containing CORT or DEX were designed to deliver 4.8 and 0.48 mg steroid/pellet • day, respectively. Animals were treated for 6 days. Glucocorticoid replacement studies Immediately after bilateral ADX animals (four or five per group) received doses of CORT (25 fig/ml) or DEX (0.25 Mg/ ml) in their 0.9% NaCl drinking solution for 5 days to approximate physiological relevant doses of glucocorticoids. Control animals were sham ADX and received water to drink. RNA isolation and quantitation Animals were killed by decapitation, tissues were removed quickly, and cytoplasmic RNA was prepared from each individual tissue. To separate nuclei from the cytoplasmic fraction, tissues were homogenized in 800 /xl ice-cold AT buffer (10 mM Tris, pH 8; 3 mM CaCl2; 2 mM MgCl2; 0.5 mM dithiothreitol; and 0.15% Triton X-100) containing 0.3 M sucrose, layered over AT buffer containing 0.4 M sucrose, and then centrifuged at 600 x g for 10 min. The supernatant was then proteinase-K treated by the addition of 100 fig/ml proteinase-K (BoehringerMannheim, Indianapolis, IN) in 0.1 vol 10 X SET buffer (10% sodium dodecyl sulfate, 50 mM EDTA, and 10 mM Tris, pH 8.0) and then incubated for 60 min at 42 C. After protein digestion the sample was phenol-chloroform extracted, cytoplasmic RNA was precipitated by the addition of 0.1 vol 5 M NH4Ac and 1 vol isopropanol, and the sample was stored at -80 C. Before total cytoplasmic RNA quantitation, samples were reconstituted in TE buffer (10 mM Tris, pH 7.4, and 1 mM EDTA), and the RNA concentration was determined by absorbance at 260 nm. Recombinant clones All RNA probes were synthesized using the Promega transcription system (Riboprobe Gemini system II, Promega, Madison, WI) to a specific activity of 1-2 x 109 cpm/Vg. A 294basepair (bp) Xbal-BamHI rat GR cDNA fragment corresponding to the 3' untranslated region was subcloned into the pGEM3 vector [original 2.2-kilobase (kb) fragment was a gift of Dr. K. Yamamoto, University of California, San Francisco] (18). Both sense RNA and radiolabeled antisense RNA probes were synthesized from this vector using either T7 or SP6 RNA polymerase, respectively. For cytoplasmic POMC mRNA measurements, a 550-bp PstI cDNA fragment spanning part of exon 3 (31) was used to synthesize a 32P-labeled antisense RNA probe and a 1800-bp X/ioI-i/mdIII fragment spanning all of exon 3, and part of the 3' untranslated region was used to synthesize
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 05:03 For personal use only. No other uses without permission. . All rights reserved.
RAT GR mRNA REGULATION complimentary sense RNA. Cyclophilin mRNA levels were determined using a Pstl-XmnI (111-bp) fragment subcloned into pBluescript KS(+) vector. The original 680-bp Pstl-Hincll cDNA was a gift from Dr. J. Douglass (32). Antisense RNA labeled with 35S was synthesized using T7 RNA polymerase.
433
pg GR sense RNA 0
** 1.4
2>8
r5.6 *
%A
HIP AP
Solution hybridization/RNA protection assay To quantitate mRNA levels, a solution hybridization/Si nuclease protection assay was employed, as previously described (33) In brief, cytoplasmic RNA (10-20 /xg for GR mRNA measurements and 0.5-1.5 /xg for POMC) or known amounts of in vitro synthesized sense RNA were hybridized to a [35S] antisense or [32P]antisense RNA probe for 18 h at 65 C, followed by Si nuclease digestion (500-700 U SI; Pharmacia, Piscataway, NJ) for 60 min at 56 C. Samples were then phenolchloroform extracted, propanol precipitated, reconstituted in TE, and run on a 5% nondenaturing polyacrylamide gel. Gels were exposed to x-ray film, and the specific mRNA- or sense RNA-antisense RNA hybrid band was excised and counted by liquid scintillation spectrophotometry. GR mRNA levels were determined by comparison to the standard curve, corrected for full-length mRNA, and then normalized to the total amount of cytoplasmic RNA loaded.
B
Results Quantitation of GR mRNA By using a solution hybridization/Si nuclease protection assay we have measured levels of GR mRNA in the anterior pituitary and hippocampus. Figure 1A is representative of a typical autoradiogram of the protected GR band observed when a [32P] antisense RNA probe (294 bases in length) is hybridized to in vitro synthesized sense RNA, hippocampal cytoplasmic RNA, or anterior pituitary cytoplasmic RNA. The hybrid band produced from both the standard curve and tissue cytoplasmic RNA ran as predicted by the length of the antisense probe, approximately 290 bp. Figure IB is the graph obtained when the counts per min in the excised protected band of the sense-[32P] antisense RNA hybrid are plotted against picograms of sense RNA put into the assay. A single protected band was obtained in both the anterior pituitary and hippocampus; levels of GR mRNA were usually higher in the hippocampus than in the anterior pituitary in untreated animals. GR mRNA and POMC mRNA were corrected for the full length of the respective mRNA and then normalized to the total amount of cytoplasmic RNA loaded. To ensure that the changes observed were not due to an artifact of the RNA isolation procedure or measurement, we measured cyclophilin mRNA levels which have been reported not to change after hormonal manipulations (32). After all treatments cyclophilin mRNA levels in both anterior pituitary and hippocampus were unchanged, as depicted in Table 1. When data are normalized to either cyto-
2
4
6
8
10
pg GR sense RNA FIG. 1. Solution hybridization/Si nuclease protection analysis of GR sense RNA and tissue cytoplasmic RNA. A, Autoradiogram depicting the specific band obtained for both in vitro synthesized GR sense RNAand cytoplasmic RNA-[32P]GR antisense RNA hybrids after solution hybridization and Si nuclease digestion (as described in Materials and Methods). Rat hippocampal (18.4 fig) and 17.3 fig rat anterior pituitary cytoplasmic RNA were loaded. B, Standard curve obtained when the counts per min from each sense-[32P]antisense GR hybrid is plotted against amount of sense RNA loaded (r2 = 0.98).
plasmic RNA or cyclophilin RNA the results were comparable; thus, all data were expressed per ng cytoplasmic RNA. Acute studies Four hours after the administration of 20 pg of either CRF or DEX the levels of GR mRNA were determined in the anterior pituitary and hippocampus by solution hybridization/Si nuclease protection assay. In the anterior pituitary of adrenal intact rats, GR mRNA levels
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 05:03 For personal use only. No other uses without permission. . All rights reserved.
RAT GR mRNA REGULATION
434
TABLE 1. Cyclophilin mRNA expression in rat anterior pituitary and hippocampus
Anterior pituitary Hippocampus B
Control
CORT
DEX
158 ± 7 698 ± 42
179 ± 24 670 ± 10
165 ± 11 676 ± 26
Control
ADX
Anterior pituitary 198 ± 24 176 ± 14 Hippocampus 647 ± 51 640 ± 23
ADX/CORT ADX/DEX 190 ± 17 532 ± 33
163 ± 19 691 ± 35
Results are expressed as counts per min in specific cyclophilinprotected band per fig cytoplasmic RNA. Treatment conditions in A were described in Fig. 2., and those in B were described in Fig. 5. There was no significant change in cyclophilin mRNA levels after any of the treatments. The results represent the mean ± SEM.
ANTERIOR PITUITARY
Endo• 1990 Voll27«Nol
In contrast to the anterior pituitary, hippocampal GR mRNA levels significantly decreased by 35% (P < 0.01) in response to acute CRF treatment, while acute DEX treatment led to a slight but not statistically significant decrease (Fig. 2B). To determine if the effects of CRF were mediated through the release of corticosteroids from the adrenal, rats were adrenalectomized 20 h before the administration of 20 ng CRF or DEX and then killed 4 h later. Although levels of GR mRNA in the anterior pituitary were not altered 20 h after ADX, CRF treatment significantly decreased GR mRNA (P < 0.05) compared to that in ADX controls, while DEX led to a significant increase of 58% (P < 0.001; Fig. 3A). After ADX there was a small but significant increase in GR mRNA in the hippocampus (22%; P < 0.05). DEX treatment, but not CRF, decreased levels of GR mRNA by 35% compared ANTERIOR PITUITARY
12 108Sham
CRF
DB
Vol. 127, No. 1 Printed in U.S.A.
Differential Regulation of Type II Corticosteroid Receptor Messenger Ribonucleic Acid Expression in the Rat Anterior Pituitary and Hippocampus* KAREN E. SHEPPARD, JAMES L. ROBERTS, AND MARIANN BLUM Fishberg Research Center in Neurobiology, Mt. Sinai Medical Center, New York, New York 10029
ABSTRACT. The present study was designed to characterize the regulation of the type II corticosteroid receptor (GR) mRNA in two tissues involved in the control of the hypothalamicpituitary-adrenal axis. We have used a solution hybridization/ Si nuclease protection assay to quantitate GR mRNA levels in the rat hippocampus and anterior pituitary after CRF, dexamethasone (DEX), or corticosterone (CORT) treatment. In general, hippocampal GR mRNA levels increased after removal of endogenous corticosteroids by surgical adrenalectomy and decreased in response to glucocorticoid treatment. More specifically, in the hippocampus 1) GR mRNA expression was decreased when adrenalectomized (ADX) animals were replaced with a relatively low dose of CORT, but not with a low dose of DEX; 2) acutely, CRF was more effective than DEX in decreasing the levels of GR mRNA in intact animals; however, under the same paradigm in ADX animals, DEX decreased the level of GR mRNA, whereas CRF was ineffective; and 3) in contrast to the decrease in GR mRNA levels observed after acute and low doses of glucocorticoid treatment, chronic treatment with
decrease in GR mRNA expression after CRF treatment is probably via the release of glucocorticoids, and that this tissue is more sensitive to endogenous glucocorticoids than DEX. Anterior pituitary GR mRNA was differentially regulated compared with that in the hippocampus. In marked contrast to GR mRNA in the hippocampus, ADX did not alter anterior pituitary GR mRNA expression, and glucocorticoid treatment led to an increase in GR mRNA levels. In the anterior pituitary 1) glucocorticoid treatment led to an increase in GR mRNA expression, when replaced with a relatively low dose of DEX, but not when replaced with a low dose of CORT; 2) acutely, neither CRF nor DEX altered levels of GR mRNA in intact animals; however, under the same paradigm DEX increased levels in ADX animals; and 3) chronic DEX or CORT treatment of intact animals elevated levels of anterior pituitary GR mRNA. In summary, these data have demonstrated tissue-specific regulation of GR mRNA in the hippocampus and anterior pituitary, which is dependent on both the dose and length of treatment and, in addition, on the glucocorticoid itself. {Endocrinology 127: 431-
either DEX or CORT did not change the level of hippocampal
439, 1990)
GR mRNA. These results suggest that in the hippocampus the
T
WO TYPES of receptors for adrenal steroids have been identified in the brain (1). The type I corticosteroid receptor, also referred to as the corticosterone (CORT)-preferring site or mineralocorticoid receptor, has similar, if not equal, apparent affinity in vitro for both the endogenous mineralocorticoid (aldosterone) and glucocorticoid (CORT in rat and cortisol in man) (2-6). However, in vivo the apparent affinity of these steroids for the type I corticosteroid receptor differs depending on the presence of tissue-modifying factors (7-9). The type II corticosteroid receptor [GR; classical dexamethasone (DEX)-binding site] has higher affinity for glucocorticoids than mineralocorticoids (5, 10). The type II corticosteroid receptor is present in nearly all mammalian tissues and has a multitude of physiological funcReceived February 5, 1990. Address all correspondence and requests for reprints to: Karen E. Sheppard, Fishberg Research Center in Neurobiology, Box 1065, Mt. Sinai Medical Center, New York, New York 10029. * This work was supported in part by a Fulbright Postdoctoral Fellowship (to K.E.S.) and NIH Grant DK-27484 (to J.L.R. and M.B.).
tions. In both the brain (in particular the hippocampus) and the anterior pituitary, one of the many actions of this receptor is its involvement in the feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis (1114). The cellular response to glucocorticoids is dependent on both the availability of steroid and the presence and cellular concentration of functional receptor (15-18). Ligand-induced down-regulation of the type II corticosteroid receptor and its increase in response to the removal of glucocorticoids by surgical adrenalectomy (ADX) have been shown by steroid binding assays (1923). Studies on corticosteroid receptor regulation by binding assays requires either the removal of endogenous ligand, which in itself can alter receptor concentration, or the complete exchange of endogenous/administered ligand for radiolabeled ligand. The manipulations needed for these assays potentially alter receptor-ligand binding characteristics and make acute studies on the effects of glucocorticoid treatment on receptor levels difficult. An-
431
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 05:03 For personal use only. No other uses without permission. . All rights reserved.
RAT GR mRNA REGULATION
432
other approach to studying the regulation of corticosteroid receptors is by examining gene expression. By measuring corticosteroid receptor gene expression, the problems associated with the binding assays can potentially be avoided and, in addition, additional information can be provided regarding the level at which the regulation is occurring. Recently, there have been several studies examining the regulation of these receptors at the mRNA level. From the studies in rats (24-27) and cultured cell lines (28-30) it appears that the regulation of GR mRNA by glucocorticoids differs depending on the cell type and mode of glucocorticoid administration. In the following study we have used a specific type II corticosteroid receptor antisense RNA probe in conjunction with a solution hybridization/Si nuclease protection assay to quantitate levels of GR mRNA in rat hippocampus and anterior pituitary after the administration of physiological and pharmacological doses of both the endogenous glucocorticoid CORT and the synthetic glucocorticoid DEX. We report that in two tissues involved in the regulation of the HPA axis (hippocampus and anterior pituitary), GR mRNA is differentially regulated in terms of 1) the direction of the response to glucocorticoids (in the hippocampus glucocorticoids decrease GR mRNA, whereas in the anterior pituitary glucocorticoids increase GR mRNA); 2) the sensitivity to either CORT or DEX (hippocampus is more sensitive to CORT, whereas anterior pituitary is more sensitive to DEX); and 3) the length of treatment (hippocampus responds rapidly but after chronic treatment there is no change in GR mRNA, whereas in the anterior pituitary chronic treatment results in an increase in GR mRNA).
Materials and Methods Animals Adult female Sprague-Dawley rats, weighing 250-300 g, purchased from Blue Spruce Farms (Altamont, NY) were used in all experiments. Animals were maintained on a 12-h light-dark cycle and fed rat chow and water ad libitum. Sham and bilateral ADX were performed under light ether anaesthesia between 1000-1100 h. After ADX, animals were given 0.9% NaCl solution to drink. Acute studies Animals (five or six per group) were sc injected with 20 ng synthetic rat (r) CRF (gift of Drs. J. Rivier and W. Vale, Salk Institute, La Jolla, CA), 20 ng DEX (Sigma Chemical Co., St. Louis, MO), or CRF vehicle (0.1% BSA and 0.1% ascorbic acid in sterile saline) and killed by decapitation 4 h later. For acute studies in adrenalectomized (ADX) animals, rats were either sham or bilaterally ADX 20 h before treatment.
E n d o • 1990 Vol 127 • No 1
Chronic glucocorticoid studies In chronic studies, animals (five or six per group) received placebo, CORT, or DEX via slow release pellets (Innovative Research of America, Toledo, OH). Placebo pellet (cholesterol, methylcellulose, and lactose), two 100-mg CORT pellets (CORT mixed with cholesterol, methylcellulose, and lactose), or one 10-mg DEX pellet (DEX mixed with cholesterol, methylcellulose, and lactose) was implanted sc behind the shoulder while the animal was under light ether anesthesia. Steroid pellets containing CORT or DEX were designed to deliver 4.8 and 0.48 mg steroid/pellet • day, respectively. Animals were treated for 6 days. Glucocorticoid replacement studies Immediately after bilateral ADX animals (four or five per group) received doses of CORT (25 fig/ml) or DEX (0.25 Mg/ ml) in their 0.9% NaCl drinking solution for 5 days to approximate physiological relevant doses of glucocorticoids. Control animals were sham ADX and received water to drink. RNA isolation and quantitation Animals were killed by decapitation, tissues were removed quickly, and cytoplasmic RNA was prepared from each individual tissue. To separate nuclei from the cytoplasmic fraction, tissues were homogenized in 800 /xl ice-cold AT buffer (10 mM Tris, pH 8; 3 mM CaCl2; 2 mM MgCl2; 0.5 mM dithiothreitol; and 0.15% Triton X-100) containing 0.3 M sucrose, layered over AT buffer containing 0.4 M sucrose, and then centrifuged at 600 x g for 10 min. The supernatant was then proteinase-K treated by the addition of 100 fig/ml proteinase-K (BoehringerMannheim, Indianapolis, IN) in 0.1 vol 10 X SET buffer (10% sodium dodecyl sulfate, 50 mM EDTA, and 10 mM Tris, pH 8.0) and then incubated for 60 min at 42 C. After protein digestion the sample was phenol-chloroform extracted, cytoplasmic RNA was precipitated by the addition of 0.1 vol 5 M NH4Ac and 1 vol isopropanol, and the sample was stored at -80 C. Before total cytoplasmic RNA quantitation, samples were reconstituted in TE buffer (10 mM Tris, pH 7.4, and 1 mM EDTA), and the RNA concentration was determined by absorbance at 260 nm. Recombinant clones All RNA probes were synthesized using the Promega transcription system (Riboprobe Gemini system II, Promega, Madison, WI) to a specific activity of 1-2 x 109 cpm/Vg. A 294basepair (bp) Xbal-BamHI rat GR cDNA fragment corresponding to the 3' untranslated region was subcloned into the pGEM3 vector [original 2.2-kilobase (kb) fragment was a gift of Dr. K. Yamamoto, University of California, San Francisco] (18). Both sense RNA and radiolabeled antisense RNA probes were synthesized from this vector using either T7 or SP6 RNA polymerase, respectively. For cytoplasmic POMC mRNA measurements, a 550-bp PstI cDNA fragment spanning part of exon 3 (31) was used to synthesize a 32P-labeled antisense RNA probe and a 1800-bp X/ioI-i/mdIII fragment spanning all of exon 3, and part of the 3' untranslated region was used to synthesize
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 05:03 For personal use only. No other uses without permission. . All rights reserved.
RAT GR mRNA REGULATION complimentary sense RNA. Cyclophilin mRNA levels were determined using a Pstl-XmnI (111-bp) fragment subcloned into pBluescript KS(+) vector. The original 680-bp Pstl-Hincll cDNA was a gift from Dr. J. Douglass (32). Antisense RNA labeled with 35S was synthesized using T7 RNA polymerase.
433
pg GR sense RNA 0
** 1.4
2>8
r5.6 *
%A
HIP AP
Solution hybridization/RNA protection assay To quantitate mRNA levels, a solution hybridization/Si nuclease protection assay was employed, as previously described (33) In brief, cytoplasmic RNA (10-20 /xg for GR mRNA measurements and 0.5-1.5 /xg for POMC) or known amounts of in vitro synthesized sense RNA were hybridized to a [35S] antisense or [32P]antisense RNA probe for 18 h at 65 C, followed by Si nuclease digestion (500-700 U SI; Pharmacia, Piscataway, NJ) for 60 min at 56 C. Samples were then phenolchloroform extracted, propanol precipitated, reconstituted in TE, and run on a 5% nondenaturing polyacrylamide gel. Gels were exposed to x-ray film, and the specific mRNA- or sense RNA-antisense RNA hybrid band was excised and counted by liquid scintillation spectrophotometry. GR mRNA levels were determined by comparison to the standard curve, corrected for full-length mRNA, and then normalized to the total amount of cytoplasmic RNA loaded.
B
Results Quantitation of GR mRNA By using a solution hybridization/Si nuclease protection assay we have measured levels of GR mRNA in the anterior pituitary and hippocampus. Figure 1A is representative of a typical autoradiogram of the protected GR band observed when a [32P] antisense RNA probe (294 bases in length) is hybridized to in vitro synthesized sense RNA, hippocampal cytoplasmic RNA, or anterior pituitary cytoplasmic RNA. The hybrid band produced from both the standard curve and tissue cytoplasmic RNA ran as predicted by the length of the antisense probe, approximately 290 bp. Figure IB is the graph obtained when the counts per min in the excised protected band of the sense-[32P] antisense RNA hybrid are plotted against picograms of sense RNA put into the assay. A single protected band was obtained in both the anterior pituitary and hippocampus; levels of GR mRNA were usually higher in the hippocampus than in the anterior pituitary in untreated animals. GR mRNA and POMC mRNA were corrected for the full length of the respective mRNA and then normalized to the total amount of cytoplasmic RNA loaded. To ensure that the changes observed were not due to an artifact of the RNA isolation procedure or measurement, we measured cyclophilin mRNA levels which have been reported not to change after hormonal manipulations (32). After all treatments cyclophilin mRNA levels in both anterior pituitary and hippocampus were unchanged, as depicted in Table 1. When data are normalized to either cyto-
2
4
6
8
10
pg GR sense RNA FIG. 1. Solution hybridization/Si nuclease protection analysis of GR sense RNA and tissue cytoplasmic RNA. A, Autoradiogram depicting the specific band obtained for both in vitro synthesized GR sense RNAand cytoplasmic RNA-[32P]GR antisense RNA hybrids after solution hybridization and Si nuclease digestion (as described in Materials and Methods). Rat hippocampal (18.4 fig) and 17.3 fig rat anterior pituitary cytoplasmic RNA were loaded. B, Standard curve obtained when the counts per min from each sense-[32P]antisense GR hybrid is plotted against amount of sense RNA loaded (r2 = 0.98).
plasmic RNA or cyclophilin RNA the results were comparable; thus, all data were expressed per ng cytoplasmic RNA. Acute studies Four hours after the administration of 20 pg of either CRF or DEX the levels of GR mRNA were determined in the anterior pituitary and hippocampus by solution hybridization/Si nuclease protection assay. In the anterior pituitary of adrenal intact rats, GR mRNA levels
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 05:03 For personal use only. No other uses without permission. . All rights reserved.
RAT GR mRNA REGULATION
434
TABLE 1. Cyclophilin mRNA expression in rat anterior pituitary and hippocampus
Anterior pituitary Hippocampus B
Control
CORT
DEX
158 ± 7 698 ± 42
179 ± 24 670 ± 10
165 ± 11 676 ± 26
Control
ADX
Anterior pituitary 198 ± 24 176 ± 14 Hippocampus 647 ± 51 640 ± 23
ADX/CORT ADX/DEX 190 ± 17 532 ± 33
163 ± 19 691 ± 35
Results are expressed as counts per min in specific cyclophilinprotected band per fig cytoplasmic RNA. Treatment conditions in A were described in Fig. 2., and those in B were described in Fig. 5. There was no significant change in cyclophilin mRNA levels after any of the treatments. The results represent the mean ± SEM.
ANTERIOR PITUITARY
Endo• 1990 Voll27«Nol
In contrast to the anterior pituitary, hippocampal GR mRNA levels significantly decreased by 35% (P < 0.01) in response to acute CRF treatment, while acute DEX treatment led to a slight but not statistically significant decrease (Fig. 2B). To determine if the effects of CRF were mediated through the release of corticosteroids from the adrenal, rats were adrenalectomized 20 h before the administration of 20 ng CRF or DEX and then killed 4 h later. Although levels of GR mRNA in the anterior pituitary were not altered 20 h after ADX, CRF treatment significantly decreased GR mRNA (P < 0.05) compared to that in ADX controls, while DEX led to a significant increase of 58% (P < 0.001; Fig. 3A). After ADX there was a small but significant increase in GR mRNA in the hippocampus (22%; P < 0.05). DEX treatment, but not CRF, decreased levels of GR mRNA by 35% compared ANTERIOR PITUITARY
12 108Sham
CRF
DB
