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Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Kelly E. Mayo, Ph.D., Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology, Hogan Science Building 3–100, 2153 Sheridan Road, Evanston, Illinois 60208. E-mail: k-mayo{at}nwu.edu
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Introduction |
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Corticosteroids are important physiological regulators of GH synthesis and secretion (12). In general, glucocorticoids exert a suppressive effect on the GH axis in mammals (13), causing growth retardation in laboratory rats (14, 15) and decreased growth rates in chronically treated children (12). In addition, patients with Cushings syndrome, which clinically presents as hypercortisolemia, have almost lost the ability to secrete GH (12). Glucocorticoids seem to exert these growth-suppressive effects by acting at multiple levels of the GH pathway (1, 12). Previously, it was demonstrated that GHRH-binding sites were down-regulated in dispersed pituitary cells from adrenalectomized rats, and hormone replacement with the synthetic glucocorticoid, dexamethasone, was able to restore GHRH binding (16, 17). Significantly, these changes in GHRH binding were not caused by altered binding affinity, suggesting that differences in receptor number were responsible for the corticosteroid effects. To characterize further the mechanisms of corticosteroid action on the GH axis, we analyzed the effects of glucocorticoids on the expression of the rat GHRH-R gene in the intact pituitary and in enzymatically dispersed pituitary cells, using RT, followed by the PCR, to measure GHRH-R mRNA levels. We demonstrate that glucocorticoids increase GHRH-R mRNA levels both in vivo and in vitro. We also demonstrate that this induction requires new RNA synthesis, indicating that glucocorticoids act to regulate gene transcription, rather than mRNA stability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Adult male Sprague-Dawley rats (Charles River, Portage, MI; and
Harlan, Indianapolis, IN), weighing 200–250 g, were housed in
facilities approved by the American Association for Accreditation of
Laboratory Animal Care, under a light:dark cycle of 14:10, with water
and food provided ad libitum. The experimental protocols
used in these studies were approved by the Northwestern University
Institutional Animal Care and Use Committee.
In vivo effects of corticosterone
Male rats were bilaterally adrenalectomized or sham operated
under light anesthesia between 0900 and 1200 h on day 1 of the
experiment. Immediately after surgery, and continuing for the duration
of the experiment, the adrenalectomized rats were maintained on 0.9%
saline in their drinking water. The animals received either 2
cholesterol pellets (average pellet weight of 340 mg) or 2
corticosterone pellets (average pellet weight of 375 mg), made
according to previously described methods (18) and implanted sc under
light metofane (Methoxyflurane, Pitman-Moore, Mundelein, IL) anesthesia
at 0900 h on day 6 of the experiment. Animals were killed by
decapitation between 1030 and 1230 h on day 9 of the experiment.
Pituitaries were removed, frozen on dry ice, and stored at -80 C until
RNA was extracted. Trunk blood was collected on ice and allowed to
clot, and serum was stored at -20 C for subsequent hormone measurement
by RIA. Adrenal gland atrophy (14) and reduced body weight (14, 15)
have previously been reported as indicators of high circulating levels
of corticosterone. Body weight was therefore monitored for each animal
throughout the course of the experiment, and the adrenal glands were
removed from the sham-operated animals and dissected, and the paired
wet weight was measured.
In vitro effects of dexamethasone
Anterior pituitary glands were obtained from 30–40 male rats at
0900 h. Primary anterior pituitary cultures were prepared, as
previously described (19), with some modifications. Briefly, the
anterior lobes were separated from the neurointermediate and posterior
lobes and placed into ice-cold, sterile HEPES buffer (25 mM
HEPES, pH 7.4; 137 mM NaCl; 5 mM KCl; 0.7
mM Na2HPO4; 10 mM
glucose) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and
0.25 µg/ml amphotericin B. The anterior pituitaries were washed in
HEPES buffer four times, then minced with sterile scalpel blades on a
siliconized glass slide into 1- to 2-mm3 pieces. The
pituitary fragments were pooled and washed twice with HEPES buffer. The
cells were dispersed two times, at 30 C for 60 min each time, in a
Bellco flask containing 25 ml enzymatic trypsin solution (HBSS
containing: 4 mM NaHCO3, 10 mM
HEPES, pH 7.4; 0.1% wt/vol trypsin; 0.2% wt/vol glucose; 0.4% wt/vol
BSA; 10 µg/ml DNAse I type IV). The cells were collected by low-speed
centrifugation (270 x g) and dissociated by
triteration in DMEM containing 10 µg/ml DNAse I type IV and 25
µg/ml soybean trypsin inhibitor. Three to five million cells were
plated into 12-well plates containing DMEM with 5% (vol/vol)
charcoal-stripped FBS (20), 100 U/ml penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml amphotericin B, then incubated at 37 C.
After 40 h, the cells were treated with ethanol vehicle or the
synthetic glucocorticoid, dexamethasone, (1 nM to 1
µM) in fresh medium and harvested at various time points.
In experiments involving the RNA polymerase inhibitor, actinomycin D
(21), the cells were pretreated with 5 µg/ml (4 µM)
actinomycin D or DMSO vehicle for 30 min, before treatment with
dexamethasone. The actinomycin D treatment was continued for the
duration of the experiment.
RNA isolation
RNA was extracted from individual pituitaries by homogenization
in guanidinium thiocyanate and purified by ultracentrifugation through
a cesium chloride density gradient (22). RNA was isolated from primary
anterior pituitary cultures by extraction of acidic guanidinium
thiocyanate lysates with phenol and chloroform (23), followed by RQ1
DNAse I digestion of the RNA.
RT-PCR
Oligonucleotide primer pairs of 22 nucleotides (RPRJA;
5'-CATCTCCTAGGTCCAAACCAGC-3' and RPRJC; 5'-GAAGTTCAGGGTCATGGCCATA-3';
50–54% G+C content) and 21 nucleotides (RPL19A,
5'-CTGAAGGTCAAAGGGAATGTG-3'; and RPL19B,
5'-GGACAGAGTCTTGATGATCTC-3'; 42–47% G+C content) were
synthesized, based on the sequences of rat GHRH-R (3) and ribosomal
protein L19 (24), respectively. Primers were designed such that the
expected products would be 489 bp for the GHRH-R and 196 bp for
ribosomal protein L19 (RPL19). We first established optimal conditions
for this assay by determining the range of the assay with regard to the
number of amplification cycles and RNA input, as previously described
(25). This revealed the appropriate range of the assay to be 22 to 35
amplification cycles for both the GHRH-R and RPL19 primers (data not
shown), and 28 cycles were utilized in all subsequent experiments.
Total RNA from intact pituitaries (2.5 µg) or primary pituitary cell
cultures (0.5 µg) were reverse transcribed at 42 C in a 20-µl
reaction using random hexamer primers and avian myeloblastosis
virus-RT. Five microliters of each reverse transcription reaction was
amplified in a 25-µl reaction in the presence of either the GHRH-R or
the RPL19 primers (125 ng each), 
32P-dCTP (0.5 µCi at
3000 Ci/mmol), and Taq DNA polymerase (0.625 units) in 10
mM Tris (pH 8.3), 50 mM KCl, 2.2 mM
MgCl2, 0.01% gelatin and overlaid with mineral oil.
Amplification was carried out for 28 cycles, using an annealing
temperature of 65 C (primers RPRJA and RPRJC) or 55 C (primers RPL19A
and RPL19B), on a Perkin Elmer thermocycler. The samples were
chloroform extracted and 10 µl of each sample were size separated by
electrophoresis on a 5% nondenaturing polyacrylamide gel. After
autoradiography, the gel was analyzed on a Molecular Dynamics
Phosphorimager using ImageQuant version 3 software (Molecular Dynamics,
Sunnyvale, CA). The intensity of the GHRH-R signal was normalized to
the RPL19 control and plotted as arbitrary phosphorimage units.
RIA
Serum corticosterone was measured using a double-antibody kit
from ICN Biomedicals employing [125I]
corticosterone-3-carboxymethyloxime as the tracer. The intraassay
variation was 2%.
Statistical analysis
Unless otherwise stated, data shown are mean plus
SEM. For the in vivo experiments, n refers to
the number of individual pituitaries for each treatment group. For the
primary pituitary cells, n refers to the number of cell culture wells
for each treatment group, and each experiment was repeated at least
twice. ANOVA was performed using CRISP statistical software (CRUNCH
Software, San Francisco, CA). Post hoc comparisons of the
effects of dexamethasone were performed using the Newman-Keuls test.
P less than 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Bilateral adrenalectomy decreased pituitary GHRH-R mRNA expression to
approximately 60% of the level observed in cholesterol-treated intact
animals (P < 0.0004). This effect was reversed by the
addition of exogenous corticosterone, resulting in a 1.9-fold increase
in GHRH-R mRNA levels relative to cholesterol-treated, adrenalectomized
rats (P < 0.0001). In addition, treatment of intact
animals with corticosterone resulted in a 1.6-fold increase in GHRH-R
mRNA levels relative to cholesterol-treated, intact animals
(P < 0.0001).
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). There was no significant difference in the weight of
the animals before treatment, regardless of surgery status. However,
the effects of surgery and treatment on the final body weight were both
statistically significant (P < 0.0001). Animals
treated with cholesterol, regardless of surgery status, gained weight
(average percent BW gain of +7.69 ± 0.72% and +12.64 ±
1.08% for sham and adrenalectomy, respectively), whereas there was a
loss in body weight or lack of weight gain in animals receiving
corticosterone (-12.14 ± 1.31% and +0.56 ± 1.03% for
sham and adrenalectomy, respectively). The level of circulating
corticosterone, as measured by RIA (66.37 µg/dl ± 19.6 for
intact rats and 50.85 µg/dl ± 1.43 for adrenalectomized rats),
was statistically elevated (P < 0.0001) when compared
to cholesterol treated animals. Additionally, the paired adrenal weight
of sham-operated rats that received corticosterone was decreased to
75% of that in cholesterol-treated animals (P <
0.0002).
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).
GHRH-R mRNA levels were significantly elevated by all doses of
dexamethasone (P < 0.0004), with a maximal increase of
approximately 5-fold in the presence of 100 nM
dexamethasone.
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). Although there was
no significant change in GHRH-R mRNA levels after 3 h of treatment
with 100 nM dexamethasone, there was a significant increase
in GHRH-R mRNA after 6 h (6.1-fold), 12 h (5.2-fold) and
24 h (4.1-fold) dexamethasone treatment, as compared with the
time-matched controls (P < 0.0001). Post
hoc comparisons of the dexamethasone-treated groups indicated that
the effect of hormone treatment on GHRH-R mRNA levels was increased
with time (P < 0.05). Parallel studies, not shown
here, indicated that GH mRNA was elevated after 12 h treatment
with 1 µM dexamethasone and 24 h exposure to 100
nM dexamethasone (P < 0.05).
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). A 4.4-fold increase in GHRH-R mRNA expression in
response to glucocorticoid treatment was observed in the absence of
actinomycin D treatment (P < 0.0001), consistent with
the inductions observed in Figs. 2
and 3. In contrast, treatment with
actinomycin D completely abolished the ability of dexamethasone to
increase GHRH-R mRNA in pituitary cells (P <
0.0001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that glucocorticoids have diverse effects at multiple levels of the GH axis. These adrenal hormones influence downstream effectors, such as IGF-I (26), regulate GH feedback mechanisms by altering the expression of the GH receptor gene (27, 28), and modify both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids can stimulate directly GH synthesis and secretion in the absence of any hypothalamic input (29). Implanting the pituitary gland under the kidney capsule removes the regulatory effects of GHRH and SS on GH expression. Under these conditions, adrenalectomy decreases pituitary GH mRNA levels, whereas subsequent treatment with dexamethasone restores GH synthesis (29). GH release (30) and GH mRNA accumulation (31) are both stimulated by glucocorticoids in cultured pituitary cells. Glucocorticoid regulation of the GH gene occurs primarily through a direct interaction of the glucocorticoid receptor with a response element within the first intron of the GH gene (32).
Glucocorticoids influence hypothalamic control over GH expression by maintaining a balance between the positive and negative influences of GHRH and SS. Previous studies have suggested that chronic exposure to glucocorticoids augments somatostatinergic tone at the level of the hypothalamus (1, 12). Expression of SS mRNA is increased in the arcuate nucleus after 48 and 72 h of dexamethasone treatment (27); however, prolonged exposure to the hormone decreases SS mRNA levels in the periventricular and paraventricular regions of the hypothalamus (28, 33). Total hypothalamic SS content is increased in response to glucocorticoid treatment during the same period (33, 34). Conversely, GHRH mRNA expression in the arcuate nucleus of the hypothalamus is decreased after prolonged treatment with dexamethasone (27, 28), and decreases in total hypothalamic GHRH content are detected after 3 days exposure to the hormone (34).
The glucocorticoid-induced changes in GHRH and SS expression at the level of the hypothalamus are similar to alterations in pituitary expression of the GHRH and SS receptors in response to glucocorticoid treatment. Some in vivo studies have suggested that glucocorticoids act by suppressing GHRH-stimulated GH release (34); however, the duration of glucocorticoid treatment has been shown to have profound effects on the GHRH response (35). Significantly, other in vivo studies have demonstrated that treatment with glucocorticoids in the presence of SS inhibitors (36) or antibodies specific for SS (37) enhances the response of GH to GHRH. In primary rat pituitary cultures, glucocorticoids augment both basal and GHRH-induced GH release (30, 34) and reduce the ability of SS to inhibit GH release in response to GHRH (30). Importantly, glucocorticoids have been shown to exert differential effects on GHRH (16, 17) and SS binding (38). The observed changes in GHRH and SS binding are not caused by altered receptor affinity, suggesting that differences in receptor numbers are responsible for the corticosteroid effects. In support of these observations, mRNAs for SS receptor (SSTR) subtypes SSTR1, SSTR2, and SSTR3 are all decreased in pituitary cell cultures after 24 h exposure to glucocorticoids (39). Our observation that GHRH-R mRNA levels are induced after 6 h of glucocorticoid treatment of primary pituitary cells and remain elevated up to 24 h after initiation of treatment further supports the hypothesis that glucocorticoids differentially regulate SS and GHRH, as well as their corresponding receptors.
Corticosteroids exert a broad spectrum of physiological effects in the animal; therefore, multiple components could be involved in eliciting the increase in GHRH-R mRNA levels that is observed in vivo. The high level of circulating corticosterone, the lack of weight gain, and the substantial decrease in adrenal gland weight in the animals that received hormone implants suggest that the paradigm used for analyzing GHRH-R mRNA regulation by corticosteroids mimics the chronic exposure previously demonstrated to inhibit both linear growth (12, 13, 14, 15) and GH release (34). Although it is possible that the increases in pituitary expression of GHRH-R mRNA are caused by the catabolic effects of corticosterone, glucocorticoids remain capable of enhancing GHRH-R gene expression in a primary pituitary cell culture system. It recently has been demonstrated that glucocorticoids directly inhibit GHRH neurons in the arcuate nucleus of the hypothalamus, whereas the inhibitory effects of this hormone on the SS neurons of the periventricular nucleus are mediated by GH (28). It is therefore possible that the changes in GHRH-R expression, in response to corticosterone that we have observed in vivo, are contributed to by GH and/or glucocorticoid-mediated changes in GHRH or SS expression in the hypothalamus. In support of this observation, it has recently been demonstrated that passive immunization with GHRH antiserum and treatment with exogenous GH both cause decreased GHRH-R mRNA expression in vivo (40). However, our finding that dexamethasone increases GHRH-R mRNA expression in cultured anterior pituitary cells suggests that glucocorticoids can act directly at the level of the pituitary to exert its effect.
Thyroid hormone also has profound effects on the GH axis (41, 42), and in many ways, these parallel the effects of glucocorticoids on the hypothalamic peptides SS and GHRH and their pituitary receptors. Thyroid ablation decreases SS mRNA, while simultaneously increasing GHRH mRNA. Similarly, GHRH-R mRNA expression is decreased in response to thyroidectomy (41) and antithyroid treatment (42); however, SSTR1 seems to be unaffected by thyroid hormone manipulations. In addition, thyroid hormone can directly stimulate GH gene transcription (31). Both glucocorticoid and thyroid hormones, therefore, act to increase somatostatinergic tone at the level of the hypothalamus and increase pituitary responsiveness to GHRH secretion by altering GHRH-R levels. This change in sensitivity could help to explain the observation that GH is expressed at an earlier developmental time, in fetal rats that have been treated with dexamethasone, than in control animals (43).
Glucocorticoids are well-characterized activators of transcription, exerting their effects through specific cellular receptors that interact directly with hormone response elements located in the regulatory regions of responsive genes (44). Steroid hormones, including glucocorticoids, also may contribute to changes in mRNA stability (45). In pituitary cells, glucocorticoids have been shown both to increase transcription of the GH gene (31) and to enhance the stability of the GH mRNA (46). It was therefore important to determine whether the effects of glucocorticoids on GHRH-R mRNA accumulation were caused by increased gene transcription or by alterations in GHRH-R mRNA stability. We have attempted to assess directly the effects of glucocorticoids on GHRH-R gene transcription by performing nuclear run-on experiments to measure gene transcription rates, but this assay lacks sufficient sensitivity, presumably because of the extremely low levels of GHRH-R expression and the heterogeneous population of cells present in primary pituitary cultures. We therefore investigated the ability of an inhibitor of transcription to block the glucocorticoid induction of GHRH-R mRNA in vitro. Actinomycin D is a well-characterized inhibitor of gene transcription that acts by intercalating into DNA, thus preventing the progression of RNA polymerase (18). Actinomycin D also can affect protein synthesis by interfering with the association of mRNAs with the ribosome (47). Although there are clearly limitations to using this potent metabolic inhibitor, short duration treatments with actinomycin D can provide important insights into transcriptional vs. posttranscriptional regulatory mechanisms. For example, actinomycin D has been used previously in pituitary primary cell cultures to assess hormonal modulation of the gonadotropin subunit mRNAs (48). We found that actinomycin D completely blocked the dexamethasone-induced increase in GHRH-R mRNA levels we had previously observed in cultured pituitary cells, consistent with the hypothesis that glucocorticoids act directly on pituitary cells to stimulate transcription of the GHRH-R gene.
Our studies have clearly defined a mechanism by which glucocorticoids can modify pituitary responsiveness to GHRH released from the hypothalamus, and thus, increase GH synthesis and secretion in vivo. Given the positive actions of glucocorticoids on both GHRH-R and GH within the pituitary somatotrope cell, the overall growth suppressive effects of this steroid are indicative of its complex actions at multiple levels of the GH axis. During the preparation of this manuscript, others reported similar effects of glucocorticoids on GHRH-R mRNA levels in vivo (49) or in vitro (50). Lam and co-workers (49) used RNA blot analysis to demonstrate that pituitary GHRH-R mRNA levels were increased in dexa-methasone-treated intact rats, whereas Tamaki and co-workers (50) used a dispersed pituitary cell system to demonstrate increased GHRH-R mRNA levels in response to dexamethasone. Although these recent studies used animal and cell culture models somewhat different than those described here, in general, these findings agree with both our in vivo and pituitary cell culture experiments demonstrating that glucocorticoids are important positive regulators of GHRH-R mRNA expression. Our observation that actinomycin D can inhibit the ability of dexamethasone to increase GHRH-R mRNA expression further suggests that this is predominantly a transcriptional response and that glucocorticoid receptors are likely to directly interact with the promoter of the GHRH-R gene to stimulate its activity.
The GHRH-R gene has been characterized recently from several species (51, 52, 53, 54), and glucocorticoid response elements have been identified in the 5' flanking region of the human GHRH-R gene (53). This observation suggests that glucocorticoids might modulate GHRH-R mRNA expression in the human pituitary gland as they do in the rat, and indeed, the human gene seems to be glucocorticoid-responsive (53). The presence of glucocorticoid response elements in the GHRH-R gene promoter is consistent with a direct interaction of the glucocorticoid receptor with this gene to regulate its transcription. Glucocorticoid receptors have been shown to colocalize in the rat pituitary, predominantly with the corticotropin and GH secreting cells (54) and, therefore, are appropriately localized to mediate transcriptional responses in somatotrope cells. Our studies demonstrating that glucocorticoids stimulate expression of the GHRH-R gene both in vivo and in vitro provide a likely explanation for the ability of this steroid to enhance pituitary responsiveness to GHRH. To understand the mechanism of glucocorticoid action in this system in more detail will clearly require the identification and characterization of the DNA elements within the GHRH-R gene that mediate glucocorticoid responsiveness.
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Acknowledgments |
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Footnotes |
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Received October 11, 1996.
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I. Bossis and T. E. Porter Ontog |
