Regulation of Growth Hormone-Releasing Hormone Receptor ...

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 6 Printed in U.S.A.

Regulation of Growth Hormone-Releasing Hormone Receptor Messenger Ribonucleic Acid Expression by Glucocorticoids in MtT-S Cells and in the Pituitary Gland of Fetal Rats* HARUO NOGAMI, KINJI INOUE, HIDEKAZU MORIYA, AKI ISHIDA, SHUZO KOBAYASHI, SETSUJI HISANO, MASATERU KATAYAMA, AND KOKI KAWAMURA Department of Anatomy (H.N, K.K) and Department of Neurosurgery (M.K), School of Medicine, Keio University, Tokyo 160, Japan; Department of Regulation Biology (K.I), Faculty of Science, Saitama University, Saitama 338, Japan; Second Department of Internal Medicine (H.M, A.I, S.K), National Defense Medical College, Saitama 359, Japan; and Department of Anatomy (S.H), Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki 305, Japan ABSTRACT Regulation of GH-releasing hormone receptor (GHRH-R) messenger RNA (mRNA) expression was studied, with the ribonuclease protection assay, in the fetal rat pituitary gland and in MtT-S clonal cells. GHRH-R mRNA was first detected on embryonic day (E)19 and increased rapidly thereafter, to reach a maximum at E21. Incubation of E17 or E18 pituitaries with 50 nM dexamethasone (DEX), a synthetic glucocorticoid, induced GHRH-R mRNA expression, suggesting that glucocorticoids play a pivotal role in the developmental expression of this mRNA. In E19 pituitaries, 24 h treatment with DEX increased GHRH-R mRNA by 60%, and GH mRNA by 76%, but did not affect pit-1 mRNA level, suggesting that the effect of DEX is specific for expressions of GH mRNA and GHRH-R mRNA. The accumulation of GHRH-R mRNA by DEX was time dependent, and it was slightly enhanced by the protein synthesis inhibitor, puromycin (100 mM). In MtT-S cells (a pituitary cell line established from an estrogeninduced tumor), DEX induced GHRH-R mRNA expression within 2 h

G

H-RELEASING hormone (GHRH), a hypothalamic polypeptide, is an important regulator of both GH secretion and the proliferation of pituitary GH-producing cells (1). During prenatal development of the hypothalamicpituitary axis in rats, hypothalamic GHRH neurons develop before pituitary GH cells. An in situ hybridization study has shown that GHRH messenger RNA (mRNA) is first expressed in the arcuate nucleus in rats at embryonic day (E)16 (2). Immunoreactive GHRH is first detected in hypothalamic extracts at E17 (3), and GHRH nerve terminals are first observed in the median eminence at E18 (4). Finally, the vascular connection between the hypothalamus and the anterior pituitary gland is established by E18 (5). These anatomical findings suggest that hypothalamic GHRH may modulate the function of the anterior pituitary gland from E18. Received October 16, 1998. Address all correspondence and requests for reprints to: Haruo Nogami, Ph.D., Department of Anatomy, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. * This work was supported, in part, by a grant from the Keio Health Counseling Center and a Special Research Grant of the Health and Science Research from the Ministry of Health and Welfare, Japan.

in a dose-dependent manner. This induction was augmented by puromycin (100 mM) or cycloheximide (3.5 mM). However, the RNA synthesis inhibitor Actinomycin D (1 mM) completely inhibited GHRH-R mRNA accumulation in response to either DEX or DEX plus puromycin, suggesting that glucocorticoids induce GHRH-R mRNA mainly through stimulation of mRNA transcription. These results suggest: that GHRH-R mRNA accumulation in the fetal pituitary gland of rats normally occurs at E19, probably because of the direct action of glucocorticoids on the pituitary gland, to stimulate GHRH-R mRNA transcription; and that the expression of glucocorticoid receptors is an important event in GH cell development in rats. Accordingly, immunocytochemical results suggest an increase in glucocorticoid receptors in immature GH cells between E17 and E18. The present results also imply that MtT-S cells may be a good model in which to further study the molecular mechanisms of the regulation of GHRH-R gene expression. (Endocrinology 140: 2763–2770, 1999)

On the other hand, GH cells are rare in the anterior pituitary gland before E18. They increase in number at E19 (6, 7). Expression of the GHRH receptor (GHRH-R) is a crucial step for the functional maturation of GH cells, because they are thus brought under hypothalamic regulation. The pituitary responds to GHRH by releasing GH, which indicates the presence of GHRH-R. GHRH-induced GH release has been detected by RIA at E18 (8) and by the reverse hemolytic plaque assay at E19 (7). The developmental pattern of GHRH-R mRNA has been studied in mice with in situ hybridization (1, 9). GHRH-R mRNA is first detected at E16.5, when GH mRNA is also first expressed and where GH mRNA and pit-1 mRNA are also detected. In the rat, GHRH-R mRNA expression was demonstrated at E19.5 (10) with an ribonuclease (RNase) protection assay. However, little is known about the mechanisms by which GHRH-R mRNA expression is induced in developing GH cells. We previously reported that a transient increase in glucocorticoid levels in the fetal circulation, which is probably caused by the increased ACTH secretion at this stage (11), induces GH mRNA expression in pituitary GH cells (12, 13). Because glucocorticoids have been shown to up-regulate

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GLUCOCORTICOID REGULATION OF GHRH-R mRNA

GHRH-R mRNA in adult rats (14), we examined in the present study whether the increased levels of glucocorticoids in the fetus induce the expression of GHRH-R mRNA in addition to the expression of GH mRNA. Our data indicate that glucocorticoids play a pivotal role in the induction of GHRH-R mRNA in fetal pituitary GH cells. The role of glucocorticoids in GHRH-R mRNA induction was also examined in MtT-S cells, a clonal cell line established from estrogen-induced pituitary tumor (15). MtT-S cells secrete only GH and display a normal GH cell-like ultrastructure, such as well-developed rough endoplasmic reticulum, Golgi apparatus, and abundant secretory granules that contain immunoreactive GH. Furthermore, they secrete an increased amount of GH in response to GHRH stimulation (15).

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Timed pregnant Sprague Dawley rats were obtained from Sankyo Laboservice Co., Ltd. (Tokyo, Japan) at day 12 of gestation and housed in a light-controlled (14-h light, 10-h dark) and temperature-controlled (22 C) room. Food and tap water were given ad libitum. The day on which spermatozoa were found in a vaginal smear was designated as day 0 of pregnancy (E0). Fetuses were dissected free from dams at E17–E21 under light ether anesthesia, and fetal pituitaries were removed under the surgical microscope and stored frozen at 280 C until RNA extraction. For organ culture studies, fetal pituitaries obtained from E17–E19 fetuses were cut into several pieces and incubated for 24 h, unless otherwise stated, in 0.5 ml of a-modification of MEM (MEM a; Gibco BRL, Grand Island, NY) supplemented with glutamine and antibiotics. The experiments were carried out in accordance with the animal experimentation guidelines of Keio University.

et al. (17) and subcloned into a pGEM4Z plasmid (Promega Corp., Madison, WI). The plasmid was linearized with HindIII digestion, and the 32P-labeled cRNA probe (185 b) was synthesized with SP-6 polymerase. A 221-bp rat pit-1 cDNA (143–363 from ATG) (18) was obtained with RT-PCR from the pituitary total RNA of adult rats and cloned into the SmaI site of a pGEM4Z plasmid. The plasmid was linearized with EcoRI digestion and transcribed with T7-polymerase to give a cRNA probe (273 b). A 100-bp (483–582 from ATG) (19) fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was obtained with RT-PCR and cloned in pBSIISK-plasmid. The plasmid was linearized with Xba-I digestion and transcribed with T7 polymerase to give a 295 b cRNA probe. Sequences of the probes were determined with the dideoxy chain termination method (20) to confirm identity with the authentic cDNA sequences. The 32P-labeled RNAs of known length (564, 370, and 185 b) were prepared and used as size markers. Total RNA was isolated from fetal pituitary glands (3–5 pituitaries were pooled for one assay) or MtT-S cells, as described previously (21), and extracted with phenol/chloroform. The RNA concentration was determined spectrophotometrically, and 6 –12 mg fetal pituitary RNA or 20 mg MtT-S RNA was subjected to mRNA determination with the RNase protection assay. Total RNA was dissolved in 20 ml hybridization buffer (400 mm NaCl, 1 mm EDTA, 40 mm piperazine diethanesulfonic acid (pH 6.4), 80% formamide) containing 2 3 104 cpm cRNA probe and incubated at 65 C for 5 min and then at 55 C for 4 h or overnight. After hybridization, 150 ml digestion buffer, composed of 300 mm sodium acetate, 5 mm EDTA, 10 mm Tris-HCl (pH 7.5), containing 10 mg/ml RNase A and 100 U/ml RNase T1 (both from Boehringer Mannheim, Mannheim, Germany), was added to the reaction mixture and incubated for 30 min at 37 C. Then, 2.5 ml proteinase K (Boehringer Mannheim, 20 mg/ml) and 10 ml of 10% sodiumdodecylsulfate was added and incubated at 37 C for an additional 15 min. The protected fragments were precipitated with ethanol and analyzed on an 8% polyacrylamide gel containing 8 m urea. The mRNA level was determined with a BAS2000 image analyzer (Fuji Photo Film Co., Ltd. Film, Tokyo, Japan).

MtT-S cell culture

Immunocytochemistry

Materials and Methods Experimental animals

MtT-S cells were grown in DMEM/Ham’s F12 medium (DMEM/F12; Gibco BRL) containing 10% horse serum, 2.5% FBS, and antibiotics (control medium). When the cells were grown in serum-containing medium, the basal level of GHRH-R mRNA expression was measured. All experiments were carried out after deinduction of mRNA expression as follows. Approximately 2 3 105 cells were placed in a 10-cm polyl-lysin-coated dish with 10 ml of control medium and were cultured for 10 –12 days, with medium changes every 3 days, after which the medium was replaced with DMEM/F12 containing charcoal-stripped serum (hormone-deficient medium) and cultured for 4 days. The medium was then replaced with serum-free DMEM/F12 and cultured for 4 days. The cells were harvested after the experiments with trypsin digestion and stored at 280 C.

Chemicals The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and used at the stated concentrations: dexamethasone (DEX, 0.01 nm–1 mm), T3 (0.1 nm), estradiol (E2, 10 nm), dibutyryl-cAMP (cAMP, 1 mm), forskolin (1 mm), puromycin (100 mm), cycloheximide (3.5 mm), actinomycin D (1 mm), and all trans-retinoic acid (RA, 1 mm). GHRH was purchased from Peptide Institute (Osaka, Japan) and used at 1 nm.

Complementary RNA (cRNA) probe and RNase protection assay A 240-bp rat GHRH-R complementary DNA (cDNA) (2–241 from ATG) (16) was obtained with RT-PCR from the pituitary RNA of adult rats and cloned into a pBSIISK-plasmid (Stratagene, La Jolla, CA). The plasmid was linearized with HindIII digestion and transcribed with T3-polymerase to give a 32P-cRNA probe (345 b) with a-32P-uridine 59-triphosphate (ICN Biomedicals, Inc., Costa Mesa, CA). A 125-bp fragment that encodes most of the sequences of the second exon of the rat GH gene was isolated from a cloned rat GH gene prepared by Takeuchi

The pituitary glands obtained from E17–E19 fetuses were fixed in 10% formalin in 0.1 m phosphate buffer, pH 7.4, and embedded in paraffin. Five-micrometer thick sections were cut and placed on glass slides. The sections were heated at 45 C overnight. Before staining, sections were deparaffinized and irradiated in a microwave oven for 10 min in 10 mm citrate buffer, pH 6.0. The sections were then incubated at 4 C overnight with a mixture of mouse monoclonal antibody to rat glucocorticoid receptor (GCR) (BuGR2, Affinity Bioreagents, Inc., NJ), and the antisera to rat pituitary hormones were diluted in PBS (0.85% NaCl in 10 mm phosphate buffer, pH 7.4), containing 0.5% skim milk to reduce nonspecific reactions. The rabbit antiserum to rat TSH b-subunit (TSH-b) and the guinea-pig antiserum to rat LH b-subunit (LH-b) were gifts from the National Hormone and Pituitary Program, NIH, MD). The rabbit antirat GH (22) and antiporcine ACTH (23) were prepared in our laboratory with rat GH B-7 (supplied by the National Hormone and Pituitary Program) and porcine ACTH (grade II, Sigma Chemical Co.) as immunogens. The working dilutions of the antisera were 1:800 (BuGR2), 1:10000 (anti-ACTH, anti-TSH b, anti-LH b, and anti-GH). The sections were rinsed with PBS and incubated with peroxidase-labeled goat IgG antimouse IgG at 37 C for 1 h. After being washed with PBS, the sections were soaked in 3,39-diaminobenzidine (DAB) solution (50 mm Tris-HCl, pH 7.5, containing 10 mg/dl DAB, 0.003% hydrogen peroxide) supplemented by 50 mg/dl ammonium nickel sulfate to stain GCRs blue. After the coloration, the sections were incubated with peroxidase-labeled goat IgG antirabbit or guinea-pig IgG at 37 C for 1 h and then soaked in a DAB solution, without ammonium nickel sulfate, to stain pituitary hormones brown.

Statistical analyses The significance of differences of the data was determined with the Student’s t test or ANOVA followed by the Student-Newman-Keuls test.


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Results Developmental and glucocorticoid regulation of GHRH-R mRNA in the fetal pituitary gland

The age-dependent expression of GHRH-R mRNA was examined in E17–E21 fetuses with the RNA protection assay (Fig. 1). GHRH-R mRNA was not detected in pituitary RNA extracts from E17 or E18 fetuses. GHRH-R mRNA was detected first at E19 and increased linearly until E21. To examine the effects of glucocorticoids on GHRH-R mRNA expression, fetal pituitary glands were removed at E17–E19 and incubated in serum-free MEM a for 24 h with or without 50 nm DEX (Fig. 2). Treatment with DEX induced a small amount of GHRH-R mRNA in the E17 pituitaries but markedly induced GHRH-R mRNA in E18 pituitaries. In the control experiment, in which pituitary glands were incubated for the same duration in a medium without DEX, no GHRH-R mRNA accumulation was noted. The increase in the GHRH-R mRNA was seen only after incubation with DEX and not after incubation with either T3 (0.1 nm) or cAMP (1 mm) (data not shown). The time course of DEX induction of GHRH-R mRNA accumulation was examined with pituitaries from E19 rats (Fig. 3). The GHRH-R mRNA level gradually increased, until 24 h, in the presence of 50 nm DEX. When E19 pituitaries were incubated in a serum-free medium without DEX, GHRH-R mRNA levels rapidly decreased to about 13% of the 0 h value after 24 h. A difference in GHRH-R mRNA levels between DEX-treated and -untreated pituitaries was observed within 4 h of the start of incubation.

FIG. 2. DEX induces the expression of GHRH-R mRNA in the fetal pituitary gland in vitro. Pituitaries were removed at the indicated stages and incubated in serum-free MEM a for 24 h with (1) or without (2) 50 nM DEX. The total RNA was extracted from these explants, and GHRH-R mRNA and GAPDH mRNA were detected. Sizes of the marker RNAs were indicated to the left. The representative autoradiogram (exposed for 7 days) was shown, in which 8 mg of the total RNA was used.

Next, we examined whether the effect of DEX on GHRH-R mRNA accumulation requires de novo protein synthesis (Fig. 4). In this experiment, E19 pituitary glands were incubated in a serum-free MEM a for 8 h. After incubation, GHRH-R mRNA levels in DEX-treated pituitaries were about 2.5 times higher than those in control cultures. Puromycin (100 mm), a protein synthesis inhibitor, did not affect GHRH-R mRNA levels when used alone. In our previous experiment, 100 mm puromycin inhibited incorporation of 35S-methionine to an acid insoluble fraction by 87%. When the pituitaries were incubated with both DEX and puromycin, induction of GHRH-R mRNA was 40% higher than that of DEX alone. These results suggest that DEX does not require ongoing protein synthesis to induce GHRH-R mRNA. Effect of DEX on GHRH-R mRNA expression in MtT-S cells FIG. 1. Expression of GHRH-R mRNA in the developing rat pituitary gland. Total RNA was extracted from the fetal pituitaries at different gestational days, as indicated. Five (E17 and E18) or three (for others) pituitaries were pooled for an assay. In this and following figures, the GHRH-R mRNA was detected by the RNase protection assay, and quantification of each of the mRNA bands was carried out using a BAS2000 image analyzer. Sizes of marker RNAs were indicated to the left. The representative autoradiogram (exposed for 7 days) was shown, in which 12 mg of the total RNA was used. The amount of GHRH-R mRNA was normalized for GAPDH mRNA, and the results were expressed as percent of the maximum (mean 6 SEM, n 5 3).

GHRH-R mRNA expression was detected, with the RNase protection assay, in MtT-S cells cultured in a control medium but not in the cells cultured in hormone-deficient or serumfree medium (Fig. 5A). The following experiments were carried out after deinduction of GHRH-R mRNA. GHRH-R mRNA was induced 24 h after treatment of deinduced MtT-S cells with DEX (50 nm) but was not induced after treatment with either T3 (0.1 nm), E2 (10 nm), cAMP (1 mm), forskolin (1 mm), or GHRH (1 nm) (Fig. 5A). The mRNA induction by DEX was dose-dependent (Fig. 5B); the increase in GHRH-R

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FIG. 4. Effects of puromycin on the induction of GHRH-R mRNA by DEX. E19 pituitaries (2–3 pituitaries/group) were incubated for 8 h in a serum-free MEM a (Cont) or in the same medium containing 50 nM DEX, 100 mM puromycin (Pum), or both (DEX 1 Pum). After incubation, total RNA was extracted, and GHRH-R mRNA and GAPDH mRNA were determined. The representative autoradiogram (exposed for 12 days) was shown, in which 10 mg of the total RNA was used. The amount of GHRH-R mRNA was normalized for GAPDH mRNA, and the results were expressed as percent of control value (mean 6 SEM, n 5 3) *, P , 0.05; NS, not significant vs. control, by ANOVA followed by Student-Newman-Keuls test.

GHRH-R mRNA responses both to DEX and to DEX-plusprotein synthesis inhibitors (Fig. 6). Specificity of the effects of DEX FIG. 3. Time course of induction of GHRH-R mRNA by DEX in the fetal pituitary gland. Pituitaries were removed from E19 fetuses and incubated in a serum-free MEM a with (F) or without (E) 50 nM DEX, for the duration indicated. Sizes of the marker RNAs were indicated to the left. The representative autoradiogram (exposed for 10 days) was shown, in which 8 mg of the total RNA was used. The amount of GHRH-R mRNA was normalized for GAPDH mRNA, and the results were expressed as percent of 0 h value (mean 6 SEM, n 5 3). *, P , 0.05; NS, not significant vs. without DEX, by the Student’s t test.

was observed within 2 h and was followed by a sharp increase in the mRNA level until 24 h later (Fig. 5C). The characteristics of GHRH-R mRNA expression in MtT-S cells were similar to those observed in the fetal pituitary gland. The effects of protein synthesis inhibitors on GHRH-R mRNA expression were also examined in MtT-S cells (Fig. 6). Treatment of deinduced MtT-S cells with 50 nm DEX for 6 h induced detectable levels of GHRH-R mRNA. This induction by DEX was significantly enhanced by puromycin (100 mm) or cycloheximide (3.5 mm), which inhibit an incorporation of 35 S-methionine to an acid insoluble fraction by 87% or 90%, respectively. These inhibitors of protein synthesis alone did not affect the levels of GHRH-R mRNA. In contrast, actinomycin D (1 mm), an RNA synthesis inhibitor, suppressed the

To analyze the effect of DEX on expression of mRNA other than GHRH-R mRNA in GH cells, E19 pituitaries were incubated with RA (1 mm), cAMP (1 mm), or DEX (50 nm) for 24 h; and the changes in pit-1 mRNA and GH mRNA levels were examined (Fig. 7). Treatment with RA increased pit-1 mRNA by 74% in the absence of DEX. DEX increased GH mRNA by 76% but did not affect the pit-1 mRNA level. cAMP did not affect pit-1 mRNA or GH mRNA levels under these experimental conditions. These results indicate that the effect of DEX is specific for GH mRNA and GHRH-R mRNA expression. Immunocytochemistry

Our results indicate the importance of GCR expression during GH cell development. If the initial induction of GHRH-R mRNA depends upon glucocorticoid stimulation, expression of GCRs should precede expression of GHRH-R in fetal GH cells. We examined this hypothesis using doublelabeling immunocytochemistry. Pituitary sections of E17– E19 fetuses were stained for GCRs and a pituitary hormone. At E17, most GCR-positive cells were ACTH cells (Fig. 8A), whereas GCR-positive LH or TSH cells were rarely found in any of the stages examined. On the other hand, cells in the


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FIG. 5. The GHRH-R mRNA expression in MtT-S cells. The amount of the total RNA used for assay was 20 mg, and the autoradiograms were exposed for 14 days. A, Basal expression of GHRH-R mRNA was detectable in MtT-S cells cultured in a control medium. This amount was almost undetectable after 4-day culture in the hormone-deficient medium. The cells were then cultured in serum-free medium for an additional 4 days. No GHRH-R mRNA expression was detected in MtT-S cells after this deinduction procedure. The MtT-S cells were used for experiments after deinduction. After 24 h incubation with the chemicals indicated, the cells were harvested, and GHRH-R mRNA and GAPDH mRNA were determined. B, DEX induced GHRH-R mRNA in MtT-S cells in a dose-dependent manner. MtT-S cells were cultured for 24 h with various concentrations of DEX. Cont, Serum-free control. C, Time course of GHRH-R mRNA accumulation in MtT-S cells in response to DEX. They were cultured with 50 nM DEX for the duration indicated, and GHRH-R mRNA and GAPDH mRNA were detected.

pituitary gland that were positive for GCR, but not for ACTH, were increased in number at E18 (Fig. 8B). At E19, many GH cells appeared, most of which were positive for GCR (data not shown). Discussion

The development of GHRH-R expression in the rat has been studied by using RIA (8) or reverse hemolytic plaque assay (7) to measure GH released from cultured fetal pituitary GH cells in response to GHRH. Experiments have found that GHRH-Rs first appear at E18 (8), E19 (7), or E20 (24). In this study, we used an RNase protection assay to examine expression of GHRH-R mRNA. If the mRNA is assumed to be translated immediately, our results indicate that GHRH-Rs develop in the rat at E19, because GHRH-R mRNA was not detected at E18 but was detected at high levels from E19 (Fig. 1). Previous reports in mice have demonstrated that GHRH-R mRNA is first expressed at E16.5, corresponding to the timing of GH mRNA expression (1, 9). In agreement with this finding, our present and previous studies (12, 13) in rats demonstrate that both GH mRNA and GHRH-R mRNA are expressed at substantial levels on the same gestational day, E19. The simultaneous expression of both GH mRNA and GHRH-R mRNA lead us to speculate that a common factor may trigger expression of the two mRNAs in the fetal pituitary gland.

Our present finding that treatment of E18 pituitaries with DEX for 24 h induced GHRH-R mRNA expression suggests that glucocorticoids are primary factors required for the initiation of GHRH-R mRNA expression in the fetal rat pituitary gland (Fig. 2). Because immunoneutralization of GHRH decreased GHRH-R mRNA levels in the pituitary gland of neonatal rats, it is conceivable that hypothalamic GHRH is required to maintain GHRH-R mRNA levels during the neonatal period (25). However, the present results of in vitro experiments demonstrate that glucocorticoids initiate GHRH-R mRNA expression in E18 GH cells in the absence of hypothalamic factors. On the other hand, it is possible that factors derived from the hypothalamus or other (as yet, unidentified) factors may be involved in the rapid elevation of pituitary GHRH-R mRNA levels at E20 and E21. Because corticosterone circulates from mother to fetus through the placenta (26, 27), serum glucocorticoid levels are believed to be in an equilibrium between the maternal and fetal compartments during gestation. Hence, glucocorticoids are supplied to the developing pituitary gland long before GHRH-Rs are expressed. However, GHRH-R mRNA is not expressed until E19. Two explanations for this can be proposed. One is a unique temporal pattern of development of GCRs in fetal GH cells. Although GCRs have been shown in the fetal pituitary gland as early as E15, cellular localization of GCRs has been demonstrated only in ACTH cells at this

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FIG. 7. Effects of DEX, RA, and cAMP on the expression of pit-1 mRNA and GH mRNA in E19 pituitary glands. E19 fetal pituitaries (2 pituitaries per group) were incubated in a serum-free MEM a (Cont) or the same medium containing DEX (50 nM), RA (1 mM), or cAMP (1 mM) for 24 h. After incubation, the total RNA was extracted from tissues; and pit-1 mRNA, GH mRNA, and GAPDH mRNA were determined. The representative autoradiogram (exposed for 10 days) was shown, in which 6 mg of the total RNA was used. The amount of GHRH-R mRNA was normalized for GAPDH mRNA, and the results were expressed as percent of controls (mean 6 SEM, n 5 3). *, P , 0.05; NS, not significant vs. control, by ANOVA followed by Student-Newman-Keuls test.

FIG. 6. Effects of protein synthesis inhibitors [Pum (100 mM) and cycloheximide (Chx, 3.5 mM)] and a transcription inhibitor [actinomycin D (AD, 1 mM)] on GHRH-R mRNA induction by DEX. MtT-S cells were incubated for 6 h in the presence of the chemicals indicated. The representative autoradiogram (exposed for 14 days) was shown, in which 20 mg of the total RNA was used. The amount of GHRH-R mRNA was normalized for GAPDH mRNA, and the results were expressed as fold increase over DEX alone (the mRNA level of DEX alone was set as 1.0). Cont, Serum-free control. Values are the mean 6 SEM (n 5 3).

stage (28). Little is known about the temporal pattern of expression of GCRs in GH progenitor cells. The present immunocytochemical results suggest that extremely few GH cells express GCRs or that GCRs are expressed at low levels in GH cells at E17. This may be a reason why pituitary GHRH-R mRNA expression was not detected at this stage. However, the distinct response of E18 pituitaries to DEX

through the expression of GHRH-R mRNA (Fig. 2) may indicate an increase in the number of GH cells that express GCRs at this stage, as suggested by results of immunocytochemical studies. Therefore, another reason should be proposed for the lack of GHRH-R mRNA expression in the E18 pituitaries in vivo, namely, a change in plasma corticosterone levels in fetuses during late gestation. Previous reports indicate that the plasma levels of corticosterone in rats increase from E17–E19 and decline thereafter (11, 29). This transient elevation of plasma corticosterone levels in the fetal rat is believed to be caused by adrenal hyperactivity induced by increased secretion of ACTH at this stage (11). Our inability to detect GHRH-R mRNA at E18 (Fig. 1) may be caused by circulating corticosterone levels at this stage being below the threshold necessary to stimulate GHRH-R mRNA expression. Accumulating data indicate that GHRH-R gene expression is regulated by multiple factors in rats. In adult animals and in adult pituitary cells in primary culture, glucocorticoids and thyroid hormones up-regulate GHRH-R (30) and its mRNA (31–34), whereas estrogen down-regulates GHRH-R mRNA (33). Aleppo et al. (35) found that a reduction or removal of serum from the culture medium markedly downregulates GHRH-R mRNA expression in the pituitary primary culture of rats and that the addition of DEX to the medium restores mRNA levels. These results indicate the dependence of basal GHRH-R mRNA expression on glucocorticoids, which was also observed in this study, in the fetal pituitary gland and in MtT-S cells (Figs. 3 and 5A). In neonatal rats, GHRH is required for GHRH-R mRNA expression (25); whereas, in adult rats, GHRH either downregulates GHRH-R mRNA expression as an acute effect (35)


FIG. 8. Immunocytochemical stainings of GCRs and ACTH in the E17 (A) and E18 (B) rat pituitary glands. Arrows indicate cells that are positive for GCR but not for ACTH antiserum. Magnification, 3300.

or up-regulates GHRH-R mRNA expression by chronic stimulation (36). The cAMP that mediates GHRH activity in GH cells has also been shown to stimulate GHRH-R mRNA expression in primary culture of the adult pituitary gland (36). However, little is known about factors regulating GHRH-R mRNA expression during the functional development of fetal GH cells. In the present study, we examined the effect of T3, E2, cAMP, forskolin, GHRH, and DEX on GHRH-R mRNA levels. Only DEX affected GHRH-R mRNA levels in fetal pituitaries and MtT-S cells (Fig. 5). Although glucocorticoids might be required for general GH cell functions, the results shown in Fig. 7 do not support this possibility. RA was found to increase pit-1 mRNA levels in the E19 pituitary gland in the absence of glucocorticoids, in agreement with a previous study in pituitary cell lines (37). On the other hand, DEX enhanced expression of both GH mRNA and GHRH-R mRNA but did not affect expression of pit-1 mRNA. Thus, the effect of glucocorticoids seems to be specific for the expression of GH mRNA and GHRH-R mRNA. It is unknown why several factors that have been shown to stimulate GHRH-R mRNA expression in the adult rat are ineffective in the fetal pituitary gland or in MtT-S cells. Only the direct effects of these factors on GHRH-R mRNA expression were observed in the present in vitro study. Glucocorticoids might act directly at the pituitary level, whereas in vivo, other factors might act indirectly. Another explanation for this inconsistency is that the regulatory mechanisms

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of GHRH-R mRNA may differ among the various developmental stages. Petersenn et al. (38) examined regulatory regions of the human GHRH-R gene and demonstrated, through transient expression studies, that transcription of the human GHRH-R gene is significantly enhanced by glucocorticoids. Although they were unable to find the consensus glucocorticoid response element in a 2-kb upstream region of the human GHRH-R gene, their results suggest the presence of unidentified positive glucocorticoid response elements within this region. Lin et al. (9) demonstrated that pit-1 is required for the stimulation of rat GHRH-R gene transcription in vitro. The present results showed, however, that the effect of DEX on GHRH-R mRNA expression was not mediated by the increased expression of pit-1 (Fig. 7). The molecular basis of glucocorticoid effects on GHRH-R mRNA expression is still unclear. To examine the mechanisms of glucocorticoid regulation of GHRH-R mRNA expression, we studied the effects of protein synthesis inhibitors and an RNA synthesis inhibitor. Puromycin unexpectedly enhanced the effect of DEX, both in fetal pituitary glands and in MtT-S cells. In contrast, actinomycin D clearly inhibited the effect of DEX, suggesting that the primary effect of DEX is the stimulation of GHRH-R gene transcription (Fig. 6). These results are similar to those of Miller and Mayo (14) in cultured pituitary cells of adult rats. Unfortunately, we were unable to demonstrate any effect of actinomycin D on the fetal pituitary because this metabolic inhibitor induced progressive cell death in our tissue fragments. Another possible cause of the increase in the pituitary GHRH-R mRNA level is an increase by glucocorticoids in the population of preexisting GHRH-R mRNA-expressing cells between E18 and E19. However, recent studies by Porter and colleagues (39, 40) do not support this supposition. They showed, by reverse hemolytic plaque assay in chicken embryo, that glucocorticoids markedly increase the number of GH cells in the pituitary gland at E12, when only a few GH cells are normally present (39, 40). The effect of glucocorticoids was not blocked by a mitosis inhibitor; and less than 10% of GH cells, newly differentiated in response to glucocorticoids, were labeled with 3H-thymidine (39). These results suggest that glucocorticoids do not stimulate proliferation of GH cells. Finally, it seems worthwhile to note the effects of puromycin on glucocorticoid-induced GHRH-R mRNA accumulation. Puromycin and cycloheximide, which inhibit protein synthesis, enhanced the effects of DEX on GHRH-R mRNA accumulation in MtT-S cells but had little effect on the level of GHRH-R mRNA when used without DEX (Fig. 6). These findings suggest the possibility that an unknown protein facilitates degradation of GHRH-R mRNA. Because GHRH-R mRNA undergoes rapid degradation in the absence of glucocorticoids, as seen in E19 pituitaries (Fig. 3), suppression of degradation would significantly increase GHRH-R mRNA levels. Another possibility is that a protein suppresses transcription of the GHRH-R gene. The downregulation of this type of suppressor would result in the activation of transcription. In summary, the present results suggest that: 1) glucocor-

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ticoids induce GHRH-R mRNA expression in the fetal pituitary gland, as previously demonstrated in adult tissues (33– 36), and that this may be the normal mechanism responsible for the onset of GHRH-R mRNA expression in immature GH cells in the fetus; and 2) GHRH-R mRNA induction by glucocorticoids is caused by stimulation of mRNA transcription.

19.

20. 21.

Acknowledgments The authors are grateful to Dr. Peter J. Sheridan, University of Texas at San Antonio, for his critical reading of this manuscript. The authors also thank Mr. M. Sone for his excellent technical assistance in immunocytochemistry.

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