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Endocrinology Vol. 141, No. 12 4396-4401
Copyright © 2000 by The Endocrine Society

ARTICLES

Retinoic Acids and Thyroid Hormone Act Synergistically with Dexamethasone to Increase Growth Hormone-Releasing Hormone Receptor Messenger Ribonucleic Acid Expression1

Haruo Nogami, Maki Matsubara, Toshio Harigaya, Masateru Katayama and Koki Kawamura

Departments of Anatomy (H.N., K.K.) and Neurosurgery (M.K.), Keio University School of Medicine, Tokyo 160-8582; and Laboratory of Functional Anatomy, Faculty of Agriculture, Meiji University, Kanagawa 214-8571, Japan

Address all correspondence and requests for reprints to: Haruo Nogami, Ph.D., Laboratory of Neuroendocrinology, Institute of Basic Medical Sciences, University of Tsukuba, 1–1-1, Tennoudai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: hnogami{at}md.tsukuba.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of all-trans-retinoic acid (RA), 9-cis-retinoic acid (9cRA), and thyroid hormone (T3) on GH-releasing hormone receptor (GHRH-R) messenger RNA (mRNA) expression were studied using ribonuclease protection assay in the fetal rat pituitary gland and in MtT/S cells, a clonal GH cell line derived from an estrogen-induced somatotropic tumor in the rat. Although RA (1 µM), 9cRA (1 µM), or T3 (1 nM) alone showed little effect on GHRH-R mRNA expression in the MtT/S cells, each of these substances was found to act synergistically with dexamethasone (DEX; 500 nM) to increase GHRH-R mRNA expression. The effects of RAs and T3 were dose dependent, with maximum effects observed at 1 µM and 1 nM, respectively. The maximum effect of RAs or T3 was not further augmented by the addition of T3 or RAs, respectively. No apparent differences were observed in this study between the actions of RA and 9cRA. The Northern analyses showed that MtT/S cells express retinoic acid receptor {alpha}2 mRNA and thyroid hormone receptor ß2 mRNA, and DEX did not affect the levels of these mRNAs. This suggests that the role of DEX in enabling RAs or T3 to up-regulate GHRH-R mRNA levels is not an induction of the expression of each specific receptor for RAs and T3. The similar enhancement of DEX induction of GHRH-R mRNA by RAs or T3 was also observed in the fetal rat pituitary gland in culture, suggesting that RA and/or T3 is involved in the mechanisms responsible for the developmentally regulated expression of GHRH-R mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH-RELEASING HORMONE (GHRH) is a hypothalamic polypeptide hormone that regulates pituitary GH secretion through binding to a specific plasma membrane receptor, GHRH receptor (GHRH-R) (1, 2). The expression of GHRH-R messenger RNA (mRNA) in the pituitary gland is known to be under multihormonal regulation. Hypothyroidism induced by either thyroidectomy (3) or administration of methimazole (4) decreased the levels of pituitary GHRH-R mRNA, whereas thyroid hormone replacement restored mRNA levels to normal. Similarly, adrenalectomy decreased both the number of GHRH-binding sites in pituitary membrane fractions (5) and the level of pituitary GHRH-R mRNA (6, 7), both of which were restored by glucocorticoid replacement. Short-term treatment of pituitary cells from adult rats with GHRH reduces the levels of GHRH-R mRNA in primary culture (8), whereas prolonged treatment with GHRH increases the level of GHRH-R mRNA (9). On the other hand, estrogen appears to down-regulate the level of pituitary GHRH-R mRNA, as ovariectomy increased and estrogen replacement reduced the level of mRNA (6). Thus, at least four hormones have been reported to be involved in the regulation of pituitary GHRH-R expression.

Although thyroid hormones have been shown to increase GHRH-R mRNA levels in the adult rat pituitary gland in vivo (3, 4) and in vitro (10), in previous studies we were unable to detect any effect of thyroid hormones on GHRH-R mRNA levels in MtT-S (11), a clonal cell line derived from an estrogen-induced pituitary tumor in the rat (12) that expresses GHRH-R mRNA. As our earlier studies showed that thyroid hormone receptor was expressed in this cell line, we decided to further examine the effects of thyroid hormone on GHRH-R mRNA expression in MtT/S cells and in a second system, the fetal rat pituitary gland in organ culture. In addition, as retinoic acids have been shown to activate GH gene transcription (13) through interacting with thyroid hormone response element (14), receptors for retinoic acid have been shown to form heterodimers with thyroid hormone receptors in a ligand-dependent manner (15, 16), and retinoic acids, metabolites of retinol (vitamin A), play crucial roles in normal growth and differentiation during the fetal period, we decided to also investigate the possible effects of two retinoic acids [all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9cRA)] on the level of GHRH-R mRNA in MtT/S cells and in the fetal rat pituitary gland in organ culture.

We report here that thyroid hormone and both retinoic acids have no effect on the level of GHRH-R mRNA in MtT/S cells or fetal rat pituitary gland in organ culture by themselves, but can effectively up-regulate the level of GHRH-R mRNA in the presence of glucocorticoid.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The MtT-S cell was supplied by Dr. Kinji Inoue (Saitama University, Saitama, Japan) and maintained in DMEM/Ham’s F-12 medium containing 10% horse serum, 2.5% FBS (Life Technologies, Inc., Gaithersburg, MD; heat inactivated before use), and antibiotics (control medium). MtT/S cells (5 x 105 cells) were cultured in a 10-cm dish with 10 ml control medium for 7 days, followed by 3–5 days of culture in serum-free DMEM/Ham’s F-12 medium to eliminate serum-dependent GHRH-R mRNA expression, which was weakly detected in MtT-S cells. This deinduction procedure reduced GHRH-R mRNA to undetectable levels. The experiments were started by replacing medium with fresh serum-free medium alone or medium containing test substances, and the cells were harvested after 24-h incubation. The cells were stored at -30 C until RNA extraction.

Animals
The timed pregnant rats (Sprague Dawley strain, Sankyo Co., Ltd., Tokyo, Japan) were obtained on days 12–14 of pregnancy and maintained in a temperature (22 C)- and light (14 h of light/day)-controlled room with free access to tap water and standard diet. On day 18 of gestation, fetuses were dissected out under ether anesthesia, and the pituitary glands were removed for organ culture under the surgical microscope. After washing in a serum free MEM{alpha} (Life Technologies, Inc.) supplemented with glutamine and antibiotics, connective tissues were removed from pituitary glands. Then, the pituitary glands were transferred to a 12-well plate (4 pituitaries/well) and incubated with 0.5 ml serum-free MEM{alpha} alone or containing test substances for 24 h. After the experiments the pituitary glands were stored at -30 C until RNA extraction. The experiments were carried out in accordance with the animal experimentation guidelines of Keio University.

Chemicals
The following chemicals were used in this study: GHRH (1–100 nM; Peptide Institute, Osaka, Japan), dexamethasone (DEX; 50–500 nM, Sigma, St. Louis, MO), T3 (1 fM to 10 nM; Sigma), estradiol (E2; 10–100 nM; Sigma), (Bu)2cAMP (1 mM; Sigma), forskolin (1 µM; Sigma), RA (Sigma; 10 pM to 10 µM), and 9cRA (100 pM to 10 µM). The stock solutions for these chemicals were prepared at a 1000-fold concentration and stored at -20 C.

RNA analysis
The total RNA preparations were prepared from MtT-S cells and fetal pituitaries by the method previously described (17). RNA was extracted with phenol/chloroform and determined by A260 absorption. The ribonuclease (RNase) protection assay was carried out as described previously (11).

Rat GHRH-R complementary DNA (cDNA; 240 bp; 2–241 from ATG) (1) obtained by RT-PCR from pituitary RNA of adult rats was cloned into the plasmid pBSIISK- (Stratagene, La Jolla, CA). The 32P-labeled complementary RNA (cRNA) probe (345 bases) was transcribed by T3 polymerase using [32P]UTP (ICN Biomedicals, Inc., Costa Mesa, CA). A 99-bp (481–579 from ATG) fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (18) was isolated and cloned in pBSIISK-. The size of 32P-labeled GAPDH cRNA was 267 or 185 bases. Sequences of the probes were determined by the dideoxy chain termination method (19) to confirm identity with the authentic cDNA sequences.

For the RNase protection assay, total RNA (10–20 µg) was ethanol precipitated and dissolved in 20 µl hybridization buffer [400 mM NaCl, 1 mM EDTA, 40 mM PIPES (pH 6.4), and 80% formamide] containing 2 x 104 cpm each of GHRH-R cRNA and GAPDH cRNA. The hybridization was carried out overnight at 55 C. After the hybridization, 150 µl RNase digestion buffer (300 mM sodium acetate, 5 mM EDTA, and 10 mM Tris-HCl, pH 7.5) containing 10 µg/ml RNase A and 100 U/ml RNase T1 (both from Roche Molecular Biochemicals, Indianapolis, IN) was added and the reaction mixture was incubated for 30 min at 37 C. The mixture was incubated at 37 C for an additional 15 min after addition of 2.5 µl proteinase K (Roche Molecular Biochemicals; 20 mg/ml) and 10 µl 10% SDS. The reaction mixture was extracted with the same volume of phenol/chloroform, the 120 µl upper aqueous phase were saved, and the RNA was ethanol precipitated. The protected RNA fragment was analyzed by electrophoresis on an 8% polyacrylamide gel containing 8 M urea. The radioactivity of the mRNA bands was determined by BAS2000 image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).

For Northern blot analyses, 20 µg total RNA prepared from MtT/S cells were subjected to the electrophoresis in 1% agarose gel containing formaldehyde. The RNA was transferred onto a nylon membrane (Hybond N+, Amersham Pharmacia Biotech, Aylesbury, UK), and the blot was hybridized overnight with 32P-labeled cDNAs for rat thyroid hormone receptor ß2 (TRß2) (20) or retinoic acid receptor {alpha}2 (RAR{alpha}2) (21). The blot was washed three times with 2 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS and then in 0.1 x SSC containing 0.1% SDS at 65 C for 1 h. The blot was exposed to x-ray film (X-OMAT AR, Eastman Kodak Co., Rochester, NY). Rat TRß2 cDNA was a gift from Dr. W. Scott Young III (NIMH, Bethesda, MD; 110–491 from ATG, 382 bp), rat RAR{alpha}2 cDNA was obtained by RT-PCR from MtT/S total RNA (147–572 from ATG, 426 bp), and rat GAPDH cDNA (18) was obtained with RT-PCR from pituitary RNA of adult rats (78–579 from ATG, 502 bp). Each cDNA fragment was 32P labeled by the random primer DNA labeling kit (Amersham Pharmacia Biotech).

RT-PCR was carried out to assess the levels of glucocorticoid receptor (GR) mRNA expression in MtT/S cells. One microgram of RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT) at 42 C for 15 min. A set of sense (5'-ATATTTGCCAATGGACTCCA-3') and antisense (5'-TTGCAGACGTTGAACTCTTG-3') primers was used to amplify a 511-bp GR cDNA (22) fragment. Another set of primers (sense, 5'-GGACATTGTTGCCATCAACG-3'; antisense, 5'- CAGCTTTCCAGAGGGGCCAT-3') was used to detect 502-bp GAPDH cDNA (18) as the internal control. The PCR condition was 94 C for 30 sec, 55 C for 2 min, and 72 C for 1 min, and the reaction was run for 25 cycles.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The MtT/S cells were incubated with RA, T3, E2, GHRH, cAMP, or forskolin at the doses indicated for 24 h in serum-free DMEM/Ham’s F-12 containing a saturating dose of DEX (500 nM). In the presence of DEX, T3 and RA showed remarkable up-regulation of GHRH-R mRNA (Fig. 1Go). T3 at a concentration of 1–100 nM increased GHRH-R mRNA approximately 7- to 8-fold over that produced by DEX alone. Similarly, 100 nM RA increased DEX induction of GHRH-R mRNA by 5-fold. On the other hand, forskolin, cAMP, GHRH, and E2 did not affect DEX induction of GHRH-R mRNA.



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  		Figure 1. Effects of forskolin, cAMP, GHRH, E2, T3, and RA on GHRH-R mRNA expression in MtT/S cells. MtT/S cells were incubated for 24 h in a serum-free medium containing 500 nM DEX without (control) or with one of the test substances indicated. In this and the following figures, GHRH-R mRNA was detected by specific RNase protection assay, and the relative abundance of the GHRH-R mRNA was determined by image analyzer. A negative control was included in which the cells were incubated in DEX-free medium [DEX(-)]. The positions of the cRNA probes are indicated to the left. Values were normalized for GAPDH mRNA, and the results were expressed as the mean ± SEM of three independent experiments [the value of DEX alone (control) was set at 1.0]. The representative autoradiogram is shown.

 
The enhancement of DEX induction of GHRH-R mRNA by T3 or RA was dose dependent (Fig. 2Go). The effect of T3 was observed at concentrations as low as 0.1 pM, and the maximum effect was seen at 1 nM. RA, at low doses (<10 nM) had a weak effect on GHRH-R mRNA, but increased the mRNA level in a dose-dependent manner at concentrations higher than 10 nM. A similar dose-dependent effect on GHRH-R mRNA expression was seen with 9cRA.



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  		Figure 2. Effects of T3, RA, or 9cRA on GHRH-R mRNA accumulation in the presence of DEX. MtT/S cells were incubated for 24 h with 500 nM DEX and different dose of T3, RA, and 9cRA. The results were expressed as the fold increase in mRNA over the level with DEX alone (mean ± SEM of three independent experiments).

 
Next, we examined whether the effects of T3 and RA are additive. Treatment of the cells for 24 h with 500 nM DEX resulted in the expression of GHRH-R mRNA, whereas T3 (1 nM) and RA (1 µM) did not when given alone (Fig. 3Go). When T3 was added to the culture with DEX, the effect of DEX was enhanced by 9-fold, and addition of RA increased it by 8-fold. However, addition of T3 and RA together with DEX to the culture resulted in no additional increase in GHRH-R mRNA levels over that observed by DEX plus T3. Thus, the effect of T3 was not enhanced further by RA and vice versa. Similarly, 9cRA enhanced the DEX induction of GHRH-R mRNA, whereas it did not affect the mRNA level by itself (Fig. 4Go). The GHRH-R mRNA level after the combined treatment with DEX, 9cRA, and T3 was not significantly higher than that obtained after treatment with DEX and 9cRA.



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  		Figure 3. Effects of DEX (500 nM), T3 (1 nM), RA (1 µM), or their combination on GHRH-R mRNA accumulation in MtT/S cells. The cells were incubated for 24 h with the substances indicated. The results were expressed as the fold increase in mRNA levels over that with DEX alone (mean ± SEM of three independent experiments). The positions of the cRNA probes are indicated to the left. DMSO, Dimethysulfoxide, a solvent of RA.

 


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  		Figure 4. Effects of DEX (500 nM), 9cRA (1 µM), and T3 (1 nM) on GHRH-R mRNA accumulation in MtT/S cells. The results were expressed as fold increase in mRNA levels over DEX alone (mean ± SEM of three independent experiments). The sizes of marker RNAs and positions of the cRNA probes are indicated to the left. DMSO, Dimethysulfoxide, a solvent of 9cRA.

 
Northern blot analysis of the total RNA obtained from MtT/S cells revealed that MtT/S cells express TRß2 mRNA and RAR{alpha}2 mRNA (Fig. 5AGo). The mRNA levels of these receptors did not change after 6 or 24 h of DEX treatment. The treatment of cells by thyroid hormone for 24 h resulted in marked decrease in TRß2 mRNA expression, but did not affect RAR{alpha}2 mRNA levels. RARß2 mRNA was detected in MtT/S cells by RT-PCR, but its level of expression was too low to detect by Northern blot analyses (data not shown). The results of RT-PCR experiments (Fig. 5Go, B and C) suggest that treatment with RAs or T3 does not affect the level of GR mRNA in MtT/S cells.



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  		Figure 5. A, Northern blot analyses of RAR{alpha}2 mRNA and TRß2 mRNA expression in MtT/S cells after DEX (500 nM) or T3 (1 nM) treatment. After probing of the receptor mRNAs, the blots were reprobed with GAPDH cDNA for the reference of RNA loading. B, Detection of GR mRNA by RT-PCR in MtT/S cells treated with DEX (500 nM), T3 (1 nM), RA (1 µM), and 9cRA (1 µM) for 24 h. SF cont, Control culture in a serum-free medium. GAPDH cDNA was also detected as an internal control. C, Increasing doses of RNA from MtT/S cells cultured in serum-free medium were subjected to RT-PCR in the same conditions as those in B, showing a dose-dependent increase in the amount of PCR product. The amount of RNA in the RT reaction was adjusted to 1 µg with transfer RNA.

 
As the synergism seen in the MtT-S cells dose not necessarily mean that the same mechanism functions in the pituitary gland, the effects of T3 and RAs were also examined in the fetal pituitary gland. Fetal pituitary on day 18 of gestation, which has been shown to have an extremely low level of GHRH-R mRNA expression, was incubated in serum-free MEM{alpha} for 24 h with DEX, T3, RA, or 9cRA alone or a combination of these. As shown in Fig. 6Go, 1Go00 nM DEX induced weak expression of GHRH-R mRNA in the explant, whereas GHRH-R mRNA remained undetectable in the control pituitaries. T3, RA, and 9cRA had no effect when added alone, whereas all of them significantly increased the effect of DEX to induce GHRH-R mRNA in the fetal pituitary gland.



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  		Figure 6. Effects of DEX (100 nM), T3 (1 nM), RA (1 µM), and 9cRA (1 µM) on GHRH-R mRNA induction in E18 fetal pituitary gland in organ culture. The fetal pituitaries (four pituitaries per group) were incubated 24 h in serum-free MEM{alpha} without (control) or with test substances as indicated. The results are expressed as the mean ± SEM of three independent experiments. The sizes of the marker RNAs and the positions of the cRNA probes are indicated to the left. tRNA, Transfer RNA (used for a negative control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results suggest that thyroid hormones, RA, and 9cRA are involved in the regulation of GHRH-R mRNA expression in MtT/S cells and the fetal pituitary gland. Although they did not have any effect on GHRH-R mRNA expression when used alone, they strongly augmented GHRH-R mRNA induction by DEX. These results indicate that DEX plays a permissive role for RAs and T3 in regulating GHRH-R mRNA expression. It is suggested that RAs and T3 may share a common mechanism in regulating GHRH-R mRNA expression, because the maximum effect of T3 or RA was not augmented further by the addition of RA or T3, respectively.

In mice, rats, and humans, GHRH-R promoter activity has been shown to be obligatorily dependent on pit-1 (23, 24, 25), a nuclear transcription factor specifically expressed in the pituitary gland (26, 27). Even though the structure of the 5'-region of the GHRH-R gene has been reported in rats (25) and humans (24, 28), limited information is available concerning the hormonal regulation of the GHRH-R gene promoter. The mechanism by which DEX acts to increase the levels of GHRH-R mRNA is thought to be augmentation of the transcription rate of the GHRH-R gene, as the transcription inhibitor, actinomycin D, completely abolishes the effect of DEX (7, 11). In fact, in humans, glucocorticoids have been shown to directly stimulate and estrogens to inhibit promoter activity (28). In contrast, in the rat, binding elements for pit-1 and GR have been reported, but no consensus sequence for TR or RAR have been found. Thus, it is presently obscure whether RAs and T3 increase GHRH-R mRNA levels by direct stimulation of mRNA transcription and/or by the suppression of mRNA degradation. Another possibility is that RA or T3 indirectly stimulates GHRH-R transcription via the activation of other cell-specific regulators of the GHRH-R gene.

In the present study using fetal pituitary glands and MtT/S cells, the effect of T3 on GHRH-R mRNA expression was observed only in the presence of DEX. These data are in conflict with a previous report using adult pituitary cells in primary culture, in which there was an increase in GHRH-R mRNA expression due to T3 alone (10). Initially, we thought that perhaps the fetal pituitary gland and MtT/S cells lacked TRs. However, as shown in Fig. 5Go, we were able to demonstrate the presence of mRNAs encoding the receptors for both thyroid hormone [TRß2, a pituitary specific class of TR (20)] and RAs (RAR{alpha}) using Northern blots. Other possibilities considered were that DEX significantly increased the amount of receptors for T3 and RAs or that T3 or RAs increased GR level. These also proved not to be the case. As shown in Fig. 5Go, DEX treatment did not alter the levels of mRNAs for RAR{alpha} and TRß2, and treatment with T3 or RAs did not affect the levels of GR mRNA in MtT/S cells. Thus, we are left with the hypotheses that there are developmental differences in the regulation of GHRH-R mRNA with respect to T3 between fetal and adult pituitary glands, or a longer period of incubation may be required for T3 to act alone. It may also be possible that the differences in the experimental procedures yielded the divergent results. In this study the effects of T3 were examined after 3–5 days of culture in a serum-free medium, eliminating the effect of glucocorticoids completely. On the other hand, in a previous study by Korytko and Cuttler (10), in which they showed a positive effect of T3, the dispersed pituitary cells were treated with T3 immediately after serum removal.

A second difference in the regulation of GHRH-R mRNA expression in our system from that previously reported involves the effect of GHRH. GHRH has been reported to increase GHRH-R mRNA expression in pituitary cells (9); however, MtT/S cells responded neither to GHRH nor to cAMP in this study (in the presence of DEX) as well as in our previous study (in the absence of DEX) (11). The cells were incubated with GHRH for 24 h in this study, whereas Miller et al. observed a maximum increase in GHRH-R mRNA in the primary culture of adult pituitary cells after 12-h incubation (9). On the contrary, Alleppo et al. (8) reported down-regulation of GHRH-R mRNA after 4-h incubation with GHRH. The more detailed examination of the time-dependent effects of GHRH in MtT/S cells may be required to solve this discrepancy.

Previous studies have shown that the expression of GHRH-R mRNA in the pituitary gland is developmentally regulated (29, 30, 31). GHRH-R mRNA expression occurs in late gestation in rodents, which coincides with the onset of GH mRNA expression (2). Expression of both of these mRNAs specific for GH cells depends upon the pituitary-specific transcription factor, pit-1 (23, 24, 32, 33). In mice, expression of pit-1 occurs on embryonic day 13.5 (E13.5) (34), and the onset of GHRH-R mRNA expression occurs on E16.5 (2). Similarly, pit-1 expression is detected on E15.5 (35) in rats, and GHRH-R mRNA expression begins on E19 (11, 30). Thus, the expression of pit-1 precedes that of GHRH-R by a few days in the murine anterior pituitary gland. This suggests that during development, pit-1 is not the sole requirement for GHRH-R mRNA expression, and that some additional factors may be necessary for the expression of this mRNA. Our previous study (11) suggested that the glucocorticoids may be one of the factors required for the induction of GHRH-R mRNA expression in fetal rats, because GHRH-R mRNA can be induced by incubating E18 rat pituitary gland in a serum-free medium containing only DEX (11). In addition, the temporal pattern of corticosterone secretion during development is consistent with a central role for glucocorticoids in the regulation of GHRH-R mRNA expression. Plasma corticosterone levels in the fetus reach a peak on E19 (36, 37), in agreement with the idea that initiation of GHRH-R mRNA expression depends upon glucocorticoids; however, corticosterone levels decline toward parturition, whereas pituitary GHRH-R mRNA levels show a linear increase from E19–E21 (11). The present results suggest that RAs and thyroid hormones may also be involved in the induction of GHRH-R mRNA expression in the fetal rat pituitary gland. RAs are supplied from the maternal compartment, and they affect a variety of physiological functions in tissue differentiation throughout the fetal period (15). The thyroid hormone concentration in the fetus shows a sharp increase from E18 due to functional maturation of the fetal thyroid gland (38). Therefore, it is conceivable that a synergism between glucocorticoids and RAs or thyroid hormones may also significantly contribute to the increased expression of GHRH-R mRNA in the rat pituitary gland during late gestation.

In conclusion, the present study demonstrated that RA, 9cRA, and T3 up-regulated GHRH-R mRNA levels in MtT/S cells in the presence of glucocorticoids, whereas each of these substances alone had no effect. Similar results were obtained in fetal pituitary glands in organ culture, suggesting that RAs and T3 might be involved in the regulation of GHRH-R mRNA expression in the rat fetus.


   Acknowledgments
 
The authors are grateful to Dr. Peter J. Sheridan, University of Texas (San Antonio, TX), for his critical reading of this manuscript.


   Footnotes
 
1 This work was supported in part by Grant-in-Aid for Scientific Research 11670020 from the Ministry of Education, Science, Sports, and Culture, Japan. Back

Received March 20, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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