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Department of Anatomy (H.N., K.K.), School of Medicine, Keio University, Tokyo 160, Japan; Department of Regulation Biology (K.I.), Faculty of Science, Saitama University, Saitama 338, Japan
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.
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These data, taken as a whole, suggest that the induction of GH mRNA by DEX in the fetal pituitary gland is not a direct effect of DEX on the GH gene but is mediated by a factor that is synthesized in the pituitary gland in response to DEX. Both immunoblot and RNase protection assays suggested that this factor is not pit-1, which is known to be required for GH mRNA expression.
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Introduction |
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Our previous studies (13, 14) showed that this phase transition is normally achieved by the elevation of endogenous glucocorticoids level (15, 16) because administration of dexamethasone (DEX), a synthetic glucocorticoid, to pregnant rats results in the early induction of GH expression in E17 or E18 fetuses. However, the molecular mechanisms underlying the induction of GH synthesis by glucocorticoids in the fetal pituitary gland have not yet been investigated.
This study used an in vitro organ culture system to investigate the mechanisms of GH induction by glucocorticoids that occurs at a particular stage of gestation (between E18 and E19).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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modification of MEM (Flow Laboratories, Irvine, UK)
supplemented with glutamine and antibiotics (control medium) in a
12-well plate. The E19 pituitaries were obtained similarly and stored
at -80 C for the positive control of Northern analyses. DEX,
T3, and puromycin (PM) (all from Sigma, St. Louis, MO) were
used at 0.5–50 nM, 0.1 to 1 nM and 100
µM, respectively. Tissues were incubated for 24 h
unless stated and thereafter either stored frozen at -80 C for protein
and mRNA analyses or fixed in 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4 at 4 C overnight for in situ
hybridization.
In situ hybridization
Tissues fixed in paraformaldehyde were soaked in 20% sucrose in
PB. The frozen section (7 µm in thickness) was placed on a
3-aminopropylethoxysilane (Shin-Etsu Chemical Co., Ltd., Tokyo,
Japan)-coated glass slide and air dried. The section was washed with
diethylpyrocarbonate (DEPC, Sigma)-treated distilled water, digested
with 0.1% pepsin (DAKO Japan Co., Ltd., Tokyo, Japan) in 0.2
N HCl at 37 C for 3 min, washed with DEPC-water, and
dehydrated with ethanol. The section was hybridized with digoxigenin
(DIG)-labeled rat GH complementary RNA (cRNA) (20 µg/ml) in 50%
formamide, 10 mM Tris, pH 7.5, 600 mM NaCl, 1
mM EDTA, 1 x Denhardt’s solution (0.02% each of
BSA, polyvinylpyrrolidone, and Ficoll), 10% dextransulfate, 0.2 mg/ml
yeast transfer RNA (tRNA), 0.1% sodium sarcosyl at 42 C overnight.
After hybridization, the section was rinsed with 2 x SSC (1
x SSC = 150 mM NaCl, 15 mM sodium
citrate), then washed with 50% formamide/2 x SSC at 55 for 60
min and incubated with 10 µg/ml of RNase A (Sigma) in 10
mM Tris-HCl, pH 7.5, 0.5 M NaCl, at 37 C for 30
min. The section was washed with 0.2 x SSC at 55 C for 60 min and
incubated with alkaline phosphatase-labeled anti-DIG antibody
(Boehringer Mannheim, GmbH, Germany), followed by alkaline phosphatase
reaction to visualize the site of GH mRNA localization. The GH
complementary DNA (cDNA) used for in situ hybridization
(pGH-1) was kindly supplied by Dr. John D. Baxter, University of
California, San Francisco, CA). The GH cDNA of about 800 bp was
subcloned into a plasmid pGEM4Z (Promega Corporation, Madison, WI) and
transcribed with either SP-6 or T7 polymerase (both from Takara Shuzo
Co., Ltd., Shiga, Japan) to obtain antisense or sense cRNA probes,
respectively.
RNA analysis
For Northern blot analysis, three to seven pituitary glands
(depending on the age of the fetus) were pooled, and total RNA was
isolated by the method described previously (17), followed by a
phenol/chloroform extraction. The amount of total RNA was determined by
densitometry and 5–10 µg of RNA were separated on a 1% agarose gel
containing formalin and transferred onto a nylon membrane (Hybond
N+, Amersham). The blot was hybridized with
32P-labeled pGH-1 as previously described (13), washed, and
exposed to x-ray film for 5–10 days. Then, the radioactive GH cDNA
probe was removed and the filters were rehybridized with a rat 18s
ribosomal RNA probe as a reference for the amount of RNA loaded on the
gel. For quantification of the relative abundance of GH mRNA, the blots
hybridized with 32P-labeled probe were analyzed with an
image analyzer (BAS2000; Fuji Film, Tokyo, Japan).
For the RNase protection assay, three pituitary glands were pooled, and total RNA was extracted as described above. A 125-bp fragment that encodes most of the sequences of the second exon of the rat GH gene was isolated from cloned rat GH gene prepared by Takeuchi et al. (18) and subcloned into a plasmid pGEM4Z (Promega Corporation, Madison, WI). The plasmid was linearized by HindIII digestion, and the 32P-labeled cRNA probe was synthesized by SP-6 polymerase using 32P-CTP (ICN Biomedicals, Inc., Costea Mesa, CA). A 221-bp rat pit-1 cDNA was obtained by RT-PCR from pituitary RNA of adult rats and cloned into the SmaI site of a plasmid pGEM4Z. This fragment contains sequences that correspond to 143–363 (from ATG) of rat pit-1 cDNA (1). The plasmid was linearized by EcoRI digestion and transcribed by T7-polymerase to give a cRNA probe. Both GH and pit-1 antisense cRNA probes contained about 60 bp of polylinker sequences. The sizes of the cRNA probes for GH and pit-1 are 185 and 283 bp, and expected sizes of protected fragments are 125 and 221 bp, respectively. Sequences of both probes were determined by the dideoxy chain termination method (19) to confirm identity with the authentic cDNA sequences. Pituitary RNA (5 µg) was dissolved in 10 µl of hybridization buffer (400 mM NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.4, 80% formamide), and combined with the same volume of cRNA probes (3–5 x 105 cpm) and incubated at 55 C for 4 h or overnight. After the hybridization, 150 µl of digestion buffer composed of 300 mM sodium acetate, 5 mM EDTA, 10 mM Tris-HCl, pH 7.5, containing 10 µg/ml of RNase A and 100 U/ml of RNase T1 (both from Boehringer Mannheim) was added to the reaction mixture and incubated for 30 min at 37 C. Then, 2.5 µl of Proteinase K (Boehringer Mannheim, 20 mg/ml) and 10 µl of 10% SDS was added and incubated at 37 C for an additional 15 min. The protected fragments were precipitated and analyzed on a 6% polyacrylamide gel containing 8 M urea.
Immunoblot analyses
Pituitary glands were homogenized in 10 µl per pituitary gland
of 0.5% Nonidet P-40/PBS (5 mM PB pH 7.4/0.9% NaCl) and
centrifuged at 15,000 rpm. The supernatant was combined with the same
volume of 0.1 M Tris-HCl, pH 6.8, 4% SDS, 2%
2-mercaptoethanol, 0.1% bromphenolblue, 20% glycerol, and heated at
95 C for 10 min. Proteins were separated on a 15% polyacrylamide gel
and electrophoretically transferred onto a nylon membrane (Immobilon-P,
Millipore Corporation, Bedford, MA). The blot was sequentially
incubated in 25% blocking reagent (Block Ace, Dainihon Seiyaku Co.,
Ltd., Tokyo, Japan)/5 mM Tris-HCl, pH 7.5 at room
temperature for 4 h, in the first specific antiserum diluted with
10% blocking reagent/5 mM Tris-HCl, pH 7.5, at 4 C for
overnight, and then in 10% blocking reagent containing 1:10000 diluted
peroxidase-labeled antirabbit IgG goat IgG fraction (MBL Co., Ltd.,
Nagoya, Japan). The GH and pit-1 were visualized with chemiluminescence
reagents (Dupont NEN, Boston, MA). The antiserum to rat GH was raised
in a rabbit with rat GHB-7, which was supplied through the National
Hormone and Pituitary Program (Baltimore, MD) and used at 1:50000
dilution. The antiserum to pit-1 was a gift from Dr. Michael G.
Rosenfeld, Howard Hughes Medical Institute, and Eukaryotic Regulatory
Biology Program, University of California, San Diego, and used at
1:4000 dilution. The specificity of both antisera was examined and
published elsewhere (14, 20).
All the experiments were carried out in triplicate or in quadruplicate, and representative data of experiments are shown.
Statistical analyses
The significance of difference of the data was determined by
ANOVA followed by Student-Newman-Keuls test.
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Results |
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). The intensity of staining varied to a
large extent between cells. Positive cells were scattered throughout
the anterior lobe. In contrast, no GH mRNA-containing cells were
detected in the E18 pituitaries incubated in control medium alone (Fig. 1A). Hybridization with the sense strand cRNA probe did not stain GH
cells in either control or DEX-treated tissue (data not shown).
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and 3). Incubation with 1
nM T3 alone had little effect on GH mRNA level;
however, the addition of both DEX and T3 had a synergistic
effect on GH mRNA induction, raising GH mRNA to a level twice as high
as that of intact E19 pituitary (Fig. 3). The induction of GH mRNA by
DEX and the synergism of DEX and T3 was also observed in
E17 pituitaries; however, the levels of GH mRNA never exceeded that of
intact E19 pituitary (Fig. 3). GH mRNA was not detectable in E16
pituitary after incubation with DEX, indicating the paucity of
DEX-responsive cells at this stage (data not shown). Induction of GH
mRNA by DEX increased in a dose-dependent manner (Fig. 2B), and
corticosterone, a major circulating glucocorticoid in rats, was also
capable of inducing GH mRNA in vitro (Fig. 2C).
Additionally, corticosterone acted synergistically with T3
to induce GH mRNA (Fig. 2C).
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). Induction of GH mRNA induction
was not observed after 5 or 10 h of incubation in medium
containing DEX, but was evident after 24 h of incubation. However,
a conspicuous GH mRNA induction occurred when tissues were exposed to
DEX for 10 h, then incubated for an additional 14 h in
DEX-free control medium. Five-hour incubation with DEX plus 19-h chase
incubation in DEX-free medium also weakly induced GH mRNA. These
results indicate that GH mRNA synthesis occurred in the absence of DEX
and suggest the presence of a factor that is induced by DEX and mediate
DEX action.
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). Incubation of E18 pituitaries
with DEX for 8 h, followed by chace incubation in control medium
for 16 h, resulted in GH mRNA induction. Addition of puromycin (54
µM) to the medium with DEX for the first 8-h incubation
period completely inhibited GH mRNA induction after the 16-h chase
incubation. However, even if puromycin was present in the first 8-h
incubation, a positive GH mRNA induction was observed when puromycin
was washed out and DEX was added to the chase incubation, indicating
that the pituitaries retained normal mechanisms for GH mRNA synthesis
after puromycin treatment. The data illustrated in Fig. 4 taken as a
whole suggest that the induction of GH mRNA in the fetal pituitary
gland by DEX requires ongoing protein synthesis and that the action of
DEX may be mediated by a factor or factors produced in the pituitary
gland in response to DEX. Because DEX is reported to enhance pit-1
production in a synergistic manner with a protein kinase C activator
(21) and pit-1 is required for GH transcription (1), the change in
pit-1 expression in the fetal pituitary gland by DEX was examined.
Immunoblot analysis indicated that incubation of E18 pituitaries with
DEX for 24 h increased pituitary GH concentration, whereas
pituitary pit-1 remained constant (Fig. 5A). In
accordance with these results, the RNase protection assay revealed that
GH mRNA but not pit-1 mRNA was enhanced by DEX treatment (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present results also indicate that the primary site of glucocorticoid action is the pituitary gland and that the participation of maternal factors, the placenta or the fetal hypothalamus, are not prerequisite for the initiation of GH production in the fetal pituitary gland. This finding is consistent with the results of Hemming et al. (22, 23), who showed with an organ culture system of E14 pituitary primordia that the development of GH cells in vitro requires cortisol. The present results extend their findings by specifying the stage of GH cell development at which glucocorticoids are required. On the other hand, thyroid hormone appears to be less important than DEX for the induction of GH expression in the fetus at this stage, despite its distinct effect on GH transcriptional activation in the adult rat (24) and in pituitary tumor cells (25, 26), a finding that suggest the presence of an intrinsic regulatory mechanisms for GH expression during the fetal period.
Because the GH mRNA level is extremely low at E18 (before
DEX-treatment), the increase in the GH mRNA level in response to DEX is
considered to be primarily due to activation of GH transcription.
However, unlike the human GH gene (27, 28), a distinct glucocorticoid
response element is not present in the 5'-upstream region of the rat GH
gene, suggesting that DEX-glucocorticoid receptor complex might not
directly modulate GH gene transcription in rats. Indeed, in other
systems, the effect of DEX on the activation of GH gene transcription
is negative or extremely weak and cannot account for the marked
increase in GH mRNA accumulation induced by this steroid (29). The
stabilization of GH mRNA is also a proposed mechanism of DEX-induced GH
mRNA accumulation (30). The data illustrated in Fig. 4, indicating that
the effect of DEX is time dependent and puromycin sensitive, lead us to
postulate the presence of a factor that is synthesized in response to
DEX and activates GH gene expression. Once this factor is accumulated
in the gland, the continuous presence of DEX is not required,
suggesting that this factor is stable with a relatively long half-life.
Another explanation for the inhibitory effect of puromycin on the GH
mRNA induction is that puromycin reduced the level of a rapidly turned
over protein, which is already present in E18 pituitary gland and is
required for the activation of GH transcription.
The nature of the factor is still unknown. Several nuclear proteins are known to interact with the promoter/enhancer region of the rat GH gene (6, 31), but only the thyroid hormone receptor, pit-1, and Zn-15 have been demonstrated to activate the rat GH gene (1, 25, 32). Because DEX induces GH mRNA expression without thyroid hormone, as demonstrated in this study, the thyroid hormone receptor cannot be the factor that mediates DEX action. Zn-15 (32) is a newly characterized nuclear protein that is expressed both in the pituitary gland and in several nonpituitary tissues. The sequences between the proximal and distal pit-1 binding sites of rat GH promoter have been shown to be important for normal GH expression in rats and are bound by Zn-15 to enhance pit-1-dependent GH transcription (32). Therefore, it is possible to postulate Zn-15 as a mediator of DEX action, although it is still unknown whether glucocorticoid is responsible for the regulation of Zn-15 synthesis.
Pit-1 is a primary requirement for GH transcription (1). In the adult pituitary, stimulation of GH secretion by several secretagogues accompany an apparent increase in the pituitary pit-1 level (33). Therefore, activation of pit-1 production may be responsible for the increase in GH transcription. Furthermore, Jong et al. (21) demonstrated the synergistic activation of pit-1 gene transcription by DEX and protein kinase C activator in GH4C1 cells. The mechanism of this reaction resembled that of the induction of GH mRNA by DEX in the fetal pituitary gland with respect to the presence of an unknown factor or factors that mediate action of DEX. However, the lack of changes in the levels of pit-1 and pit-1 mRNA after DEX, irrespective of the conspicuous increases in the levels of GH and GH mRNA, suggests that the change in the pit-1 level is not involved in the mechanisms of DEX action in the fetal pituitary gland.
The mechanism by which GH expression is stimulated by a DEX-induced factor is obscure. The factor may directly activate GH transcription through the interaction with an unknown cis-element on the GH promoter or enhancer region or may exert its effect by modifying the nuclear protein(s) to alter their ability to stimulate GH transcription. The factor might also enhance GH transcription through modification of local chromatin structure. In vivo footprinting experiments revealed that the promoter region (GHF-1/pit-1 binding site) of the GH gene of GH-producing cells are sensitive to nuclease digestion, whereas that of non-GH-producing cells is not (6). Another possibility is that the DEX-induced factor removes inhibitory controls for GH expression. Activins (34), insulin (35), and insulin-like growth factor (36) are all known to suppress pituitary GH expression. If the GH gene is inactivated before E19 by one or more of these substances, the removal of these negative control by DEX-induced factor would enhance GH expression.
As glucocorticoid receptor has been demonstrated in multiple types of pituitary cells, the types of cells that are targets of DEX were not specified in this study. If immature GH cells are targets of DEX, the mechanisms of DEX action might be as described above. On the other hand, DEX might stimulate other types of pituitary cells to secrete a substance, which, in turn, stimulates GH expression in a paracrine fashion. Expression of glucocorticoid receptor mRNA in the anterior pituitary gland was noted as early as E15 in rats, and at E17, when no GH cells have yet developed, some of the cells expressing glucocorticoid receptor are demonstrated by immunocytochemistry to be ACTH cells (37).
In conclusion, the present results suggest that glucocorticoid plays a principal role in the induction of GH mRNA in the E18 pituitary gland. The induction of GH mRNA by glucocorticoids is mediated by an unknown factor that is produced in the pituitary gland in response to glucocorticoid stimulation and is probably involved in the activation of GH gene transcription.
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Acknowledgments |
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Footnotes |
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Received October 21, 1996.
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