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Endocrinology Vol. 145, No. 7 3523-3531
Copyright © 2004 by The Endocrine Society

Pituitary Expression of Type I and Type II Glucocorticoid Receptors during Chicken Embryonic Development and Their Involvement in Growth Hormone Cell Differentiation

Ioannis Bossis, Shotaro Nishimura, Michael Muchow and Tom E. Porter

Department of Animal and Avian Sciences (I.B., M.M., T.E.P.) and Molecular and Cell Biology Program (M.M., T.E.P.), University of Maryland, College Park, Maryland 20742; and Department of Animal and Marine Bioresource Sciences (S.N.), Faculty of Agriculture, Kyushu University, Fukuoka-shi 812-8581, Japan

Address all correspondence and requests for reprints to: Dr. Tom E. Porter, Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742. E-mail: teporter{at}umd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids can induce somatotroph differentiation in vitro and in vivo during chick embryonic and rat fetal development. In the present study, we identified the nuclear receptors involved in somatotroph differentiation and examined their ontogeny and cellular distribution during pituitary development in the chicken embryo. Several steroids were tested for their ability to induce GH cell differentiation. Only glucocorticoids and aldosterone were effective at low nanomolar concentrations, suggesting involvement of both type I (mineralocorticoid) and type II (glucocorticoid) receptors (MR and GR, respectively). ZK98299 and spironolactone (GR and MR antagonists, respectively) when used alone were unable to block corticosterone or aldosterone (2 nM)-induced somatotroph differentiation. However, ZK98299 and spironolactone in combination abolished corticosterone or aldosterone (2 nM)-induced somatotroph differentiation. When used separately, both antagonists attenuated induction of GH mRNA by corticosterone. Spironolactone alone blocked somatotroph differentiation induced by 0.2 nM corticosterone or aldosterone, indicating that corticosteroids at subnanomolar concentrations act only through the MR. GR protein was detected in pituitary extracts as early as embryonic d 8, whereas MR protein was readily detectable only around d 12. GR were expressed in greater than 95% of all pituitary cells, whereas MR were expressed in about 40% of all pituitary cells. Dual-label immunofluorescence revealed that the majority of somatotrophs on d 12 expressed MR. Given the high affinity of corticosteroids for MR and that corticosteroid concentrations during embryonic development are in the subnanomolar range, expression of MR may constitute a significant developmental event during somatotroph differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MATURE PITUITARY gland is a highly differentiated organ composed of at least five distinct cell phenotypes, namely somatotrophs, lactotrophs, thyrotrophs, gonadotrophs, and corticotrophs. In recent years considerable progress has been made in identifying pituitary-specific transcription factors involved in early pituitary differentiation, such as Lhx-3, Lhx-4, P-OTX, Rpx, and Prop1, and in the terminal differentiation and cell-specific expression of hormones from these cells, such as SF1, GATA-2, and Pit-1 (1, 2). In contrast, limited progress has been made in identifying systemic factors such as steroids that, through activation of their specific intracellular receptors, can influence pituitary cell differentiation and production of hormones on their own or in a synergistic manner with pituitary-specific transcription factors.

We demonstrated previously that a significant increase in the population of GH-secreting cells occurs between d 13 and d 16 of chicken embryonic development, although occasional somatotrophs can be detected as early as embryonic day (e) 10 by reverse hemolytic plaque assay (3) or immunocytochemistry (4). Somatotrophs do not appear in cultures of e12 chicken pituitary cells without an extrapituitary signal (5), and GH-expressing cells can be induced in these cultures with corticosterone (CORT) or serum from e16 embryos but not with serum from e12 embryos (5, 6). Treatment of chicken embryos with corticosterone on e11 increased the number of GH-secreting cells on e13, and this response involved increased expression of GH mRNA in the caudal portion of the anterior pituitary gland (7, 8). Corticosterone increased GH mRNA levels in cultures of e12 pituitary cells, which was due to an increase in the number of cells expressing GH mRNA (9, 10). In addition, induction of GH cells could not be induced by administration of corticosterone before d 11 of chicken embryonic development, and the corticosterone-induced premature increase in GH cells in vivo does not affect abundance of somatotrophs later in development (8). Similar results were found in fetal rats, in which glucocorticoids were demonstrated to induce somatotroph differentiation in vitro and in vivo (11, 12, 13, 14). These findings indicate that during chick and rat development, corticosteroids are involved in the final steps of GH cell differentiation and that the corticosteroid-induced increase in GH gene expression and GH secretion is restricted to a pool of cells that are already committed to become somatotrophs.

Low levels of corticosterone (the primary glucocorticoid in avian species) can be detected in the circulation by d 10 of embryonic development (15, 16), and adrenals from d 8 embryos but not from d 6 have secretory capabilities after culture in vitro for 48 h (17). Around d 13–14, an increase in plasma corticosterone concentrations is observed that coincides with increased mitotic activity in the adrenals and acquisition of responsiveness to ACTH (15, 18, 19). These observations indicate that somatotroph recruitment may occur in response to increased adrenal corticosteroid production and that administration of glucocorticoids before this endogenous increase results in premature somatotroph differentiation.

It has long been recognized that lipophilic hormones, including steroids, play a crucial role in several developmental, differentiative, and homeostatic processes. They bind and activate intracellular receptors, which are direct modulators of transcription (20, 21). The glucocorticoid receptor (GR) was one of the first eukaryotic transcription factors to be identified. It is widely expressed in a variety of cell types, and it is structurally similar to other nuclear receptors such as the progesterone, estrogen, androgen, and mineralocorticoid receptor (MR) (21, 22). The actions of glucocorticoids are thought to be mediated primarily by GR. However, in preliminary experiments with e12 pituitary cells, we were unable to abolish corticosterone-induced somatotroph differentiation using specific and high-affinity GR antagonists such as RU32486 and ZK98299. For this reason, we pursued identifying the nuclear receptors involved in somatotroph differentiation and examined their ontogeny and cellular distribution during pituitary development in the chicken embryo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Unless stated otherwise, all chemicals used were obtained from Sigma Chemical Co. (St. Louis, MO), and cell culture reagents were purchased from Life Technologies, Inc. Invitrogen (Grand Island, NY). RU32486 was a gift from Roussel Uclaf (Paris, France) and ZK98299 kindly supplied by Schering AG (Berlin, Germany). A polyclonal antichicken GH antibody was made in this laboratory using recombinant chicken GH as immunogen and validated previously (3). Monoclonal antiidiotypic antibody MA1–620 that cross-reacts with the chicken MRs (MR{alpha} and -ß) was purchased from Affinity BioReagents (Golden, CO). Antirat GR monoclonal antibody GR49–4 that specifically recognizes chicken GR was kindly provided from Dr. Lily Vardimon (The George S. Wise Faculty of Life Sciences, Tel Aviv, Israel) with permission from Dr. Heiner Westphal (National Institutes of Health, Bethesda, MD). For ACTH measurements, a double-antibody RIA against human ACTH (Diagnostic Products Corp., Los Angeles, CA) that was previously validated for chicken ACTH was used (23). Antimouse and antirabbit conjugated second antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA), and radioisotopes were purchased from NEN Life Science Products (Boston, MA).

Animals and cell culture
All embryonated chicken eggs used in the present studies were Avian x Avian broiler strain purchased from Allen’s Hatchery (Seaford, DE). All studies with chicken embryos were approved by the Institutional Animal Care and Use Committee at the University of Maryland. Eggs were placed in a humidified incubator at 37.5 C, and that day is designated as e0. On d 12 of incubation (e12), the eggs were removed from the incubator, and their pituitary glands were isolated under a dissecting microscope. Depending on the experiment, 15–60 pituitaries were pooled, and dispersed cells were obtained by mild trypsin digestion and mechanical agitation as previously described (3). The cells were cultured in serum-free medium consisting of a 1:1 mixture of phenol red-free M199 and Ham’s F-12 nutrient mixture supplemented with 0.1% BSA, 5 µg/ml human transferrin, 5 µg/ml bovine insulin, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate.

Immunocytochemistry (ICC)
GH immunostaining was performed directly in cell culture plates. Culture plates were washed twice with PBS and then fixed with 3.7% formaldehyde in PBS for 20 min. After permeabilization with 0.1% Triton X-100/0.1% Tween 20 in PBS for 2 min and blocking with 5% normal goat serum for 30 min, the plates were incubated overnight at 4 C with GH antiserum (1:5000) in PBS 2% goat serum. Somatotrophs were detected using the rabbit Vectastain ABC and VIP substrate kits (Vector Laboratories, Burlingame, CA). Cells were counted with an inverted light microscope and positively stained cells expressed as a percentage of all cells. For GR immunostaining, freshly isolated e12 pituitary cells were allowed to adhere for 2 h before being fixed for 10 min with 1% glutaraldehyde, 0.1% Nonidet P-40 in 0.2 M HEPES (pH 7.5) prewarmed to 40 C. Subsequently cells were incubated for 10 min with 100 mM glycine to quench free aldehydes, washed several times with PBS, blocked with 5% normal goat serum in PBS for 30 min, and incubated overnight with monoclonal antibody GR49–4 (1:1000) in PBS 2% goat serum. GR-positive cells were visualized using a mouse ABC kit as described above. For dual-label fluorescence ICC against MR and GH, e12 cells were cultured in poly-L-lysine-coated slides for 24 h with CORT to induce somatotrophs and fixed with 2% freshly depolymerized formaldehyde and 2% saturated picric acid in 0.3 M phosphate buffer (pH 7.4) as previously described (24). Cells were permeabilized with 0.1% Triton X-100 in PBS for 2 min and blocked with 3% BSA for 30 min before being incubated overnight at 4 C with GH antiserum (1:5000) and monoclonal antibody MA1–620 (1:250) in PBS 2% BSA. After several washes with PBS, the slides were incubated with fluorescein-conjugated goat antirabbit and Texas Red-conjugated goat antimouse IgGs. The slides were mounted in Vectashield (Vector Laboratories) and visualized using an Axiophot2 fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ). Fluorescence images were automatically captured and merged using IPLab Spectrum software (Scananalytics, Inc., Fairfax, VA).

Reverse hemolytic plaque assays (RHPAs)
The RHPA was performed according to the protocol described in detail previously (3). Briefly, pituitary cells (1.0 x 105 cells/ml) were mixed with an equal volume of an 18% suspension of protein A-coated ovine erythrocytes and infused by capillary action into poly-L-lysine-coated Cunningham chambers. Cells were allowed to attach for 45 min (37.5 C; 95% air/5% CO2) and rinsed with DMEM. Chambers were then infused with DMEM containing GH antiserum (1:40) and human GHRH (10–7 M), and replicate chambers were incubated for 20 h (three chambers per treatment). Plaque formation was induced with guinea pig complement (1:40 in DMEM), and the cells were fixed with 2% glutaraldehyde in 0.9% saline and stained with methyl green. GH-secreting cells were defined as those surrounded by a zone of hemolysis at least one lysed ovine erythrocyte in radius.

Quantitative in situ hybridization plate assay (ISHPA)
GH mRNA was directly quantified in cell culture plates as described and validated previously (10). Briefly, cells were fixed with 10% analytical grade formalin in PBS (pH 7.0) for 20 min and subsequently permeabilized with 0.2% (vol/vol) Triton X-100 for 15 min to allow probe penetration. Subsequently, 500 µl of prewarmed hybridization buffer containing 32P-labeled GH riboprobe was added to each well, and the plates were sealed with plastic tape and hybridized overnight in a hybridization oven at 55 C. The hybridization buffer consisted of 50% formamide, 7.5% polyethylene glycol 8000, 300 mM NaCl, 1x Denhardt’s solution (0.02% BSA, 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone), 0.1% Triton X-100, 500 µg/ml tRNA, 50 µg/ml heparin, 2 mM EDTA (pH 8), 20 mM Tris-HCl (pH 7.6). At the end of hybridization, the plates were washed with 1x saline sodium citrate (SSC) [20x SSC is 3 M NaCl, 0.3 M sodium citrate (pH 7)] and incubated for 20 min with Rnase A (10 µg/ml) in Rnase A buffer [10 mM Tris-HCl (pH 7.6); 1 mM EDTA (pH 8); 500 mM NaCl]. High-stringency washes were performed with 2x SSC for 20 min at 55 C and twice with 0.2x SSC for 15 min at 60 C. The hybridized probe in each well was subsequently recovered with proteinase K digestion (250 µg/ml) in proteinase K buffer [20 mM Tris-HCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS) (pH 8)] for 2 h at 50 C and transferred to scintillation vials for counting.

Tissue extracts and Western blotting
Pituitary glands from d 8, 10, and 12 embryos were isolated with the aid of a dissecting stereoscope, homogenized in protein extraction buffer [15 mM HEPES (pH 7.9), 10% glycerol, 400 mM NaCl, 50 mM KCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1x proteinase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany), 2 mM EDTA] in a Dounce homogenizer, briefly sonicated, and clarified by centrifugation at 16,000 x g for 20 min. Protein concentrations in pituitary extracts were quantified by the BCA assay (Pierce, Rockford, IL) according to the manufacturer’s directions. Fifty micrograms from each sample were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in a Tris-buffered saline solution with 5% nonfat dry milk and incubated with antibodies overnight at 4 C. Immunoreactive signals were detected by incubation with horseradish peroxidase-conjugated secondary antibodies followed by chemiluminescent detection (SuperSignal West Dura substrate kit, Pierce).

Statistical analysis
Each experiment was replicated three times. Data are reported as the mean ± SEM from the replicate trials and analyzed using the GLM procedure of SAS (SAS Institute, Cary, NC). In most cases, log-transformed data were used to remedy for nonhomogeneity of variance. Tukey’s test was used to compare differences between treatments. Differences were considered significant at P < 0.05. Western blots and ICC images are presented from representative experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GR-specific antagonists do not block CORT-induced GH cell differentiation
We reported that corticosterone can induce somatotroph differentiation (6, 7, 8, 9, 10). Classically, the effects of glucocorticoids are mediated primarily by nuclear GR. Therefore, we tested whether RU32486 and ZK98299 can block CORT-induced GH cell differentiation. In contrast to humans, these antiprogestins do not bind the chicken progesterone receptor (25, 26, 27) but display high affinity for the GR. The e12 chicken pituitary cells in culture were pretreated with RU32486 (10 µM) or ZK98229 (10 µM) for 1 h before addition of CORT (2 nM) and incubated for 36 h. At the end of incubation, somatotroph induction was estimated by ICC for GH. CORT treatment resulted in a substantial increase in somatotrophs. However, neither RU32486 nor ZK98229 was able to block this effect (Fig. 1Go). The results in Fig. 1EGo are the percentage of pituitary cells that stained for GH. Whereas neither antagonist blocked the increase in the percentage of GH cells induced by CORT, we noted that the staining intensity for GH in cells treated with ZK98299 was diminished (compare Fig. 1Go, D and B).



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  		FIG. 1. Effects of the GR antagonists RU32486 and ZK98299 on corticosteroid-induced GH cells. The e12 pituitary cells were incubated for 36 h with CORT alone (2 nM) or in combination with RU32486 (10 µM) and ZK98299 (10 µM). At the end of incubation, ICC was counted in quadruplicate wells for each experiment. Representative ICC results are presented for control (CNT; A), CORT alone (B), CORT + RU32486 (C), and CORT + ZK98299 (D). The results are also expressed as percentage of total pituitary cells and represent the means from three independent trials (E). Means denoted with a different letter are significantly different (P < 0.05). All treatments indicated with a b were significantly greater than the control, denoted with an a.

 
To verify that RU32486 and ZK98299 act as GR antagonists in chickens, we tested whether they can abrogate the inhibitory effects of CORT on ACTH secretion in vitro. Pituitary cells were obtained from e20 embryos and treated with CORT, corticotropin-releasing factor (CRF), and CORT plus CRF alone or in the presence of GR antagonists. Indeed, ZK98299 completely blocked the CORT-mediated inhibition of basal (Fig. 2AGo) and CRF-stimulated ACTH secretion (Fig. 2BGo), whereas RU32486 was a partial inhibitor.



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  		FIG. 2. Effects of RU32486 and ZK98299 on CORT-mediated inhibition of basal and CRH (CRF)-stimulated ACTH secretion. The e20 pituitary cells were incubated in the absence (A) or presence (B) of CRF and with CORT alone and in the presence or absence of either RU32486 or ZK98299 for 36 h. At the end of incubation, ACTH was measured in the medium by RIA. Each treatment was evaluated in quadruplicate wells and the results are the means from three independent trials. Values identified with different letters are significantly different from one another (P < 0.05). Values denoted with an a are greater than those denoted with a b, which in turn are greater than those denoted with a c.

 
Both types I and II GRs are involved in GH cell differentiation
The inability of GR-specific antagonists to block CORT-induced somatotroph differentiation prompted us to hypothesize that another class of steroid receptor may also be involved. Therefore, we tested progesterone, estradiol, testosterone, and aldosterone for their ability to induce somatotroph differentiation in cultures of e12 pituitary cells. Progesterone, estradiol, and testosterone failed to exhibit any effect at high nanomolar concentrations (100 nM). However, both corticosterone and aldosterone were able to induce differentiation at 2 nM (Fig. 3Go). The ability of these corticosteroids to induce somatotroph differentiation was evaluated further by treating e12 pituitary cells for 48 h with corticosterone or aldosterone (10–11 to 10–7 M) and then assessing GH production by ICC and GH secretion by RHPA. Both steroids increased the abundance of somatotrophs in a dose-dependent manner (Fig. 4Go). However, greater concentrations were required to induce GH-secreting cells than GH-containing cells. This may reflect differences in sensitivity of the two assays or that GH is not released until substantial hormone is induced. Nonetheless, both steroids were again effective at inducing GH-containing cells at low nanomolar concentrations. This observation suggested that the type I GR (MR) may be involved. To test this hypothesis, a MR antagonist (spironolactone) was tested for its ability to block corticosterone or aldosterone-induced somatotroph differentiation alone or in combination with the GR antagonist ZK98299. ZK98299 and spironolactone alone were unable to block corticosterone or aldosterone (2 nM)-induced somatotroph differentiation in e12 pituitary cells. However, ZK98299 and spironolactone in combination were able to abolish either corticosterone or aldosterone (2 nM)-induced somatotroph differentiation (Fig. 5Go). These results indicate that corticosterone and aldosterone, at the concentration used, bind to both GR and MR and that activation of either receptor is sufficient for somatotroph differentiation. Spironolactone alone was able to block somatotroph differentiation induced by 0.2 nM aldosterone or corticosterone, indicating that these corticosteroids at subnanomolar concentrations bind only to the MR.



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  		FIG. 3. Effects of different steroids on somatotroph differentiation. The e12 pituitary cells were incubated with 2 or 100 nM of CORT, progesterone (PROG), estradiol (ESTR), testosterone (TEST), and aldosterone (ALDO). At the end of a 36-h incubation, ICC for GH-containing cells was performed. A minimum of 200 cells was counted in quadruplicate wells for each experiment. The results are expressed as percentage of total pituitary cells and represent the means from three independent trials. Values identified with an asterisk (*) are significantly different from control (P < 0.05).

 


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  		FIG. 4. Effects of corticosterone and aldosterone on GH cell induction. The e12 pituitary cells were treated for 48 h with corticosterone and aldosterone at the concentrations indicated. Cells were then harvested and subjected to ICC (A) or RHPA (B) to detect GH-containing cells and GH-secreting cells, respectively. The results shown are from three independent experiments. Means denoted with an asterisk (*) are significantly different from basal (P < 0.05).

 


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  		FIG. 5. Effects of the GR antagonist ZK98299 and the MR antagonist spironolactone (SPIRO) on CORT- and aldosterone-induced somatotroph differentiation. The e12 chicken pituitary cells were incubated for 36 h with CORT (2 or 0.2 nM) and aldosterone (2 or 0.2 nM) alone or in combinations with SPIRO (10 µM) and ZK98299 (10 µM). At the end of incubation, ICC for GH-containing cells was performed. A minimum of 200 cells was counted on quadruplicate wells for each experiment. The results are expressed as percentage of total pituitary cells and represent the means ± SEM from three independent trials. Values denoted with an a are significantly different from those denoted with b (P < 0.05).

 
In the aforementioned studies, involvement of GR and MR in somatotroph differentiation was assessed using ICC and RHPA. Even though these techniques are highly informative at the single-cell level, they cannot estimate the contribution of GR and MR to activation of the GH gene. Nevertheless, the presence of either ZK98299 or spironolactone appeared to reduce the staining intensity of somatotrophs induced by corticosterone or aldosterone. To better evaluate these effects, in situ GH mRNA quantitation was performed as previously described (10). Corticosterone treatment of e12 cells for 36 h resulted in an approximately 25-fold increase in GH mRNA relative to untreated cells (Fig. 6Go). Coincubation with ZK98299 attenuated induction of GH mRNA by 50%, whereas spirolonolactone did so by 65%. Coincubation with both ZK98299 and spironolactone attenuated induction of GH mRNA by more than 90%. These results indicate that both GR and MR are involved in activation of the GH gene in either an independent or cooperative manner. However, selective inhibition of either receptor type results in reduced transactivation.



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  		FIG. 6. Effects of CORT, SPIRO, and ZK98299 on GH gene expression as assessed by quantitative in situ hybridization plate assays for GH mRNA (ISHPA). The e12 chicken pituitary cells were incubated for 36 h with CORT (2 nM) alone or in combinations with SPIRO (10 µM) and ZK98299 (10 µM). At the end of incubation, GH mRNA was quantified across treatments using ISHPA. Values are expressed as means ± SEM of counts per minute of four independent experiments. See text for further details on the ISHPA procedure. Values denoted with an a are significantly greater than those denoted with a b (P < 0.05), which in turn are greater than those denoted with a c.

 
Geldanamycin, a heat shock protein (HSP) 90-binding benzoquinone ansamycin, inhibits corticosterone-mediated somatotroph differentiation
Based on the classical model, glucocorticoids bind to intracellular receptors and trigger genomic effects. It has recently been realized that steroids can affect intracellular signaling through nongenomic mechanisms, likely via specific membrane receptors (reviewed in Ref.28). In the present study, we used geldanamycin to address whether corticosterone-induced somatotroph differentiation is mediated by genomic or nongenomic mechanisms. In the absence of a ligand, both mammalian and avian GR and MR are complexed with HSPs (HSP90, HSP70, HSP56) that maintain the receptors in an inactive state (29, 30, 31). Upon ligand binding, the receptors undergo key steric changes during which many of the HSPs and other factors are dissociated and the molecule acquires a dimer conformation (32). HSPs are in fact necessary to stabilize the receptors, enable high-affinity ligand binding, and increase the affinity of corticosteroid receptors for glucocorticoid response elements in the nucleus (33, 34). Geldanamycin binds to HSP90 with high affinity and disrupts its function, thus preventing the efficient binding of glucocorticoids to MR and GR (33, 35). Based on these properties, geldanamycin has been used to distinguish whether a glucocorticoid effect is a nuclear receptor-dependent as well as transcription-dependent genomic effect (36).

Incubation of e12 pituitary cells in the present study with geldanamycin at submicromolar concentrations (10 nM) completely abolished the differentiative effects of corticosteroids (Fig. 7Go). Preliminary experiments were conducted to determine the lowest concentration of geldanamycin that completely blocked the effect of corticosterone. We also found that geldanamycin blocked aldosterone induction of GH cells in preliminary trials (not shown). Because geldanamycin completely blocked the effect of 2 nM corticosterone, our results indicate that both GR and MR effects required interactions with HSP90. This finding suggests that corticosteroid effects on GH cell induction are mediated by genomic rather than nongenomic actions.



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  		FIG. 7. Effect of geldanamycin (GELD), a HSP 90-binding benzoquinone ansamycin, on CORT-mediated somatotroph differentiation. The e12 pituitary cells were incubated for 36 h with CORT (2 nM) alone or in combination with GELD (10 nM). At the end of incubation, ICC for GH-containing cells was performed. A minimum of 200 cells was counted in quadruplicate wells for each experiment. The results are expressed as percentage of total pituitary cells and represent the means ± SEM from three independent trials. Values denoted with an asterisk (*) are significantly different (P < 0.05) from control (CNT).

 
Ontogeny of GR and MR in the embryonic pituitary
To investigate developmental expression and relative abundance of GR and MR, we collected pituitaries from e8, e10, and e12 embryos. Western blotting indicated high GR levels as early as e8. No dramatic appearance of GR occurred between e8 and e12 (Fig. 8AGo). In contrast, MR was not detectable on e8. Low levels of MRß were first detected on e10 and significantly increased on e12, whereas MR{alpha} was first detected on e12 (Fig. 8BGo).



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  		FIG. 8. Ontogeny of the chicken GR (A) and MR (B) during embryonic development. Western blot analysis was performed on protein extracts from pooled pituitaries of e8, e10, and e12 embryos. Results are representative of three independent experiments.

 
Cellular distribution of GR and MR among embryonic pituitary cells
Distribution of GR and MR was initially determined in freshly isolated e12 pituitary cells without any steroid treatment. Detection of GR in whole cells was proven to be a tedious task. We were unable to detect GR in chicken pituitary cells using formaldehyde, alcohol, or acetone-based fixatives (results not shown). It has been well documented that loss of GR can occur as a result of insufficient fixation (reviewed in Ref.37). Particularly in formaldehyde or alcohol-fixed cells, GR leakage during fixation or subsequent permeabilization steps is unavoidable. However, using glutaraldehyde in a rapid fixation-permeabilization protocol, we were able to identify strong nuclear GR immunoreactivity in greater than 95% of the cells, suggesting universal GR expression in the pituitary gland (Fig. 9AGo). In contrast, MR was readily detectable, and immunoreactivity was found only in approximately 40% of e12 pituitary cells (Fig. 9BGo). In addition, dual-fluorescence immunocytochemistry on e12 pituitary cells treated for 24 h with corticosterone indicated expression of MR in greater than 90% of GH-containing cells (Fig. 9CGo).



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  		FIG. 9. ICC for the chicken GR (A) and MR (B), and dual-fluorescence ICC for GH and MR in pituitary cells from e12 embryos (C). See text for additional details.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we provide evidence indicating that corticosteroid-induced somatotroph differentiation in chicken embryonic pituitary cells is mediated by both types I and II glucocorticoid receptors, MR and GR. The critical role of GR in regulation of several physiological processes, including cell proliferation and differentiation, has long been recognized. However, involvement of MR in these processes is poorly understood. Nevertheless, it is becoming increasingly evident that the importance of MR extends beyond the classical regulation of salt and water balance (38). Our findings are the first in any species to indicate that somatotroph differentiation and GH gene expression involve both GR and MR.

The actions of corticosteroids are mediated through binding to two distinct intracellular transcription factors, MR and GR. Activated by corticosteroids, these receptors regulate transcription positively or negatively either by direct binding to DNA or by protein-protein interactions (20). Specificity of receptor action in response to corticosteroids is achieved primarily by different expression patterns and the fact that corticosteroids have higher affinity for MR (dissociation constant ~0.1–0.5 nM) than GR (dissociation constant ~2–5 nM) (39, 40). In cells expressing both receptors, low concentrations of corticosteroids will predominantly activate the MR, whereas higher corticosteroid levels are required for GR occupation (41, 42, 43). Even though the present study was not designed to precisely estimate the affinity of avian GR and MR for corticosteroids, we did observe that induction of GH cells by both aldosterone and corticosterone at subnanomolar concentrations could be blocked by the MR antagonist spironolactone, suggesting they were binding only to MR at these concentrations. This observation also suggests a prominent role of MR in activation of corticosteroid responsive genes during early embryonic development, when concentrations of corticosteroids are very low (15, 16).

Glucocorticoids are typically 100- to 1000-fold more abundant than aldosterone in the plasma. This should lead to permanent occupancy of the MR by glucocorticoid hormones. However, glucocorticoid action on target tissue depends not only on circulating levels but also on intracellular concentrations. The main regulators of intracellular glucocorticoid levels in mammals are 11ß-hydroxysteroid dehydrogenase (11ßHSD)-1 and -2. 11ßHSD-1 acts predominantly as a reductase in vivo, facilitating glucocorticoid action by converting circulating receptor-inactive 11-ketogluco-corticoids to active glucocorticoids. In contrast, 11ßHSD-2 acts exclusively as an 11ß-dehydrogenase and decreases intracellular glucocorticoids by converting them to their receptor-inactive 11-ketometabolites (44, 45, 46). This effectively determines selectivity of MR for circulating aldosterone. However, in avian species 11ßHSD-2 may not provide selectivity of MR for aldosterone. Limited evidence indicates that this function may be served by 20HSD (47, 48). Expression of 11ßHSD-1, 11ßHSD-2, or 20HSD in the chicken anterior pituitary has not been studied at any stage of development. Thus, at present the contribution of these enzymes to corticosterone induction of GH cells cannot be defined. Despite high-affinity constants of both aldosterone and corticosterone for the chicken MR, kinetic experiments have shown that the off-rate of aldosterone from the chicken MR was much lower than that of glucocorticoids, indicating an intrinsic discriminating property of MR (49). Therefore, in addition to 11ßHSD in mammals or 20HSD in birds, the MR itself plays an active role in the mechanism of aldosterone selectivity.

We have previously shown that glucocorticoids cannot induce somatotroph differentiation before e11 (8). Several studies have also suggested that cell responsiveness to glucocorticoids often increases during embryonic development (50, 51, 52). In addition, the level of corticosteroid receptor expression in a specific cell population is a limiting factor in the process of gene induction and can affect the magnitude of biological effects (53, 54, 55). In the present study, we observed high levels of GR expression in the pituitary gland as early as e8. Thus, absence of GR expression cannot account for the apparent unresponsiveness of embryonic pituitary cells to glucocorticoids before e11. In contrast, we observed that the ontogeny of MR in embryonic pituitaries is correlated with developmental acquisition of responsiveness to glucocorticoids and onset of somatotroph differentiation. At this point, however, we can only speculate how expression of MR in a cell population that already possesses high levels of GR can result in increased responsiveness to corticosteroids.

Ligand-activated GR and MR bind to and activate transcription similarly from a consensus simple hormone response element in the promoter region of target genes (56). Effective binding to these hormone response elements requires receptor dimerization. It was previously thought that steroid receptors form homodimers exclusively (57, 58). Evidence has shown, however, that GR and MR can form heterodimers. These GR/MR heterodimers display increased DNA binding and transactivation properties, compared with either MR or GR homodimers (59). Interestingly, the increased potency of GR/MR heterodimers over GR and MR homodimers is more pronounced at low corticosteroid concentrations (60). In addition, expression of certain genes may be predominantly or exclusively regulated by GR/MR heterodimers, and this may depend on potential protein-protein interactions of the heterodimer with other transcription factors (61). The results obtained from our study are in agreement and suggest that activation of the GH gene in embryonic pituitary cells by low nanomolar corticosteroid levels may require GR/MR heterodimers.

There are no prior studies for GR and MR immunoreactivity in the pituitary gland of any avian species. In rodents, however, the pituitary gland and certain brain areas such as the cerebral cortex, hypothalamic paraventricular nucleus, and the hippocampus are considered GR-rich tissues (62, 63). The GR has been detected in the pituitary gland as early as e13 (64) in mice and by e15 in rats (65). However, few studies have addressed colocalization of GR and hormone-producing cells, and the reports are somewhat conflicting. Initially, it was reported that only ACTH and GH cells in rats showed GR immunoreactivity (66). More recently, studies have shown GR immunoreactivity in almost all hormone-producing cells in the rat anterior pituitary and especially somatotrophs, corticotrophs, and folliculostelate cells, but the percentage of colocalization of GR and particular hormones differ from report to report (65, 67, 68). In the present study, we observed high GR immunoreactivity in almost all pituitary cells. This might be due to species differences, variations in fixation and detection protocols, and affinity of the antibody used. Use of a very high-affinity monoclonal antibody may permit detection of low levels of GR, as might be the case with certain hormone-secreting cell types.

In contrast to the rather ubiquitously expressed GR, the MR is much less abundant and often is undetectable in most tissues (69, 70, 71). In mouse embryonic pituitary glands, the MR can first be detected around e13.5, but expression significantly increases around e14.5 (70). In the present study, we observed a similar ontogeny of the MR in chicken embryonic pituitary glands. Interestingly, in both mice and chickens, expression of the GR in the pituitary gland precedes that of MR. However, expression of MR coincides with the onset of somatotroph differentiation. Colocalization studies of MR with hormone-producing cells in the pituitary gland has not been reported so far for any species. In the present study, we clearly demonstrate expression of MR in about 40% of all pituitary cells and in almost all somatotrophs. This observation in combination with our pharmacological studies indicates involvement of the MR in somatotroph differentiation and/or regulation of GH production. This is the first report that MR plays a significant role in corticosteroid regulation of GH production in any species.


   Footnotes
 
This work was supported by United States Department of Agriculture Grant 00-035206-9463 (to T.E.P.).

Abbreviations: CORT, Corticosterone; CRF, corticotropin-releasing factor; e, embryonic day; GR, glucocorticoid receptor; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; HSP, heat shock protein; ICC, immunocytochemistry; ISHPA, in situ hybridization plate assay; MR, mineralocorticoid receptor; RHPA, reverse hemolytic plaque assay; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate.

Received February 6, 2004.

Accepted for publication March 26, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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