The pituitary function of androgen receptor constitutes a glucocorticoid production circuit

Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.     

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus): effect of seawater exposure and cortisol treatment

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.     

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 microg/mouse, i.p.) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Social dominance regulates androgen and estrogen receptor gene expression

In Astatotilapia burtoni, dominant males have higher levels of sex steroid hormones than subordinate males. Because of the complex regulatory interactions between steroid hormones and receptors, we asked whether dominance is also associated with variation in sex steroid receptor gene expression. Using quantitative PCR, we compared the expression of specific subtypes of androgen (AR) and estrogen (ER) receptor genes between dominant and subordinated males in 3 divisions of the brain, the pituitary, and the testes. We measured mRNA levels of AR-alpha, AR-beta, ER-alpha, ER-betaa, and ER-betab, gonadotropin-releasing hormone 1 (GnRH1), and GnRH receptor 1 (GnRH-R1) relative to 18S rRNA. In the anterior part of the brain, we found that dominant males had higher mRNA expression of AR-alpha, AR-beta, ER-betaa, and ER-betab, but not ER-alpha, compared to subordinate males. This effect of dominance was reflected in a positive correlation between testes size and AR-alpha, AR-beta, ER-betaa, and ER-betab in the anterior brain. In addition, mRNA levels of all ARs and ERs in the anterior brain were positively correlated with mRNA level of GnRH1. In the middle and posterior portions of the brain, as well as the testes, steroid receptor mRNA levels were similar among dominants and subordinates. In the pituitary, ER-alpha mRNA level was positively correlated with testes size and AR-alpha mRNA was positively correlated with GnRH-R1 mRNA level. These data suggest that dominant male brains could be more sensitive to sex steroids, which may contribute to the increased complexity of the behavioral repertoires of dominant males.     

Abnormal glucocorticoid receptor gene and mRNA in primary cortisol resistance

Abnormal steroid hormone receptors have been implicated as causing several forms of primary steroid hormone resistance in humans, but as yet no abnormality has been described at the gene level. We describe the analysis of the mRNA and genomic DNA from the Epstein-Barr (EB) virus transformed cells of two siblings with Primary Cortisol Resistance. The cells of the propositus and his brother show a decreased level of glucocorticoid receptor (GR) mRNA, and the genomic DNA of both individuals shows an altered restriction enzyme pattern with the restriction enzyme Bgl II, one of eleven restriction enzymes tested. The genomic differences could be detected with a probe specific for the putative steroid binding domain of the human GR gene.     

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase1 (3β-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.     

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Rα) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Rα cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Rα, IL-18 and GH showed that IL-18Rα was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway. This work was supported by a Grant-in-Aid for Scientific Research (B) (No.13460122) from the Ministry of Education, Science and Culture of Japan     

Immunohistochemical localization of carboxypeptidases D, E, and Z in pituitary adenomas and normal human pituitary.

Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.     

Intermediate filament expression in non-neoplastic pituitary cells.

Fifty-one non-neoplastic human pituitary glands, including examples with Crooke's hyalinization or amyloidosis, were examined by an immunoperoxidase method using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein (GFAP), desmin, actin, S-100 protein and a variety of pituitary hormones. It was confirmed that most of the epithelial cells in the pituitary gland express keratin immunoreactivity. These cells included endocrine cells in the anterior lobe, endocrine cells and squamous metaplastic cells in the pars tuberalis, columnar and ciliated epithelia forming follicular structures and salivary-type epithelium in the pars intermedia, and anterior lobe cells infiltrating the posterior lobe. This study also demonstrated that keratin and NFs may be co-expressed in endocrine cells in the pituitary anterior lobe, that keratin, vimentin and GFAP may be co-expressed in the epithelial cells forming cyst-like follicle in the pars intermedia, and that vimentin and GFAP may be co-expressed in folliculo-stellate cells and pituicytes. In addition, the GFAP and S-100 protein-negative high columnar epithelium in the pars intermedia tended to be positive for adrenocorticotropic hormone and melanocyte stimulating hormone, while the low columnar epithelium with the co-expression of GFAP and S-100 protein was negative for pituitary hormones.