The best characterized effect of glucose-dependent insulinotropic polypeptide (GIP) is its stimulatory effect on insulin secretion by pancreatic β-cells. Recently, it was demonstrated that some cases of primary adrenal Cushing’s syndrome were secondary to the ectopic expression of non-mutated GIP receptor (GIP-R) in bilateral adrenal hyperplasias or unilateral adrenal adenomas, resulting in food-dependent steroidogenesis. Using a human multiple-expression tissue array, GIP-R was found to be expressed in a large number of human adult and fetal tissues, but not in the adrenal gland. The analysis of the promoter region of human (h) GIP-R gene revealed six consensus sequences important in regulating the reporter gene activity and capable of binding to Sp1 and Sp3 transcription factors. Data obtained by gene array and semi-quantitative RT-PCR showed an increase in the expression of Sp3 and CRSP9 (co-regulator of Sp1 transcription factor, subunit 9) in the adrenal adenomas or bilateral macronodular hyperplasias of patients with GIP-dependent Cushing’s syndrome; they were, however, also increased in some patients with non-GIP-dependent cortisol-secreting adenomas or with ACTH-dependent Cushing’s disease. This study represents the first step in our understanding of the mechanisms involved in the regulation of the expression of the hGIP-R gene.
Valérie Baldacchino, Sylvie Oble, Patrick-Olivier Décarie, Isabelle Bourdeau, Pavel Hamet, Johanne Tremblay, and André Lacroix
Eduard Muráni, Siriluck Ponsuksili, Richard B D'Eath, Simon P Turner, Gary Evans, Ludger Thölking, Esra Kurt, Ronald Klont, Aline Foury, Pierre Mormède, and Klaus Wimmers
To gain insight into the adrenal stress response, we analysed differential mRNA expression of genes associated with psychosocial stress in the pig (Sus scrofa domestica). Various levels of psychosocial stress were induced by mixing groups of unfamiliar pigs with different aggressiveness. We selected two experimental groups for comparison, each comprising eight animals, which differed significantly in aggressive behaviour and plasma cortisol levels. To identify differentially expressed genes, we compared the adrenal transcriptome of these two groups of pigs, using the Affymetrix GeneChip porcine Genome Array. Bioinformatic analysis revealed that psychosocial stress induced upregulation of transcripts enriched for functions associated with cholesterol accumulation and downregulation of transcripts enriched for functions associated with cell growth and death. These responses are similar to those induced by ACTH stimulation. Nevertheless, the majority of the differentially expressed genes were so far not described as ACTH responsive. Some, such as GAL and GALP, may have responded to sympathoadrenal stimulation. Several of the differentially expressed transcripts, such as AGT, are associated with processes modulating steroidogenic response of adrenocortical cells to ACTH. One of the most significant findings was upregulation of LOC100039095, comprising a precursor of the microRNA miR-202, pointing to a previously unrecognised layer of regulation of adrenal steroidogenesis by microRNA. Our study, performed under entirely physiological conditions, complements previous studies focusing either on a single adrenal tissue and/or on a single stimulus, and contributes to understanding of the fine-tuning of adrenal stress response.
Alexander Dierks, Urs D Lichtenauer, Simone Sackmann, Ariadni Spyroglou, Igor Shapiro, Marcel Geyer, Jenny Manonopoulou, Martin Reincke, Constanze Hantel, and Felix Beuschlein
Potassium and angiotensin II are the main stimulators of aldosterone secretion from the adrenal cortex. As potassium-induced in vivo gene regulation in the adrenal cortex has not been studied in detail, we applied a stepwise screening approach: first, we investigated the effects of chronic potassium substitution in mice. Microarray analysis of adrenal glands revealed a set of genes (set A) that were counter-regulated in a high potassium (HP) and low potassium substitution group, while others (set B) were highly upregulated in the HP intake group. In a second step, time dependency of expression changes of these pre-defined genes was studied following short-term potassium stimulation experiments in vivo. Thirdly, dose dependency of potassium-induced gene regulation was investigated in vitro. Finally, to provide indirect evidence for the potential relevance of the detected changes for autonomous aldosterone secretion, expression analysis of aldosterone-producing adenomas was compared with normal adrenal glands. While most investigated genes were similarly regulated following long- and short-term potassium stimulation in vivo, observed changes were reproducible in NCI h295R adrenocortical cells mostly for the set of genes identified in the HP group (set B). Similarly, in Conn's adenomas, mostly genes from set B displayed changes in expression pattern in comparison to normal adrenal glands, while genes from set A were mostly unchanged. Thus, while in vivo models can help in identifying genes potentially involved in potassium-dependent aldosterone secretion, these findings also underline the necessity to interpret potassium-induced gene regulation on the basis of the experimental setting.
Hong-Wei Chang, Chao-Yuan Huang, Shao-Yu Yang, Vin-Cent Wu, Tzong-Shinn Chu, Yung-Ming Chen, Bor-Shen Hsieh, and Kwan-Dun Wu
Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia are the two characteristic types of primary aldosteronism. Dysregulation of adrenal cortical cell proliferation contributes to both diseases. We previously demonstrated that APA expressed less dopamine D2 receptor than the respective non-tumor tissue and might contribute to the overproduction of aldosterone. As activation of D2 receptor inhibits the proliferation of various cells, downregulation of D2 receptor in APA may play a role in the tumorigenesis of APA. In this study, we demonstrate that D2 receptor plays a role in angiotensin II (AII)-stimulated adrenal cortical cell proliferation. The D2 receptor agonist, bromocriptine, inhibited AII-stimulated cell proliferation in primary cultures of the normal human adrenal cortex and APA through attenuating AII-induced phosphorylation of PK-stimulated cyclin D1 protein expression and cell proliferation. D2 receptor also inhibited AII-induced ERK1/2 phosphorylation. Our results demonstrate that, in addition to inhibiting aldosterone synthesis/production, D2 receptor exerts an anti-proliferative effect in adrenal cortical and APA cells by attenuating PKCμ and ERK phosphorylation. The lower level of expression of D2 receptor in APA may augment cell proliferation and plays a crucial role in the tumorigenesis of APA. Our novel finding suggests a new therapeutic target for primary aldosteronism.
Kazutaka Nanba, Andrew X Chen, Adina F Turcu, and William E Rainey
The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic–pituitary–adrenal axis.
P. Netchitailo, A. Larcher, F. Leboulenger, M. Feuilloley, D. Philibert, and H. Vaudry
To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 μg/ml) induced a 45–80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.
Tomoko Tanaka, Shigeki Gondo, Taijiro Okabe, Kenji Ohe, Hisao Shirohzu, Hidetaka Morinaga, Masatoshi Nomura, Kenzaburo Tani, Ryoichi Takayanagi, Hajime Nawata, and Toshihiko Yanase
Steroidogenic factor 1/adrenal 4 binding protein (SF-1/Ad4BP) is an essential nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. Mesenchymal bone marrow cells (BMCs) contain pluripotent progenitor cells, which differentiate into multiple lineages. In a previous study, we reported that adenovirus-mediated forced expression of SF-1 could transform mouse primary long-term cultured BMCs into steroidogenic cells. For future clinical application, trials using human BMCs would be indispensable. In this study, we examined whether SF-1 could transform human BMCs into steroidogenic cells and compared the steroid profile of these cellswith that of mouse steroidogenic BMCs. Primary cultured human BMCs infected with adenovirus containing bovine SF-1 cDNA could produce progesterone, corticosterone, cortisol, dehydroepiandrosterone, testosterone, and estradiol. Such a mixed character of adrenal and gonadal steroid production in human BMCs was supported by the expressions of P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c21, P450c11, P450c17, 17β-HSD, and P450arom mRNAs. Unlike mouse steroidogenic BMCs, introduction of SF-1 into human BMCs caused dramatic inductions of both ACTH and LH receptors, thus leading to good responsiveness of the cells to ACTH and LH respectively. Importantly, among several factors that are known to be closely associated with adrenal and/or gonadal development, introduction of only SF-1 enabled the human BMCs to express P450scc and to produce cortisol and testosterone, suggesting that SF-1 is truly a master regulator for the production of steroidogenic cells from human BMCs.
Edson F Nogueira, Yewei Xing, Claudia A V Morris, and William E Rainey
Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. We previously defined eight transcription factors that are rapidly induced following Ang II treatment using three in vitro adrenocortical cell models. Herein, we investigated the function of these transcription factors in the regulation of the enzymes needed for aldosterone production. H295R adrenal cells were co-transfected with expression vectors for each transcription factor and promoter/reporter constructs prepared for genes encoding the enzymes needed for aldosterone production. NGFI-B family members induced promoter activity of 3-beta-hydroxysteroid-dehydrogenase type 2 (HSD3B2), 21-hydroxylase (CYP21A2), and aldosterone synthase (CYP11B2). The importance of NGFI-B in the regulation of CYP11B2 was also demonstrated by reduced CYP11B2 transcription in the presence of a dominant-negative-NGFI-B. A pharmacological approach was used to characterize the Ang II pathways regulating transcription of NGFI-B family genes. Transcription of NGFI-B members were decreased following inhibition of Ang II type 1 receptor (AT1R), protein kinase C (PKC), calcium/calmodulin-dependent kinases (CaMK), and Src tyrosine kinase (SRC). Taken together, these results suggest that Ang II binding to the AT1R increases activity of PKC, CaMK, and SRC, which act to increase expression of the family of NGFI-B genes as well as CYP11B2. Ang II induction of the NGFI-B family members represents an important pathway to increase the capacity of adrenal cells to produce aldosterone.
S. Barker, S. M. Laird, M. M. Ho, G. P. Vinson, and J. P. Hinson
We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization.
Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30 000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. Invivo treatment with ACTH (100 μg/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro.
In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60 000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 μmol/l) or dibutyryl-cAMP (100 μmol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2. As in the in-vivo treatments, the increase in IZAg1 was associated with a corresponding increase in corticosterone and 18-OH-DOC production.
These findings suggest that the IZAb recognizes a protein (IZAg2) which occurs in unstimulated adrenal cells. On stimulation by steroidogenic agents, this protein becomes processed to yield a smaller protein (IZAg1) which is associated with enhanced adrenal steroidogenesis.
Matthew Stagl, Mary Bozsik, Christopher Karow, David Wertz, Ian Kloehn, Savin Pillai, Paul J Gasser, Marieke R Gilmartin, and Jennifer A Evans
Glucocorticoid production is gated at the molecular level by the circadian clock in the adrenal gland. Stress influences daily rhythms in behavior and physiology, but it remains unclear how stress affects the function of the adrenal clock itself. Here, we examine the influence of stress on adrenal clock function by tracking PERIOD2::LUCIFERASE (PER2::LUC) rhythms in vitro. Relative to non-stressed controls, adrenals from stressed mice displayed marked changes in PER2::LUC rhythms. Interestingly, the effect of stress on adrenal rhythms varied by sex and the type of stress experienced in vivo. To investigate the basis of sex differences in the adrenal response to stress, we next stimulated male and female adrenals in vitro with adrenocorticotropic hormone (ACTH). ACTH shifted phase and increased amplitude of adrenal PER2::LUC rhythms. Both phase and amplitude responses were larger in female adrenals than in male adrenals, an observation consistent with previously described sex differences in the physiological response to stress. Lastly, we reversed the sex difference in adrenal clock function using stress and sex hormone manipulations to test its role in driving adrenal responses to ACTH. We find that adrenal responsiveness to ACTH is inversely proportional to the amplitude of adrenal PER2::LUC rhythms. This suggests that larger ACTH responses from female adrenals may be driven by their lower amplitude molecular rhythms. Collectively, these results indicate a reciprocal relationship between stress and the adrenal clock, with stress influencing adrenal clock function and the state of the adrenal clock gating the response to stress in a sexually dimorphic manner.