Timing of adrenal regression controlled by synergistic interaction between Sf1 SUMOylation and Dax1.

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function. A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient-Sf12KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone).

Development of adrenal cortex zonation.

The human adult adrenal cortex is composed of the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), which are responsible for production of mineralocorticoids, glucocorticoids, and adrenal androgens, respectively. The final completion of cortical zonation in humans does not occur until puberty with the establishment of the zR and its production of adrenal androgens; a process called adrenarche. The maintenance of the adrenal cortex involves the centripetal displacement and differentiation of peripheral Sonic hedgehog-positive progenitors cells into zG cells that later transition to zF cells and subsequently zR cells.

Adrenal cell aldosterone production is stimulated by very-low-density lipoprotein (VLDL).

Very low-density lipoproteins (VLDL) are a class of large lipoprotein synthesized in the liver. The key function of VLDL, in vivo, is to carry triglyceride from the liver to adipose tissue. As a steroidogenic organ, the adrenal gland mainly uses lipoproteins as sources of cholesterol. Although VLDL receptors have been detected in the human adrenal, the function of VLDL in the adrenal gland remains unknown. Herein, we used primary cultures of human and bovine adrenal cells and the adrenocortical cell line H295R as models to determine the effects of VLDL on adrenal steroidogenesis. Our studies revealed that VLDL significantly increased aldosterone synthesis in all of the models tested. This increase was largely due to VLDL's stimulation of the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2). VLDL increased CYP11B2 mRNA expression in a concentration-dependent manner. Effects of VLDL on CYP11B2 transcript levels were not additive with angiotensin II or potassium but were additive with the cAMP pathway agonists ACTH and forskolin. Nifedipine completely inhibited the effects of VLDL on CYP11B2 mRNA, suggesting that calcium is the main signal transduction pathway used by VLDL in adrenal cells. Indeed, VLDL increased cytosolic free calcium levels. An in vivo study conducted in sucrose-fed rats showed a positive correlation between elevated triglyceride (VLDL) levels in plasma and CYP11B2 expression in the adrenal. In conclusion, we have shown that VLDL can stimulate aldosterone synthesis in adrenocortical cells by increasing StAR and CYP11B2 expression, an event likely mediated by a calcium-initiated signaling cascade.

Differential gene expression in the adrenals of normal and anencephalic fetuses and studies focused on the Fras-1-related extracellular matrix protein (FREM2) gene.

PRECIS: Many genes are differentially expressed in normal compared to anencephalic human fetal adrenals (HFAs), especially the Fras-1-related extracellular matrix protein (FREM2) gene. FREM2 expression appears to be regulated by adrenocorticotrophic hormone (ACTH). CONTEXT: The expression profiles of genes responsible for cortical growth and zonation in the HFA gland are poorly characterized. The neural tube disorder anencephaly is associated with fetal adrenal hypoplasia with a large size reduction of the fetal zone of the HFA. OBJECTIVE: To determine gene expression profile differences in the adrenals of anencephalic compared to normal HFAs to identify genes that may play important roles in adrenal development. DESIGN AND METHODS: Fresh tissues were obtained at the time of autopsy from normal and anencephalic human fetuses delivered at mid-gestation. The following techniques were used: cell culture, messenger RNA (mRNA) extraction, microarray analysis, complementary DNA (cDNA) synthesis, quantitative real-time reverse transcriptase polymerase chain reaction (QT-PCR). RESULTS: We identified over 40 genes expressed at levels 4-fold or greater in the normal versus anencephalic HFAs and that 28 genes were expressed at increased levels in the anencephalic HFA. The expression of FREM2 at approximately 40-fold greater levels in the normal HFA compared to the HFA of anencephalic fetuses was confirmed by QT-PCR. Expression of FREM2 in the kidney was not significantly different between normal and anencephalic fetuses. In cultured HFA cells, ACTH treatment for 48 hours increased the expression of FREM2 and a gene responsive to ACTH, CYP17, but not tyrosine hydroxylase. CONCLUSIONS: Abnormal expression of many genes may be involved in the adrenal hypoplasia seen in anencephaly. FREM2 appears to be regulated by ACTH and is the most differentially expressed gene, which may be important in the development and function of the HFA, particularly the fetal zone of the HFA.

The effects of ACTH on steroid metabolomic profiles in human adrenal cells.

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48  h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11ß-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11ß-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.

Aldosterone production in human adrenocortical cells is stimulated by high-density lipoprotein 2 (HDL2) through increased expression of aldosterone synthase (CYP11B2).

Adrenal aldosterone production is regulated by physiological agonists at the level of early and late rate-limiting steps. Numerous studies have focused on the role of lipoproteins including high-density lipoprotein (HDL) as cholesterol providers in this process; however, recent research suggests that HDL can also act as a signaling molecule. Herein, we used the human H295R adrenocortical cell model to study the effects of HDL on adrenal aldosterone production and CYP11B2 expression. HDL, especially HDL2, stimulated aldosterone synthesis by increasing expression of CYP11B2. HDL treatment increased CYP11B2 mRNA in both a concentration- and time-dependent manner, with a maximal 19-fold increase (24 h, 250 µg/ml of HDL). Effects of HDL on CYP11B2 were not additive with natural agonists including angiotensin II or K(+). HDL effects were likely mediated by a calcium signaling cascade, because a calcium channel blocker and a calmodulin kinase inhibitor abolished the CYP11B2-stimulating effects. Of the two subfractions of HDL, HDL2 was more potent than HDL3 in stimulating aldosterone and CYP11B2. Further studies are needed to identify the active components of HDL, which regulate aldosterone production.

Human adrenal cells that express both 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) contribute to adrenal androstenedione production.

Androstenedione is one of several weak androgens produced in the human adrenal gland. 3ß-Hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) are both required for androstenedione production. However, previous studies demonstrated the expression of HSD3B2 within the zona glomerulosa (ZG) and fasciculata (ZF) but low levels in the zona reticularis. In contrast, CYB5A expression increases in the zona reticularis (ZR) in human adrenal glands. Although their colocalization has been reported in gonadal theca and Leydig cells this has not been studied in the human adrenal. Therefore, we immonolocalized HSD3B2 and CYB5A in normal human adrenal glands and first demonstrated their co-expression in the cortical cells located at the border between the ZF and ZR in normal human adrenal. Results of in vitro studies using the human adrenal H295R cells treated with the HSD3B2 inhibitor, trilostane, also demonstrated a markedly decreased androstenedione production. Decreasing CYB5A mRNA using its corresponding siRNA also resulted in significant inhibition of androstenedione production in the H295R cells. These findings together indicate that there are a group of cells co-expressing HSD3B2 and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex, and these hybrid cortical cells may play an important role in androstenedione production in human adrenal gland.

ACTH is a potent regulator of gene expression in human adrenal cells.

The adrenal glands are the primary source of minerocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it has been established that ACTH can stimulate steroidogenesis, the effects of ACTH on overall gene expression in human adrenal cells have not been established. In this study, we defined the effects of chronic ACTH treatment on global gene expression in primary cultures of both adult adrenal (AA) and fetal adrenal (FA) cells. Microarray analysis indicated that 48 h of ACTH treatment caused 30 AA genes and 84 FA genes to increase by greater than fourfold, with 20 genes common in both cell cultures. Among these genes were six encoding enzymes involved in steroid biosynthesis, the ACTH receptor and its accessory protein, melanocortin 2 receptor accessory protein (ACTH receptor accessory protein). Real-time quantitative PCR confirmed the eight most upregulated and one downregulated common genes between two cell types. These data provide a group of ACTH-regulated genes including many that have not been previously studied with regard to adrenal function. These genes represent candidates for regulation of adrenal differentiation and steroid hormone biosynthesis.

The mediator complex subunit 1 enhances transcription of genes needed for adrenal androgen production.

There are three enzymes involved in the biosynthesis of the adrenal androgen dehydroepiandrosterone (DHEA) sulfate. Cholesterol side-chain cleavage (CYP11A1) and 17alpha-hydroxylase/17,20-lyase (CYP17) metabolize cholesterol into DHEA, whereas steroid sulfotransferase family 2A1 (SULT2A1) is responsible for conversion of DHEA to DHEA sulfate. We previously examined the mechanisms regulating CYP11A1, CYP17, and SULT2A1 transcription and found that each is regulated, in part, by the transcription factor GATA-6. Previous studies suggested that mediator complex subunit 1 (MED1, also called PPARBP or TRAP220) is a cofactor involved in not only the regulation of nuclear receptors but also the activation of GATA-6 transcription. Herein we demonstrated a role for MED1 in the regulation of CYP11A1, CYP17, and SULT2A1 transcription. Transient transfection assays with SULT2A1 deletion and mutation promoter constructs allowed the determination of specific the GATA-6 binding cis-regulatory elements necessary for transactivation of SULT2A1 transcription. Binding of MED1 and GATA-6 was confirmed by coimmunoprecipitation/Western analysis and chromatin immunoprecipitation assay. We demonstrated expression of MED1 mRNA and protein in the human adrenal and determined that knockdown of MED1 expression via specific small interfering RNA attenuated CYP11A1, CYP17, and SULT2A1 expression levels in H295R cells. In addition, we demonstrated that MED1 enhanced GATA-6 stimulated transcription of promoter constructs for each of these genes. Moreover, the activity of MED1 for SULT2A1 promoter was mediated by GATA-6 via the -190 GATA-binding site. These data support the hypothesis that MED1 and GATA-6 are key regulators of SULT2A1 expression, and they play important roles in adrenal androgen production.

Type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) contributes to testosterone production in the adrenal reticularis.

CONTEXT: The human adrenal gland produces small amounts of testosterone that are increased under pathological conditions. However, the mechanisms through which the adrenal gland produces testosterone are poorly defined. OBJECTIVE: Our objective was to define the role of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in human adrenal production of testosterone. DESIGN AND METHODS: Adrenal vein sampling was used to confirm ACTH stimulation of adrenal testosterone production. Adrenal expression of AKR1C3 was studied using microarray, quantitative real-time RT-PCR, and immunohistochemical analyses. AKR1C3 knockdown was accomplished in cultured adrenal cells (H295R) using small interfering RNA, followed by measurement of testosterone production. RESULTS: Acute ACTH administration significantly increased adrenal vein testosterone levels. Examination of the enzymes required for the conversion of androstenedione to testosterone using microarray analysis, quantitative real-time RT-PCR, and immunohistochemistry demonstrated that AKR1C3 was present in the adrenal gland and predominantly expressed in the zona reticularis. Decreasing adrenal cell expression of AKR1C3 mRNA and protein inhibited testosterone production in the H295R adrenal cell line. CONCLUSIONS: The human adrenal gland directly secretes small, but significant, amounts of testosterone that increases in diseases of androgen excess. AKR1C3 is expressed in the human adrenal gland, with higher levels in the zona reticularis than in the zona fasciculata. AKR1C3, through its ability to convert androstenedione to testosterone, is likely responsible for adrenal testosterone production.

Role of angiotensin II-induced rapid response genes in the regulation of enzymes needed for aldosterone synthesis.

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.

G protein-coupled receptor expression in the adult and fetal adrenal glands.

Hormonal regulation of adrenal function occurs primarily through G protein-coupled receptors (GPCR), which may play different roles in fetal vs. adult adrenal glands. In this study, we compared the transcript levels of GPCR between fetal and adult adrenal and found that gonadotropin-releasing hormone receptor (GnRHR), latrophilin 3 receptor, G protein-coupled receptor 37, angiotensin II receptor type 2, latrophilin 2 receptor and melanocortin receptor were expressed at significantly higher levels in fetal adrenal. High GnRHR protein expression was also detected in fetal adrenal using immunohistochemical analysis. To define potential ligand sources for fetal adrenal GnRHR, we demonstrated that GnRH1 mRNA was expressed at high levels in the placenta, while fetal adrenal had high expression of GnRH2. In summary, certain GPCR particularly GnRHR were highly expressed in fetal adrenal and the expression of GnRH mRNA in the placenta and the fetal adrenal raises the possibility of endocrine and/or paracrine/autocrine influences on fetal adrenal function. However, the exact function of GnRHR in fetal adrenal remains to be determined.

The farnesoid X receptor regulates transcription of 3beta-hydroxysteroid dehydrogenase type 2 in human adrenal cells.

Recent studies have shown that the adrenal cortex expresses high levels of farnesoid X receptor (FXR), but its function remains unknown. Herein, using microarray technology, we tried to identify candidate FXR targeting genes in the adrenal glands, and showed that FXR regulated 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) expression in human adrenocortical cells. We further demonstrated that FXR stimulated HSD3B2 promoter activity and have defined the cis-element responsible for FXR regulation of HSD3B2 transcription. Transfection of H295R adrenocortical cells with FXR expression vector effectively increased FXR expression levels and additional treatment with chenodeoxycholic acid (CDCA) caused a 25-fold increase in the mRNA for organic solute transporter alpha (OSTalpha), a known FXR target gene. HSD3B2 mRNA levels also increased following CDCA treatment in a concentration-dependent manner. Cells transfected with a HSD3B2 promoter construct and FXR expression vector responded to CDCA with a 20-fold increase in reporter activity compared to control. Analysis of constructs containing sequential deletions of the HSD3B2 promoter suggested a putative regulatory element between -166 and -101. Mutation of an inverted repeat between -137 and -124 completely blocked CDCA/FXR induced reporter activity. Chromatin immunoprecipitation assays further confirmed the presence of a FXR response element in the HSD3B2 promoter. In view of the emerging role of FXR agonists as therapeutic treatment of diabetes and certain liver diseases, the effects of such agonists on other FXR expressing tissues should be considered. Our findings suggest that in human adrenal cells, FXR increases transcription and expression of HSD3B2. Alterations in this enzyme would influence the capacity of the adrenal gland to produce corticosteroids.

Metastin stimulates aldosterone synthesis in human adrenal cells.

Kisspeptins, including metastin, are encoded by the KiSS-1 gene and play an important role in regulating the hypothalamic gonadotropin-releasing hormone (GnRH) system via G protein-coupled receptor 54 (GPR54, also called KiSS-1R). Normally, metastin (also called Kp-54) levels are quite low, except during pregnancy, when levels increase 1000-fold over those found in men and nonpregnant women. However, the potential hormonal role of metastin in the fetal and maternal circulation is unknown. In this study, the authors examine the levels of GPR54 mRNA expression in human adult and fetal adrenals using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In addition, they examine the effects of metastin on steroidogenesis and steroidogenic enzyme mRNA levels in fetal adrenal cells and in the H295R adrenocortical cell line using enzyme immunoassay and RT-PCR techniques. The authors demonstrate that GPR54 mRNA is significantly higher (50-fold) in human fetal adrenals than in adult adrenals. Immunohistochemical studies have demonstrated that the GPR54 protein is predominantly expressed in the neocortex of human fetal adrenals in the third trimester. Metastin increases aldosterone production (approximately 2-fold) in both fetal neocortex adrenal cells and H295R adrenal cells, with a maximal increase seen at 100 nM. In addition, metastin increased angiotensin II (Ang II)-stimulated aldosterone production by approximately 1.5-fold. Metastin also increased the ability of the H295R cells to metabolize exogenously added pregnenolone to aldosterone but had no effect on the expression of aldosterone synthase (CYP11B2). These results suggest that the high fetal/maternal levels of metastin seen during pregnancy may affect adrenal production of aldosterone.