Human Fetal Adrenal Gland Research Articles

MOXD1 is a lineage-specific gene and a tumor suppressor in neuroblastoma.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads.

Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 gestational weeks (GW) via 10× Genomics single-cell transcriptome techniques, reconstructed their location information by spatial transcriptomics. Relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple non-steroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the window of sexual differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

S-15-04 Knowledge gaps: case studies from human fetal ovary and adrenal gland

S-15-04 Knowledge gaps: case studies from human fetal ovary and adrenal gland

The effects of selected inhibitors on human fetal adrenal steroidogenesis differs under basal and ACTH-stimulated conditions

BackgroundDisordered fetal adrenal steroidogenesis can cause marked clinical effects including virilization of female fetuses. In postnatal life, adrenal disorders can be life-threatening due to the risk of adrenal crisis and must be carefully managed. However, testing explicit adrenal steroidogenic inhibitory effects of therapeutic drugs is challenging due to species-specific characteristics, and particularly the impact of adrenocorticotropic hormone (ACTH) stimulation on drugs targeting steroidogenesis has not previously been examined in human adrenal tissue. Therefore, this study aimed to examine the effects of selected steroidogenic inhibitors on human fetal adrenal (HFA) steroid hormone production under basal and ACTH-stimulated conditions.MethodsThis study used an established HFA ex vivo culture model to examine treatment effects in 78 adrenals from 50 human fetuses (gestational weeks 8–12). Inhibitors were selected to affect enzymes critical for different steps in classic adrenal steroidogenic pathways, including CYP17A1 (Abiraterone acetate), CYP11B1/2 (Osilodrostat), and a suggested CYP21A2 inhibitor (Efavirenz). Treatment effects were examined under basal and ACTH-stimulated conditions in tissue from the same fetus and determined by quantifying the secretion of adrenal steroids in the culture media using liquid chromatography-tandem mass spectrometry. Statistical analysis was performed on ln-transformed data using one-way ANOVA for repeated measures followed by Tukey’s multiple comparisons test.ResultsTreatment with Abiraterone acetate and Osilodrostat resulted in potent inhibition of CYP17A1 and CYP11B1/2, respectively, while treatment with Efavirenz reduced testosterone secretion under basal conditions. ACTH-stimulation affected the inhibitory effects of all investigated drugs. Thus, treatment effects of Abiraterone acetate were more pronounced under stimulated conditions, while Efavirenz treatment caused a non-specific inhibition on steroidogenesis. ACTH-stimulation prevented the Osilodrostat-mediated CYP11B1 inhibition observed under basal conditions.ConclusionsOur results show that the effects of steroidogenic inhibitors differ under basal and ACTH-stimulated conditions in the HFA ex vivo culture model. This could suggest that in vivo effects of therapeutic drugs targeting steroidogenesis may vary in conditions where patients have suppressed or high ACTH levels, respectively. This study further demonstrates that ex vivo cultured HFAs can be used to evaluate steroidogenic inhibitors and thereby provide novel information about the local effects of existing and emerging drugs that targets steroidogenesis.

Possible Relevance of Soluble Luteinizing Hormone Receptor during Development and Adulthood in Boys and Men.

Simple SummaryThe reproductive hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are both agonists for the luteinizing hormone receptor (LHCGR) and essential for male reproduction during development and adulthood. LHCGR is expressed and stimulates testosterone production from the testicular Leydig cells. In this study, we demonstrate the presence of soluble LHCGR in blood, urine, and seminal fluid in both healthy boys and men, and patients with aberrations in sex-chromosomes. We show how circulating levels of sLHCGR are associated with pubertal development, testicular function, and semen quality and demonstrate that LHCGR is released from fetal human non-gonadal tissue. sLHCGR is released into serum by testis and other organs, which suggests possible extra-gonadal effects of LH or hCG in boys and men.Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are agonists for the luteinizing hormone receptor (LHCGR) which regulates male reproductive function. LHCGR may be released into body fluids. We wish to determine whether soluble LHCGR is a marker for gonadal function. Cross-sectional, longitudinal, and intervention studies on 195 healthy boys and men and 396 men with infertility, anorchia, or Klinefelter Syndrome (KS) were used to correlate LHCGR measured in serum, seminal fluid, urine, and hepatic/renal artery and vein with gonadal function. LHCGR was determined in fluids from in vitro and in vivo models of human testicular tissue and cell lines, xenograft mouse models, and human fetal kidney and adrenal glands. Western blot showed LHCGR fragments in serum and gonadal tissue of similar size using three different antibodies. The LHCGR-ELISA had no species cross-reactivity or unspecific reaction in mouse serum even after human xenografting. Instead, sLHCGR was released into the media after the culture of a human fetal kidney and adrenal glands. Serum sLHCGR decreased markedly during puberty in healthy boys (p = 0.0001). In healthy men, serum sLHCGR was inversely associated with the Inhibin B/FSH ratio (β −0.004, p = 0.027). In infertile men, seminal fluid sLHCGR was inversely associated with serum FSH (β 0.006, p = 0.009), sperm concentration (β −3.5, p = 0.003) and total sperm count (β −3.2, p = 0.007). The injection of hCG lowered sLHCGR in serum and urine of healthy men (p < 0.01). In conclusion, sLHCGR is released into body-fluids and linked with pubertal development and gonadal function. Circulating sLHCGR in anorchid men suggests that sLHCGR in serum may originate from and possibly exert actions in non-gonadal tissues. (ClinicalTrials: NTC01411527, NCT01304927, NCT03418896).

Establishment of a Novel Human Fetal Adrenal Culture Model that Supports de Novo and Manipulated Steroidogenesis.

Disorders affecting adrenal steroidogenesis promote an imbalance in the normally tightly controlled secretion of mineralocorticoids, glucocorticoids, and androgens. This may lead to differences/disorders of sex development in the fetus, as seen in virilized girls with congenital adrenal hyperplasia (CAH). Despite the important endocrine function of human fetal adrenals, neither normal nor dysregulated adrenal steroidogenesis is understood in detail. Due to significant differences in adrenal steroidogenesis between human and model species (except higher primates), we aimed to establish a human fetal adrenal model that enables examination of both de novo and manipulated adrenal steroidogenesis. Human adrenal tissue from 54 1st trimester fetuses were cultured ex vivo as intact tissue fragments for 7 or 14 days. Model validation included examination of postculture tissue morphology, viability, apoptosis, and quantification of steroid hormones secreted to the culture media measured by liquid chromatography-tandem mass spectrometry. The culture approach maintained cell viability, preserved cell populations of all fetal adrenal zones, and recapitulated de novo adrenal steroidogenesis based on continued secretion of steroidogenic intermediates, glucocorticoids, and androgens. Adrenocorticotropic hormone and ketoconazole treatment of ex vivo cultured human fetal adrenal tissue resulted in the stimulation of steroidogenesis and inhibition of androgen secretion, respectively, demonstrating a treatment-specific response. Together, these data indicate that ex vivo culture of human fetal adrenal tissue constitutes a novel approach to investigate local effects of pharmaceutical exposures or emerging therapeutic options targeting imbalanced steroidogenesis in adrenal disorders, including CAH.

Single-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma

Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB.

SUN-039 Characterization of Human Adrenal Steroidogenesis during Fetal Development

Context: The endocrine function of human fetal adrenals (HFA) is activated already during first trimester, but changes in adrenal steroidogenesis during fetal life are not well characterized. Objective: This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from 1st and 2nd trimester. Design and Setting: Male and female HFA samples from gestational week (GW) 8-19 were examined, including a total of 101 samples from 83 fetuses. Main Outcome Measure(s): Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS. Results: Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2 and SULT2A1 was significantly higher in 2nd trimester compared with 1st trimester (P<0.05), while expression levels of 3β-HSD2 and ARK1C3 were unaltered between GW 8-19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout GW 8-19 with most enzymes expressed primarily in the fetal zone, except 3β-HSD1/2 which was mainly expressed in the definitive zone. The abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1,071-2,723 ng/g tissue, in average) and testosterone levels the lowest (2-14 ng/g tissue, in average). Conclusions: The expression profiles of HFA steroidogenic enzymes are distinct from 1st to 2nd trimester, with no major differences between male and female samples. The intra-adrenal steroid hormone concentrations confirms that cortisol is produced throughout 1st and 2nd trimester, suggesting continued regulation of the HPA axis during this entire period.

The transient cortical zone in the adrenal gland: the mystery of the adrenal X-zone.

The X-zone is a transient cortical region enriched in eosinophilic cells located in the cortical-medullary boundary of the mouse adrenal gland. Similar to the X-zone, the fetal zone in human adrenals is also a transient cortical compartment, comprising the majority of the human fetal adrenal gland. During adrenal development, fetal cortical cells are gradually replaced by newly formed adult cortical cells that develop into outer definitive zones. In mice, the regression of this fetal cell population is sexually dimorphic. Many mouse models with mutations associated with endocrine factors have been reported with X-zone phenotypes. Increasing findings indicate that the cell fate of this aged cell population of the adrenal cortex can be manipulated by many hormonal and nonhormonal factors. This review summarizes the current knowledge of this transient adrenocortical zone with an emphasis on genes and signaling pathways that affect X-zone cells.

Characterization of Human Adrenal Steroidogenesis During Fetal Development.

ContextThe endocrine function of human fetal adrenals (HFAs) is activated already during first trimester, but adrenal steroidogenesis during fetal life is not well characterized.ObjectiveThis study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from first and second trimesters.Design and SettingMale and female HFA from gestational weeks (GWs) 8 to 19 were examined, including a total of 101 samples from 83 fetuses.Main Outcome Measure(s)Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS.ResultsTranscriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2, and SULT2A1 were significantly higher in second trimester compared to first trimester (P < 0.05), whereas expression levels of 3β-HSD2 and ARK1C3 were unaltered between GWs 8 and 19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout the investigated period, with most enzymes expressed primarily in the fetal zone, except 3β-HSD1/2, which was expressed mainly in the definitive zone. Abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1071 to 2723 ng/g tissue), and testosterone levels were the lowest (2 to 14 ng/g tissue).ConclusionsThe expression profiles of HFA steroidogenic enzymes are distinct from first to second trimester, with no major differences between male and female samples. Intra-adrenal steroid hormone concentrations confirm that cortisol is produced throughout first and second trimesters, suggesting continued regulation of the hypothalamus-pituitary-adrenal axis during this entire period.

The role of epithelial growth factors and insulin growth factors in the adrenal neoplasms.

Human fetal and adult adrenal gland express both insulin growth factor-1 (IGF-1) and IGF-2, their receptors (IGF-Rs) and a variety of specific IGF binding proteins suggesting their potential role in the regulation of adrenal growth and function. IGF-2 overexpression is essential for the growth of monoclonal lesions, such as large benign adenomas (ACA) and adrenocortical carcinomas (ACC) and has been found to contribute to tumorigenesis in Beckwith-Wiedemann syndrome. IGF-2 is the most highly expressed gene observed in more than 85% of ACCs. However, no significant differences in clinical, biological and transcriptomic traits were found between tumors with high and low expression of IGF-2. On the contrary, the expression of IGF-1R, mediating the IGF-2 effects in vivo, was more discriminant between malignant (overexpression) and benign tumors. Data on the role of epithelial growth factor (EGF) and its receptor (EGF-R) in adrenocortical tumorigenesis are controversial. Several studies have shown EGF-R overexpression in ACCs but not in benign ACAs, suggesting that EGF-R could potentially be used as a marker for the differential diagnosis of ACAs and ACCs. Although, in vitro and animal studies provide promising results in the therapeutic role of IGF and EGF pathway inhibitors, the available data in humans are still not encouraging. Herein, we aim to present recent data on the role of IGF and EGF pathways in adrenal development and tumorigenesis and their potential implication in the treatment of the ACC, a rare malignancy with very poor prognosis.

The human fetal adrenal produces cortisol but no detectable aldosterone throughout the second trimester

BackgroundHuman fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone, estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention through secretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objective of this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzyme expression in normal second trimester human fetuses.MethodsThis was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to 109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study design was balanced to show effects of maternal smoking.ResultsConcentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and mass spectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in all adrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 and CYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-level expression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetal plasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 and NR5A1 were increased by maternal smoking.ConclusionsThe human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectable aldosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen in extreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which could have knock-on effects on post-natal health.

Kisspeptin Is a Novel Regulator of Human Fetal Adrenocortical Development and Function: A Finding With Important Implications for the Human Fetoplacental Unit.

Context:The human fetal adrenal (HFA) is an integral component of the fetoplacental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in fetal adrenal function remains unknown.Objective:To determine the role of kisspeptin in the developing HFA.Design:Experiments using H295R and primary HFA cells as in vitro models of the fetal adrenal. Association of plasma kisspeptin levels with HFA size in a longitudinal clinical study.Setting:Academic research center and tertiary fetal medicine unit.Participants:Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan.Main Outcome Measures:The spatiotemporal expression of Kiss1R in the HFA. The production of dehydroepiandrosterone sulfate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone. Fetal adrenal volume (FAV) and kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks’ gestation).Results:Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner fetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated fetal weight.Conclusions:Kisspeptin plays a key role in the regulation of the HFA and thus the fetoplacental unit, particularly in the second trimester of pregnancy.

Androgenic potential of human fetal adrenals at the end of the first trimester.

The onset of steroidogenesis in human fetal adrenal glands (HFA) during the first trimester is poorly investigated. An unresolved question is the capacity of the HFA to produce potent androgen DHT via conventional and/or the backdoor pathway(s) at the end of first trimester, when androgen-responsive organs are developed. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes and transcription factors in HFA at gestational weeks (GW) 9–12 with focus on their androgenic potential. Steroids in the HFA were analyzed by gas chromatography/mass spectrometry. The expression of steroidogenic enzymes and transcription factors in the HFA at GW9–12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that during GW9–12 HFA produced steroids of the ∆5, ∆4 and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17α-OH-dihydroprogesterone, androsterone and androstanedione without further conversion to DHT. The only androgens identified in the HFA were testosterone and androsterone, a precursor in the biosynthesis of DHT. We also observed higher levels of CYP17A1 but low expression of 3βHSD2 at GW11–12 in the HFA. Elevated levels of CYP17A1 were associated with an increased expression of SF-1 and GATA-6. Altogether, our data demonstrate that of those steroids analyzed, the only potent androgen directly produced by the HFA at GW9–12 was testosterone. The onset of steroidogenesis in the HFA is a complex process that is regulated by the coordinated action of related transcription factors.

Role of urocortin in pregnancy: An update and future perspectives.

The activities of corticotropin-releasing factor (CRF) and related peptides are mediated a number of receptors with seven transmembrane domains that are coupled to the Gs and Gq proteins. These receptors are known as CRF-Rs. In vitro studies have evidenced that urocortin (UCN) and CRF provoke an increase in the contractility of the uterus which is induced by endometrial prostaglandin F2a. Furthermore, through trophoblasts, it stimulates the secretion of adrenocorticotropic hormone (ACTH) and prostaglandin PGE2 and has a vasodilatory effect on the placenta. While it is well known that the placenta produces considerable quantities of CRF, several studies have, however, excluded that the placenta can generate significant quantities of UCN. In the short term, the human fetal adrenal gland produces more cortisol and dehydroepiandrosterone sulfate. The gestational tissues express UCN3 and UCN2 mRNA in cytotrophoblast and syncytiotrophoblast cells, while UCN2 is only to be found in the maternal and fetal vessels and amniotic cells. Nevertheless, gestational tissues express UCN2 and UCN3 differentially and do not stimulate placental ACTH secretion. In term pregnancies, maternal plasma levels of CRF and UCN are lower than at the beginning of pregnancy and are correlated to labor onset. Conversely, they do not decrease in post-term pregnancies. This evidence would seem to indicate that the fine-regulated expression of these neuropeptides is important in determining the duration of human gestation. In this scenario, low concentrations of UCN in the amniotic fluid at mid-term may be considered a sign of predisposition to preterm birth.

Human foetal adrenal gland development and effects of maternal smoking

Human foetal adrenal gland development and effects of maternal smoking

LRH-1 May Rescue SF-1 Deficiency for Steroidogenesis: An in vitro and in vivo Study

Steroidogenic factor 1 (NR5A1/SF-1) mutations usually manifest in 46,XY individuals with variable degrees of disordered sex development and in 46,XX women with ovarian insufficiency. So far, there is no genotype-phenotype correlation. The broad spectrum of phenotype with NR5A1 mutations may be due to a second hit in a gene with similar function to NR5A1/SF-1. Liver receptor homologue-1 (LRH-1/NR5A2) might be a good candidate. We performed in vitro studies for the interplay between SF-1, LRH-1 and DAX-1, expression profiles in human steroidogenic tissues, and NR5A2 genetic studies in a cohort (11 patients, 8 relatives, 11 families) harboring heterozygote NR5A1/SF-1 mutations. LRH-1 isoforms transactivate the CYP17A1 and HSD3B2 promoters similarly to SF-1 and compensate for SF-1 deficiency. DAX-1 inhibits SF-1- and LRH-1-mediated transactivation. LRH-1 is found expressed in human adult and fetal adrenals and testes. However, no NR5A2/LRH-1 mutations were detected in 14 individuals with heterozygote NR5A1/SF-1 mutations. These findings demonstrate that in vitro LRH-1 can act like SF-1 and compensate for its deficiency. Expression of LRH-1 in fetal testis suggests a role in male gonadal development. However, as we found no NR5A2/LRH-1 mutations, the ‘second genetic hit' in SF-1 patients explaining the broad phenotypic variability remains elusive.

Sonic Hedgehog Signaling Is Active in Human Adrenal Cortex Development and Deregulated in Adrenocortical Tumors

The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability. The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

QRFP induces aldosterone production via PKC and T-type calcium channel-mediated pathways in human adrenocortical cells: evidence for a novel role of GPR103

Hormonal regulation of adrenal function occurs primarily through activation of GPCRs. GPCRs are central to many of the body's endocrine and neurotransmitter pathways. Recently, it was shown that activation of GPR103 by its ligand QRFP induced feeding, locomotor activity, and metabolic rate, and QRFP is bioactive in adipose tissue of obese individuals. Given that the adrenal gland is a pivotal organ for energy balance and homeostasis, we hypothesized that GPR103 and QRFP are involved in steroidogenic responses. Using qRT-PCR and immunohistochemistry, we mapped both GPR103 and QRFP in human fetal and adult adrenal gland as well as rat adrenals. Both were primarily localized in the adrenal cortex but not in the medulla. Activation of GPR103 in human adrenocortical H295R cells led to a decrease in forskolin-increased cAMP and an increase of intracellular Ca(2+) levels. In addition, treatment of H295R cells with QRFP induced aldosterone and cortisol secretion as measured by ELISA. These increases were accompanied by increased expression and activity of StAR, CYB11B1, and CYP11B2 as assessed by qRT-PCR and luciferase reporter assay, respectively. Using specific inhibitors, we also demonstrated that aldosterone induction involves MAPK, PKC, and/or T-type Ca(2+) channel-dependent pathways. These novel data demonstrate that QRFP induces adrenal steroidogenesis in vitro by regulating key steroidogenic enzymes involving MAPK/PKC and Ca(2+) signaling pathways.