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CONTEXT: Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme 11ß-hydroxylase (CYP11B1) executes the final step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyandrostenedione and 11-hydroxytestosterone, respectively. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated androgens (11-oxyandrogens) are similar in males and females of reproductive ages, despite enormous differences in T. OBJECTIVE: To assess the gonadal contribution to the circulating pool of 11-oxyandrogens. METHODS: We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxyandrogens in: (I) paired gonadal and peripheral vein blood samples obtained during gonadal venograms from 11 patients (7 women), median age 37 (range 31-51 years); and (II) 17 women, median age 57 (range 41-81 years) before and after bilateral salpingo-oophorectomy (BSO). We also compared CYP11B1, 17α-hydroxylase/17,20-lyase (CYP17A1), and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA expression in adrenal, ovarian, and testicular tissue. RESULTS: A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins vs. periphery (p < 0.05 for all), while four 11-oxyandrogens were similar between matched gonadal and peripheral vein samples. Equally, in women who underwent BSO, A4 (median [interquartile range]: 59.7 [47.7-67.6] ng/dL vs. 32.7 [27.4-47.8] ng/dL, p < 0.001) and T (24.1 [16.4-32.3] vs.15.5 [13.7-19.0] ng/dL, p < 0.001) declined, while 11-oxyandrogens remained stable. Gonadal tissue displayed negligible CYP11B1 mRNA. CONCLUSION: Despite producing substantial amounts of A4 and T, human gonads are not relevant sources of 11-oxyandrogens.
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The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45â¯nM and 0.30â¯nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6â¯nM and 56.0â¯nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20â¯nM (CV1-MRLuc) to â¼2000â¯nM, while the EC50 for aldosterone remained unchanged. The addition of 18ß-glycyrrhetinic acid (18ß-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to â¼70â¯nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.
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Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.
Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Proteínas de Transporte de Cátions , Hiperaldosteronismo , Masculino , Humanos , Aldosterona/genética , Adenoma Adrenocortical/genética , Hiperaldosteronismo/genética , Adenoma/genética , Adenoma/complicações , Mutação , Zinco/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas de Transporte de Cátions/genéticaRESUMO
Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Epigênese Genética , Cromatina/genéticaRESUMO
CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)âguided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (nâ =â 12 men, 65 women) with either OCS (nâ =â 32) or MACE (nâ =â 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Síndrome de Cushing/genética , Hidrocortisona/sangue , Neoplasias do Córtex Suprarrenal/sangue , Neoplasias do Córtex Suprarrenal/complicações , Neoplasias do Córtex Suprarrenal/diagnóstico , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/cirurgia , Adrenalectomia , Adenoma Adrenocortical/sangue , Adenoma Adrenocortical/complicações , Adenoma Adrenocortical/diagnóstico , Adulto , Cromograninas/genética , Síndrome de Cushing/sangue , Síndrome de Cushing/diagnóstico , Síndrome de Cushing/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Hidrocortisona/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Gravidade do Paciente , beta Catenina/genéticaRESUMO
The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.
Assuntos
Córtex Suprarrenal , Adenoma Adrenocortical , Humanos , Zona Reticular/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/metabolismo , Corticosteroides/metabolismo , Linhagem CelularRESUMO
Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/metabolismo , Transcriptoma/efeitos dos fármacos , Angiotensina II/metabolismo , Células Cultivadas , Humanos , Hidrocortisona/metabolismoRESUMO
BACKGROUND: Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized. PARTICIPANTS AND METHODS: Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18-30 years, and 10 older, 60-80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11ß-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11ß-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed. RESULTS: 11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance. CONCLUSIONS: 11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.
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Androgênios/sangue , Ritmo Circadiano/fisiologia , Sulfatos/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/sangue , Androgênios/química , Análise Química do Sangue/métodos , Voluntários Saudáveis , Humanos , Hidroxiesteroides/sangue , Cetosteroides/sangue , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Adulto JovemRESUMO
Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.
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Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.
Assuntos
Adenoma/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Síndrome de Cushing/metabolismo , Esteroides/metabolismo , Adenoma/sangue , Neoplasias do Córtex Suprarrenal/sangue , Adulto , Cromatografia Líquida , Síndrome de Cushing/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Progesterona Redutase/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroides/sangue , Espectrometria de Massas em TandemRESUMO
Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.
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Córtex Suprarrenal/química , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Córtex Suprarrenal/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Fixação de TecidosRESUMO
Background: Somatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Objective: To analyze the transcript expression of type 1 angiotensin II receptors (AGTR1), ACTH receptors (MC2R), and melanocortin 2 receptor accessory protein (MRAP) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from APAs with mutations in: KCNJ5 (n = 14), ATP1A1 (n = 14), CACNA1D (n = 14), and ATP2B3 (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of MC2R, MRAP, AGTR1, aldosterone synthase (CYP11B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11ß-hydroxylase (CYP11B1) were quantified using quantitative RT-PCR and normalized to ß-actin. Results: Compared to adjacent normal adrenal tissue, APAs had higher transcript levels of CYP11B2 (2,216.4 [1,112.0, 2,813.5]-fold, p < 0.001), MC2R (2.88 [2.00, 4.52]-fold, p < 0.001), and AGTR1 (1.80 [1.02, 2.80]-fold, p < 0.001]), and lower transcript levels of MRAP, CYP17A1, and CYP11B1 (0.28-0.36, p < 0.001 for all). MC2R and CYP11B2 transcripts were lower in APAs with KCNJ5 vs. other mutations (p < 0.01 for both). MC2R expression correlated positively with that of AGTR1 in APAs harboring KCNJ5 and CACNA1D mutations, and with MRAP expression in APAs harboring ATPase mutations. Conclusions: While MC2R and AGTR1 are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with ATPase-mutations are more responsive to ACTH than KCNJ5-mutated APAs.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Aldosterona/metabolismo , Proteínas de Membrana/biossíntese , Mutação , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 2 de Melanocortina/biossíntese , Adenoma , Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Melanocortina/genética , Receptores da Corticotropina/metabolismo , Esteroide 11-beta-Hidroxilase/biossíntese , Esteroide 11-beta-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/biossíntese , Esteroide 17-alfa-Hidroxilase/genética , Adulto JovemRESUMO
BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.
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Córtex Suprarrenal/patologia , Córtex Suprarrenal/fisiologia , Envelhecimento/fisiologia , Adolescente , Córtex Suprarrenal/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Estudos de Casos e Controles , Citocromo P-450 CYP11B2/metabolismo , Citocromos b5/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Progesterona Redutase/metabolismo , Esteroide 11-beta-Hidroxilase/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Adulto Jovem , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia , Zona Reticular/metabolismo , Zona Reticular/patologiaRESUMO
Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in CLCN2 in 2 APAs that were negative for currently known aldosterone-driver mutations. The CLCN2 gene encodes the voltage-gated chloride channel ClC-2. CLCN2 germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in CLCN2 were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the CLCN2 somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second CLCN2 mutation, which affects the same region of the gene, has not been reported previously (c.64-2_74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA.
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Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.
Assuntos
Neoplasias do Córtex Suprarrenal/classificação , Neoplasias do Córtex Suprarrenal/diagnóstico , Biomarcadores Tumorais/genética , Inclusão em Parafina/métodos , RNA-Seq/métodos , Transcriptoma , Neoplasias do Córtex Suprarrenal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Seguimentos , Formaldeído/química , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
CONTEXT: The gonads are the major source of sex steroids during reproductive ages. The gonadal function declines abruptly in women and gradually in men. The adrenals produce 11-oxygenated androgens (11-oxyandrogens), which start rising during adrenarche. Following menopause, 11-oxyandrogens levels remain similar to reproductive ages. OBJECTIVE: To compare the circulating 11-oxyandrogen concentrations in men and women across adult ages. METHODS: We used mass spectrometry to measure testosterone (T), androstenedione (A4), 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 and cytochrome b5 in adrenal tissue from 19 men, age 23-78 years. RESULTS: We included 590 patients (319 men), aged 18 to 97 years, and 84% white. 11KT and 11KA4 were stable across ages in women, but they declined in men (0.21 and 0.06 ng/dL/year, respectively; P < 0.05). 11OHA4 and 11OHT increased modestly with age in women (0.6 and 0.09 ng/dL/year, respectively; P < 0.01), and both remained stable across ages in men. As body mass index (BMI) increased, 11KA4 decreased in women, and 11KT increased in men, both suggesting higher 17ß-hydroxysteroid dehydrogenase activity in obese individuals. A4 and T declined with age and A4 with BMI in both sexes; T declined with BMI in men. Adrenal androgenic enzyme expressions in aging men were similar to those observed in women. CONCLUSIONS: In contrast with traditional androgens, the production of 11OHA4 and 11OHT is sustained with aging in both sexes. The bioactive androgen 11KT declines in aging men but not in women.
Assuntos
Envelhecimento/sangue , Androgênios/sangue , Androstenodiona/análogos & derivados , Hidroxitestosteronas/sangue , Testosterona/análogos & derivados , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Androstenodiona/sangue , Androstenodiona/fisiologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Testosterona/sangue , Testosterona/fisiologia , Adulto JovemRESUMO
Primary aldosteronism (PA) is characterized by autonomous aldosterone production by renin-independent mechanisms and is most commonly sporadic. While 60-70% of sporadic PA can be attributed to bilateral hyperaldosteronism, the remaining 30-40% is caused by a unilateral aldosterone-producing adenoma (APA). Somatic mutations in or near the selectivity filter the KCNJ5 gene (encoding the potassium channel GIRK4) have been implicated in the pathogenesis of both sporadic and familial PA. Several studies using tumor tissue, peripheral and adrenal vein samples from PA patients have demonstrated that along with aldosterone, the hybrid steroids 18-hydroxycortisol (18OHF) and 18-oxocortisol (18oxoF) are a hallmark of APA harboring KCNJ5 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of KCNJ5 mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling.
RESUMO
The adrenal gland is a source of sex steroid precursors, and its activity is particularly relevant during fetal development and adrenarche. Following puberty, the synthesis of androgens by the adrenal gland has been considered of little physiologic importance. Dehydroepiandrosterone (DHEA) and its sulfate, DHEAS, are the major adrenal androgen precursors, but they are biologically inactive. The second most abundant unconjugated androgen produced by the human adrenals is 11ß-hydroxyandrostenedione (11OHA4). 11-Ketotestosterone, a downstream metabolite of 11OHA4 (which is mostly produced in peripheral tissues), and its 5α-reduced product, 11-ketodihydrotestosterone, are bioactive androgens, with potencies equivalent to those of testosterone and dihydrotestosterone. These adrenal-derived androgens all share an oxygen atom on carbon 11, so we have collectively termed them 11-oxyandrogens. Over the past decade, these androgens have emerged as major components of several disorders of androgen excess, such as congenital adrenal hyperplasia, premature adrenarche and polycystic ovary syndrome, as well as in androgen-dependent tumours, such as castration-resistant prostate cancer. Moreover, in contrast to the more extensively studied, traditional androgens, circulating concentrations of 11-oxyandrogens do not demonstrate an age-dependent decline. This Review focuses on the rapidly expanding knowledge regarding the implications of 11-oxyandrogens in human physiology and disease.
Assuntos
Glândulas Suprarrenais/metabolismo , Androgênios/metabolismo , Doenças do Sistema Endócrino/metabolismo , Hiperplasia Suprarrenal Congênita/metabolismo , Androgênios/química , Feminino , Humanos , Masculino , Oxigênio/química , Síndrome do Ovário Policístico/metabolismo , Neoplasias da Próstata/metabolismo , Puberdade Precoce/metabolismoRESUMO
Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence among patients with resistant hypertension. Despite the recent discovery of somatic variants in aldosterone-producing adenoma (APA)-associated PA, causes for PA due to bilateral aldosterone production (bilateral hyperaldosteronism; BHA) remain unknown. Herein, we identified rare gene variants in ATP2B4, in a cohort of patients with BHA. ATP2B4 belongs to the same family of Ca-ATPases as ATP2B3, which is involved in the pathogenesis of APA. Endogenous ATP2B4 expression was characterized in adrenal tissue, and the gene variants were functionally analyzed for effects on aldosterone synthase (CYP11B2) expression, steroid production in basal and agonist-stimulated conditions, and for changes in biophysical properties of channel properties. Knockdown of ATP2B4 in HAC15 exhibited reduced angiotensin II stimulation in one of four shRNA clones. Stable HAC15 cell lines with doxycycline (dox) - inducible wild-type and variant forms of ATP2B4 - were generated, and dox-induced upregulation of ATP2B4 mRNA and protein was confirmed. However, ATP2B4 variants did not alter basal or agonist-stimulated CYP11B2 expression. Whole-cell recordings in HAC15 cells indicated robust endogenous ATP2B4 conductance in native cells but reduced conductance with overexpressed WT and variant ATP2B4. The previously defined PA-causing ATP2B3 variant served as a positive control and exhibited elevated CYP11B2 mRNA. In conclusion, while this study did not confirm a pathogenic role for ATP2B4 variants in BHA, we describe the sequencing analysis for familial and sporadic BHA and outline a template for the thorough in vitro characterization of gene variants.
Assuntos
Eletrofisiologia/métodos , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hiperaldosteronismo/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Linhagem Celular Tumoral , Feminino , Humanos , MasculinoRESUMO
Driver somatic mutations for aldosterone excess have been found in ≈90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. In the present study, we identified a novel somatic CACNA1H mutation (c.T4289C, p.I1430T) in an APA without any currently known aldosterone-driver mutations using CYP11B2 immunohistochemistry-guided whole exome sequencing. The CACNA1H gene encodes a voltage-dependent T-type calcium channel alpha-1H subunit. Germline variants in this gene are known as a cause of familial hyperaldosteronism IV. Targeted next-generation sequencing detected identical CACNA1H variants in 2 additional APAs in a cohort of the University of Michigan, resulting in a prevalence of 4% (3/75) in APAs. We tested the functional effect of the variant on adrenal cell aldosterone production and CYP11B2 mRNA expression using the human adrenocortical HAC15 cell line with a doxycycline-inducible CACNA1HI1430T mutation. Doxycycline treatment increased CYP11B2 mRNA levels as well as aldosterone production, supporting a pathological role of the CACNA1H p.I1430T mutation on the development of primary aldosteronism. In conclusion, somatic CACNA1H mutation is a genetic cause of APAs. Although the prevalence of this mutation is low, this study will provide better understanding of molecular mechanism of inappropriate aldosterone production in APAs.