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1.
Hypertension ; 74(5): 1152-1159, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31564164

RESUMO

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10-24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.


Assuntos
Aldosterona/biossíntese , Anoctaminas/genética , Regulação da Expressão Gênica , Hiperaldosteronismo/genética , Hipertensão/fisiopatologia , Transdução de Sinais/genética , Zona Glomerulosa/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Análise de Variância , Comunicação Celular/genética , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Hiperaldosteronismo/patologia , Hipertensão/etiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise Serial de Tecidos , Técnicas de Cultura de Tecidos , Transcriptoma/genética , Regulação para Cima , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/patologia
2.
Eur J Endocrinol ; 181(1): 69-78, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096184

RESUMO

Objective: The pathophysiology of aldosterone-producing adenomas (APAs) has been intensively investigated using genetic and epigenetic approaches. However, the role of miRNAs in APA is not fully understood. The present study profiled miRNAs in APAs as an exploratory approach to elucidate their pathophysiological roles in APAs. Design: Tissues of APAs and other adrenocortical adenomas were obtained from patients who underwent adrenalectomy. Methods: Candidate miRNAs differentially detected from samples were examined by whole miRNA sequencing. The expression of candidate miRNAs in APA tissues were further validated by real-time quantitative polymerase chain reaction (qPCR). Further, differential miRNA expression between APAs with and without KCNJ5 somatic mutations was examined. Prediction of miRNA target genes was performed by bioinformatics analysis. For specific miRNAs, correlation analysis between the levels of their target genes and CYP11B2 was analyzed in APA tissues. Results: Our study determined differential expression of six miRNAs in APA or APA with KCNJ5 mutations. We further demonstrated that miR299 levels were negatively correlated with mRNA levels of CACNB2, which encodes the beta-subunit of the L-type calcium channel. Additionally, we found significant correlations among miR299, CACNB2, and CYP11B2 levels in APA tissues. Conclusions: Our study suggests the possible pathophysiological involvement of specific miRNAs in calcium signaling and aldosterone hypersecretion in APAs. Further studies, including in vitro analyses, are required to clarify these findings.


Assuntos
Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Citocromo P-450 CYP11B2/genética , Perfilação da Expressão Gênica , MicroRNAs/fisiologia , Adrenalectomia , Adenoma Adrenocortical/metabolismo , Idoso , Canais de Cálcio Tipo L/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Expressão Gênica/fisiologia , Humanos , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade , Mutação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real
3.
Curr Opin Nephrol Hypertens ; 28(2): 105-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30608249

RESUMO

PURPOSE OF REVIEW: Immunohistochemistry for aldosterone synthase (CYP11B2) has markedly provided a comprehensive picture of the adrenocortical diseases, particularly primary aldosteronism. The findings from CYP11B2-immunohistochemistry are consistent with the clinical courses of most patients with primary aldosteronism. We herein review the updated pathophysiology and usefulness of the method for understanding individual patients with different subtypes of primary aldosteronism. RECENT FINDINGS: After our discovery of aldosterone-producing cell clusters (APCCs) using the immunohistochemistry for CYP11B2, we found possible APCC-to-APA transitional lesions (pAATLs) in a few cases that had been hitherto classified as unilateral hyperplasia or multiple nodules. On the basis of morphological and functional features of pAATLs as well as distributions of somatic mutations within the lesions, we have made a hypothesis that APCC grows to APA via pAATL for one of developmental courses of APA. Recently, we successfully performed in-situ detection of aldosterone on adrenal tissue sections using a state-of-the-art technique, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-imaging). This method revealed in-situ synthesis of aldosterone in APCCs and APAs in addition to several other steroids. SUMMARY: CYP11B2 immunohistochemistry revealed the pathophysiology of aldosterone production in the past decade, especially formation of APCC in normal adrenals and pAATL that is a possible lesion developing from APCC to APA. The term 'idiopathic hyperaldosteronism' may soon become obsolete.


Assuntos
Adenoma/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Citocromo P-450 CYP11B2/metabolismo , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/metabolismo , Adenoma/patologia , Neoplasias do Córtex Suprarrenal/patologia , Glândulas Suprarrenais/patologia , Adulto , Idoso , Feminino , Humanos , Hiperaldosteronismo/etiologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
Int J Cardiol ; 275: 114-119, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30384979

RESUMO

BACKGROUND: Myocarditis may be self-limited but has been identified as an important contributor to downstream cardiomyopathy. Aldosterone mediates myocardial damage in various conditions, but has not been considered specifically as a therapeutic target for inflammatory damage in acute myocarditis. We sought to demonstrate local aldosterone synthesis in human myocardium affected by acute myocarditis. METHODS: We evaluated myocardial samples obtained via endomyocardial biopsy (EMB) for expression of CYP11B2, the final and key enzyme for aldosterone synthesis, from patients with acute myocarditis and from stable heart transplant recipients with no evidence of rejection as negative controls. Excised adrenal glands from patients with aldosterone-secreting adenomas were used as positive controls. An experienced cardiovascular pathologist blinded to clinical information rated CYP11B2 stains as negative, positive, or borderline, also recording location of any CYP11B2-positivity. RESULTS: Sixteen patients' EMB samples showing definite acute myocarditis were identified (50% female). CYP11B2 was positive in 13/16 cases (81%), typically showing diffuse intracardiomyocyte cytoplasmic staining, vs. 2/16 borderline stains in transplant controls (p < 0.001 myocarditis vs. negative controls). All 3 adrenalectomy samples stained positive for CYP11B2 (diffuse intracellular staining). Importantly, no myocarditis or transplant patients were on aldosterone antagonist therapy at the time of biopsy. CONCLUSIONS: In this proof-of-concept study, myocardium from patients with acute myocarditis demonstrates evidence and high prevalence of local aldosterone synthesis by immunohistochemistry that showed high accuracy, specificity, and sensitivity. Aldosterone warrants consideration as a specific target for therapy in patients with myocardial damage due to inflammation towards strategies that reduce downstream complications.


Assuntos
Aldosterona/biossíntese , Miocardite/metabolismo , Miocárdio/metabolismo , Doença Aguda , Adulto , Biomarcadores/metabolismo , Biópsia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miocardite/patologia , Miocárdio/patologia
5.
Urol J ; 16(3): 318-321, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120761

RESUMO

We encountered 2 patients (a 33-year-old woman and a 66-year-old man) with an aldosterone-producing adenoma (APA) and a left accessory spleen. The patients' primary symptoms were hypertension and hypokalemia, and both had elevated serum aldosterone levels. Preoperative computed tomography a left suprarenal retroperitoneal mass and laparoscopic left adrenalectomy was performed in both cases. The postoperative microscopic examination revealed splenic tissue. Both patients experienced relief of their hypertension and hypokalemia, with an uneventful recovery.


Assuntos
Adenoma/complicações , Neoplasias das Glândulas Suprarrenais/complicações , Hiperaldosteronismo/etiologia , Baço/anormalidades , Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Aldosterona/biossíntese , Feminino , Humanos , Masculino
6.
J Steroid Biochem Mol Biol ; 185: 137-141, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30125658

RESUMO

Angiotensin 1-7 (Ang 1-7), which is a protein cleaved from angiotensin II (A-II), binds to the MAS receptor. Ang 1-7 has been demonstrated to exert protective effects against A-II-mediated cardiac, atherosclerotic, and renal damages. The aims of our study were to demonstrate the inhibitory role of Ang 1-7 in A-II-mediated aldosterone production by interacting with the MAS receptor in human adrenocortical carcinoma (HAC15) cells, and clarify the intracellular signaling mechanisms underlying the inhibition of aldosterone production by Ang 1-7. Ang 1-7 significantly suppressed A-II-stimulated aldosterone production, and partially abrogated A-II-induced upregulation of CYP11B2 expression. Treatment with a selective Ang 1-7 antagonist abrogated Ang 1-7-mediated inhibition of aldosterone production in HAC15 cells. Incubation of A-II-treated HAC15 cells with conditioned medium containing Ang 1-7 was demonstrated to suppress A-II-mediated aldosterone production and CYP11B2 expression. Proteomic analysis showed that Ang 1-7 predominantly inhibited the phosphorylation of JAK-STAT proteins in A-II stimulated HAC15 cells. Treatment of HAC15 cells with a STAT3 inhibitor partially but significantly repressed A-II-mediated aldosterone production by 63.2%. Similarly, treatment with a STAT5 inhibitor significantly abrogated A-II-stimulated aldosterone production in HAC15 cells by 60.7%. In conclusion, we demonstrated that Ang 1-7 negatively regulates A-II-mediated aldosterone production, and the observed inhibition of aldosterone production was associated with JAK/STAT signaling in human adrenal cells. Therefore, activation of Ang 1-7 or stimulation of the MAS receptor, which inhibits aldosterone production, is a promising therapeutic approach for the prevention of cardiovascular events that can directly affect the target organs.


Assuntos
Aldosterona/biossíntese , Angiotensina II/metabolismo , Angiotensina I/metabolismo , Janus Quinases/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição STAT/antagonistas & inibidores , Carcinoma Adrenocortical/metabolismo , Angiotensina I/antagonistas & inibidores , Doenças Cardiovasculares/tratamento farmacológico , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Citocromo P-450 CYP11B2/biossíntese , Humanos , Fragmentos de Peptídeos/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
7.
Hypertension ; 73(2): 469-480, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30580688

RESUMO

Primary aldosteronism is a disease of excessive production of adrenal steroid hormones and the most common cause of endocrine hypertension. Primary aldosteronism results mainly from bilateral adrenal hyperplasia or unilateral aldosterone-producing adenoma (APA). Primary aldosteronism cause at the molecular level is incompletely understood and a targeted treatment preventing excessive adrenal steroid production is not available. Here, we perform deep quantitative proteomic and phosphoproteomic profiling of 6 pairs of APA and adjacent nontumoral adrenal cortex. We show that increased steroidogenesis in APA is accompanied by upregulation of steroidogenic enzymes (HSD3B2, CYP21A2, CYP11B2) and of proteins involved in cholesterol uptake (LSR). We demonstrate that HSD3B2 is phosphorylated at Ser95 or 96 and identify a novel phosphorylation site, Ser489, in CYP21A2, suggesting that steroidogenic enzymes are regulated by phosphorylation. Our analysis also reveals altered ECM (extracellular matrix) composition in APA that affects ECM-cell surface interactions and actin cytoskeleton rearrangements. We show that RHOC, a GTPase controlling actin organization in response to extracellular stimuli, is upregulated in APA and promotes expression of the aldosterone synthase gene CYP11B2. Our data also indicate deregulation of protein N-glycosylation and GABAergic signaling in APAs. Finally, we find that mTORC1 (mammalian target of rapamycin complex 1) signaling is the major pathway deregulated in APA. Our study provides a rich resource for future research on the molecular mechanisms of primary aldosteronism.


Assuntos
Adenoma/metabolismo , Aldosterona/biossíntese , Proteômica/métodos , Matriz Extracelular/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismo
8.
Hypertension ; 72(4): 874-880, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30354720

RESUMO

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.


Assuntos
Aldosterona , Canais de Cálcio Tipo L/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo , Hipertensão , Zona Glomerulosa , Aldosterona/biossíntese , Aldosterona/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Hiperaldosteronismo/complicações , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Hiperplasia , Hipertensão/etiologia , Hipertensão/metabolismo , Imuno-Histoquímica , Masculino , Mutação , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
9.
J Clin Endocrinol Metab ; 103(10): 3869-3876, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085035

RESUMO

Context: Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown. Objective: To investigate the prevalence and spectrum of somatic mutations in APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)‒guided next-generation sequencing (NGS) approach. Methods: Formalin-fixed paraffin-embedded adrenal tissue from white American patients with primary aldosteronism who underwent adrenalectomy at the University of Michigan was used. Genomic DNA was isolated from 75 APAs (identified by CYP11B2 IHC). NGS was performed to identify somatic mutations by sequencing the entire coding region of a panel of genes mutated in APAs. Results: Somatic mutations were identified in 66 of 75 APAs (88%). Of the APAs with somatic mutations, six were smaller than coexisting CYP11B2-negative adrenocortical adenomas. The most frequently mutated gene was KCNJ5 (43%), followed by CACNA1D (21%), ATP1A1 (17%), ATP2B3 (4%), and CTNNB1 (3%). In addition to identification of previously reported mutations, we identified five previously unreported mutations (two in KCNJ5, one in ATP1A1, one in ATP2B3, and one in CACNA1D genes). KCNJ5 mutations were more frequent in women (70% vs 24% in men). Conclusion: Comprehensive NGS of CYP11B2-expressing adrenal tumors identified somatic mutations in aldosterone-driving genes in 88% of APAs, a higher rate than in previous studies using conventional approaches.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Mutação , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/cirurgia , Adrenalectomia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Adenoma Adrenocortical/cirurgia , Adulto , Canais de Cálcio Tipo L/genética , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPase Trocadora de Sódio-Potássio/genética
10.
J Clin Endocrinol Metab ; 103(9): 3477-3485, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30020487

RESUMO

Context: Aldosterone biosynthesis is regulated principally by ACTH and gene mutations as well as by angiotensin II and serum potassium. In addition, previous studies have reported the potential effects of KCNJ5 mutations in aldosterone-producing adenoma (APA) on cardiovascular diseases. However, responsiveness to ACTH in APAs according to potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5) mutations remains unknown. Objective: To investigate KCNJ5 genotype-specific differences in aldosterone biosynthesis in response to ACTH stimulation. Design and Setting: A cross-sectional study through retrieval of clinical records. Participants: One hundred forty-one patients aged ≥20 years with APA were examined. Main Outcome Measures: Associations between KCNJ5 mutations and clinical parameters reflecting the renin-angiotensin system [saline infusion test (SIT)] and ACTH pathways [dexamethasone suppression test (DST)]. Results: KCNJ5 mutations were detected in 107 cases. In the crude comparison, patients with mutations in KCNJ5 had higher plasma aldosterone concentrations (PACs) both at baseline and after the SIT. PAC after the DST showed a significant inverse association with KCNJ5 genotypes after controlling for age, sex, tumor size, and PAC after the SIT. Immunohistochemical analysis of 101 cases revealed more abundant immunoreactivity of CYP11B1 and CYP17 in the KCNJ5-mutated group than in the KCNJ5 wild-type group. Conclusion: This report of marked suppression of PAC by dexamethasone in patients with KCNJ5-mutated APAs indicates that such APAs respond to endogenous ACTH more readily than APAs in nonmutated cases. Further molecular and epidemiologic studies are required to validate our results and clarify the clinical effectiveness of the DST for predicting KCNJ5 mutations before adrenalectomy.


Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Adenoma Adrenocortical/tratamento farmacológico , Aldosterona/biossíntese , Antineoplásicos Hormonais/administração & dosagem , Dexametasona/administração & dosagem , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Hormônio Adrenocorticotrópico/sangue , Adulto , Aldosterona/sangue , Estudos Transversais , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Sistema Renina-Angiotensina/efeitos dos fármacos , Resultado do Tratamento
11.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 49(3): 469-473, 2018 May.
Artigo em Chinês | MEDLINE | ID: mdl-30014653

RESUMO

OBJECTIVE: To assess the efficacy of screening and confirmatory tests of primary aldosteronism (PA) in diagnosing aldosterone producing adenoma (APA). METHODS: Clinical data of 167 hypertensive patients were retrospectively reviewed,including 93 patients with APA and 74 patients with essential hypertension (EH). The area under curves (AUC) of receiver operating characteristic (ROC) curves were compared among the five indicators: supine plasma aldosterone concentration (PAC),absolute PAC values and PAC drop rates post saline infusion test (SIT) and captopril challenge test (CCT). RESULTS: APA patients had higher supine PAC,higher percentage of third degree hypertension,and lower serum potassium level than EH patients (P<0.05). Compared with EH patients,APA patients had lower PAC change rates,post posture change and SIT (P<0.05),but similar post CCT (P>0.05). The AUC of supine PAC reached 0.975. Higher AUC was found in absolute PAC values post SIT compared with PAC droop rates (0.984 vs. 0.680,P<0.001). Similar results were also found with CCT (0.949 vs. 0.538,P<0.001). A cut-off of supine aldosterone renin ratio (ARR) >30 and supine PAC>17.8 ng/dL had 96.8% sensitivity and 90.5% specificity. A cut-off of 14.59 ng/dL PAC post SIT had 90.2% sensitivity and 97.3% specificity. A cut-off of 19.11 ng/dL PAC post CCT had 88.8% sensitivity and 95.9% specificity. CONCLUSION: Screening tests using supine ARR>30 plus supine PAC>17.83 ng/dL are preferred with high sensitivity and specificity. The absolute values of PAC post SIT and CCT are recommended for confirming APA.


Assuntos
Adenoma/metabolismo , Aldosterona/biossíntese , Hiperaldosteronismo/diagnóstico , Humanos , Hipertensão/complicações , Renina , Estudos Retrospectivos
12.
Mol Cell Endocrinol ; 478: 1-9, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29959979

RESUMO

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.


Assuntos
Córtex Suprarrenal/citologia , Aldosterona/biossíntese , Sinalização do Cálcio , Receptor Muscarínico M3/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Fluorescência , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/genética
13.
Int J Mol Sci ; 19(6)2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29874863

RESUMO

Mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) display a phenotype of hyperaldosteronism, due to the upregulation of type VI 3ß-hydroxyl-steroid dehydrogenase (Hsd3b6), the murine counterpart to the human type I 3ß-hydroxyl-steroid dehydrogenase (HSD3B1) gene. In the present study, we evaluated the role of CRY1 and CRY2 genes, and their potential interplay with HSD3B isoforms in adrenal pathophysiology in man. Forty-six sporadic aldosterone-producing adenomas (APAs) and 20 paired adrenal samples were included, with the human adrenocortical cells HAC15 used as the in vitro model. In our cohort of sporadic APAs, CRY1 expression was 1.7-fold [0.75⁻2.26] higher (p = 0.016), while CRY2 showed a 20% lower expression [0.80, 0.52⁻1.08] (p = 0.04) in APAs when compared with the corresponding adjacent adrenal cortex. Type II 3ß-hydroxyl-steroid dehydrogenase (HSD3B2) was 317-fold [200⁻573] more expressed than HSD3B1, and is the main HSD3B isoform in APAs. Both dehydrogenases were more expressed in APAs when compared with the adjacent cortex (5.7-fold and 3.5-fold, respectively, p < 0.001 and p = 0.001) and HSD3B1 was significantly more expressed in APAs composed mainly of zona glomerulosa-like cells. Treatment with angiotensin II (AngII) resulted in a significant upregulation of CRY1 (1.7 ± 0.25-fold, p < 0.001) at 6 h, and downregulation of CRY2 at 12 h (0.6 ± 0.1-fold, p < 0.001), through activation of the AngII type 1 receptor. Independent silencing of CRY1 and CRY2 genes in HAC15 cells resulted in a mild upregulation of HSD3B2 without affecting HSD3B1 expression. In conclusion, our results support the hypothesis that CRY1 and CRY2, being AngII-regulated genes, and showing a differential expression in APAs when compared with the adjacent adrenal cortex, might be involved in adrenal cell function, and in the regulation of aldosterone production.


Assuntos
Adenoma/genética , Criptocromos/genética , Hipertensão/genética , Complexos Multienzimáticos/genética , Progesterona Redutase/genética , Esteroide Isomerases/genética , Adenoma/metabolismo , Adenoma/patologia , Aldosterona/biossíntese , Angiotensina II/genética , Animais , Linhagem Celular Tumoral , Criptocromos/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Hipertensão/patologia , Camundongos
14.
J Am Heart Assoc ; 7(10)2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739797

RESUMO

BACKGROUND: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We demonstrate that stimulatory signals can change the DNA methylation status around transcription factor binding sites and a transcription start site and activate expression of the aldosterone synthase gene (CYP11B2). METHODS AND RESULTS: DNA methylation of CYP11B2 was analyzed in aldosterone-producing adenomas, nonfunctioning adrenal adenomas, and adrenal glands and compared with the gene expression levels. CpG dinucleotides in the CYP11B2 promoter were found to be hypormethylated in tissues with high expression, but not in those with low expression, of CYP11B2. Methylation of the CYP11B2 promoter fused to a reporter gene decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminished their DNA-binding activity. A methylated-CpG-binding protein MECP2 interacted directly with the methylated CYP11B2 promoter. In rats, low salt intake led to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with angiotensin II type 1 receptor antagonist decreased CYP11B2 expression and led to DNA hypermethylation. CONCLUSIONS: DNA demethylation may switch the phenotype of CYP11B2 expression from an inactive to an active state and regulate aldosterone biosynthesis.


Assuntos
Glândulas Suprarrenais/enzimologia , Aldosterona/biossíntese , Angiotensina II/metabolismo , Citocromo P-450 CYP11B2/genética , Metilação de DNA , Epigênese Genética , Sódio/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ilhas de CpG , Citocromo P-450 CYP11B2/metabolismo , Metilação de DNA/efeitos dos fármacos , Dieta Hipossódica , Epigênese Genética/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Regiões Promotoras Genéticas , Ratos Wistar , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
15.
Int J Mol Sci ; 19(5)2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29738496

RESUMO

Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied. However, coregulators supporting the Nurr1 function still remain elusive. In this study, we performed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), a recently developed endogenous coregulator purification method, in human adrenocortical H295R cells and identified PARP1 as one of the top Nurr1-interacting proteins. Nurr1-PARP1 interaction was verified by co-immunoprecipitation. In addition, both siRNA knockdown of PARP1 and treatment of AG14361, a specific PARP1 inhibitor suppressed the angiotensin II-mediated target gene induction in H295R cells. Furthermore, PARP1 inhibitor also suppressed the aldosterone secretion in response to the angiotensin II. Together, these results suggest PARP1 is a prime coregulator for Nurr1.


Assuntos
Aldosterona/biossíntese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Poli(ADP-Ribose) Polimerase-1/genética , Mapas de Interação de Proteínas/genética , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Aldosterona/genética , Aldosterona/metabolismo , Angiotensina II/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Espectrometria de Massas , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Interferente Pequeno/genética , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo
16.
Sci Rep ; 8(1): 5025, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29567944

RESUMO

Development of metabolic syndrome is associated with hyperactivity of the HPA axis characterized by elevated levels of circulating adrenal hormones including cortisol and aldosterone. However, the molecular mechanism leading to the dysregulation of the HPA axis is not well elucidated. In this study, we found that insulin regulates adrenal steroidogenesis by increasing the expression and activity of steroidogenic factor 1 (SF-1) both in vitro and in vivo and this insulin effect was partly through inhibition of FoxO1. Specifically, insulin increased the protein and RNA levels of SF-1 and steroidogenic target genes. Further, adrenal SF-1 expression was significantly increased by hyperactivation of insulin signaling in mice. Together with the elevated SF-1 expression in adrenal glands, hyperactivation of insulin signaling led to increased aldosterone and corticosterone levels. On the other hand, suppressing the insulin signaling using streptozotocin markedly reduced the expression of adrenal SF-1 in mice. In addition, overexpression of FoxO1 significantly suppressed SF-1 and its steroidogenic target genes implying that the positive effect of insulin on SF-1 activity might be through suppression of FoxO1 in the adrenal gland. Taken together, these results indicate that insulin regulates adrenal steroidogenesis through coordinated control of SF-1 and FoxO1.


Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/biossíntese , Corticosterona/biossíntese , Diabetes Mellitus Experimental/metabolismo , Proteína Forkhead Box O1/metabolismo , Insulina/metabolismo , Fator Esteroidogênico 1/metabolismo , Córtex Suprarrenal/citologia , Aldosterona/sangue , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Corticosterona/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Dieta Hiperlipídica/efeitos adversos , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologia , Sistema Hipófise-Suprarrenal/fisiologia , RNA Interferente Pequeno/metabolismo , Fator Esteroidogênico 1/genética , Estreptozocina/toxicidade
17.
Curr Opin Endocrinol Diabetes Obes ; 25(3): 147-154, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29432258

RESUMO

PURPOSE OF REVIEW: Primary aldosteronism is the most common form of secondary hypertension. Early diagnosis and treatment are key to cure of hypertension and prevention of cardiovascular complications. Recent genetic discoveries have improved our understanding on the pathophysiology of aldosterone production and triggered the development of new diagnostic procedures and targeted treatments for primary aldosteronism. RECENT FINDINGS: Different inherited genetic abnormalities distinguish specific forms of familial hyperaldosteronism. Somatic mutations are found not only in aldosterone-producing adenoma (APA), leading to primary aldosteronism, but also in aldosterone producing cell clusters of normal and micronodules from image-negative adrenal glands. Genetic knowledge has allowed the discovery of surrogate biomarkers and specific pharmacological inhibitors. Ageing appears to be associated with dysregulated and relatively autonomous aldosterone production. SUMMARY: New biochemical markers and pharmacological approaches may allow preoperative identification of somatic mutation carriers and use of targeted treatments.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Aldosterona/genética , Hiperaldosteronismo/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/metabolismo , Fatores Etários , Envelhecimento , Aldosterona/fisiologia , Citocromo P-450 CYP11B2/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/terapia , Hipertensão/etiologia , Masculino , Mutação , Fatores Sexuais , Síndrome
18.
Endocrinology ; 159(3): 1352-1359, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29293917

RESUMO

Because blunted expression of the twik-related acid-sensitive K+ channel 2 (TASK-2) is a common feature of aldosterone-producing adenoma (APA) causing primary aldosteronism (PA), we sequenced the promoter region of the TASK-2 gene (KCNK5) in APAs (n = 76), primary hypertensive patients (n = 98), and 20-year-old healthy volunteers (n = 71), searching for variants that could affect expression of this channel. We found TASK-2 promoter mutations in 25% of the APAs: C999T in 6.6%, G595A in 5.3%, G36A in 5.3%, and C562T, Gins468, G265C, C1247T, G1140T, and C1399T in 1.3% each. The C999T mutation was found in only one of the 98 primary hypertensive patients, but mutations were detected also in 12% of volunteers: 4 carried the C999T, 3 G1288C, 1 the G1140T mutation, and 1 the 468ins mutation. After a 16-year follow-up, none of these patients developed hypertension or PA. The effect of C999T mutation was investigated in H295R cells using reporter vectors with the mutated or the wild-type (WT) TASK-2 promoters. TASK-2 gene expression was decreased by 31% ± 18% (P = 0.01) in mutated compared with WT APA. Likewise, in transfected H295R cells, the C999T mutation decreased TASK-2 transcriptional activity by 35% (normalized luciferase signal fold change: 0.65 ± 0.25, P < 0.001). Thus, mutations in the promoter region of the TASK-2 gene can account for the low expression in ∼25% of APAs. As they did not result in hypertension or PA during long-term follow-up in healthy participants, these mutations do not seem to be a factor in causing PA by themselves.


Assuntos
Hiperaldosteronismo/genética , Mutação , Canais de Potássio de Domínios Poros em Tandem/genética , Adenoma Adrenocortical/metabolismo , Adulto , Aldosterona/biossíntese , Sítios de Ligação , Feminino , Mutação em Linhagem Germinativa , Humanos , Hipertensão/genética , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo
19.
J Clin Endocrinol Metab ; 103(3): 965-971, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29294065

RESUMO

Context: Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation. Objective: Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels. Methods: Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells. Results: The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells. Conclusions: We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Metilação de DNA/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Adulto , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais Cultivadas
20.
Eur J Endocrinol ; 178(3): R101-R111, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29348113

RESUMO

Aldosterone is the main mineralocorticoid hormone in humans and plays a key role in maintaining water and electrolyte homeostasis. Primary aldosteronism (PA), characterized by autonomous aldosterone overproduction by the adrenal glands, affects 6% of the general hypertensive population and can be either sporadic or familial. Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH) are the two most frequent subtypes of sporadic PA and 4 forms of familial hyperaldosteronism (FH-I to FH-IV) have been identified. Over the last six years, the introduction of next-generation sequencing has significantly improved our understanding of the molecular mechanisms responsible for autonomous aldosterone overproduction in both sporadic and familial PA. Somatic mutations in four genes (KCNJ5, ATP1A1, ATP2B3 and CACNA1D), differently implicated in intracellular ion homeostasis, have been identified in nearly 60% of the sporadic APAs. Germline mutations in KCNJ5 and CACNA1H cause FH-III and FH-IV, respectively, while germline mutations in CACNA1D cause the rare PASNA syndrome, featuring primary aldosteronism seizures and neurological abnormalities. Further studies are warranted to identify the molecular mechanisms underlying BAH and FH-II, the most common forms of sporadic and familial PA whose molecular basis is yet to be uncovered.


Assuntos
Canais de Cálcio Tipo L/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPase Trocadora de Sódio-Potássio/genética , Aldosterona/biossíntese , Variação Genética , Humanos
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