RESUMO
Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/ß-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike ß-catenin gain-of-function models, which induce high Wnt/ß-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/ß-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing ß-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/ß-catenin activation, which is regulated by ZNRF3.
Assuntos
Córtex Suprarrenal/metabolismo , Homeostase/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/crescimento & desenvolvimento , Doenças do Córtex Suprarrenal/fisiopatologia , Animais , Proliferação de Células/genética , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Modelos Animais , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.
Assuntos
Diafragma/metabolismo , Macrófagos/metabolismo , Doenças Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculo Quadríceps/metabolismo , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animais , Compostos de Bário , Cloretos , Citocinas/genética , Citocinas/metabolismo , Diafragma/imunologia , Diafragma/patologia , Modelos Animais de Doenças , Feminino , Fibrose , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos mdx , Camundongos Knockout , Doenças Musculares/induzido quimicamente , Doenças Musculares/imunologia , Doenças Musculares/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/patologia , Músculo Quadríceps/imunologia , Músculo Quadríceps/patologia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Mineralocorticoides/genética , Transdução de SinaisRESUMO
Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic ß-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.
RESUMO
The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.
Assuntos
Adenoma/patologia , Aldosterona/metabolismo , Proliferação de Células , ATPase Trocadora de Sódio-Potássio/genética , Adenoma/metabolismo , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Mutação , Ouabaína/farmacologia , Fosforilação/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcriptoma , Quinases da Família src/metabolismoRESUMO
The CYP11B2 enzyme is the terminal enzyme in the biosynthesis of aldosterone. Immunohistochemistry using antibodies against CYP11B2 defines cells of the adrenal ZG that synthesize aldosterone. CYP11B2 expression is normally stimulated by angiotensin II, but becomes autonomous in primary hyperaldosteronism, in most cases driven by recently discovered somatic mutations of ion channels or pumps. Cells expressing CYP11B2 in young normal humans form a continuous band beneath the adrenal capsule; in older individuals they form discrete clusters, aldosterone-producing cell clusters (APCC), surrounded by non-aldosterone producing cells in the outer layer of the adrenal gland. Aldosterone-producing adenomas may exhibit a uniform or heterogeneous expression of CYP11B2. APCC frequently persist in the adrenal with an aldosterone-producing adenoma suggesting autonomous CYP11B2 expression in these cells as well. This was confirmed by finding known mutations that drive aldosterone production in adenomas in the APCC of clinically normal people. Unilateral aldosteronism may also be due to multiple CYP11B2-expressing nodules of various sizes or a continuous band of hyperplastic ZG cells expressing CYP11B2. Use of CYP11B2 antibodies to identify areas for sequencing has greatly facilitated the detection of aldosterone-driving mutations.
Assuntos
Glândulas Suprarrenais/metabolismo , Citocromo P-450 CYP11B2/metabolismo , Hiperaldosteronismo/metabolismo , Imuno-Histoquímica/métodos , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Glândulas Suprarrenais/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Aldosterona/metabolismo , Humanos , Hiperaldosteronismo/patologiaRESUMO
Primary aldosteronism is the most common form of secondary hypertension with a prevalence of 5-10% in hypertensive patients. Aldosterone-producing adenoma (APA) is a subtype of primary aldosteronism, and somatic mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, CLCN2, or CTNNB1 were identified and recognized to drive aldosterone production and/or contribute to tumorigenesis in APA. Mutations of KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CLCN2 are known to activate calcium signaling, and its activation potentiate CYP11B2 (aldosterone synthesis) transcription in adrenal cells. Transcriptome analyses combined with bioinformatics using APA samples were conductive for each gene mutation mediated pivotal pathway, gene ontology, and clustering. Several important intracellular molecules in increase aldosterone production were detected by transcriptome analysis, and additional functional analyses demonstrated intracellular molecular mechanisms of aldosterone production which focused on calcium signal, CYP11B2 transcription and translation. Furthermore, DNA methylation analysis revealed that promoter region of CYP11B2 was entirely hypomethylated, but that of other steroidogenic enzymes were not in APA. Integration of transcriptome and DNA methylome analysis clarified some DNA methylation associated gene expression, and the transcripts have a role for aldosterone production. In this article, we reviewed the intracellular molecular mechanisms of aldosterone production in APA, and discussed future challenges for basic studies leading to clinical practice.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , Hiperaldosteronismo/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Canais de Cloro CLC-2 , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Canais de Cloreto/genética , Citocromo P-450 CYP11B2/genética , Metilação de DNA/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Perfilação da Expressão Gênica , Humanos , Hiperaldosteronismo/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Regiões Promotoras Genéticas , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Glucocorticoids (GCs) are secreted into the blood by the adrenal glands and are also locally-produced by organs such as the lymphoid organs (bone marrow, thymus, and spleen). Corticosterone is the primary circulating GC in many species, including mice, rats and birds. Within lymphoid organs, corticosterone can be locally produced from the inactive metabolite, 11-dehydrocorticosterone (DHC). However, very little is known about endogenous DHC levels, and no immunoassays are currently available to measure DHC. Here, we developed an easy-to-use and inexpensive immunoassay to measure DHC that is accurate, precise, sensitive, and specific. The DHC immunoassay was validated in multiple ways, including comparison with a mass spectrometry assay. After assay validations, we demonstrated the usefulness of this immunoassay by measuring DHC (and corticosterone) in mice, rats and song sparrows. Overall, corticosterone levels were higher than DHC levels across species. In Study 1, using mice, we measured steroids in whole blood and lymphoid organs at postnatal day (PND) 5, PND23, and PND90. Corticosterone and DHC showed distinct tissue-specific patterns across development. In Studies 2 and 3, we measured circulating corticosterone and DHC in adult rats and song sparrows, before and after restraint stress. In rats and song sparrows, restraint stress rapidly increased circulating levels of both steroids. This novel DHC immunoassay revealed major changes in DHC concentrations during development and in response to stress, which have important implications for understanding GC physiology, effects of stress on immune function, and regulation of local GC levels.
Assuntos
Envelhecimento/metabolismo , Corticosterona/análogos & derivados , Caracteres Sexuais , Aves Canoras/sangue , Estresse Fisiológico , Animais , Anticorpos/metabolismo , Corticosterona/sangue , Corticosterona/química , Reações Cruzadas , Feminino , Glucocorticoides/química , Glucocorticoides/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ratos Long-Evans , Padrões de ReferênciaRESUMO
FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11ß-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Aldosterona/metabolismo , Animais , Citocromo P-450 CYP11B2/biossíntese , Citocromo P-450 CYP11B2/genética , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Camundongos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologiaRESUMO
Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.
Assuntos
Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Mutação , Córtex Suprarrenal/metabolismo , Citocromo P-450 CYP11B2/metabolismo , DNA/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Humanos , Hiperaldosteronismo/etiologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Análise de Sequência de DNA , Análise de Sequência de RNA , Transcriptoma , Zona GlomerulosaRESUMO
Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.
Assuntos
Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Mineralocorticoides/agonistas , Adolescente , Adulto , Aldosterona/farmacologia , Western Blotting , Células Cultivadas , Eplerenona , Humanos , Masculino , Distrofia Muscular de Duchenne , Prednisolona/farmacologia , Espironolactona/análogos & derivados , Espironolactona/farmacologia , Adulto JovemRESUMO
Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations.
Assuntos
Glândulas Suprarrenais/metabolismo , Citocromo P-450 CYP11B2/genética , Hiperaldosteronismo/genética , Adenoma/genética , Adenoma/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/patologia , Aldosterona/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Hiperaldosteronismo/metabolismo , Hiperaldosteronismo/patologia , Hiperplasia/genética , Hiperplasia/metabolismoRESUMO
BACKGROUND: In obesity, the excessive synthesis of aldosterone contributes to the development and progression of metabolic and cardiovascular dysfunctions. Obesity-induced hyperaldosteronism is independent of the known regulators of aldosterone secretion, but reliant on unidentified adipocyte-derived factors. We hypothesized that the adipokine leptin is a direct regulator of aldosterone synthase (CYP11B2) expression and aldosterone release and promotes cardiovascular dysfunction via aldosterone-dependent mechanisms. METHODS AND RESULTS: Immunostaining of human adrenal cross-sections and adrenocortical cells revealed that adrenocortical cells coexpress CYP11B2 and leptin receptors. Measurements of adrenal CYP11B2 expression and plasma aldosterone levels showed that increases in endogenous (obesity) or exogenous (infusion) leptin dose-dependently raised CYP11B2 expression and aldosterone without elevating plasma angiotensin II, potassium or corticosterone. Neither angiotensin II receptors blockade nor α and ß adrenergic receptors inhibition blunted leptin-induced aldosterone secretion. Identical results were obtained in cultured adrenocortical cells. Enhanced leptin signaling elevated CYP11B2 expression and plasma aldosterone, whereas deficiency in leptin or leptin receptors blunted obesity-induced increases in CYP11B2 and aldosterone, ruling out a role for obesity per se. Leptin increased intracellular calcium, elevated calmodulin and calmodulin-kinase II expression, whereas calcium chelation blunted leptin-mediated increases in CYP11B2, in adrenocortical cells. Mineralocorticoid receptor blockade blunted leptin-induced endothelial dysfunction and increases in cardiac fibrotic markers. CONCLUSIONS: Leptin is a newly described regulator of aldosterone synthesis that acts directly on adrenal glomerulosa cells to increase CYP11B2 expression and enhance aldosterone production via calcium-dependent mechanisms. Furthermore, leptin-mediated aldosterone secretion contributes to cardiovascular disease by promoting endothelial dysfunction and the expression of profibrotic markers in the heart.
Assuntos
Adipócitos/metabolismo , Aldosterona/metabolismo , Endotélio Vascular/metabolismo , Cardiopatias/metabolismo , Cardiopatias/patologia , Leptina/fisiologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibrose/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Ratos , Ratos Endogâmicos WKY , Ratos ZuckerRESUMO
Early treatment with heart failure drugs lisinopril and spironolactone improves skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. The angiotensin converting enzyme inhibitor lisinopril and mineralocorticoid receptor (MR) antagonist spironolactone indirectly and directly target MR. The presence and function of MR in skeletal muscle have not been explored. MR mRNA and protein are present in all tested skeletal muscles from both wild-type mice and DMD mouse models. MR expression is cell autonomous in both undifferentiated myoblasts and differentiated myotubes from mouse and human skeletal muscle cultures. To test for MR function in skeletal muscle, global gene expression analysis was conducted on human myotubes treated with MR agonist (aldosterone; EC50 1.3 nM) or antagonist (spironolactone; IC50 1.6 nM), and 53 gene expression differences were identified. Five differences were conserved in quadriceps muscles from dystrophic mice treated with spironolactone plus lisinopril (IC50 0.1 nM) compared with untreated controls. Genes down-regulated more than 2-fold by MR antagonism included FOS, ANKRD1, and GADD45B, with known roles in skeletal muscle, in addition to NPR3 and SERPINA3, bona fide targets of MR in other tissues. MR is a novel drug target in skeletal muscle and use of clinically safe antagonists may be beneficial for muscle diseases.
Assuntos
Aldosterona/farmacologia , Lisinopril/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares , Receptores de Melanocortina , Espironolactona/farmacologia , Animais , Linhagem Celular , Humanos , Camundongos , Proteínas Musculares/agonistas , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/metabolismo , Doenças Musculares/tratamento farmacológico , Doenças Musculares/metabolismo , Receptores de Melanocortina/agonistas , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). METHODS: We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10(-7)M aldosterone for 1h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10(-7)M aldosterone for 3h by microarray. RESULTS: 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10(-9)M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. CONCLUSIONS: We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.
Assuntos
Genoma/genética , Túbulos Renais Distais/metabolismo , Receptores de Mineralocorticoides/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Aldosterona/farmacologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Imediatamente Precoces/genética , Túbulos Renais Distais/citologia , Túbulos Renais Distais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Análise de Sequência com Séries de Oligonucleotídeos , Podócitos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Mineralocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Ligação a Tacrolimo/genética , Tensinas , Fatores de Transcrição/genéticaRESUMO
Activation of mineralocorticoid receptors (MR) of the hypothalamic paraventricular nucleus (PVN) increases sympathetic excitation. To determine whether MR and glucocorticoid receptors (GR) are expressed in preautonomic neurons of the PVN and how they relate to endogenous aldosterone levels in healthy rats, retrograde tracer was injected into the intermediolateral cell column at T4 to identify preautonomic neurons in the PVN. Expression of MR, GR, 11-ß hydroxysteroid dehydrogenase1 and 2 (11ß-HSD1, 2), and hexose-6-phosphate dehydrogenase (H6PD) required for 11ß-HSD1 reductase activity was assessed by immunohistochemistry. RT-PCR and Western blot analysis were used to determine MR gene and protein expression. Most preautonomic neurons were in the caudal mediocellular region of PVN, and most expressed MR; none expressed GR. 11ß-HSD1, but not 11ß-HSD2 nor H6PD immunoreactivity, was detected in the PVN. In rats with chronic low or high sodium intakes, the low-sodium diet was associated with significantly higher plasma aldosterone, MR mRNA and protein expression, and c-Fos immunoreactivity within labeled preautonomic neurons. Plasma corticosterone and sodium and expression of tonicity-responsive enhancer binding protein in the PVN did not differ between groups, suggesting osmotic adaptation to the altered sodium intake. These results suggest that MR within preautonomic neurons in the PVN directly participate in the regulation of sympathetic nervous system drive, and aldosterone may be a relevant ligand for MR in preautonomic neurons of the PVN under physiological conditions. Dehydrogenase activity of 11ß-HSD1 occurs in the absence of H6PD, which regenerates NADP(+) from NADPH and may increase MR gene expression under physiological conditions.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Adaptação Fisiológica , Ração Animal , Animais , Dieta , Regulação da Expressão Gênica/fisiologia , Genes fos/genética , Genes fos/fisiologia , Imuno-Histoquímica/métodos , Neurônios/classificação , Ratos , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We describe herein the European Reference Network on Rare Endocrine Conditions clinical practice guideline on diagnosis and management of familial forms of hyperaldosteronism. The guideline panel consisted of 10 experts in primary aldosteronism, endocrine hypertension, paediatric endocrinology, and cardiology as well as a methodologist. A systematic literature search was conducted, and because of the rarity of the condition, most recommendations were based on expert opinion and small patient series. The guideline includes a brief description of the genetics and molecular pathophysiology associated with each condition, the patients to be screened, and how to screen. Diagnostic and treatment approaches for patients with genetically determined diagnosis are presented. The recommendations apply to patients with genetically proven familial hyperaldosteronism and not to families with more than one case of primary aldosteronism without demonstration of a responsible pathogenic variant.
Assuntos
Hiperaldosteronismo , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Hiperaldosteronismo/terapia , Humanos , Europa (Continente) , Doenças Raras/diagnóstico , Doenças Raras/genética , Doenças Raras/terapiaRESUMO
BACKGROUND: Myosin phosphatase targeting subunit 2 (MYPT2) is an important subunit of cardiac MLC (myosin light chain) phosphatase, which plays a crucial role in regulating the phosphorylation of MLC to phospho-MLC (p-MLC). A recent study demonstrated mineralocorticoid receptor-related hypertension is associated with RhoA/Rho-associated kinase/MYPT1 signaling upregulation in smooth muscle cells. Our purpose is to investigate the effect of MYPT2 on cardiac function and fibrosis in mineralocorticoid receptor-related hypertension. METHODS AND RESULTS: HL-1 murine cardiomyocytes were incubated with different concentrations or durations of aldosterone. After 24-hour stimulation, aldosterone increased CTGF (connective tissue growth factor) and MYPT2 and decreased p-MLC in a dose-dependent manner. MYPT2 knockdown decreased CTGF. Cardiac-specific MYPT2-knockout (c-MYPT2-/-) mice exhibited decreased type 1 phosphatase catalytic subunit ß and increased p-MLC. A disease model of mouse was induced by subcutaneous aldosterone and 8% NaCl food for 4 weeks after uninephrectomy. Blood pressure elevation and left ventricular hypertrophy were observed in both c-MYPT2-/- and MYPT2+/+ mice, with no difference in heart weights or nuclear localization of mineralocorticoid receptor in cardiomyocytes. However, c-MYPT2-/- mice had higher ejection fraction and fractional shortening on echocardiography after aldosterone treatment. Histopathology revealed less fibrosis, reduced CTGF, and increased p-MLC in c-MYPT2-/- mice. Basal global radial strain and global longitudinal strain were higher in c-MYPT2-/- than in MYPT2+/+ mice. After aldosterone treatment, both global radial strain and global longitudinal strain remained higher in c-MYPT2-/- mice compared with MYPT2+/+ mice. CONCLUSIONS: Cardiac-specific MYPT2 knockout leads to decreased myosin light chain phosphatase and increased p-MLC. MYPT2 deletion prevented cardiac fibrosis and dysfunction in a model of mineralocorticoid receptor-associated hypertension.
Assuntos
Hipertensão , Fosfatase de Miosina-de-Cadeia-Leve , Animais , Camundongos , Aldosterona/farmacologia , Aldosterona/metabolismo , Fibrose , Hipertensão/genética , Hipertensão/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismoRESUMO
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.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Aldosterona , Antagonistas de Receptores de Mineralocorticoides , Receptores de Mineralocorticoides , Receptores de Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/agonistas , Humanos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , Aldosterona/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Animais , Hidrocortisona/metabolismo , Hidrocortisona/farmacologia , Linhagem CelularRESUMO
The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.
Assuntos
Aldosterona , Receptores de Mineralocorticoides , Animais , Camundongos , Ratos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Ligantes , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Proteína Regulatória Associada a mTOR , RNA Guia de Sistemas CRISPR-Cas , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hyperaldosteronism is often associated with inappropriate aldosterone production and aldosterone synthase (Cyp11b2) expression. Normally, Cyp11b2 expression is limited to the adrenal zona glomerulosa (ZG) and regulated by angiotensin II which signals through Gq protein-coupled receptors. As cells migrate inwards, they differentiate into 11ß-hydroxylase-expressing zona fasciculata (ZF) cells lacking Cyp11b2. The mechanism causing ZG-specific aldosterone biosynthesis is still unclear. We investigated the effect of chronic Gq signaling using transgenic mice with a clozapine N-oxide (CNO)-activated human M3 muscarinic receptor (DREADD) coupled to Gq (hM3Dq) that was expressed throughout the adrenal cortex. CNO raised circulating aldosterone in the presence of a high sodium diet with greater response seen in females compared to males. Immunohistochemistry and transcriptomics indicated disrupted zonal Cyp11b2 expression while Wnt signaling remained unchanged. Chronic Gq-DREADD signaling also induced an intra-adrenal RAAS in CNO-treated mice. Chronic Gq signaling disrupted adrenal cortex zonal aldosterone production associated with ZF expression of Cyp11b2.