RESUMO
BACKGROUND: Understanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment. METHODS: We used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options. RESULTS: Our human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor. CONCLUSIONS: These studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/genética , Proteínas de Homeodomínio/genética , Peptídeos/farmacologia , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Animais , Proliferação de Células/genética , Feminino , Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular , Peptídeos/genéticaRESUMO
Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.
Assuntos
Córtex Suprarrenal/enzimologia , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Transdução de Sinais , Córtex Suprarrenal/metabolismo , Animais , Diferenciação Celular , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esteroides/metabolismo , Zona Fasciculada/citologia , Zona Fasciculada/enzimologia , Zona Fasciculada/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/enzimologia , Zona Glomerulosa/metabolismoRESUMO
SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Processamento de Proteína Pós-Traducional/fisiologia , Sumoilação/fisiologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/ultraestrutura , Neoplasias do Córtex Suprarrenal/patologia , Hormônio Adrenocorticotrópico/administração & dosagem , Animais , Complexo de Carney/metabolismo , Linhagem Celular Tumoral , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Preparações de Ação Retardada , Dexametasona/análogos & derivados , Dexametasona/farmacologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Fator de Transcrição E2F1/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Biologia Computacional , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Humanos , Indóis/farmacologia , Camundongos Knockout , Análise Multivariada , Modelos de Riscos Proporcionais , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Securina/genéticaRESUMO
Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Bases de Dados de Ácidos Nucleicos , Progressão da Doença , Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Transgênicos , Interferência de RNA , Fatores de Risco , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.
Assuntos
Doenças do Córtex Suprarrenal/metabolismo , Doenças do Córtex Suprarrenal/patologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteína de Morte Celular Associada a bcl/metabolismo , Doenças do Córtex Suprarrenal/genética , Hormônio Adrenocorticotrópico/administração & dosagem , Hormônio Adrenocorticotrópico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/ß-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that ß-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/ß-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/biossíntese , Hiperaldosteronismo/metabolismo , Via de Sinalização Wnt , Neoplasias do Córtex Suprarrenal/complicações , Adenoma Adrenocortical/complicações , Adulto , Aldosterona/sangue , Aldosterona/metabolismo , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperaldosteronismo/etiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismoRESUMO
Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing's syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 alpha-regulatory subunit (R1alpha) of the cAMP-dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing's syndrome. To demonstrate the implication of R1alpha loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing's syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1alpha loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1alpha is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD.
Assuntos
Córtex Suprarrenal/metabolismo , Síndrome de Cushing/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Proliferação de Células , Síndrome de Cushing/embriologia , Síndrome de Cushing/genética , Síndrome de Cushing/patologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Esteroide 17-alfa-Hidroxilase/metabolismoRESUMO
In the adrenal gland, adrenocorticotropin (ACTH) acting through the cAMP protein kinase (PKA) transduction pathway is the main regulator of genes involved in glucocorticoid synthesis. The prolactin (PRL) receptor is expressed in the adrenal cortex of most mammals, but experimental proof that PRL ensures direct control on glucocorticoid synthesis in rodents remains elusive. To unravel the physiological importance of PRL in adrenocortical functions, we measured steroidogenic capacity of Prlr-deficient mice (Prlr(-/-)) and explored the influence of JAK/STAT signaling, the major PRL transduction pathway, on the steroidogenic activity of adrenocortical cell cultures. We demonstrate that lack of Prlr does not affect basal (nor stress-induced) corticosterone levels in mice. PRL triggers JAK2/STAT5-dependent transcription in adrenal cells, but this does not influence corticosterone release. In contrast, pharmacological or siRNA-mediated inhibition of JAK2 reveals its essential role in both basal and ACTH/cAMP-induced steroidogenesis. We demonstrate that nuclear JAK2 regulates the amount of active transcription factor CREB (cAMP response element-binding protein) through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional activation of the rate-limiting steroidogenic Star gene. Hence, we describe a novel link between PKA and JAK2 by which nuclear JAK2 signaling controls adrenal steroidogenesis by increasing the stability of CREB.
Assuntos
Glândulas Suprarrenais/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Janus Quinase 2/metabolismo , Esteroides/biossíntese , Transcrição Gênica , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Corticosterona/biossíntese , AMP Cíclico/farmacologia , Humanos , Masculino , Camundongos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Prolactina/farmacologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ratos , Ovinos , Transcrição Gênica/efeitos dos fármacosRESUMO
Adrenocortical carcinoma is a rare but aggressive cancer with unknown aetiology. Constitutive activation of beta-catenin is the most frequent alteration in benign and malignant adrenocortical tumours in patients. Here, we show that constitutive activation of beta-catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated spindle-shaped cells hyperplasia as well as dysplasia of the cortex and medulla. Over a 17 months time course, transgenic adrenals developed malignant characteristics such as uncontrolled neovascularization and loco-regional metastatic invasion. These oncogenic events were accompanied by ectopic differentiation of glomerulosa at the expense of fasciculata cells, which caused primary hyperaldosteronism. Altogether these observations demonstrate that constitutively active beta-catenin is an adrenal oncogene which triggers benign aldosterone-secreting tumour development and promotes malignancy.
Assuntos
Córtex Suprarrenal/patologia , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , beta Catenina/metabolismo , Córtex Suprarrenal/metabolismo , Neoplasias das Glândulas Suprarrenais/fisiopatologia , Aldosterona/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Metástase NeoplásicaRESUMO
Carney complex is a rare familial multineoplastic syndrome predisposing to endocrine and nonendocrine tumors due to inactivating mutations of PRKAR1A, leading to perturbations of the cAMPâprotein kinase A signaling pathway. Skin lesions are the most common manifestation of Carney complex, including lentigines, blue nevi, and cutaneous myxomas in unusual locations such as oral and genital mucosa. Unlike endocrine disorders, the pathogenesis of skin lesions remains unexplained. In this study, we show that embryonic invalidation of the Prkar1a gene in steroidogenic factor-1âexpressing cells leads to the development of familial skin pigmentation alterations, reminiscent of those in patients with Carney complex. Immunohistological and molecular analyses, coupled with genetic monitoring of recombinant cell lineages in mouse skin, suggest that familial lentiginosis and myxomas occur in skin areas specifically enriched in dermal melanocytes. In lentigines- and blue neviâprone areas from mutant mice and patients, Prkar1a/PRKAR1A invalidation occurs in a subset of dermal fibroblasts capable of inducing, under the influence of protein kinase A signaling, the production of promelanogenic EDN3 and hepatocyte GF signals. Our model strongly suggests that the origin of the typical Carney complex cutaneous lesions is the result of noncell-autonomous promelanogenic activity of a dermal fibroblast population sharing a community of origin with steroidogenic factor-1 lineage.
Assuntos
Complexo de Carney , Lentigo , Mixoma , Nevo Azul , Dermatopatias , Animais , Camundongos , Complexo de Carney/genética , Complexo de Carney/patologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Mixoma/genética , Mixoma/patologia , Síndrome , Lentigo/patologiaRESUMO
Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/patologia , Androgênios , Animais , Feminino , Masculino , Camundongos , PrognósticoRESUMO
SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.
Assuntos
Transdiferenciação Celular , Cisteína Endopeptidases , Glucocorticoides , Animais , Camundongos , Córtex Suprarrenal/metabolismo , Corticosteroides/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , beta Catenina/metabolismo , Transdiferenciação Celular/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Glucocorticoides/metabolismo , Via de Sinalização WntRESUMO
Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.
Assuntos
Síndrome de Cushing , Animais , Domínio Catalítico/genética , Síndrome de Cushing/genética , Síndrome de Cushing/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hidrocortisona/metabolismo , Camundongos , ProteômicaRESUMO
The hypothalamic-pituitary-adrenal axis is the primary neuroendocrine system activated to re-establish homeostasis during periods of stress, including critical illness and major surgery. During critical illness, evidence suggests that locally induced inflammation of the adrenal gland could facilitate immune-adrenal cross-talk and, in turn, modulate cortisol secretion. It has been hypothesized that immune cells are necessary to mediate the effect of inflammatory stimuli on the steroidogenic pathway that has been observed in vivo. To test this hypothesis, we developed and characterized a trans-well co-culture model of THP1 (human monocytic cell)-derived macrophages and ATC7 murine zona fasciculata adrenocortical cells. We found that co-culture of ATC7 and THP1 cells results in a significant increase in the basal levels of IL-6 mRNA in ATC7 cells, and this effect was potentiated by treatment with LPS. Addition of LPS to co-cultures of ATC7 and THP1 significantly decreased the expression of key adrenal steroidogenic enzymes (including StAR and DAX-1), and this was also found in ATC7 cells treated with pro-inflammatory cytokines. Moreover, 24-h treatment with the synthetic glucocorticoid dexamethasone prevented the effects of LPS stimulation on IL-6, StAR and DAX-1 mRNA in ATC7 cells co-cultured with THP1 cells. Our data suggest that the expression of IL-6 and steroidogenic genes in response to LPS depends on the activation of intra-adrenal immune cells. Moreover, we also show that the effects of LPS can be modulated by glucocorticoids in a time- and dose-dependent manner with potential implications for clinical practice.
Assuntos
Sistema Imunitário/metabolismo , Modelos Biológicos , Monócitos/citologia , Zona Fasciculada/citologia , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroides/metabolismo , Células THP-1 , Fatores de TempoRESUMO
Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.
Assuntos
Complexo de Carney/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células de Sertoli/citologia , Neoplasias Testiculares/metabolismo , Proteína Wnt4/metabolismo , Animais , Apoptose , Complexo de Carney/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Comunicação Parácrina , Fenótipo , Pigmentação , Túbulos Seminíferos/metabolismo , Testículo/metabolismo , TranscriptomaRESUMO
Studies in vivo have suggested the involvement of CREB-regulated transcription coactivator (CRTC)2 on ACTH-induced transcription of the key steroidogenic protein, Steroidogenic Acute Regulatory (StAR). The present study uses two ACTH-responsive adrenocortical cell lines, to examine the role of CRTC on Star transcription. Here we show that ACTH-induced Star primary transcript, or heteronuclear RNA (hnRNA), parallels rapid increases in nuclear levels of the 3 isoforms of CRTC; CRTC1, CRTC2 and CRTC3. Furthermore, ACTH promotes recruitment of CRTC2 and CRTC3 by the Star promoter and siRNA knockdown of either CRTC3 or CRTC2 attenuates the increases in ACTH-induced Star hnRNA. Using pharmacological inhibitors of PKA, MAP kinase and calcineurin, we show that the effects of ACTH on Star transcription and CRTC nuclear translocation depend predominantly on the PKA pathway. The data provides evidence that CRTC2 and CRTC3, contribute to activation of Star transcription by ACTH, and that PKA/CRTC-dependent pathways are part of the multifactorial mechanisms regulating Star transcription.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Hormônios/farmacologia , Fosfoproteínas/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Feminino , Camundongos , Regiões Promotoras Genéticas , Transporte Proteico/efeitos dos fármacos , RNA Nuclear Heterogêneo/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Cholesterol is the obligate precursor to adrenal steroids but is cytotoxic at high concentrations. Here, we show the role of the liver X receptors (LXRalpha and LXRbeta) in preventing accumulation of free cholesterol in mouse adrenal glands by controlling expression of genes involved in all aspects of cholesterol utilization, including the steroidogenic acute regulatory protein, StAR, a novel LXR target. Under chronic dietary stress, adrenal glands from Lxralphabeta-/- mice accumulated free cholesterol. In contrast, wild-type animals maintained cholesterol homeostasis through basal expression of genes involved in cholesterol efflux and storage (ABC transporter A1 [ABCA1], apoE, SREBP-1c) while preventing steroidogenic gene (StAR) expression. Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased. Basally, Lxralphabeta-/- mice exhibited a marked decrease in ABCA1 and a derepression of StAR expression, causing a net decrease in cholesterol efflux and an increase in steroidogenesis. These changes occurred under conditions that prevented the acute stress response and resulted in a phenotype more specific to the loss of LXRalpha, including hypercorticosteronemia, cholesterol ester accumulation, and adrenomegaly. These results imply LXRalpha provides a safety valve to limit free cholesterol levels as a basal protective mechanism in the adrenal gland, where cholesterol is under constant flux.
Assuntos
Glândulas Suprarrenais/metabolismo , Colesterol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/patologia , Animais , Células Cultivadas , Corticosterona/sangue , Proteínas de Ligação a DNA/genética , Homeostase , Humanos , Hipertrofia , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão , Compostos Orgânicos/metabolismo , Receptores Nucleares Órfãos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismoRESUMO
The Cre-loxP system combined with gene targeting strategies has proven to be very useful for gene inactivation in specific tissues and/or cell types. To achieve adrenal cortex specific recombination in vivo, we used a 0.5-kb fragment of the 5'-flanking region of the akr1b7 gene to drive Cre expression in adrenocortical cells. The resulting 0.5 akr1b7-Cre mice express Cre in all steroidogenic zones of the adrenal cortex but not in the gonads. Although recombination of the ROSA26R reporter locus was not observed in all cortical cells, we provide evidence that Cre is expressed in all the cells of the cortex in adult mice. In addition, Cre activity was found in collecting ducts and maturing glomeruli of the kidney. This line is the first to show specific Cre expression in the adrenal cortex in the absence of Cre expression in the gonads. This transgene thus provides a valuable tool for specific gene recombination in the adrenal cortex and kidney.
Assuntos
Córtex Suprarrenal/enzimologia , Integrases/metabolismo , Camundongos Transgênicos , Córtex Suprarrenal/citologia , Córtex Suprarrenal/embriologia , Animais , Feminino , Gônadas/citologia , Gônadas/enzimologia , Integrases/genética , Rim/citologia , Rim/enzimologia , Masculino , CamundongosRESUMO
The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies.