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1.
J Biol Chem ; 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988243

RESUMO

Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders.

2.
Front Immunol ; 10: 2449, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824476

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3'UTR, were decreased (-3.9 to -3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence ß-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.

3.
Basic Res Cardiol ; 114(6): 47, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673803

RESUMO

A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte transverse tubular system (t-system), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-system is altered in GRKO hearts and if GR signalling is required for t-system preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-system density and increased distances between ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC). Because t-system remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-system density than cells treated with dexamethasone (EC50 1.1 nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR-LTCC coupling and synchrony of intracellular Ca2+ release, but did not alter expression levels of t-system-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-system preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-system loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-system. These findings suggest that GR signalling and autophagy are critically involved in t-system preservation and remodelling in the heart.

4.
Br J Pharmacol ; 176 Suppl 1: S229-S246, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710718

RESUMO

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14750. Nuclear hormone receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

5.
Br J Pharmacol ; 176 Suppl 1: S1-S20, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710719

RESUMO

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14747. In addition to this overview, in which are identified Other protein targets which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

6.
J Am Heart Assoc ; 8(15): e011012, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31311395

RESUMO

Background The contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, we sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, we conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model). Methods and Results Deletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. Functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA-Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR-deficient cardiomyocytes presented a response comparable with controls. Conclusions These data suggest that GR regulates calcium responses in a sex-biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

7.
J Biol Chem ; 294(29): 11225-11239, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31167788

RESUMO

Glucocorticoids are among the most widely used drugs to treat many autoimmune and inflammatory diseases. Although much research has been focused on investigating glucocorticoid activity, it remains unclear how glucocorticoids regulate distinct processes in different cells. Glucocorticoids exert their effects through the glucocorticoid receptor (GR), which, upon glucocorticoid binding, interacts with regulatory proteins, affecting its activity and function. These protein-protein interactions are necessary for the resolution of glucocorticoid-dependent physiological and pharmacological processes. In this study, we discovered a novel protein interaction between the glucocorticoid receptor and ß-arrestin-1, a scaffold protein with a well-established role in G protein-coupled receptor signaling. Using co-immunoprecipitation and in situ proximity ligation assays in A549 cells, we observed that ß-arrestin-1 and unliganded GR interact in the cytoplasm and that, following glucocorticoid binding, the protein complex is found in the nucleus. We show that siRNA-mediated ß-arrestin-1 knockdown alters GR protein turnover by up-regulating the E3 ubiquitin ligase Pellino-1, which catalyzes GR ubiquitination and thereby marks the receptor for proteasomal degradation. The enhanced GR turnover observed in ß-arrestin-1-deficient cells limits the duration of the glucocorticoid response on GR target genes. These results demonstrate that ß-arrestin-1 is a crucial player for the stability of the glucocorticoid receptor. The GR/ß-arrestin-1 interaction uncovered here may help unravel mechanisms that contribute to the cell type-specific activities of glucocorticoids.

8.
Sci Signal ; 12(577)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992401

RESUMO

Stress is increasingly associated with heart dysfunction and is linked to higher mortality rates in patients with cardiometabolic disease. Glucocorticoids are primary stress hormones that regulate homeostasis through two nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), both of which are present in cardiomyocytes. To examine the specific and coordinated roles that these receptors play in mediating the direct effects of stress on the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction and died prematurely from heart failure. In contrast, the cardioMRKO mice exhibited normal heart morphology and function. Despite the presence of myocardial stress, the cardioGRMRdKO mice were resistant to the cardiac remodeling, left ventricular dysfunction, and early death observed in the cardioGRKO mice. Gene expression analysis revealed the loss of gene changes associated with impaired Ca2+ handling, increased oxidative stress, and enhanced cell death and the presence of gene changes that limited the hypertrophic response and promoted cardiomyocyte survival in the double knockout hearts. Reexpression of MR in cardioGRMRdKO hearts reversed many of the cardioprotective gene changes and resulted in cardiac failure. These findings reveal a critical role for balanced cardiomyocyte GR and MR stress signaling in cardiovascular health. Therapies that shift stress signaling in the heart to favor more GR and less MR activity may provide an improved approach for treating heart disease.

9.
Commun Biol ; 2: 104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911679

RESUMO

Excessive or chronic stress can lead to a variety of diseases due to aberrant activation of the glucocorticoid receptor (GR), a ligand activated transcription factor. Pregnancy represents a particular window of sensitivity in which excessive stress can have adverse outcomes, particularly on the developing fetus. Here we show maternal hepatic stress hormone responsiveness is diminished via epigenetic silencing of the glucocorticoid receptor during pregnancy. Provocatively, reinstallation of GR to hepatocytes during pregnancy by adeno-associated viral transduction dysregulates genes involved in proliferation, resulting in impaired pregnancy-induced hepatomegaly. Disruption of the maternal hepatic adaptation to pregnancy results in in utero growth restriction (IUGR). These data demonstrate pregnancy antagonizes the liver-specific effects of stress hormone signaling in the maternal compartment to ultimately support the healthy development of embryos.

10.
J Clin Invest ; 129(3): 1345-1358, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30652972

RESUMO

In the stomach, chronic inflammation causes metaplasia and creates a favorable environment for the evolution of gastric cancer. Glucocorticoids are steroid hormones that repress proinflammatory stimuli, but their role in the stomach is unknown. In this study, we show that endogenous glucocorticoids are required to maintain gastric homeostasis. Removal of circulating glucocorticoids in mice by adrenalectomy resulted in the rapid onset of spontaneous gastric inflammation, oxyntic atrophy, and spasmolytic polypeptide-expressing metaplasia (SPEM), a putative precursor of gastric cancer. SPEM and oxyntic atrophy occurred independently of lymphocytes. However, depletion of monocytes and macrophages by clodronate treatment or inhibition of gastric monocyte infiltration using the Cx3cr1 knockout mouse model prevented SPEM development. Our results highlight the requirement for endogenous glucocorticoid signaling within the stomach to prevent spontaneous gastric inflammation and metaplasia, and suggest that glucocorticoid deficiency may lead to gastric cancer development.

11.
BMC Med Genet ; 19(1): 175, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30241514

RESUMO

BACKGROUND: We recently identified a role for the muscle-specific ubiquitin ligase MuRF1 in right-sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1-/- mice exposed to CH are resistant to right ventricular (RV) dysfunction whereas MuRF1 Tg + mice exhibit impaired function indicative of heart failure. The present study was undertaken to understand the underlying transcriptional alterations in the RV of MuRF1-/- and MuRF1 Tg + mice. METHODS: Microarray analysis was performed on RNA isolated from the RV of MuRF1-/-, MuRF1 Tg+, and wild-type control mice exposed to CH. RESULTS: MuRF1-/- RV differentially expressed 590 genes in response to CH. Analysis of the top 66 genes (> 2-fold or < - 2-fold) revealed significant associations with oxidoreductase, transcription regulation, and transmembrane component annotations. The significant genes had promoters enriched for HOXD12, HOXC13, and RREB-1 protein transcription factor binding sites. MuRF1 Tg + RV differentially expressed 150 genes in response to CH. Analysis of the top 45 genes (> 3-fold or < - 3-fold) revealed significant associations with oxidoreductase-metabolic, glycoprotein-transmembrane-integral proteins, and alternative splicing/splice variant annotations. The significant genes were enriched for promoters with ZIC1 protein transcription factor binding sites. CONCLUSIONS: The differentially expressed genes in MuRF1-/- and MuRF1 Tg + RV after CH have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally-enhanced MuRF1-/- hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects of MuRF1 through regulating Rreb-1, and they reveal mechanisms by which MuRF1 may transcriptionally regulate anti-oxidant systems in the face of right heart failure.


Assuntos
Insuficiência Cardíaca/genética , Hipóxia/genética , Proteínas Musculares/genética , Transcrição Genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Disfunção Ventricular Direita/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Anotação de Sequência Molecular , Proteínas Musculares/deficiência , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/deficiência , Ubiquitina-Proteína Ligases/deficiência , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/fisiopatologia
12.
Front Mol Neurosci ; 11: 251, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30127715

RESUMO

Studies conducted in rodents subjected to chronic stress and some observations in humans after psychosocial stress, have allowed to establish a link between stress and the susceptibility to many complex diseases, including mood disorders. The studies in rodents have revealed that chronic exposure to stress negatively affects synaptic plasticity by triggering changes in the production of trophic factors, subunit levels of glutamate ionotropic receptors, neuron morphology, and neurogenesis in the adult hippocampus. These modifications may account for the impairment in learning and memory processes observed in chronically stressed animals. It is plausible then, that stress modifies the interplay between signal transduction cascades and gene expression regulation in the hippocampus, therefore leading to altered neuroplasticity and functioning of neural circuits. Considering that miRNAs play an important role in post-transcriptional-regulation of gene expression and participate in several hippocampus-dependent functions; we evaluated the consequences of chronic stress on the expression of miRNAs in dorsal (anterior) portion of the hippocampus, which participates in memory formation in rodents. Here, we show that male rats exposed to daily restraint stress (2.5 h/day) during 7 and 14 days display a differential profile of miRNA levels in dorsal hippocampus and remarkably, we found that some of these miRNAs belong to the miR-379-410 cluster. We confirmed a rise in miR-92a and miR-485 levels after 14 days of stress by qPCR, an effect that was not mimicked by chronic administration of corticosterone (14 days). Our in silico study identified the top-10 biological functions influenced by miR-92a, nine of which were shared with miR-485: Nervous system development and function, Tissue development, Behavior, Embryonic development, Organ development, Organismal development, Organismal survival, Tissue morphology, and Organ morphology. Furthermore, our in silico study provided a landscape of potential miRNA-92a and miR-485 targets, along with relevant canonical pathways related to axonal guidance signaling and cAMP signaling, which may influence the functioning of several neuroplastic substrates in dorsal hippocampus. Additionally, the combined effect of miR-92a and miR-485 on transcription factors, along with histone-modifying enzymes, may have a functional relevance by producing changes in gene regulatory networks that modify the neuroplastic capacity of the adult dorsal hippocampus under stress.

13.
Mol Cancer Res ; 16(11): 1761-1772, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29991529

RESUMO

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF-dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors (n = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread.Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761-72. ©2018 AACR.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/metabolismo , Paclitaxel/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Glucocorticoides/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Células MCF-7 , Fenótipo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
14.
J Clin Endocrinol Metab ; 103(10): 3630-3639, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30020469

RESUMO

Background: The glucocorticoid receptor (GR) consists of two alternatively spliced isoforms: GRα, which activates gene transcription, and GRß, a dominant-negative receptor. Theoretically, inactivating variants of GRß could result in glucocorticoid hypersensitivity. Design: A 46-year-old woman presented for evaluation of adrenal insufficiency prompted by low plasma cortisol levels and multiple unexplained symptoms but without clinical evidence of glucocorticoid insufficiency. To explain these findings, extensive clinical, genetic, and molecular studies were performed. Methods: Standard clinical methods assessed the patient's hypothalamic-pituitary-adrenal axis. Validated molecular techniques were used for receptor sequencing, stable transfections, stimulation of candidate genes, cDNA arrays, Ingenuity Pathway Analysis, volcano analysis, and isolation and analysis of the patient's mononuclear cells. Results: Clinical studies excluded primary or secondary adrenal insufficiency, established consistently low basal cortisol levels, and demonstrated hypersensitivity to ultra-low-dose dexamethasone. Receptor sequencing identified two variants of GR9ß (A3669G and G3134T) as well as the known Bcl1 polymorphism. Reductionist studies using stable osteosarcoma cell lines transfected with the GRß variants demonstrated glucocorticoid hypersensitivity of transcribed genes on cDNA array analysis. The patient's monocytes responded to hydrocortisone with exaggerated stimulation of the candidate genes GILZ and FKBP5. Conclusion: Two variants of the dominant-negative GRß, in conjunction with a common Bcl1 intron variant, resulted in hypersensitivity to endogenous and exogenous glucocorticoids and, as a reflection of severity, low circulating cortisol levels without clinical evidence of glucocorticoid insufficiency. This prismatic case exemplifies the unique effects of variants of a dominant-negative receptor.


Assuntos
Glucocorticoides/farmacologia , Hipersensibilidade/epidemiologia , Hipersensibilidade/genética , Mutação , Receptores de Glucocorticoides/genética , Biomarcadores/sangue , Feminino , Humanos , Hidrocortisona/sangue , Hipersensibilidade/tratamento farmacológico , Incidência , Pessoa de Meia-Idade , Prognóstico
15.
FASEB J ; 32(10): 5258-5271, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29672221

RESUMO

Glucocorticoids are primary stress hormones, and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced from a single gene by alternative translation initiation; however, the role individual isoforms play in tissue-specific responses to glucocorticoids is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. GR-C3 knockin mice die at birth due to respiratory distress. Microarray analysis of fibroblasts from wild-type and GR-C3 mice indicated that most genes regulated by GR-C3 were unique to this isoform. Antenatal glucocorticoid administration rescued GR-C3 knockin mice from neonatal death. Dual-energy X-ray absorptiometry revealed no major alterations in body composition for rescued knockin mice. Rescued female, but not male, GR-C3 mice exhibited increased wheel running activity in the light portion of the day. LPS administration induced premature mortality in rescued GR-C3 knockin mice, and gene expression studies revealed a deficiency in the ability of GR-C3 to repress a large cohort of immune and inflammatory response genes. These findings demonstrate that specific GR translational isoforms can influence development, circadian rhythm, and inflammation through the regulation of distinct gene networks.-Oakley, R. H., Ramamoorthy, S., Foley, J. F., Busada, J. T., Lu, N. Z., Cidlowski, J. A. Glucocorticoid receptor isoform-specific regulation of development, circadian rhythm, and inflammation in mice.


Assuntos
Ritmo Circadiano , Receptores de Glucocorticoides/biossíntese , Caracteres Sexuais , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Glucocorticoides/farmacologia , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores de Glucocorticoides/genética
16.
Environ Health Perspect ; 126(4): 047002, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29624291

RESUMO

BACKGROUND: Female reproductive tract development is sensitive to the endocrine-disrupting potential of environmental estrogens. Early-life exposure to the dietary phytoestrogen genistein impairs fertility and persistently alters the transcriptome in the oviduct and uterus of rodents. Glucocorticoid signaling, which has recently been shown to be essential for normal fertility in the female mouse uterus, is antagonized by genistein. OBJECTIVE: Our goal was to determine whether early-life exposure to genistein disrupts glucocorticoid signaling in the mouse uterus, which may contribute to infertility. METHODS: Female C57Bl/6 mice were exposed to either 50 mg/kg per day genistein, 10 µg/kg per day estradiol, or vehicle (corn oil) on postnatal days 1-5 (PND1-5), and then treated with the synthetic glucocorticoid dexamethasone (Dex: 1 mg/kg) or vehicle (saline) on PND5, at weaning on PND21, or as adults on PND56 following adrenalectomy and ovariectomy to evaluate glucocorticoid responsiveness. Uteri were isolated following treatment for gene expression or chromatin immunoprecipitation. RESULTS: Neonatal exposure to genistein altered the uterine transcriptome of adult mice and caused substantial changes to the transcriptional response to glucocorticoids. Although expression of the glucocorticoid receptor was not affected, genistein exposure disrupted glucocorticoid receptor recruitment to specific regulatory sites in target genes. Many genes involved in chromatin remodeling were dysregulated in genistein-exposed mice, suggesting that epigenetic reprograming may contribute to the altered glucocorticoid response of the uterus following early-life exposure to genistein. These changes affected the biological activity of glucocorticoids within the uterus, as glucocorticoids antagonized the proliferative effects of estradiol in the uterus of control mice but not genistein-exposed mice. CONCLUSIONS: Our findings suggest that disruption of glucocorticoid signaling due to early-life exposure to environmental estrogens may in part render the uterus unable to support implantation. https://doi.org/10.1289/EHP1575.


Assuntos
Dexametasona/metabolismo , Genisteína/efeitos adversos , Glucocorticoides/metabolismo , Fitoestrógenos/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Útero/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Camundongos , Camundongos Endogâmicos C57BL
17.
Cell Rep ; 22(10): 2690-2701, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514097

RESUMO

Glucocorticoids (GCs) are master regulators of systemic metabolism. Intriguingly, Cushing's syndrome, a disorder of excessive GCs, phenocopies several menopause-induced metabolic pathologies. Here, we show that the glucocorticoid receptor (GR) drives steatosis in hypogonadal female mice because hepatocyte-specific GR knockout mice are refractory to developing ovariectomy-induced steatosis. Intriguingly, transcriptional profiling revealed that ovariectomy elicits hepatic GC hypersensitivity globally. Hypogonadism-induced GC hypersensitivity results from a loss of systemic but not hepatic estrogen (E2) signaling, given that hepatocyte-specific E2 receptor deletion does not confer GC hypersensitivity. Mechanistically, enhanced chromatin recruitment and ligand-dependent hyperphosphorylation of GR underlie ovariectomy-induced glucocorticoid hypersensitivity. The dysregulated glucocorticoid-mediated signaling present in hypogonadal females is a product of increased follicle-stimulating hormone (FSH) production because FSH treatment in ovary-intact mice recapitulates glucocorticoid hypersensitivity similar to hypogonadal female mice. Our findings uncover a regulatory axis between estradiol, FSH, and hepatic glucocorticoid receptor signaling that, when disrupted, as in menopause, promotes hepatic steatosis.


Assuntos
Estrogênios/deficiência , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Receptores de Glucocorticoides/metabolismo , Animais , Cromatina/metabolismo , Síndrome de Cushing/metabolismo , Estradiol/deficiência , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Ligantes , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ovariectomia , Ovário/metabolismo , Ovário/patologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
18.
Mol Cell Biol ; 38(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29437838

RESUMO

Glucocorticoid receptor ß (GRß) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRß functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRß complexed with the antagonist RU-486. The structure reveals that GRß binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRß are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRß do not contribute to RU-486 binding. Intriguingly, the GRß/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRß is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRß could repress transcription.


Assuntos
Receptores de Glucocorticoides/metabolismo , Sequência de Aminoácidos , Escherichia coli/metabolismo , Glucocorticoides/metabolismo , Humanos
19.
Invest Ophthalmol Vis Sci ; 59(1): 1-12, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29297055

RESUMO

Purpose: Corticosteroids remain the mainstay of treatment for inflammatory diseases almost 80 years after their first clinical use. Topical ophthalmic formulations of corticosteroids have been available to treat disease of the anterior segment of the eye, but the approval of corticosteroids to treat vitreoretinal diseases, including vein occlusion, diabetic macular edema, and uveitis, has occurred only recently. Although most diseases respond to corticosteroid therapy, some patients are resistant to this therapy and side effects, including cataract and elevated intraocular pressure, can limit their use. The purpose of this review is to detail the basic science of corticosteroids focusing on differences in potency, drug delivery, pharmacokinetics, and gene activation, and how these differences affect safety and efficacy in the treatment of diabetic macular edema. Methods: A review was conducted of basic science and pharmacology of the corticosteroids used to treat diabetic macular edema. Results: Clinically available corticosteroids not only have differing potency and pharmacokinetics, but also activate different genes in different target tissues. These differences are associated with distinct efficacy, pharmacokinetic, and safety profiles. It is important to understand these differences in selecting corticosteroids to treat diabetic macular edema. Conclusions: Recent advances in our understanding of the basic science of corticosteroids can explain clinical differences in these agents regarding efficacy and safety. Importantly, this understanding should allow the future discovery of additional novel corticosteroids to treat diabetic macular edema.


Assuntos
Retinopatia Diabética/complicações , Glucocorticoides/administração & dosagem , Edema Macular/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Humanos , Injeções Intravítreas , Edema Macular/etiologia
20.
Cell Death Differ ; 25(3): 486-541, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29362479

RESUMO

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.


Assuntos
Morte Celular , Animais , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Necrose/metabolismo , Necrose/patologia
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