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Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.
Assuntos
RNA , Transcriptoma , RNA/genética , Sítios de Ligação , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Anticorpos/química , ImunoprecipitaçãoRESUMO
FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo-electron microscopy to elucidate structures of activated FFAR1 bound to a Gq mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved "DRY" and "NPXXY" motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.
Assuntos
Ácidos Graxos , Insulina , Insulina/metabolismo , Ligantes , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Graxos não Esterificados , GlucoseRESUMO
A structurally novel class of benzo- or pyrido-fused 1,3-dihydro-2H-imidazole-2-imines was designed and evaluated in an inositol phosphate accumulation assay for Gq signaling to measure agonistic activation of the orexin receptor type 2 (OX2R). These compounds were synthesized in 4-9 steps overall from readily available starting materials. Analogs that contain a stereogenic methyl or cyclopropyl substituent at the benzylic center, and a correctly configured alkyl ether, alkoxyalkyl ether, cyanoalkyl ether, or α-hydroxyacetamido substituted homobenzylic sidechain were identified as the most potent activators of OX2R coupled Gq signaling. Our results also indicate that agonistic activity was stereospecific at both the benzylic and homobenzylic stereogenic centra. We identified methoxyethoxy-substituted pyrido-fused dihydroimidazolimine analog 63c containing a stereogenic benzylic methyl group was the most potent agonist, registering a respectable EC50 of 339 nM and a maximal response (Emax) of 96 % in this assay. In vivo pharmacokinetic analysis indicated good brain exposure for several analogs. Our combined results provide important information towards a structurally novel class of orexin receptor agonists distinct from current chemotypes.
Assuntos
Imidazóis , Iminas , Receptores de Orexina/agonistas , Iminas/farmacologia , Imidazóis/farmacologia , Piridinas , ÉteresRESUMO
The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ9-tetrahydrocannabinol (THC). The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain, epilepsy, obesity, and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.
RESUMO
Nuclear receptors play a central role in both energy metabolism and cardiomyocyte death and survival in the heart. Recent evidence suggests they may also influence cardiomyocyte endowment. Although several members of the nuclear receptor family play key roles in heart maturation (including thyroid hormone receptors) and cardiac metabolism, here, the focus will be on the corticosteroid receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The heart is an important target for the actions of corticosteroids, yet the homeostatic role of GR and MR in the healthy heart has been elusive. However, MR antagonists are important in the treatment of heart failure, a condition associated with mitochondrial dysfunction and energy failure in cardiomyocytes leading to mitochondria-initiated cardiomyocyte death (Ingwall and Weiss, Circ Res 95:135-145, 2014; Ingwall , Cardiovasc Res 81:412-419, 2009; Zhou and Tian , J Clin Invest 128:3716-3726, 2018). In contrast, animal studies suggest GR activation in cardiomyocytes has a cardioprotective role, including in heart failure.
Assuntos
Insuficiência Cardíaca , Receptores de Mineralocorticoides , Animais , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Receptores de Glucocorticoides/fisiologia , Receptores dos Hormônios Tireóideos/metabolismoRESUMO
The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24 h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. KEY POINTS: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.
Assuntos
Glucocorticoides , Nascimento Prematuro , Animais , Dexametasona/farmacologia , Ácidos Graxos , Feminino , Coração Fetal , Glucocorticoides/farmacologia , Camundongos , Miócitos Cardíacos , Gravidez , Receptores de Glucocorticoides/genética , OvinosRESUMO
The CB1 receptor mediates the central nervous system response to cannabinoids, and is a drug target for pain, anxiety and seizures. CB1 also responds to allosteric modulators, which influence cannabinoid binding and efficacy. To understand the mechanism of these compounds, we solved the crystal structure of CB1 with the negative allosteric modulator (NAM) ORG27569 and the agonist CP55940. The structure reveals that the NAM binds to an extrahelical site within the inner leaflet of the membrane, which overlaps with a conserved site of cholesterol interaction in many G protein-coupled receptors (GPCRs). The ternary structure with ORG27569 and CP55940 captures an intermediate state of the receptor, in which aromatic residues at the base of the agonist-binding pocket adopt an inactive conformation despite the large contraction of the orthosteric pocket. The structure illustrates a potential strategy for drug modulation of CB1 and other class A GPCRs.
Assuntos
Receptor CB1 de Canabinoide/metabolismo , Regulação Alostérica , Cristalização , Cicloexanóis/farmacologia , Humanos , Ligação Proteica , Receptor CB1 de Canabinoide/agonistasRESUMO
The NK1 tachykinin G-protein-coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NK1R, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NK1R (hNK1R) modulators have shown promise in clinical trials for migraine, depression, and emesis. However, the only currently approved drugs targeting hNK1R are inhibitors for chemotherapy-induced nausea and vomiting (CINV). To better understand the molecular basis of ligand recognition and selectivity, we solved the crystal structure of hNK1R bound to the inhibitor L760735, a close analog of the drug aprepitant. Our crystal structure reveals the basis for antagonist interaction in the deep and narrow orthosteric pocket of the receptor. We used our structure as a template for computational docking and molecular-dynamics simulations to dissect the energetic importance of binding pocket interactions and model the binding of aprepitant. The structure of hNK1R is a valuable tool in the further development of tachykinin receptor modulators for multiple clinical applications.
Assuntos
Morfolinas/metabolismo , Receptores da Neurocinina-1/química , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Morfolinas/química , Ligação Proteica , Conformação Proteica , Substância P/químicaRESUMO
Glucocorticoid action on target tissues is determined by the density of "nuclear" receptors and intracellular metabolism by the two isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) which catalyze interconversion of active cortisol and corticosterone with inert cortisone and 11-dehydrocorticosterone. 11ß-HSD type 1, a predominant reductase in most intact cells, catalyzes the regeneration of active glucocorticoids, thus amplifying cellular action. 11ß-HSD1 is widely expressed in liver, adipose tissue, muscle, pancreatic islets, adult brain, inflammatory cells, and gonads. 11ß-HSD1 is selectively elevated in adipose tissue in obesity where it contributes to metabolic complications. Similarly, 11ß-HSD1 is elevated in the ageing brain where it exacerbates glucocorticoid-associated cognitive decline. Deficiency or selective inhibition of 11ß-HSD1 improves multiple metabolic syndrome parameters in rodent models and human clinical trials and similarly improves cognitive function with ageing. The efficacy of inhibitors in human therapy remains unclear. 11ß-HSD2 is a high-affinity dehydrogenase that inactivates glucocorticoids. In the distal nephron, 11ß-HSD2 ensures that only aldosterone is an agonist at mineralocorticoid receptors (MR). 11ß-HSD2 inhibition or genetic deficiency causes apparent mineralocorticoid excess and hypertension due to inappropriate glucocorticoid activation of renal MR. The placenta and fetus also highly express 11ß-HSD2 which, by inactivating glucocorticoids, prevents premature maturation of fetal tissues and consequent developmental "programming." The role of 11ß-HSD2 as a marker of programming is being explored. The 11ß-HSDs thus illuminate the emerging biology of intracrine control, afford important insights into human pathogenesis, and offer new tissue-restricted therapeutic avenues.
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases/genética , Animais , Regulação Enzimológica da Expressão Gênica , Glucocorticoides/química , Humanos , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Distribuição TecidualRESUMO
There are few biomarkers to predict efficacy of glucocorticoid treatment in childhood acute lymphoblastic leukemia (ALL) at diagnosis. Here, we demonstrate reciprocal regulation of 11beta-hydroxysteroid dehydrogenase (11ß-HSD), may predict the apoptotic response of ALL to glucocorticoid treatment. Our data may be useful to refine glucocorticoid treatment, to retain benefit while minimizing side effects.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/fisiologia , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prednisolona/uso terapêutico , Adolescente , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Resultado do TratamentoRESUMO
RATIONALE: The activity of the glucocorticoid activating enzyme 11ß-hydroxysteroid dehydrogenase type-1 (11ßHSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11ßHSD1 converts inert 11-dehydrocorticosterone (11-DHC) into the active form, corticosterone (CORT). A sensitive, specific liquid chromatography/tandem mass spectrometry method was sought to simultaneously quantify total 11-DHC and total and free CORT in murine plasma for simple assessment of 11ßHSD1 activity in murine models. METHODS: Mass spectrometry parameters were optimised and a method for the chromatographic separation of CORT and 11-DHC was developed. Murine plasma was prepared by 10:1 chloroform liquid-liquid extraction (LLE) for analysis. Limits of quantitation (LOQs), linearity and other method criteria were assessed, according to bioanalytical method validation guidelines. RESULTS: Reliable separation of 11-DHC and CORT was achieved using an ACE Excel 2 C18-AR (2.1 × 150 mm; 2 µm) fused core column at 25°C, with an acidified water/acetonitrile gradient over 10 min. Analytes were detected by multiple reaction monitoring after positive electrospray ionisation (m/z 345.1.1 â 121.2, m/z 347.1 â 121.1 for 11-DHC and CORT, respectively). The LOQs were 0.25 and 0.20 ng/mL for 11-DHC and CORT, respectively. CONCLUSIONS: This LC/MS method is suitable for the reliable analysis of 11-DHC and CORT following simple LLE of murine plasma, bringing preclinical analysis in line with recommendations for clinical endocrinology and biochemistry.
Assuntos
Cromatografia Líquida/métodos , Corticosterona/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Animais , Corticosterona/sangue , Corticosterona/química , Corticosterona/isolamento & purificação , Limite de Detecção , Modelos Lineares , Extração Líquido-Líquido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glucocorticoides/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adenoviridae/genética , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Glucocorticoides/antagonistas & inibidores , Humanos , Camundongos , Mifepristona/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta-hydroxysteroid dehydrogenase-1 (11ßHSD1). Although 11ßHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet-induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride-induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11ßHSD1 deficiency (global, Hsd11b1-/- , and hepatic myofibroblast-specific, Hsd11b1fl/fl /Pdgfrb-cre) 11ßHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte-specific Hsd11b1fl/fl /albumin-cre mice did not differ from that of controls, ruling out 11ßHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11ßHSD1 inhibitor [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone. HSCs from Hsd11b1-/- and Hsd11b1fl/fl /Pdgfrb-cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4-(2-chlorophenyl-4-fluoro-1-piperidinyl][5-(1H-pyrazol-4-yl)-3-thienyl]-methanone administration prior to chemical injury recapitulated findings in Hsd11b1-/- mice, including greater fibrosis. CONCLUSION: 11ßHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11ßHSD1 inhibitors on liver injury and repair are likely to be context-dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia. (Hepatology 2018;67:2167-2181).
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenases/deficiência , Cirrose Hepática/etiologia , Miofibroblastos/fisiologia , Animais , Modelos Animais de Doenças , Hepatócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Fetoplacental glucocorticoid overexposure is a significant mechanism underlying fetal growth restriction and the programming of adverse health outcomes in the adult. Placental glucocorticoid inactivation by 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) plays a key role. We previously discovered that Hsd11b2(-/-) mice, lacking 11ß-HSD2, show marked underdevelopment of the placental vasculature. We now explore the consequences for fetal cardiovascular development and whether this is reversible. We studied Hsd11b2(+/+), Hsd11b2(+/-), and Hsd11b2(-/-) littermates from heterozygous (Hsd11b(+/-)) matings at embryonic day (E)14.5 and E17.5, where all three genotypes were present to control for maternal effects. Using high-resolution ultrasound, we found that umbilical vein blood velocity in Hsd11b2(-/-) fetuses did not undergo the normal gestational increase seen in Hsd11b2(+/+) littermates. Similarly, the resistance index in the umbilical artery did not show the normal gestational decline. Surprisingly, given that 11ß-HSD2 absence is predicted to initiate early maturation, the E/A wave ratio was reduced at E17.5 in Hsd11b2(-/-) fetuses, suggesting impaired cardiac function. Pravastatin administration from E6.5, which increases placental vascular endothelial growth factor A and, thus, vascularization, increased placental fetal capillary volume, ameliorated the aberrant umbilical cord velocity, normalized fetal weight, and improved the cardiac function of Hsd11b2(-/-) fetuses. This improved cardiac function occurred despite persisting indications of increased glucocorticoid exposure in the Hsd11b2(-/-) fetal heart. Thus, the pravastatin-induced enhancement of fetal capillaries within the placenta and the resultant hemodynamic changes correspond with restored fetal cardiac function. Statins may represent a useful therapeutic approach to intrauterine growth retardation due to placental vascular hypofunction.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/fisiologia , Retardo do Crescimento Fetal/prevenção & controle , Glucocorticoides/metabolismo , Cardiopatias/prevenção & controle , Doenças Placentárias/prevenção & controle , Pravastatina/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Cardiopatias/metabolismo , Cardiopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Placentárias/metabolismo , Doenças Placentárias/patologia , Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chronically elevated glucocorticoid levels impair cognition and are pro-inflammatory in the brain. Deficiency or inhibition of 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1), which converts inactive into active glucocorticoids, protects against glucocorticoid-associated chronic stress- or age-related cognitive impairment. Here, we hypothesised that 11ß-HSD1 deficiency attenuates the brain cytokine response to inflammation. Because inflammation is associated with altered energy metabolism, we also examined the effects of 11ß-HSD1 deficiency upon hippocampal energy metabolism. Inflammation was induced in 11ß-HSD1 deficient (Hsd11b1Del/Del) and C57BL/6 control mice by intraperitoneal injection of lipopolysaccharide (LPS). LPS reduced circulating neutrophil and monocyte numbers and increased plasma corticosterone levels equally in C57BL/6 and Hsd11b1Del/Del mice, suggesting a similar peripheral inflammatory response. However, the induction of pro-inflammatory cytokine mRNAs in the hippocampus was attenuated in Hsd11b1Del/Del mice. Principal component analysis of mRNA expression revealed a distinct metabolic response to LPS in hippocampus of Hsd11b1Del/Del mice. Expression of Pfkfb3 and Ldha, key contributors to the Warburg effect, showed greater induction in Hsd11b1Del/Del mice. Consistent with increased glycolytic flux, levels of 3-phosphoglyceraldehyde and dihydroxyacetone phosphate were reduced in hippocampus of LPS injected Hsd11b1Del/Del mice. Expression of Sdha and Sdhb, encoding subunits of succinate dehydrogenase/complex II that determines mitochondrial reserve respiratory capacity, was induced specifically in hippocampus of LPS injected Hsd11b1Del/Del mice, together with increased levels of its product, fumarate. These data suggest 11ß-HSD1 deficiency attenuates the hippocampal pro-inflammatory response to LPS, associated with increased capacity for aerobic glycolysis and mitochondrial ATP generation. This may provide better metabolic support and be neuroprotective during systemic inflammation or aging.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Metabolismo Energético/fisiologia , Hipocampo/metabolismo , Inflamação/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Corticosterona/sangue , Hipocampo/efeitos dos fármacos , Comportamento de Doença/efeitos dos fármacos , Comportamento de Doença/fisiologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismoRESUMO
The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.
Assuntos
Transformação Celular Neoplásica/metabolismo , Segregação de Cromossomos , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Camundongos , Camundongos Mutantes , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genéticaRESUMO
Genome-wide association studies have consistently implicated the interleukin-15 (IL-15) gene in acute lymphoblastic leukemia (ALL) biology, including associations with disease susceptibility, and increased risk of central nervous system (CNS) involvement. However, whether pre-B ALL blasts directly respond to IL-15 is unknown. Here, we show that most pre-B ALL primary samples and cell lines express IL-15 and components of its receptor and that primary pre-B ALL cells show increased growth in culture in response to IL-15. Investigation of mechanisms of action using IL-15-responsive SD-1 cells shows this growth advantage is maximal under low-serum conditions, mimicking those found in cerebrospinal fluid. IL-15 also upregulates PSGL-1 and CXCR3, molecules associated with CNS trafficking. Investigation of downstream signaling pathways indicates that IL-15 induces signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinase (ERK) 1/2, and to a lesser extent phosphatidylinositol 3-kinase (PI3K) and nuclear factor κB (NF-κB) phosphorylation. The IL-15-mediated growth advantage is abolished by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), PI3K, and NF-κB inhibitors but preserved in the presence of STAT5 inhibition. Together, these observations provide a mechanistic link between increased levels of IL-15 expression and leukemogenesis, high-risk disease, and CNS relapse and suggest potential therapeutic targets.
Assuntos
Sistema Nervoso Central/imunologia , Interleucina-15/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-15/genética , Glicoproteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidor 1 de Ativador de Plasminogênio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores CXCR3/genética , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Regulação para CimaRESUMO
Glucocorticoids are vital for the structural and functional maturation of foetal organs, yet excessive foetal exposure is detrimental to adult cardiovascular health. To elucidate the role of glucocorticoid signalling in late-gestation cardiovascular maturation, we have generated mice with conditional disruption of glucocorticoid receptor (GR) in cardiomyocytes and vascular smooth muscle cells using smooth muscle protein 22-driven Cre recombinase (SMGRKO mice) and compared them with mice with global deficiency in GR (GR(-/-)). Echocardiography shows impaired heart function in both SMGRKO and GR(-/-) mice at embryonic day (E)17.5, associated with generalized oedema. Cardiac ultrastructure is markedly disrupted in both SMGRKO and GR(-/-) mice at E17.5, with short, disorganized myofibrils and cardiomyocytes that fail to align in the compact myocardium. Failure to induce critical genes involved in contractile function, calcium handling and energy metabolism underpins this common phenotype. However, although hearts of GR(-/-) mice are smaller, with 22% reduced ventricular volume at E17.5, SMGRKO hearts are normally sized. Moreover, while levels of mRNA encoding atrial natriuretic peptide are reduced in E17.5 GR(-/-) hearts, they are normal in foetal SMGRKO hearts. These data demonstrate that structural, functional and biochemical maturation of the foetal heart is dependent on glucocorticoid signalling within cardiomyocytes and vascular smooth muscle, though some aspects of heart maturation (size, ANP expression) are independent of GR at these key sites.
Assuntos
Coração Fetal/crescimento & desenvolvimento , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Animais , Corticosterona/sangue , Corticosterona/fisiologia , Coração Fetal/fisiologia , Coração/embriologia , Coração/fisiologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Contração Miocárdica , Miocárdio/ultraestrutura , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miofibrilas/ultraestruturaRESUMO
BACKGROUND: The current estimated average requirement (EAR) and RDA for protein of 0.66 and 0.8 g â kg-1 â d-1, respectively, for adults, including older men, are based on nitrogen balance data analyzed by monolinear regression. Recent studies in young men and older women that used the indicator amino acid oxidation (IAAO) technique suggest that those values may be too low. This observation is supported by 2-phase linear crossover analysis of the nitrogen balance data. OBJECTIVE: The main objective of this study was to determine the protein requirement for older men by using the IAAO technique. METHODS: Six men aged >65 y were studied; each individual was tested 7 times with protein intakes ranging from 0.2 to 2.0 g â kg-1 â d-1 in random order for a total of 42 studies. The diets provided energy at 1.5 times the resting energy expenditure and were isocaloric. Protein was consumed hourly for 8 h as an amino acid mixture with the composition of egg protein with L-[1-13C]phenylalanine as the indicator amino acid. The group mean protein requirement was determined by applying a mixed-effects change-point regression analysis to F13CO2 (label tracer oxidation in breath 13CO2), which identified a breakpoint in F13CO2 in response to graded intakes of protein. RESULTS: The estimated protein requirement and RDA for older men were 0.94 and 1.24 g â kg-1 â d-1, respectively, which are not different from values we published using the same method in young men and older women. CONCLUSIONS: The current intake recommendations for older adults for dietary protein of 0.66 g â kg-1 â d-1 for the EAR and 0.8 g â kg-1 â d-1 for the RDA appear to be underestimated by â¼30%. Future longer-term studies should be conducted to validate these results. This trial was registered at clinicaltrials.gov as NCT01948492.