RESUMO
Monogenic lipodystrophies are a heterogeneous group of rare disorders characterized by a lack of adipose tissue (AT), all of which predispose patients to the development of insulin resistance and its related metabolic sequelae. The extent of AT loss ranges from partial, as in familial partial lipodystrophy (FPLD), to a total absence of metabolically active AT in congenital generalized lipodystrophy (CGL) and is generally associated with the severity of metabolic complications. Significant genetic, allelic, phenotypic, and clinical heterogeneity exists among the lipodystrophies. Patients with FPLD3 due to mutations in the PPARG gene, which encodes a key transcriptional regulator of adipocyte development and function, provide a particularly striking example of this heterogeneity. We will present several gene-gene and gene-environment factors and mechanisms that are critical for adequate PPARγ expression and activity in AT and discuss how these interactions potentially contribute to the observed spectrum of FPLD3 phenotypes. Comparable mechanisms may play a role in other types of lipodystrophies too, and their elucidation may further improve our molecular understanding of AT dysfunction.
Assuntos
Regulação da Expressão Gênica , Interação Gene-Ambiente , Lipodistrofia Parcial Familiar/genética , Mutação , PPAR gama/genética , Tecido Adiposo/metabolismo , Animais , HumanosRESUMO
Childhood obesity coincides with increased numbers of circulating classical CD14++CD16- and intermediate CD14++CD16+ monocytes. Monocytes are key players in the development and exacerbation of atherosclerosis, which prompts the question as to whether the monocytosis in childhood obesity contributes to atherogenesis over the years. Here, we dissected the monocyte gene expression profile in childhood obesity using an Illumina microarray platform on sorted monocytes of 35 obese children and 16 lean controls. Obese children displayed a distinctive monocyte gene expression profile compared to lean controls. Upon validation with quantitative PCR, we studied the association of the top 5 differentially regulated monocyte genes in childhood obesity with obesity and complexity of coronary atherosclerosis (SYNTAX score) in a cohort of 351 adults at risk for ischemic cardiovascular disease. The downregulation of monocyte IMPDH2 and TMEM134 in childhood obesity was also observed in obese adults. Moreover, downregulation of monocyte TMEM134 was associated with a higher SYNTAX atherosclerosis score in adults. In conclusion, childhood obesity entails monocyte gene expression alterations associated with obesity and enhanced complexity of coronary atherosclerosis in adults.
Assuntos
Doença da Artéria Coronariana/patologia , Monócitos/metabolismo , Obesidade Infantil/patologia , Adolescente , Adulto , Índice de Massa Corporal , Estudos de Casos e Controles , Criança , Estudos de Coortes , Angiografia Coronária , Doença da Artéria Coronariana/genética , Regulação para Baixo , Feminino , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Monócitos/citologia , Obesidade Infantil/genética , Risco , Índice de Gravidade de Doença , TranscriptomaRESUMO
HYPOTHESIS: Childhood obesity is accompanied by low-grade systemic inflammation, which contributes to the development of insulin resistance and cardiovascular complications later in life. As vitamin D exhibits profound immunomodulatory functions and vitamin D deficiency is highly prevalent in childhood obesity, we hypothesized that vitamin D deficiency in childhood obesity coincides with enhanced systemic inflammation and reduced insulin sensitivity. METHODS: In a cross-sectional study of 64 obese and 32 healthy children aged 6-16 years, comprehensive profiling of 32 circulating inflammatory mediators was performed, together with assessment of 25-hydroxyvitamin D (25(OH)D) levels and measures for insulin sensitivity. RESULTS: Severe vitamin D insufficiency, which is further referred to as vitamin D deficiency, was defined as a 25(OH)D level ≤37.5 nmol l(-1), and was highly prevalent in obese (56%) versus healthy control children (16%). Throughout the study, 25(OH)D-deficient children were compared with the other children, including 25(OH)D insufficient (37.5-50 nmol l(-1)) and 25(OH)D sufficient children (≥50 nmol l(-1)). First, 25(OH)D-deficient obese children showed a lower insulin sensitivity than other obese children, as measured by a lower quantitative insulin sensitivity check index. Second, the association between 25(OH)D deficiency and insulin resistance in childhood obesity was confirmed with multiple regression analysis. Third, 25(OH)D-deficient obese children showed higher levels of the inflammatory mediators cathepsin S, chemerin and soluble vascular adhesion molecule (sVCAM), compared with the other obese children. Finally, hierarchical cluster analysis revealed an over-representation of 25(OH)D deficiency in obese children expressing inflammatory mediator clusters with high levels of cathepsin S, sVCAM and chemerin. CONCLUSION: 25(OH)D deficiency in childhood obesity was associated with enhanced systemic inflammation and reduced insulin sensitivity. The high cathepsin S and sVCAM levels may reflect activation of a pro-inflammatory, pro-diabetic and atherogenic pathway, which could be inhibited by vitamin D supplementation.
Assuntos
Doença da Artéria Coronariana/prevenção & controle , Mediadores da Inflamação/sangue , Inflamação/etiologia , Resistência à Insulina , Obesidade Infantil/complicações , Deficiência de Vitamina D/complicações , Adolescente , Glicemia/metabolismo , Índice de Massa Corporal , Quimiocinas/sangue , Criança , Análise por Conglomerados , Estudos Transversais , Suplementos Nutricionais , Feminino , Humanos , Inflamação/sangue , Inflamação/imunologia , Resistência à Insulina/imunologia , Masculino , Obesidade Infantil/sangue , Obesidade Infantil/imunologia , Prevalência , Vitamina D/análogos & derivados , Vitamina D/uso terapêutico , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/imunologia , Vitaminas/uso terapêuticoRESUMO
Adipose tissue (AT) represents a microenvironment where intersection takes place between immune processes and metabolic pathways. A variety of immune cells have been characterized in AT over the past decades, with the most recent addition of invariant natural killer T (iNKT) cells. As members of the T cell family, iNKT cells represent a subset that exhibits both innate and adaptive characteristics and directs ensuing immune responses. In disease conditions, iNKT cells have established roles that include disorders in the autoimmune spectrum in malignancies and infectious diseases. Recent work supports a role for iNKT cells in the maintenance of AT homeostasis through both immune and metabolic pathways. The deficiency of iNKT cells can result in AT metabolic disruptions and insulin resistance. In this review, we summarize recent work on iNKT cells in immune regulation, with an emphasis on AT-resident iNKT cells, and identify the potential mechanisms by which adipocytes can mediate iNKT cell activity.
Assuntos
Tecido Adiposo/imunologia , Doenças Metabólicas/imunologia , Células T Matadoras Naturais/imunologia , Tecido Adiposo/metabolismo , Animais , Apresentação de Antígeno , Antígenos CD1d/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Resistência à Insulina/imunologia , Lipídeos/imunologia , Macrófagos/imunologia , Doenças Metabólicas/metabolismo , Camundongos , Modelos Imunológicos , Células T Matadoras Naturais/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Obesity is associated with a prothrombotic state, which may contribute to the increased risk of thrombotic events. OBJECTIVE: To assess the effects of (pre)adipocyte-derived adipokines on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) production by hepatocytes. METHODS: HepG2 hepatocytes were incubated with conditioned media (CM) derived from preadipocytes and adipocytes, which had been untreated or prestimulated with tumor necrosis factor (TNF)-α, interleukin (IL)-1ß or IL-6. After 24 h, supernatants and cell lysates were harvested for measurement of fibrinogen, PAI-1 and TF. RESULTS: (Pre)adipocyte CM significantly enhanced the production of PAI-1 by HepG2 cells 2.5- to 4.4-fold. CM from cytokine-stimulated (pre)adipocytes significantly induced fibrinogen secretion 1.5- to 4.2-fold. TF production was not affected by the CM. After specific depletion of TNF-α, IL-1ß or IL-6 from the CM, IL-6 was shown to be the most prominent stimulus of fibrinogen secretion and IL-1ß of PAI-1 secretion. In addition, fibrinogen, PAI-1 and tissue factor production was evaluated by direct stimulation of HepG2 cells with TNF-α, IL-1ß or IL-6. IL-6 enhanced fibrinogen synthesis 4.3-fold (P<0.01), whereas IL-1ß induced PAI-1 production 5.0-fold (P<0.01). Gene expression analyses showed that TNF-α and IL-1ß stimulate the adipocyte expression of TNF-α, IL-1ß and IL-6. Cytokine stimulation of adipocytes may thus have induced an inflammatory response, which may have stimulated fibrinogen and PAI-1 production by HepG2 cells more potently. CONCLUSIONS: SGBS (pre)adipocytes release cytokines that increase the production of fibrinogen and PAI-1 by HepG2 cells. IL-6 and IL-1ß produced by (pre)adipocytes were the strongest inducers of fibrinogen and PAI-1 secretion, respectively.
RESUMO
AIMS/HYPOTHESIS: In adults, circulating inflammatory mediators and activated CD14(++) monocytes link obesity to its metabolic and cardiovascular complications. However, it is largely unknown whether these inflammatory changes already occur in childhood obesity. To survey inflammatory changes during the early stages of obesity, we performed a comprehensive analysis of circulating inflammatory mediators, monocyte populations and their function in childhood obesity. METHODS: In lean and obese children aged 6 to 16 years (n = 96), 35 circulating inflammatory mediators including adipokines were measured. Hierarchical cluster analysis of the inflammatory mediator profiles was performed to investigate associations between inflammatory mediator clusters and clinical variables. Whole-blood monocyte phenotyping and functional testing with the toll-like receptor 4 ligand, lipopolysaccharide, were also executed. RESULTS: First, next to leptin, the circulating mediators chemerin, tissue inhibitor of metalloproteinase 1, EGF and TNF receptor 2 were identified as novel inflammatory mediators that are increased in childhood obesity. Second, cluster analysis of the circulating mediators distinguished two obesity clusters, two leanness clusters and one mixed cluster. All clusters showed distinct inflammatory mediator profiles, together with differences in insulin sensitivity and other clinical variables. Third, childhood obesity was associated with increased CD14(++) monocyte numbers and an activated phenotype of the CD14(++) monocyte subsets. CONCLUSIONS/INTERPRETATION: Inflammatory mediator clusters were associated with insulin resistance in obese and lean children. The activation of CD14(++) monocyte subsets, which is associated with increased development of atherosclerosis in obese adults, was also readily detected in obese children. Our results indicate that inflammatory mechanisms linking obesity to its metabolic and cardiovascular complications are already activated in childhood obesity.
Assuntos
Mediadores da Inflamação/sangue , Inflamação/sangue , Inflamação/patologia , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/patologia , Obesidade/sangue , Obesidade/patologia , Adolescente , Estudos de Casos e Controles , Contagem de Células , Quimiocinas/sangue , Criança , Análise por Conglomerados , Comorbidade , Fator de Crescimento Epidérmico/sangue , Feminino , Humanos , Inflamação/epidemiologia , Peptídeos e Proteínas de Sinalização Intercelular , Leptina/sangue , Masculino , Monócitos/imunologia , Obesidade/epidemiologia , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Análise de Regressão , Inibidor Tecidual de Metaloproteinase-1/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
BACKGROUND: Adipose tissue dysfunction is associated with inflammation, type 2 diabetes mellitus and vascular diseases. Visceral adipose tissue (VAT)-derived adipokines, which are released in the portal circulation may influence liver metabolism. OBJECTIVES: (1) To estimate the contribution of VAT and subcutaneous adipose tissue (SAT) on adipokine levels by measuring differences in adipokine concentrations between the portal draining inferior mesenteric vein and the subclavian vein. (2) To determine the relation of both VAT and SAT quantity and composition to mesenteric and systemic concentrations of adipokines. DESIGN: Cross-sectional cohort study. SUBJECTS: A total of 32 patients undergoing abdominal aortic surgery. MEASUREMENTS: A panel of 18 adipokines was measured in perioperatively obtained blood samples from the subclavian vein and the inferior mesenteric vein. Adipocyte size, macrophage infiltration and capillary density were measured in subcutaneous and mesenteric adipose tissue biopsies; SAT and VAT areas were measured on computed tomography images. RESULTS: Serum interferon-γ-inducible protein 10 (IP-10) and hepatocyte growth factor (HGF) concentrations were significantly higher in the inferior mesenteric vein vs the subclavian vein. SAT area (ß -18; 95% confidence interval (CI) -35 to -2), subcutaneous adipocyte size (ß -488; 95% CI -938 to -38) and SAT macrophages quantity (ß -1439; 95% CI -2387 to -491) were negatively associated with adiponectin levels in the systemic circulation. SAT area was related to systemic concentrations of leptin. Mesenteric adiponectin concentrations were related to VAT area (ß -20; 95% CI -35 to -5) and visceral adipocyte size (ß -1076; 95% CI -1624 to -527). VAT area, adipocyte size and capillary density were related to systemic adiponectin concentrations. CONCLUSION: SAT and VAT quantities as well as morphologic characteristics of both adipose tissue depots are related to systemic and mesenteric adipokine concentrations. There were no differences in adipokine concentrations between the mesenteric and subclavian vein, except for higher IP-10 and HGF concentrations in the inferior mesenteric vein, indicating a possible contribution of VAT to IP-10 and HGF levels.
Assuntos
Adipocinas/metabolismo , Inflamação/metabolismo , Gordura Intra-Abdominal/metabolismo , Fígado/metabolismo , Veias Mesentéricas/metabolismo , Veia Subclávia/metabolismo , Gordura Subcutânea/metabolismo , Idoso , Quimiocina CXCL10/sangue , Estudos de Coortes , Estudos Transversais , Feminino , Fator de Crescimento de Hepatócito/sangue , Humanos , Gordura Intra-Abdominal/patologia , Fígado/patologia , Masculino , Veias Mesentéricas/patologia , Veia Subclávia/patologia , Gordura Subcutânea/patologiaRESUMO
CONTEXT: Type 1 diabetes (T1D) is considered a proinflammatory condition. Adipose tissue involvement seems evident because adiponectin levels correlate with disease remission and administration of leptin suppresses the low-grade systemic inflammation in mice with T1D. Whether adipose tissue involvement in T1D already occurs at a young age is yet unknown. OBJECTIVE: The aim was to explore the extent of adipokine alterations in pediatric T1D and gain more insight into the mechanisms underlying the involvement of adipose tissue. DESIGN AND PARTICIPANTS: First, plasma adipokine profiling (24 adipokines) of 20 children with onset T1D, 20 children with long-standing T1D, and 17 healthy controls was performed using a recently developed and validated multiplex immunoassay. Second, the effects of diabetic plasma factors on preadipocyte proliferation and differentiation were studied in vitro. RESULTS: In children with onset and long-standing T1D, plasma adipokine profiling showed increased levels of various adipokines acting at the crossroads of adipose tissue function and inflammation, including CCL2/monocyte chemoattractant protein-1 and the novel adipokines cathepsin S, chemerin, and tissue inhibitor of metalloproteinase-1 (P < 0.05). Furthermore, onset and long-standing diabetic plasma significantly induced preadipocyte proliferation and adipocyte differentiation in vitro (P < 0.05). Two candidate plasma factors, glucose and the saturated fatty acid palmitic acid, did not affect proliferation or adipocyte differentiation in vitro but were found to increase CCL2 (monocyte chemoattractant protein-1) secretion by adipocytes. CONCLUSIONS: The adipogenic effects of diabetic plasma in vitro and the altered adipokine levels in vivo suggest adipose tissue involvement in the low-grade inflammation associated with T1D, already in pediatric patients.
Assuntos
Adipócitos/fisiologia , Adipocinas/sangue , Diferenciação Celular , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/fisiopatologia , Adipócitos/patologia , Adolescente , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Criança , Estudos de Coortes , Meios de Cultivo Condicionados/farmacologia , Citocinas/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/fisiologia , Feminino , Humanos , Hiperglicemia/sangue , Hiperglicemia/complicações , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Masculino , Cultura Primária de CélulasRESUMO
Elevated plasma triglyceride levels, as often seen in obese subjects, are independently associated with an increased risk of cardiovascular diseases. By secreting adipokines (such as adiponectin and leptin) and other proteins (such as lipoprotein lipase and cholesteryl ester transferase protein), adipose tissue affects triglyceride metabolism. In obesity, adipocyte hypertrophy leads to many changes in adipocyte function and production of anti- and pro-inflammatory cytokines. Furthermore, free fatty acids are released into the circulation contributing to insulin resistance. Adipose tissue dysfunction will eventually lead to abnormalities in lipid metabolism, such as hypertriglyceridemia (due to increased hepatic very-low-density lipoprotein production and decreased triglyceride hydrolysis), small dense low-density lipoprotein particles, remnant lipoproteins and low high-density lipoprotein cholesterol levels, all associated with a higher risk for the development of cardiovascular diseases. The clinical implications of elevated plasma triglycerides are still a matter of debate. Understanding the pathophysiology of adipose tissue dysfunction in obesity, which is becoming a pandemic condition, is essential for designing appropriate therapeutic interventions. Lifestyle changes are important to improve adipose tissue function in obese patients. Pharmacological interventions to improve adipose tissue function need further evaluation. Although statins are not very potent in reducing plasma triglycerides, they remain the mainstay of therapy for cardiovascular risk reduction in high-risk patients.
Assuntos
Tecido Adiposo/fisiopatologia , Hipertrigliceridemia/fisiopatologia , Aterosclerose/etiologia , Humanos , Hipertrigliceridemia/complicações , Hipertrigliceridemia/terapiaRESUMO
AIMS/HYPOTHESIS: Familial partial lipodystrophy (FPLD) is a rare metabolic disorder with clinical features that may not be readily recognised. As FPLD patients require a specific therapeutic approach, early identification is warranted. In the present study we aimed to identify cases of FPLD among non-obese patients with type 2 diabetes mellitus and marked insulin resistance. METHODS: We searched the databases of three diabetic outpatient clinics for patients with marked insulin resistance, arbitrarily defined as the use of ≥100 U insulin/day, and BMI ≤ 27 kg/m(2). In all patients, metabolic variables and anthropomorphic measurements were evaluated and DNA was sequenced for mutations in the genes encoding lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC). RESULTS: Out of 5,221 diabetic individuals, 24 patients fulfilled all criteria. Twelve patients were willing to participate, of whom five showed clinical features of lipodystrophy. In three of these patients the clinical diagnosis of FPLD was confirmed by the presence of mutations in LMNA or PPARG; one patient harboured a novel heterozygous mutation (Y151C) in PPARG. The Y151C mutant displayed impaired DNA-binding capacity and hence reduced transcriptional activity compared with wild-type PPARγ. Dominant-negative activity was absent. CONCLUSION/INTERPRETATION: The combination of BMI ≤ 27 kg/m(2) and the use of >100 U insulin/day increases the chance of identifying lipodystrophy. Thus careful assessment of clinical features of FPLD should be considered in these patients, allowing earlier therapeutic interventions.
Assuntos
Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/fisiologia , Lipodistrofia Parcial Familiar/diagnóstico , Lipodistrofia Parcial Familiar/genética , PPAR gama/genética , Adulto , Idoso , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Humanos , Insulina/administração & dosagem , Insulina/uso terapêutico , Resistência à Insulina/genética , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
The mechanisms of receptor- and cell-specific effects of the adrenal corticosteroid hormones via mineralo- (MRs) and glucocorticoid receptors (GRs) are still poorly understood. Because the expression levels of two splice variants of the steroid receptor coactivator-1 (SRC-1) 1a and 1e, can differ significantly in certain cell populations, we tested the hypothesis that their relative abundance could determine cell- and receptor-specific effects of corticosteroid receptor-mediated transcription. In transient transfections, we demonstrate three novel types of SRC-1a- and SRC-1e-specific effects for corticosteroid receptors. One is promoter dependence: SRC-1e much more potently coactivated transcription from several multiple response element-containing promoters. Mammalian 1-hydrid studies indicated that this likely does not involve promoter-specific coactivator recruitment. Endogenous phenylethanolamine-N-methyltransferase mRNA induction via GRs was also differentially affected by the splice variants. Another type is receptor specificity: responses mediated by the N-terminal part of the MR, but not the GR, were augmented by SRC-1e at synergizing response elements. SRC fragment SRC(988-1240) by the MR but not the GR N-terminal fragment in a 1-hybrid assay. The last type, for GRs, is ligand dependence. Due to effects on partial agonism of RU486-activated GRs, different ratios of SRC-1a and 1e can lead to large differences in the extent of antagonism of RU486 on GR-mediated transcription. Furthermore, we show that SRC-1e but not SRC-1a mRNA expression was regulated in the pituitary by corticosterone. We conclude that the cellular differences in SRC-1a to SRC-1e ratio demonstrated in vivo might be involved in cell-specific responses to corticosteroids in a promoter- and ligand-dependent way.
Assuntos
Receptores de Esteroides/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Processamento Alternativo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Corticosterona/metabolismo , Genes Reporter , Histona Acetiltransferases , Humanos , Hibridização In Situ , Ligantes , Modelos Biológicos , Coativador 1 de Receptor Nuclear , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
The adenovirus E1A protein regulates transcription of cellular genes via its interaction with the transcriptional coactivators p300/CBP. The collagenase promoter activated by the c-Jun protein is repressed by E1A. Here we show that E1A repression is specific for c-Jun, as E1A does not repress the collagenase promoter activated by the homologous transcription factor EB1. Using chimeras of c-Jun and EB1, we demonstrate that a 12 amino acid region in the basic region of the c-Jun DNA-binding domain is essential for repression by E1A. Since repression requires the binding of p300 to E1A, we studied the involvement of p300 acetyltransferase activity in the repression mechanism. We demonstrate that c-Jun is acetylated in vivo, and mutational analysis identified Lys271 in the c-Jun basic region to be essential for repression of the collagenase promoter by E1A. In addition, Lys271 is acetylated both in vitro and in vivo. These results suggest that the specific repression of the collagenase promoter by E1A involves acetylation of c-Jun.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteínas Virais , Acetilação/efeitos dos fármacos , Proteínas E1A de Adenovirus/farmacologia , Substituição de Aminoácidos , Animais , Carcinógenos/farmacologia , Linhagem Celular , Colagenases/biossíntese , Colagenases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Proteína p300 Associada a E1A , Indução Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Lisina/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutagênese Sítio-Dirigida , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/farmacologia , Retina/citologia , Retina/efeitos dos fármacos , Retina/embriologia , Retina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/genética , TransfecçãoRESUMO
The estrogen receptor (ER) suppresses transcriptional activity of the RelA subunit of nuclear factor-kappaB in a hormone-dependent manner by a mechanism involving both the receptor DNA binding domain and ligand binding domain (LBD). In this study we examine the role of the ER LBD in mediating ligand-dependent RelA transrepression. Both ERalpha and ERbeta inhibit RelA in response to 17beta-estradiol but not in the presence of antihormones. We have identified residues within the ERalpha LBD that are responsible for receptor dimerization and show that dimerization is necessary for transactivation and transrepression. Moreover we have generated mutant receptors that have lost their ability to inhibit RelA but retain their capacity to stimulate transcription and conversely mutants that are transcriptionally defective but capable of antagonizing RelA. Overexpression of p160 and cAMP-response element-binding protein-binding protein/p300 co-activators failed to relieve repression of RelA, which is consistent with the demonstration that RelA inhibition can occur independently of these co-activators. These findings suggest it is unlikely that sequestration of these cofactors required for ER transcriptional activation can account for hormone-dependent antagonism of RelA. The identification of ER mutants that discriminate between transactivation and transrepression implies that distinct surfaces within the LBD are involved in mediating these two receptor functions.
Assuntos
Mutação , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Ativação Transcricional , Animais , Western Blotting , Células COS , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dimerização , Estradiol/farmacologia , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Ligantes , Ligases/antagonistas & inibidores , Ligases/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Transativadores/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
Steroid receptor co-activator (SRC1) is one of a number of transcriptional co-activators that are capable of potentiating the activity of nuclear receptors including the oestrogen receptor (ER). Here we report that two isoforms, SRC1a and SRC1e, which diverge at their C-termini, are functionally distinct as they differ in their abilities to enhance the activity of the ER in intact cells. SRC1e enhanced the ability of the ER to stimulate transcription to a greater extent than SRC1a, which had negligible effects on certain promoters. To elucidate the basis of this functional difference, we compared the nuclear receptor-binding properties and mapped the transcriptional activation domains in the two SRC1 isoforms. Both isoforms share a triplet of nuclear receptor-binding motifs (LXXLL motifs) for binding to functional ER dimers, and an activation domain which co-localizes with the CBP-binding domain, while SRC1a contains a unique LXXLL motif in its C-terminus. Although this LXXLL motif increases the affinity for the ER in vitro, it does not appear to be responsible for the functional difference between the two isoforms. This difference is due to a second activation domain that is CBP independent and is suppressed in the SRC1a isoform. Thus, SRC1 exists as functionally distinct isoforms which are likely to play different roles in ER-mediated transcription.
Assuntos
Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Células COS , Histona Acetiltransferases , Humanos , Dados de Sequência Molecular , Coativador 1 de Receptor Nuclear , Ligação Proteica , Receptores de Estrogênio/química , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The RelA subunit of NF-kappaB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor.
Assuntos
NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Animais , Sítios de Ligação , Células COS , DNA/metabolismo , Humanos , Conformação Proteica , Receptores de Glucocorticoides/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição RelA , Ativação TranscricionalRESUMO
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.
Assuntos
Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Hipolipemiantes/metabolismo , Ligantes , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ácido Araquidônico/metabolismo , Células HeLa , Histona Acetiltransferases , Humanos , Coativador 1 de Receptor Nuclear , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Ativação Transcricional , Xenopus laevisRESUMO
The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.
Assuntos
Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteína de Ligação a CREB , Sequência Conservada , DNA/metabolismo , Histona Acetiltransferases , Humanos , Ligantes , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Coativador 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear , Proteína 1 de Interação com Receptor Nuclear , Estrutura Secundária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/química , TransfecçãoRESUMO
Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.
Assuntos
Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Histona Acetiltransferases , Humanos , Lisina/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Coativador 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear , Proteína 1 de Interação com Receptor Nuclear , Oligodesoxirribonucleotídeos/genética , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Homologia de Sequência de Aminoácidos , Ativação TranscricionalRESUMO
The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription.
Assuntos
Mutação , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Ligação a RNA , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Sequência Conservada , DNA/metabolismo , Células HeLa , Histona Acetiltransferases , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Coativador 1 de Receptor Nuclear , Plasmídeos/genética , Ligação Proteica , Receptores de Estrogênio/química , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Tirosina/genéticaRESUMO
The syndrome of resistance to thyroid hormone is characterized by elevated serum free thyroid hormones, failure to suppress pituitary thyrotropin secretion, and variable peripheral refractoriness to hormone action. Here we describe a novel leucine to valine mutation in codon 454 (L454V) of the thyroid hormone beta receptor (TR beta) in this disorder, resulting in a mutant receptor with unusual functional properties. Although the mutant protein binds ligand comparably to wild-type receptor and forms homo- and heterodimers on direct repeat, everted repeat, or palindromic thyroid response elements, its ability to activate transcription via these elements is markedly impaired. The hydrophobic leucine residue lies within an amphipathic alpha-helix at the carboxyl terminus of TR beta and the position of the homologous residue in the crystal structure of TR alpha indicates that its side chain is solvent-exposed and might interact with other proteins. We find that two putative transcriptional mediators (RIP140 and SRC-1) exhibit hormone-dependent association with wild-type TR. In comparison, the interaction of this natural mutant (L454V) and artificial mutants (L454A, E457A) with RIP140 and SRC-1 is markedly reduced. Furthermore, coexpression of SRC-1 is able to restore the transcriptional activity of the L454V mutant receptor, indicating that the interaction of this residue with accessory proteins is critical for transcriptional activation. Finally, the occurrence of the L454V mutation in resistance to thyroid hormone, together with impaired negative regulation of the thyroid-stimulating hormone alpha promoter by this mutant, suggests that the amphipathic alpha-helix also mediates hormone-dependent transcriptional inhibition, perhaps via interaction with these or other accessory factors.