RESUMO
Alterations of symbiosis between microbiota and intestinal epithelial cells (IEC) are associated with intestinal and systemic pathologies. Interactions between bacterial products (MAMPs) and Toll-like receptors (TLRs) are known to be mandatory for IEC homeostasis, but how TLRs may time homeostatic functions with circadian changes is unknown. Our functional and molecular dissections of the IEC circadian clock demonstrate that its integrity is required for microbiota-IEC dialog. In IEC, the antiphasic expression of the RORα activator and RevErbα repressor clock output regulators generates a circadian rhythmic TLR expression that converts the temporally arrhythmic microbiota signaling into circadian rhythmic JNK and IKKß activities, which prevents RevErbα activation by PPARα that would disrupt the circadian clock. Moreover, through activation of AP1 and NF-κB, these activities, together with RORα and RevErbα, enable timing homeostatic functions of numerous genes with IEC circadian events. Interestingly, microbiota signaling deficiencies induce a prediabetic syndrome due to ileal corticosterone overproduction consequent to clock disruption.
Assuntos
Íleo/microbiologia , Íleo/fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Metagenoma , Receptores Toll-Like/imunologia , Animais , Corticosterona/metabolismo , Íleo/imunologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.
Assuntos
Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Redes e Vias Metabólicas , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Dieta Hiperlipídica , Jejum/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Ativação TranscricionalRESUMO
Time-restricted feeding (RF) is known to shift the phasing of gene expression in most primary metabolic tissues, whereas a time misalignment between the suprachiasmatic nucleus circadian clock (SCNCC) and its peripheral CCs (PCC's) is known to induce various pathophysiological conditions, including a metabolic syndrome. We now report that a unique "light therapy," involving different light intensities (TZT0-ZT12150-TZT0-ZT12700 lx, TZT0-ZT1275-TZT0-ZT12150 lx, and TZT0-ZT12350-TZT0-ZT12700 lx), realigns the RF-generated misalignment between the SCNCC and the PCC's. Using such high-light regime, we show that through shifting the SCNCC and its activity, it is possible in a RF and "night-shifted mouse model" to prevent/correct pathophysiologies (e.g., a metabolic syndrome, a loss of memory, cardiovascular abnormalities). Our data indicate that such a "high-light regime" could be used as a unique chronotherapy, for those working on night shifts or suffering from jet-lag, in order to realign their SCNCC and PCC's, thereby preventing the generation of pathophysiological conditions.
Assuntos
Relógios Circadianos , Núcleo Supraquiasmático , Animais , Relógios Circadianos/fisiologia , Camundongos , Núcleo Supraquiasmático/metabolismo , Síndrome Metabólica/terapia , Síndrome Metabólica/metabolismo , Fototerapia/métodos , Masculino , Camundongos Endogâmicos C57BL , Ritmo Circadiano/fisiologia , LuzRESUMO
The glucocorticoid (GC) receptor (GR), when liganded to GC, activates transcription through direct binding to simple (+)GRE DNA binding sequences (DBS). GC-induced direct repression via GR binding to complex "negative" GREs (nGREs) has been reported. However, GR-mediated transrepression was generally ascribed to indirect "tethered" interaction with other DNA-bound factors. We report that GC-induces direct transrepression via the binding of GR to simple DBS (IR nGREs) unrelated to (+)GRE. These DBS act on agonist-liganded GR, promoting the assembly of cis-acting GR-SMRT/NCoR repressing complexes. IR nGREs are present in over 1000 mouse/human ortholog genes, which are repressed by GC in vivo. Thus variations in the levels of a single ligand can coordinately turn genes on or off depending in their response element DBS, allowing an additional level of regulation in GR signaling. This mechanism suits GR signaling remarkably well, given that adrenal secretion of GC fluctuates in a circadian and stress-related fashion.
Assuntos
Receptores de Glucocorticoides/agonistas , Proteínas Repressoras/metabolismo , Elementos de Resposta , Animais , Citocinas , Dermatite Atópica/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Regiões Promotoras Genéticas , Transcrição Gênica , Linfopoietina do Estroma do TimoRESUMO
The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.
Assuntos
Relógios Circadianos , Relógios Circadianos/genética , Fatores de Transcrição/metabolismo , Expressão Gênica , Ritmo Circadiano/genética , Cromatina/genética , Homólogo 5 da Proteína Cromobox , DNA , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismoRESUMO
We demonstrate that there is a tight functional relationship between two highly evolutionary conserved cell processes, i.e., the circadian clock (CC) and the circadian DNA demethylation-methylation of cognate deoxyCpG-rich islands. We have discovered that every circadian clock-controlled output gene (CCG), but not the core clock nor its immediate-output genes, contains a single cognate intronic deoxyCpG-rich island, the demethylation-methylation of which is controlled by the CC. During the transcriptional activation period, these intronic islands are demethylated and, upon dimerization of two YY1 protein binding sites located upstream to the transcriptional enhancer and downstream from the deoxyCpG-rich island, store activating components initially assembled on a cognate active enhancer (a RORE, a D-box or an E-box), in keeping with the generation of a transcriptionally active condensate that boosts the initiation of transcription of their cognate pre-mRNAs. We report how these single intronic deoxyCpG-rich islands are instrumental in such a circadian activation/repression transcriptional process.
Assuntos
Relógios Circadianos , Relógios Circadianos/genética , Regiões Promotoras Genéticas , Ritmo Circadiano/genética , Sequências Reguladoras de Ácido Nucleico , Proteínas CLOCK/genética , DesmetilaçãoRESUMO
Regulatory T cells (T(reg) cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in T(reg) cell development. Mice that lacked all Nr4a factors could not produce T(reg) cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the T(reg) cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4(+) T cell fates in the thymus and thus contribute to immune homeostasis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Esteroides/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/genética , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genes Precoces , Homeostase , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Timócitos/metabolismoRESUMO
Estrogen receptor alpha (ERα) has been recognized now for several decades as playing a key role in reproduction and exerting functions in numerous nonreproductive tissues. In this review, we attempt to summarize the in vitro studies that are the basis of our current understanding of the mechanisms of action of ERα as a nuclear receptor and the key roles played by its two activation functions (AFs) in its transcriptional activities. We then depict the consequences of the selective inactivation of these AFs in mouse models, focusing on the prominent roles played by ERα in the reproductive tract and in the vascular system. Evidence has accumulated over the two last decades that ERα is also associated with the plasma membrane and activates non-nuclear signaling from this site. These rapid/nongenomic/membrane-initiated steroid signals (MISS) have been characterized in a variety of cell lines, and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS and the generation of mice expressing an ERα protein impeded for membrane localization have begun to unravel the physiological role of MISS in vivo. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators based on the integration of the physiological and pathophysiological roles of MISS actions of estrogens.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Transdução de Sinais , Animais , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Genótipo , Humanos , Camundongos Transgênicos , Fenótipo , Moduladores Seletivos de Receptor Estrogênico/farmacologiaRESUMO
BACKGROUND & AIMS: Liver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC-oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis - a rising global unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.
RESUMO
Building a document-level classifier for COVID-19 on radiology reports could help assist providers in their daily clinical routine, as well as create large numbers of labels for computer vision models. We have developed such a classifier by fine-tuning a BERT-like model initialized from RadBERT, its continuous pre-training on radiology reports that can be used on all radiology-related tasks. RadBERT outperforms all biomedical pre-trainings on this COVID-19 task (P<0.01) and helps our fine-tuned model achieve an 88.9 macro-averaged F1-score, when evaluated on both X-ray and CT reports. To build this model, we rely on a multi-institutional dataset re-sampled and enriched with concurrent lung diseases, helping the model to resist to distribution shifts. In addition, we explore a variety of fine-tuning and hyperparameter optimization techniques that accelerate fine-tuning convergence, stabilize performance, and improve accuracy, especially when data or computational resources are limited. Finally, we provide a set of visualization tools and explainability methods to better understand the performance of the model, and support its practical use in the clinical setting. Our approach offers a ready-to-use COVID-19 classifier and can be applied similarly to other radiology report classification tasks.
Assuntos
COVID-19 , Radiologia , Humanos , Relatório de Pesquisa , Processamento de Linguagem NaturalRESUMO
We previously reported that the nonsteroidal compound CpdX, which was initially characterized 20 y ago as a possible gestagen and, shortly afterward, as a possible drug for treatments of inflammatory diseases, selectively triggers the NFκB/AP1-mediated tethered indirect transrepression function of the glucocorticoid receptor (GR), and could therefore be a selective glucocorticoid receptor agonistic modulator (SEGRAM). We now demonstrate that, upon administration to the mouse, CpdX and one of its deuterated derivatives, CpdX-D3, repress as efficiently as a synthetic glucocorticoid (e.g., Dexamethasone) an induced skin atopic dermatitis, an induced psoriasis-like inflammation, a house dust mite (HDM)-induced asthma-like allergic lung inflammation, a collagen-induced arthritis, an induced ulcerative colitis, and an ovalbumin-induced allergic conjunctivitis. Interestingly, in the cases of an HDM-induced asthma-like allergic lung inflammation and of a collagen-induced arthritis, the CpdX antiinflammatory activity was selectively exerted by one of the two CpdX enantiomers, namely, CpdX(eA) or CpdX-D3(eA).
Assuntos
Anti-Inflamatórios/farmacologia , Glucocorticoides/farmacologia , Inflamação/tratamento farmacológico , Receptores de Glucocorticoides/genética , Animais , Anti-Inflamatórios/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/genética , Artrite Experimental/patologia , Asma/tratamento farmacológico , Asma/genética , Asma/patologia , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Alérgica/genética , Conjuntivite Alérgica/patologia , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/patologia , Dexametasona/farmacologia , Modelos Animais de Doenças , Glucocorticoides/genética , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , NF-kappa B/genética , Ovalbumina/toxicidade , Progestinas/química , Progestinas/farmacologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/química , Pele/efeitos dos fármacos , Pele/patologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Seventy years after the discovery of their anti-inflammatory properties, glucocorticoids (GCs) remain the mainstay treatment for major allergic and inflammatory disorders, such as atopic dermatitis, asthma, rheumatoid arthritis, colitis, and conjunctivitis, among others. However, their long-term therapeutical administration is limited by major debilitating side effects, e.g., skin atrophy, osteoporosis, Addison-like adrenal insufficiency, fatty liver, and type 2 diabetes syndrome, as well as growth inhibition in children. These undesirable side effects are mostly related to GC-induced activation of both the direct transactivation and the direct transrepression functions of the GC receptor (GR), whereas the activation of its GC-induced indirect tethered transrepression function results in beneficial anti-inflammatory effects. We have reported in the accompanying paper that the nonsteroidal compound CpdX as well as its deuterated form CpdX-D3 selectively activate the GR indirect transrepression function and are as effective as synthetic GCs at repressing inflammations generated in several mouse models of major pathologies. We now demonstrate that these CpdX compounds are bona fide selective GC receptor agonistic modulators (SEGRAMs) as none of the known GC-induced debilitating side effects were observed in the mouse upon 3-mo CpdX treatments. We notably report that, unlike that of GCs, the administration of CpdX to ovariectomized (OVX) mice does not induce a fatty liver nor type 2 diabetes, which indicates that CpdX could be used in postmenopausal women as an efficient "harmless" GC substitute.
Assuntos
Inflamação/tratamento farmacológico , Osteoporose/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Esteroides/farmacologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Glucocorticoides/farmacologia , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Osteoporose/genética , Osteoporose/patologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/químicaRESUMO
Estrogen treatment increases bone mass and reduces fat mass but is associated with adverse effects in postmenopausal women. Knowledge regarding tissue-specific estrogen signaling is important to aid the development of new tissue-specific treatments. We hypothesized that the posttranslational modification phosphorylation in estrogen receptor alpha (ERα) may modulate ERα activity in a tissue-dependent manner. Phosphorylation of site S122 in ERα has been shown in vitro to affect ERα activity, but the tissue-specific role in vivo is unknown. We herein developed and phenotyped a novel mouse model with a point mutation at the phosphorylation site 122 in ERα (S122A). Female S122A mice had increased fat mass and serum insulin levels but unchanged serum sex steroid levels, uterus weight, bone mass, thymus weight, and lymphocyte maturation compared to WT mice. In conclusion, phosphorylation site S122 in ERα has a tissue-dependent role with an impact specifically on fat mass in female mice. This study is the first to demonstrate in vivo that a phosphorylation site in a transactivation domain in a nuclear steroid receptor modulates the receptor activity in a tissue-dependent manner.
Assuntos
Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Fosforilação/genética , Animais , Densidade Óssea/genética , Osso e Ossos/metabolismo , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/genética , Mutação Puntual/genética , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17ß-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the C451A-ERα, the R264A-ERα females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in R264A-ERα mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. CONCLUSIONS: These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions.
Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Terapia de Reposição de Estrogênios , Estrogênios/farmacologia , Fertilidade/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Mutação Puntual , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Ciclo Estral/efeitos dos fármacos , Feminino , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Ovariectomia , Reepitelização/efeitos dos fármacos , Transdução de Sinais , Fatores de Tempo , Útero/efeitos dos fármacos , Útero/metabolismo , Remodelação Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Recent studies have underscored the critical role of retinoic acid (RA) in the development of lineage-committed CD4 and CD8 T cells in vivo. We have shown that under acute graft-versus-host disease (GVHD) inflammatory conditions, RA is upregulated in the intestine and is proinflammatory, as GVHD lethality was attenuated when donor allogeneic T cells selectively expressed a dominant negative RA receptor α that blunted RA signaling. RA can function in an autocrine and paracrine fashion, and as such, the host cell lineage responsible for the production of RA metabolism and the specific RA-metabolizing enzymes that potentiate GVHD severity are unknown. In this study, we demonstrate that enhancing RA degradation in the host and to a lesser extent donor hematopoietic cells by overexpressing the RA-catabolizing enzyme CYP26A1 reduced GVHD. RA production is facilitated by retinaldehyde isoform-2 (RALDH2) preferentially expressed in dendritic cells (DCs). Conditionally deleted RA-synthesizing enzyme RALDH2 in host or to a lesser extent donor DCs reduced GVHD lethality. Improved survival in recipients with RALDH2-deleted DCs was associated with increased T cell death, impaired T effector function, increased regulatory T cell frequency, and augmented coinhibitory molecule expression on donor CD4+ T cells. In contrast, retinaldehydrogenase isoform-1 (RALDH1) is dominantly expressed in intestinal epithelial cells. Unexpectedly, conditional host intestinal epithelial cells RALDH1 deletion failed to reduce GVHD. These data demonstrate the critical role of both donor and especially host RALDH2+ DCs in driving murine GVHD and suggest RALDH2 inhibition or CYP26A1 induction as novel therapeutic strategies to prevent GVHD.
Assuntos
Aldeído Oxirredutases/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Aldeído Oxirredutases/genética , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/patologia , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/imunologia , Tretinoína/imunologiaRESUMO
We previously reported that selective ablation of the nuclear receptors retinoid X receptor (RXR)-α and RXR-ß in mouse epidermal keratinocytes (RXR-αßep-/-) or a topical application of active vitamin D3 (VD3) and/or all-trans retinoic acid (RA) on wild-type mouse skin induces a human atopic dermatitis-like phenotype that is triggered by an increased expression of the thymic stromal lymphopoietin (TSLP) proinflammatory cytokine. We demonstrate here that in epidermal keratinocytes, unliganded heterodimers of vitamin D receptor (VDR)/RXR-α and retinoic acid receptor-γ (RAR-γ)/RXR-ß are bound as repressing complexes to their cognate DNA-binding sequence(s) (DBS) in the TSLP promoter regulatory region. Treatments with either an agonistic VD3 analog or RA dissociate the repressing complexes and recruit coactivator complexes and RNA polymerase II, thereby inducing transcription. Furthermore, we identified several functional NF-κB, activator protein 1 (AP1), STAT, and Smad DBS in the TSLP promoter region. Interestingly, many of these transcription factors and DBS present in the TSLP promoter region are differentially used in intestinal epithelial cell(s) (IEC). Collectively, our study reveals that, in vivo within their heterodimers, the RXR and RAR isotypes are not functionally redundant, and it also unveils the combinatorial mechanisms involved in the tissue-selective regulation of TSLP transcription in epidermal keratinocytes and IEC.
Assuntos
Citocinas/genética , Epiderme/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Pele/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Citocinas/metabolismo , Mucosa Intestinal/citologia , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Ligação Proteica , Multimerização Proteica , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Fatores de Transcrição/metabolismo , Linfopoietina do Estroma do Timo , Receptor gama de Ácido RetinoicoRESUMO
Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated repression, which can be mediated by a LBD-truncated GR, whereas it is mandatory for NCoR1-mediated repression through an interaction with K579 in the LBD.
Assuntos
Glucocorticoides/farmacologia , Histona Desacetilases/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Histona Desacetilases/genética , Camundongos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/genética , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , SumoilaçãoRESUMO
Upon binding of a glucocorticoid (GC), the GC receptor (GR) can exert one of three transcriptional regulatory functions. We recently reported that SUMOylation of the GR at position K293 in humans (K310 in mice) within the N-terminal domain is indispensable for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression. We now demonstrate that the integrity of this GR SUMOylation site is mandatory for the formation of a GR-small ubiquitin-related modifiers (SUMOs)-SMRT/NCoR1-HDAC3 repressing complex, which is indispensable for NF-κB/AP1-mediated GC-induced tethered indirect transrepression in vitro. Using GR K310R mutant mice or mice containing the N-terminal truncated GR isoform GRα-D3 lacking the K310 SUMOylation site, revealed a more severe skin inflammation than in WT mice. Importantly, cotreatment with dexamethasone (Dex) could not efficiently suppress a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in these mutant mice, whereas it was clearly decreased in WT mice. In addition, in mice selectively ablated in skin keratinocytes for either nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors or histone deacetylase 3 (HDAC3), Dex-induced tethered transrepression and the formation of a repressing complex on DNA-bound NF-κB/AP1 were impaired. We previously suggested that GR ligands that would lack both (+)GRE-mediated transactivation and IR nGRE-mediated direct transrepression activities of GCs may preferentially exert the therapeutically beneficial GC antiinflammatory properties. Interestingly, we now identified a nonsteroidal antiinflammatory selective GR agonist (SEGRA) that selectively lacks both Dex-induced (+)GRE-mediated transactivation and IR nGRE-mediated direct transrepression functions, while still exerting a tethered indirect transrepression activity and could therefore be clinically lesser debilitating on long-term GC therapy.
Assuntos
Dexametasona/farmacologia , Histona Desacetilases/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Ligantes , Camundongos , Camundongos Mutantes , SumoilaçãoRESUMO
The molecular mechanisms underlying the events through which alterations in diurnal activities impinge on peripheral circadian clocks (PCCs), and reciprocally how the PCCs affect metabolism, thereby generating pathologies, are still poorly understood. Here, we deciphered how switching the diurnal feeding from the active to the rest phase, i.e., restricted feeding (RF), immediately creates a hypoinsulinemia during the active phase, which initiates a metabolic reprogramming by increasing FFA and glucagon levels. In turn, peroxisome proliferator-activated receptor alpha (PPARα) activation by free fatty acid (FFA), and cAMP response element-binding protein (CREB) activation by glucagon, lead to further metabolic alterations during the circadian active phase, as well as to aberrant activation of expression of the PCC components nuclear receptor subfamily 1, group D, member 1 (Nr1d1/RevErbα), Period (Per1 and Per2). Moreover, hypoinsulinemia leads to an increase in glycogen synthase kinase 3ß (GSK3ß) activity that, through phosphorylation, stabilizes and increases the level of the RevErbα protein during the active phase. This increase then leads to an untimely repression of expression of the genes containing a RORE DNA binding sequence (DBS), including the Bmal1 gene, thereby initiating in RF mice a 12-h PCC shift to which the CREB-mediated activation of Per1, Per2 by glucagon modestly contributes. We also show that the reported corticosterone extraproduction during the RF active phase reflects an adrenal aberrant activation of CREB signaling, which selectively delays the activation of the PPARα-RevErbα axis in muscle and heart and accounts for the retarded shift of their PCCs.
Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar , Fatores de Transcrição ARNTL/genética , Animais , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Feminino , Regulação da Expressão Gênica , Glucagon/metabolismo , Homeostase , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculos/metabolismo , Mutação , PPAR alfa/metabolismo , Proteínas Circadianas Period/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The light-entrained master central circadian clock (CC) located in the suprachiasmatic nucleus (SCN) not only controls the diurnal alternance of the active phase (the light period of the human light-dark cycle, but the mouse dark period) and the rest phase (the human dark period, but the mouse light period), but also synchronizes the ubiquitous peripheral CCs (PCCs) with these phases to maintain homeostasis. We recently elucidated in mice the molecular signals through which metabolic alterations induced on an unusual feeding schedule, taking place during the rest phase [i.e., restricted feeding (RF)], creates a 12-h PCC shift. Importantly, a previous study showed that the SCN CC is unaltered during RF, which creates a misalignment between the RF-shifted PCCs and the SCN CC-controlled phases of activity and rest. However, the molecular basis of SCN CC insensitivity to RF and its possible pathological consequences are mostly unknown. Here we deciphered, at the molecular level, how RF creates this misalignment. We demonstrate that the PPARα and glucagon receptors, the two instrumental transducers in the RF-induced shift of PCCs, are not expressed in the SCN, thereby preventing on RF a shift of the master SCN CC and creating the misalignment. Most importantly, this RF-induced misalignment leads to a misexpression (with respect to their normal physiological phase of expression) of numerous CC-controlled homeostatic genes, which in the long term generates in RF mice a number of metabolic pathologies including diabetes, obesity, and metabolic syndrome, which have been reported in humans engaged in shift work schedules.