RESUMEN
Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Regulación de la Expresión Génica , Intestino Delgado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Linfocitos T/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Receptor de Androstano Constitutivo , Enfermedad de Crohn/metabolismo , Femenino , Ileítis/metabolismo , Inflamación/metabolismo , Interleucina-10/biosíntesis , Interleucina-10/genética , Intestino Delgado/citología , RatonesRESUMEN
Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown3,4. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined3,4. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.
Asunto(s)
Adipocitos/metabolismo , Hemo/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Progesterona/metabolismo , Animales , Homeostasis , Humanos , Espacio Intracelular/metabolismo , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Progesterona/deficiencia , Receptores de Progesterona/genética , Transcripción GenéticaRESUMEN
T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células Th17/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Sitios Genéticos , Células HEK293 , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Ratones , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , RNA-Seq , Elementos de Respuesta/genética , Células Th17/metabolismo , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.
Asunto(s)
Materiales Biomiméticos/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Distrofia Muscular de Duchenne/tratamiento farmacológico , Neumonía/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Materiales Biomiméticos/química , Línea Celular , Femenino , Células HEK293 , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Inflamación/tratamiento farmacológico , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Necrosis/tratamiento farmacológico , Dominios Proteicos , Células RAW 264.7RESUMEN
The nuclear receptors REV-ERBα and REV-ERBß have been demonstrated to play key roles in the regulation of numerous physiological functions, such as metabolism and the circadian rhythm. Recent studies have established the REV-ERBs' roles in immunity, including macrophage and T cell responses. In contrast, their roles in dendritic cells have not been well defined. Dendritic cells are potent antigen presenting cells, connecting microbial sensing and innate immunity to adaptive immune responses. We demonstrate that both REV-ERBα and REV-ERBß expression is upregulated during the course of bone marrow derived dendritic cell (BMDC) differentiation. BMDCs from REV-ERBα and REV-ERBß deficient mice showed enhanced expression of maturation markers like CD86, MHCII, and proinflammatory cytokines. Conversely, treatment of BMDCs with a REV-ERB-specific agonist, SR9009, inhibited the expression of maturation markers and proinflammatory cytokines. Our study suggests the REV-ERBs act as negative regulators of dendritic cell development and activation. These results indicate that pharmacological modulation of REV-ERB activity could be an attractive strategy to modulate DC activation status and for DC-based therapies.
Asunto(s)
Células de la Médula Ósea/citología , Células Dendríticas/citología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genética , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Pirrolidinas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Proteínas Represoras/agonistas , Tiofenos/farmacologíaRESUMEN
Pathological proliferation of retinal blood vessels commonly causes vision impairment in proliferative retinopathies, including retinopathy of prematurity. Dysregulated crosstalk between the vasculature and retinal neurons is increasingly recognized as a major factor contributing to the pathogenesis of vascular diseases. Class 3 semaphorins (SEMA3s), a group of neuron-secreted axonal and vascular guidance factors, suppress pathological vascular growth in retinopathy. However, the upstream transcriptional regulators that mediate the function of SEMA3s in vascular growth are poorly understood. Here we showed that retinoic acid receptor-related orphan receptor α (RORα), a nuclear receptor and transcription factor, is a novel transcriptional regulator of SEMA3E-mediated neurovascular coupling in a mouse model of oxygen-induced proliferative retinopathy. We found that genetic deficiency of RORα substantially induced Sema3e expression in retinopathy. Both RORα and SEMA3E were expressed in retinal ganglion cells. RORα directly bound to a specific ROR response element on the promoter of Sema3e and negatively regulated Sema3e promoter-driven luciferase expression. Suppression of Sema3e using adeno-associated virus 2 carrying short hairpin RNA targeting Sema3e promoted disoriented pathological neovascularization and partially abolished the inhibitory vascular effects of RORα deficiency in retinopathy. Our findings suggest that RORα is a novel transcriptional regulator of SEMA3E-mediated neurovascular coupling in pathological retinal angiogenesis.-Sun, Y., Liu, C.-H., Wang, Z., Meng, S. S., Burnim, S. B., SanGiovanni, J. P., Kamenecka, T. M., Solt, L. A., Chen, J. RORα modulates semaphorin 3E transcription and neurovascular interaction in pathological retinal angiogenesis.
Asunto(s)
Glicoproteínas/genética , Proteínas de la Membrana/genética , Neovascularización Patológica/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Neovascularización Retiniana/metabolismo , Vasos Retinianos/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas del Citoesqueleto , Células Endoteliales/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Neovascularización Patológica/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Células Ganglionares de la Retina , Neovascularización Retiniana/genética , SemaforinasRESUMEN
We sought to develop RORß-selective probe molecules in order to investigate the function of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To accomplish this, we modified a potent dual RORß/RORγ inverse agonist from the primary literature with the goal of improving selectivity for RORß vs RORγ. Truncation of the Western portion of the molecule ablated activity at RORγ and led to a potent series of RORß modulators. Continued exploration of this series investigated alternate replacement cores for the aminothiazole ring. Numerous suitable replacements were found during the course of our SAR investigations and are reported herein.
Asunto(s)
Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Tiofenos/farmacología , Humanos , Espectrometría de Masas/métodosRESUMEN
Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-ß have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.
Asunto(s)
Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/fisiología , Metabolismo Energético/efectos de los fármacos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Pirrolidinas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Tiofenos/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/genética , Relojes Biológicos/fisiología , Ritmo Circadiano/genética , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Metaboloma/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Obesidad/inducido químicamente , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Pathologic ocular angiogenesis is a leading cause of blindness, influenced by both dysregulated lipid metabolism and inflammation. Retinoic-acid-receptor-related orphan receptor alpha (RORα) is a lipid-sensing nuclear receptor with diverse biologic function including regulation of lipid metabolism and inflammation; however, its role in pathologic retinal angiogenesis remains poorly understood. Using a mouse model of oxygen-induced proliferative retinopathy, we showed that RORα expression was significantly increased and genetic deficiency of RORα substantially suppressed pathologic retinal neovascularization. Loss of RORα led to decreased levels of proinflammatory cytokines and increased levels of antiinflammatory cytokines in retinopathy. RORα directly suppressed the gene transcription of suppressors of cytokine signaling 3 (SOCS3), a critical negative regulator of inflammation. Inhibition of SOCS3 abolished the antiinflammatory and vasoprotective effects of RORα deficiency in vitro and in vivo. Moreover, treatment with a RORα inverse agonist SR1001 effectively protected against pathologic neovascularization in both oxygen-induced retinopathy and another angiogenic model of very-low-density lipoprotein receptor (Vldlr)-deficient (Vldlr (-/-) ) mice with spontaneous subretinal neovascularization, whereas a RORα agonist worsened oxygen-induced retinopathy. Our data demonstrate that RORα is a novel regulator of pathologic retinal neovascularization, and RORα inhibition may represent a new way to treat ocular neovascularization.
Asunto(s)
Neovascularización Patológica , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Retina/patología , Neovascularización Retiniana , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Antiinflamatorios/química , Proliferación Celular , Inmunoprecipitación de Cromatina , Citocinas/metabolismo , Heterocigoto , Homocigoto , Inflamación/patología , Lípidos/química , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Oxígeno/química , Unión Proteica , ARN Mensajero/metabolismo , Receptores de LDL/genética , Proteína 3 Supresora de la Señalización de CitocinasRESUMEN
An effective adaptive immune response relies on the ability of lymphocytes to rapidly act upon a variety of insults. In T lymphocytes, this response includes cell growth, clonal expansion, differentiation, and cytokine production, all of which place a significant energy burden on the cell. Recent evidence shows that T-cell metabolic reprogramming is an essential component of the adaptive immune response and specific metabolic pathways dictate T-cell fate decisions, including the development of TH 17 versus T regulatory (Treg) cells. TH 17 cells have garnered significant attention due to their roles in the pathology of immune-mediated inflammatory diseases. Attempts to characterize TH 17 cells have demonstrated that they are highly dynamic, adjusting their function to environmental cues, which dictate their metabolic program. In this review, we highlight recent data demonstrating the impact of cellular metabolism on the TH 17/Treg balance and present factors that mediate TH 17-cell metabolism. Some examples of these include the differential impact of the mTOR signaling complexes on T-helper-cell differentiation, hypoxia inducible factor 1 alpha (HIF1α) promotion of glycolysis to favor TH 17-cell development, and ACC1-dependent de novo fatty acid synthesis favoring TH 17-cell development over Treg cells. Finally, we discuss the potential therapeutic options and the implications of modulating TH 17-cell metabolism for the treatment of TH 17-mediated diseases.
Asunto(s)
Diferenciación Celular/inmunología , Glucólisis/fisiología , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Acetiltransferasas/metabolismo , Animales , Recuento de Linfocito CD4 , Proliferación Celular , Ácidos Grasos/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Células Th17/citología , Células Th17/inmunologíaRESUMEN
T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Sulfonamidas/farmacología , Células Th17/citología , Células Th17/inmunología , Tiazoles/farmacología , Animales , Autoinmunidad/inmunología , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células Th17/efectos de los fármacos , Células Th17/metabolismoRESUMEN
Nuclear receptors are ligand-activated transcription factors and include the receptors for steroid hormones, lipophilic vitamins, sterols, and bile acids. These receptors serve as targets for development of myriad drugs that target a range of disorders. Classically defined ligands that bind to the ligand-binding domain of nuclear receptors, whether they are endogenous or synthetic, either activate receptor activity (agonists) or block activation (antagonists) and due to the ability to alter activity of the receptors are often termed receptor "modulators." The complex pharmacology of nuclear receptors has provided a class of ligands distinct from these simple modulators where ligands display agonist/partial agonist/antagonist function in a tissue or gene selective manner. This class of ligands is defined as selective modulators. Here, we review the development and pharmacology of a range of selective nuclear receptor modulators.
Asunto(s)
Descubrimiento de Drogas/métodos , Receptores Citoplasmáticos y Nucleares , Animales , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiologíaRESUMEN
REV-ERBα and REV-ERBß are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERBß can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERBß with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERBß indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERBß crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.
Asunto(s)
Porfirinas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Hemo/metabolismo , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/química , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Porfirinas/agonistas , Unión Proteica , Estructura Terciaria de Proteína , Protoporfirinas/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genéticaRESUMEN
The nuclear receptors for heme, REV-ERBα and REV-ERBß, play important roles in the regulation of metabolism and inflammation. Recently it was demonstrated that reduced REV-ERBα expression in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model. A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Atherosclerotic plaque size was significantly reduced (p < 0.05) in mice administered SR9009 (100 mg/kg) for seven weeks compared to control mice (n = 10 per group). SR9009 treatment of bone marrow-derived mouse macrophages (BMDM) reduced the polarization of BMDMs to proinflammatory M1 macrophage while increasing the polarization of BMDMs to anti-inflammatory M2 macrophages. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de LDL/genética , Receptores de LDL/fisiologíaRESUMEN
Choroidal neovascularization (CNV) causes acute vision loss in neovascular age-related macular degeneration (AMD). Genetic variations of the nuclear receptor RAR-related orphan receptor alpha (RORα) have been linked with neovascular AMD, yet its specific role in pathological CNV development is not entirely clear. In this study, we showed that Rora was highly expressed in the mouse choroid compared with the retina, and genetic loss of RORα in Staggerer mice (Rorasg/sg) led to increased expression levels of Vegfr2 and Tnfa in the choroid and retinal pigment epithelium (RPE) complex. In a mouse model of laser-induced CNV, RORα expression was highly increased in the choroidal/RPE complex post-laser, and loss of RORα in Rorasg/sg eyes significantly worsened CNV with increased lesion size and vascular leakage, associated with increased levels of VEGFR2 and TNFα proteins. Pharmacological inhibition of RORα also worsened CNV. In addition, both genetic deficiency and inhibition of RORα substantially increased vascular growth in isolated mouse choroidal explants ex vivo. RORα inhibition also promoted angiogenic function of human choroidal endothelial cell culture. Together, our results suggest that RORα negatively regulates pathological CNV development in part by modulating angiogenic response of the choroidal endothelium and inflammatory environment in the choroid/RPE complex.
Asunto(s)
Neovascularización Coroidal , Degeneración Macular Húmeda , Ratones , Humanos , Animales , Inhibidores de la Angiogénesis , Factor A de Crecimiento Endotelial Vascular/metabolismo , Agudeza Visual , Degeneración Macular Húmeda/complicaciones , Neovascularización Coroidal/genética , Neovascularización Coroidal/tratamiento farmacológico , Rayos Láser , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Autoimmune diseases affect 50 million Americans, predominantly women, and are thought to be one of the top 10 leading causes of death among women in age groups up to 65 years. A central role for TH17 cells has been highlighted by genome-wide association studies (GWAS) linking genes preferentially expressed in TH17 cells to several human autoimmune diseases. We and others have reported that the nuclear receptors REV-ERBα and ß are cell-intrinsic repressors of TH17 cell development and pathogenicity and might therefore be therapeutic targets for intervention. Herein, we describe detailed SAR studies of a novel REV-ERBα-selective scaffold. Metabolic stability of the ligands was optimized allowing for in vivo interrogation of the receptor in a mouse model of multiple sclerosis (EAE) with a ligand (34). Reduction in frequency and number of T-cells in the CNS as well as key REV-ERB target genes is a measure of target engagement in vivo.
Asunto(s)
Estudio de Asociación del Genoma Completo , Esclerosis Múltiple , Ratones , Animales , Humanos , Femenino , Masculino , Factores de Transcripción/genética , Diferenciación Celular , Esclerosis Múltiple/tratamiento farmacológico , Relación Estructura-Actividad , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismoRESUMEN
The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA binding affinity and a 5 °C decrease in thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in an only 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to those of other Pitx2 homeodomain mutations.
Asunto(s)
Fenómenos Biofísicos , Enfermedades de la Conjuntiva/genética , Enfermedades de la Córnea/genética , Quiste Dermoide/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Genes Reporteros/genética , Histidina , Proteínas de Homeodominio/genética , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Luciferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Terciaria de Proteína , Soluciones , Termodinámica , Factores de Transcripción/genética , Proteína del Homeodomínio PITX2RESUMEN
The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.
Asunto(s)
Membrana Celular/metabolismo , Metabolismo Energético/genética , Homeostasis/genética , Receptor de Melanocortina Tipo 3/genética , Receptor de Melanocortina Tipo 3/metabolismo , Alelos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Codón/genética , Femenino , Técnicas de Inactivación de Genes , Sitios Genéticos/genética , Genotipo , Masculino , Metaboloma/genética , Ratones , Ratones Transgénicos , Obesidad/genética , Fenotipo , Receptor de Melanocortina Tipo 3/deficienciaRESUMEN
The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas Portadoras/fisiología , Línea Celular , Células HeLa , Proteínas de Andamiaje Homer , Humanos , Proteínas I-kappa B/fisiología , Sinapsis Inmunológicas/enzimología , Sinapsis Inmunológicas/inmunología , Células Jurkat , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Fracciones Subcelulares/inmunología , Fracciones Subcelulares/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
T cells rapidly transition from a quiescent state into active proliferation and effector function upon exposure to cognate antigen. These processes are tightly controlled by signal transduction pathways that influence changes in chromatin remodeling, gene transcription, and metabolism, all of which collectively drive specific T cell memory or effector cell development. Dysregulation of any of these events can mediate disease and the past several years has shown unprecedented novel approaches to understand these events, down to the single-cell level. The massive explosion of sequencing approaches to assess the genome and transcriptome at the single cell level has transformed our understanding of T cell activation, developmental potential, and effector function under normal and various disease states. Despite these advances, there remains a significant dearth of information regarding how these events are translated to the protein level. For example, resolution of protein isoforms and/or specific post-translational modifications mediating T cell function remains obscure. The application of proteomics can change that, enabling significant insights into molecular mechanisms that regulate T cell function. However, unlike genomic approaches that have enabled exquisite visualization of T cell dynamics at the mRNA and chromatin level, proteomic approaches, including those at the single-cell level, has significantly lagged. In this review, we describe recent studies that have enabled a better understanding of how protein synthesis and degradation change during T cell activation and acquisition of effector function. We also highlight technical advances and how these could be applied to T cell biology. Finally, we discuss future needs to expand upon our current knowledge of T cell proteomes and disease.