RESUMEN
The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion.
Asunto(s)
Adipogénesis , Tejido Adiposo/citología , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células 3T3-L1 , Tejido Adiposo/metabolismo , Envejecimiento , Animales , Células Dendríticas/citología , Metabolismo Energético , Femenino , Eliminación de Gen , Glucosa/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Homeostasis , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/citología , Células Mieloides/metabolismoRESUMEN
Macrophage metalloelastase, a matrix metallopeptidase (MMP12) predominantly expressed by mature tissue macrophages, is implicated in pathological processes. However, physiological functions for MMP12 have not been described. Because mRNA levels for the enzyme increase markedly in adipose tissue of obese mice, we investigated the role of MMP12 in adipose tissue expansion and insulin resistance. In humans, MMP12 expression correlated positively and significantly with insulin resistance, TNF-α expression, and the number of CD14(+)CD206(+) macrophages in adipose tissue. MMP12 was the most abundant matrix metallopeptidase detected by proteomic analysis of conditioned medium of M2 macrophages and dendritic cells. In contrast, it was detected only at low levels in bone marrow derived macrophages and M1 macrophages. When mice received a high-fat diet, adipose tissue mass increased and CD11b(+)F4/80(+)CD11c(-) macrophages accumulated to a greater extent in MMP12-deficient (Mmp12(-/-)) mice than in wild-type mice (Mmp12(+/+)). Despite being markedly more obese, fat-fed Mmp12(-/-) mice were more insulin sensitive than fat-fed Mmp12(+/+) mice. Expression of inducible nitric oxide synthase (Nos2) by Mmp12(-/-) macrophages was significantly impaired both in vivo and in vitro, suggesting that MMP12 might mediate nitric oxide production during inflammation. We propose that MMP12 acts as a double-edged sword by promoting insulin resistance while combatting adipose tissue expansion.
Asunto(s)
Tejido Adiposo/enzimología , Insulina/metabolismo , Macrófagos/enzimología , Metaloproteinasa 12 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Obesidad/enzimología , Tejido Adiposo/crecimiento & desarrollo , Tejido Adiposo/metabolismo , Adulto , Animales , Femenino , Humanos , Técnicas In Vitro , Resistencia a la Insulina , Macrófagos/metabolismo , Masculino , Metaloproteinasa 12 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/metabolismo , Obesidad/genética , Obesidad/metabolismo , Adulto JovenRESUMEN
Testicular nuclear receptor 4 (TR4), also known as NR2C2, belongs to the nuclear receptor superfamily and shares high homology with the testicular nuclear receptor 2. The natural ligands of TR4 remained unclear until the recent discoveries of several energy/lipid sensors including the polyunsaturated fatty acid metabolites, 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, and their synthetic ligands, thiazolidinediones, used for treatment of diabetes. TR4 is widely expressed throughout the body and particularly concentrated in the testis, prostate, cerebellum, and hippocampus. It has been shown to play important roles in cerebellar development, forebrain myelination, folliculogenesis, gluconeogenesis, lipogenesis, muscle development, bone development, and prostate cancer progression. Here we provide a comprehensive summary of TR4 signaling including its upstream ligands/activators/suppressors, transcriptional coactivators/repressors, downstream targets, and their in vivo functions with potential impacts on TR4-related diseases. Importantly, TR4 shares similar ligands/activators with another key nuclear receptor, peroxisome proliferator-activated receptor γ, which raised several interesting questions about how these 2 nuclear receptors may collaborate with or counteract each other's function in their related diseases. Clear dissection of such molecular mechanisms and their differential roles in various diseases may help researchers to design new potential drugs with better efficacy and fewer side effects to battle TR4 and peroxisome proliferator-activated receptor γ involved diseases.
Asunto(s)
Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal , Envejecimiento , Animales , Enfermedades Cardiovasculares/genética , Humanos , Infertilidad/genética , Síndrome Metabólico/genética , Ratones Noqueados , Neoplasias/genética , Procesamiento Proteico-Postraduccional , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/fisiologíaRESUMEN
Clinical investigations highlight the increased incidence of metabolic syndrome in prostate cancer (PCa) patients receiving androgen deprivation therapy (ADT). Studies using global androgen receptor (AR) knockout mice demonstrate that AR deficiency results in the development of insulin resistance in males. However, mechanisms by which AR in individual organs coordinately regulates insulin sensitivity remain unexplored. Here we tested the hypothesis that functional AR in the brain contributes to whole-body insulin sensitivity regulation and to the metabolic abnormalities developed in AR-deficient male mice. The mouse model selectively lacking AR in the central nervous system and AR-expressing GT1-7 neuronal cells were established and used to delineate molecular mechanisms in insulin signaling modulated by AR. Neuronal AR deficiency leads to reduced insulin sensitivity in middle-aged mice. Neuronal AR regulates hypothalamic insulin signaling by repressing nuclear factor-κB (NF-κB)-mediated induction of protein-tyrosine phosphatase 1B (PTP1B). Hypothalamic insulin resistance leads to hepatic insulin resistance, lipid accumulation, and visceral obesity. The functional deficiency of AR in the hypothalamus leads to male mice being more susceptible to the effects of high-fat diet consumption on PTP1B expression and NF-κB activation. These findings suggest that in men with PCa undergoing ADT, reduction of AR function in the brain may contribute to insulin resistance and visceral obesity. Pharmacotherapies targeting neuronal AR and NF-κB may be developed to combat the metabolic syndrome in men receiving ADT and in elderly men with age-associated hypogonadism.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hipotálamo/metabolismo , Resistencia a la Insulina/fisiología , FN-kappa B/metabolismo , Neuronas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/biosíntesis , Receptores Androgénicos/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Dieta Alta en Grasa , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina/genética , Leptina/sangre , Leptina/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad Abdominal/genética , Obesidad Abdominal/metabolismo , Receptores Androgénicos/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. METHODS: We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. RESULTS: We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. CONCLUSIONS: Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.
Asunto(s)
Remodelación Ósea , Osteoblastos/metabolismo , Osteocalcina , Osteoporosis/metabolismo , Regiones Promotoras Genéticas , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Regulación hacia Arriba , Animales , Animales Recién Nacidos , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Huesos/metabolismo , Huesos/patología , Diferenciación Celular , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Noqueados , Osteoblastos/patología , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis , Osteoporosis/patología , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genéticaRESUMEN
Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.
Asunto(s)
Diferenciación Celular/inmunología , Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Células Mieloides/inmunología , Proteoma/metabolismo , Animales , Cromatografía Liquida , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Proteoma/genética , Proteómica , Espectrometría de Masas en TándemRESUMEN
The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/antagonistas & inhibidores , Radiación Ionizante , Animales , Apoptosis/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Reparación del ADN/efectos de la radiación , Exones , Fibroblastos/metabolismo , Intrones , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/genética , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Successful reproductive efforts require the establishment of a situation favorable for reproduction that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related to reproduction have not been completely revealed. METHODS: Male TR4 knockout (TR4 -/-) and wild type mice were used for the sexual behavior and penile dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were analyzed by quantitative RT-PCR. Reporter gene assays were performed. RESULTS: We describe an unexpected finding of priapism in TR4 -/- mice. As a transcriptional factor, we demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein levels, consequently may lead to erectile dysfunction. In addition, male TR4 -/- mice display defects in sexual and social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation. Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior and stress response. CONCLUSIONS: Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male fertility via regulating behavior and internal physiological events.
Asunto(s)
Fertilidad , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/fisiología , Pene/metabolismo , Priapismo/metabolismo , Conducta Sexual Animal , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Masculino , Ratones , Ratones Noqueados , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Músculo Liso/patología , Músculo Liso/fisiopatología , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/genética , Pene/crecimiento & desarrollo , Pene/patología , Pene/fisiopatología , Priapismo/patología , Priapismo/fisiopatología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Índice de Severidad de la Enfermedad , Activación TranscripcionalRESUMEN
UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.
Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Regulación de la Expresión Génica , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Animales , Línea Celular , Daño del ADN , Reparación del ADN , Humanos , Ratones , Ratones Transgénicos , Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Transducción de Señal , Transcripción Genética , Rayos UltravioletaRESUMEN
OBJECTIVE: TR4 is a nuclear receptor without clear pathophysiological roles. We investigated the roles of hepatic TR4 in the regulation of lipogenesis and insulin sensitivity in vivo and in vitro. RESEARCH DESIGN AND METHODS: TR4 activity and phosphorylation assays were carried out using hepatocytes and various TR4 wild-type and mutant constructs. Liver tissues from TR4 knockout, C57BL/6, and db/db mice were examined to investigate TR4 target gene stearoyl-CoA desaturase (SCD) 1 regulation. RESULTS: TR4 transactivation is inhibited via phosphorylation by metformin-induced AMP-activated protein kinase (AMPK) at the amino acid serine 351, which results in the suppression of SCD1 gene expression. Additional mechanistic dissection finds TR4-transactivated SCD1 promoter activity via direct binding to the TR4-responsive element located at -243 to -255 on the promoter region. The pathophysiological consequences of the metforminâAMPKâTR4âSCD1 pathway are examined via TR4 knockout mice and primary hepatocytes with either knockdown or overexpression of TR4. The results show that the suppression of SCD1 via loss of TR4 resulted in reduced fat mass and increased insulin sensitivity with increased ß-oxidation and decreased lipogenic gene expression. CONCLUSIONS: The pathway from metforminâAMPKâTR4âSCD1âinsulin sensitivity suggests that TR4 may function as an important modulator to control lipid metabolism, which sheds light on the use of small molecules to modulate TR4 activity as a new alternative approach to battle the metabolic syndrome.
Asunto(s)
Hígado/metabolismo , Metformina/farmacología , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Prueba de Tolerancia a la Glucosa , Inmunoprecipitación , Insulina/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Estearoil-CoA Desaturasa/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Early studies suggest that TR4 nuclear receptor is a key transcriptional factor regulating various biological activities, including reproduction, cerebella development, and metabolism. Here we report that mice lacking TR4 (TR4(-/-)) exhibited increasing genome instability and defective oxidative stress defense, which are associated with premature aging phenotypes. At the cellular level, we observed rapid cellular growth arrest and less resistance to oxidative stress and DNA damage in TR4(-/-) mouse embryonic fibroblasts (MEFs) in vitro. Restoring TR4 or supplying the antioxidant N-acetyl-l-cysteine (NAC) to TR4(-/-) MEFs reduced the DNA damage and slowed down cellular growth arrest. Focused qPCR array revealed alteration of gene profiles in the DNA damage response (DDR) and anti-reactive oxygen species (ROS) pathways in TR4(-/-) MEFs, which further supports the hypothesis that the premature aging in TR4(-/-) mice might stem from oxidative DNA damage caused by increased oxidative stress or compromised genome integrity. Together, our finding identifies a novel role of TR4 in mediating the interplay between oxidative stress defense and aging.
Asunto(s)
Envejecimiento Prematuro/genética , Antioxidantes/metabolismo , Sistema Inmunológico/metabolismo , Estrés Oxidativo/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Envejecimiento Prematuro/metabolismo , Animales , Antioxidantes/fisiología , Células Cultivadas , Senescencia Celular/genética , Senescencia Celular/fisiología , Daño del ADN/genética , Daño del ADN/fisiología , Femenino , Inestabilidad Genómica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/fisiología , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In earlier studies, we had suggested that the fasting signal induces TR4 orphan nuclear receptor expression in vivo. The detailed mechanism(s), however, remain unclear. In this study, we found that cAMP/PKA, the mediator of fasting and glucagon signals, could induce TR4 gene expression that in turn modulates gluconeogenesis. Mechanistic dissection by in vitro studies in hepatocytes demonstrated that cAMP/PKA might trigger C/EBP alpha and beta binding to the selective cAMP response element, which is located at the TR4 promoter, thus inducing TR4 transcription. We also demonstrated that the binding activity of C/EBPs to the TR4 promoter is increased in response to cAMP treatment. Together, our data identified a new signaling pathway from the fasting signal --> cAMP/PKA --> C/EBP alpha and beta --> TR4 --> gluconeogenesis in hepatocytes; and suggested that TR4 could be an important regulator to control glucose homeostasis. The identification of activator(s)/inhibitor(s) or ligand(s) of TR4 may provide us an alternative way to control gluconeogenesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/farmacología , Regiones Promotoras Genéticas , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Activación Transcripcional , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , AMP Cíclico/fisiología , Regulación de la Expresión Génica , Gluconeogénesis/genética , Hepatocitos/metabolismo , Humanos , Modelos Biológicos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Activación Transcripcional/efectos de los fármacosRESUMEN
The testicular orphan nuclear receptors (TRs) 2 and 4 act as either transcriptional activators or regulatory proteins of other nuclear receptor superfamily members. With no identified cognate ligands, their physiological roles remain unclear. Here we showed the phenotypes of TR2(-/-):TR4(-/-) mutant embryos, which reveal that the loss of TR2 and TR4 causes early embryonic lethality and increased cell death. We also found that TR2 and TR4 are expressed in blastocysts and embryonic stem (ES) cells, and can act as transcriptional activators in ES cells. The results on further investigating the roles of TR2 and TR4 in ES cells showed that TR2 and TR4 were differentially expressed when ES cells were induced into different specialized cell types, and their expression is regulated by retinoic acid. Knocking down TR2 and TR4 mRNAs decreased the expression of Oct-3/4 and Nanog genes. Mechanism dissection suggests that TR2 and TR4 may affect the Oct-3/4 gene by binding to a direct repeat-1 element located in its promoter region, which is influenced by retinoic acid. Together, our findings highlight possible roles for TR2 and TR4 in early embryonic development by regulating key genes involved in stem cell self-renewal, commitment, and differentiation.
Asunto(s)
Desarrollo Embrionario/genética , Células Madre Embrionarias/metabolismo , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/fisiología , Adipogénesis/genética , Animales , Blastocisto/metabolismo , Blastocisto/fisiología , Diferenciación Celular/genética , Células Cultivadas , Cruzamientos Genéticos , Embrión de Mamíferos , Células Madre Embrionarias/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Neurogénesis/genética , Miembro 1 del Grupo C de la Subfamilia 2 de Receptores Nucleares , Osteogénesis/genética , Embarazo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Testículo/metabolismo , Tretinoina/farmacologíaRESUMEN
UNLABELLED: Early studies demonstrated that whole-body androgen receptor (AR)-knockout mice with hypogonadism exhibit insulin resistance. However, details about the mechanisms underlying how androgen/AR signaling regulates insulin sensitivity in individual organs remain unclear. We therefore generated hepatic AR-knockout (H-AR(-/y)) mice and found that male H-AR(-/y) mice, but not female H-AR(-/-) mice, fed a high-fat diet developed hepatic steatosis and insulin resistance, and aging male H-AR(-/y) mice fed chow exhibited moderate hepatic steatosis. We hypothesized that increased hepatic steatosis in obese male H-AR(-/y) mice resulted from decreased fatty acid beta-oxidation, increased de novo lipid synthesis arising from decreased PPARalpha, increased sterol regulatory element binding protein 1c, and associated changes in target gene expression. Reduced insulin sensitivity in fat-fed H-AR(-/y) mice was associated with decreased phosphoinositide-3 kinase activity and increased phosphenolpyruvate carboxykinase expression and correlated with increased protein-tyrosine phosphatase 1B expression. CONCLUSION: Together, our results suggest that hepatic AR may play a vital role in preventing the development of insulin resistance and hepatic steatosis. AR agonists that specifically target hepatic AR might be developed to provide a better strategy for treatment of metabolic syndrome in men.
Asunto(s)
Hígado Graso/etiología , Hígado Graso/metabolismo , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Receptores Androgénicos/metabolismo , Envejecimiento/metabolismo , Animales , Grasas de la Dieta/efectos adversos , Femenino , Glucosa/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Metabolismo de los Lípidos/fisiología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/complicaciones , Obesidad/etiología , PPAR alfa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Receptores Androgénicos/genética , Caracteres SexualesRESUMEN
Early studies reveal that testicular orphan nuclear receptor 4 (TR4) modulates signaling pathways that control various cell functions. However, how TR4 activity is regulated without the involvement of specific ligand(s) remains unclear. Here we identify a daf-16 family protein-binding element (DBE; 5'-TGTTTAC-3') in the TR4 promoter that can be recognized by the forkhead transcriptional factor FOXO3a, a key stress-responsive factor, through which TR4 gene expression is activated. The interaction between DBE and FOXO3a was confirmed using EMSA and chromatin immunoprecipitation assays. Activation of FOXO3a by oxidative stress and phosphatidylinositol 3-kinase inhibitor induced TR4 expression; in contrast, suppression of FOXO3a by small interfering RNA can reduce oxidative stress-induced TR4 expression. The biological consequence of the FOXO3a-induced TR4 by oxidative stress is to protect against stress-induced cell death in which cells with reduced FOXO3a are less resistant to oxidative stress, and addition of functional TR4 can increase stress resistance. These results suggest that this new identified oxidative stress-FOXO3a-TR4 pathway is a fundamentally important mechanism regulating stress resistance and cell survival.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Ensayo de Cambio de Movilidad Electroforética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Microscopía Fluorescente , Morfolinas/farmacología , Oxidantes/farmacología , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Testículo/metabolismoRESUMEN
Insulin resistance occurs through an inadequate response to insulin by insulin target organs such as liver, muscle, and adipose tissue with consequent insufficient glucose uptake. In previous studies we demonstrated that whole body androgen receptor (AR) knockout (AR(-/y)) mice develop obesity and exhibit insulin and leptin resistance at advanced age. By examining adipose tissue-specific AR knockout (A-AR(-/y)) mice, we found A-AR(-/y) mice were hyperleptinemic but showed no leptin resistance, although body weight and adiposity index of A-AR(-/y) mice were identical with those of male wild-type control mice. Hypotriglyceridemia and hypocholesterolemia found in nonobese A-AR(-/y) mice suggested a beneficial effect of high leptin levels independent of fat deposition. Further examination showed that androgen-AR signaling in adipose tissue plays a direct regulatory role in leptin expression via enhanced estrogen receptor transactivation activity due to elevated intraadipose estrogens. The present study in A-AR(-/y) mice suggests a differential tissue-specific role of AR in energy balance control in males.
Asunto(s)
Tejido Adiposo/metabolismo , Leptina/sangre , Enfermedades Metabólicas/genética , Receptores Androgénicos/genética , Células 3T3-L1 , Animales , Distribución de la Grasa Corporal , Células Cultivadas , Embrión de Mamíferos , Metabolismo Energético/genética , Fibroblastos/metabolismo , Integrasas/genética , Leptina/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/sangre , Masculino , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/sangre , Obesidad/genética , Especificidad de Órganos/genética , Oxidación-Reducción , Receptores Androgénicos/metabolismoRESUMEN
Testicular orphan nuclear receptor 4 (TR4) plays essential roles for normal spermatogenesis in male mice. However, its roles in female fertility and ovarian function remain largely unknown. Here we found female mice lacking TR4 (TR4-/-) displayed subfertility and irregular estrous cycles. TR4-/- female mice ovaries were smaller with fewer or no preovulatory follicles and corpora lutea. After superovulation, TR4-/- female mice produced fewer oocytes, preovulatory follicles, and corpora lutea. In addition, more intensive granulosa apoptosis was found in TR4-/- ovaries. Functional analyses suggest that subfertility in TR4-/- female mice can be due to an ovarian defect with impaired folliculogenesis rather than a deficiency in pituitary gonadotropins. Molecular mechanism dissection of defective folliculogenesis found TR4 might induce LH receptor (LHR) gene expression via direct binding to its 5' promoter. The consequence of reduced LHR expression in TR4-/- female mice might then result in reduced gonadal sex hormones via reduced expression of enzymes involved in steroidogenesis. Together, our results showed TR4 might play essential roles in normal folliculogenesis by influencing LHR signals. Modulation of TR4 expression and/or activation via its upstream signals or unidentified ligand(s) might allow us to develop small molecule(s) to control folliculogenesis.
Asunto(s)
Infertilidad Femenina/fisiopatología , Folículo Ovárico/patología , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/fisiología , Animales , Apoptosis/genética , Apoptosis/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Estradiol/metabolismo , Femenino , Genitales Femeninos/metabolismo , Genitales Femeninos/patología , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Infertilidad Femenina/genética , Masculino , Ratones , Ratones Noqueados , Folículo Ovárico/metabolismo , Ovario/metabolismo , Ovario/patología , Progesterona/metabolismo , Receptores de HL/genética , Receptores de HL/fisiología , Receptores de Esteroides/deficiencia , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/deficiencia , Receptores de Hormona Tiroidea/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: Regulation of phosphoenolpyruvate carboxykinase (PEPCK), the key gene in gluconeogenesis, is critical for glucose homeostasis in response to quick nutritional depletion and/or hormonal alteration. RESEARCH DESIGN/METHODS AND RESULTS: Here, we identified the testicular orphan nuclear receptor 4 (TR4) as a key PEPCK regulator modulating PEPCK gene via a transcriptional mechanism. TR4 transactivates the 490-bp PEPCK promoter-containing luciferase reporter gene activity by direct binding to the TR4 responsive element (TR4RE) located at -451 to -439 in the promoter region. Binding to TR4RE was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Eliminating TR4 via knockout and RNA interference (RNAi) in hepatocytes significantly reduced the PEPCK gene expression and glucose production in response to glucose depletion. In contrast, ectopic expression of TR4 increased PEPCK gene expression and hepatic glucose production in human and mouse hepatoma cells. Mice lacking TR4 also display reduction of PEPCK expression with impaired gluconeogenesis. CONCLUSIONS: Together, both in vitro and in vivo data demonstrate the identification of a new pathway, TR4 --> PEPCK --> gluconeogenesis --> blood glucose, which may allow us to modulate metabolic programs via the control of a new key player, TR4, a member of the nuclear receptor superfamily.
Asunto(s)
Gluconeogénesis/fisiología , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Animales , Células Cultivadas , Regulación de la Expresión Génica , Hepatocitos/fisiología , Masculino , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Interferencia de ARN , Ratas , Receptores de Esteroides/deficiencia , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/deficiencia , Receptores de Hormona Tiroidea/fisiología , Transcripción Genética , TransfecciónRESUMEN
While other plasma lipoproteins are exclusively expressed in liver and intestine, apoliprotein E (apoE) is ubiquitously synthesized in many tissues. To understand the molecular mechanism of non-tissue-specific apoE expression, we tested the testicular orphan receptor 4 (TR4) effect on apoE expression in different cell lines, such as HepG2, COS-1, and H1299 cells. Gel shift assay and 5' promoter activity analyses identified one distinct hormone response element (TR4RE-DR0-apoE at -303 to -292 bp) that binds to TR4 and results in full induction of apoE gene transcription. TR4 also forms a complex with Sp1 to synergistically induce apoE expression via a region containing the TR4RE-DR0-apoE and the Sp1 binding site (-169 to -140 bp). Induction of apoE expression by TR4 was also confirmed at the mRNA and protein levels in H1299 cells. Together, our data demonstrate that TR4 can enhance apoE gene expression via binding to TR4RE-DR0 in apoE 5' promoter and this TR4 binding is essential for synergistic interaction with another transcription factor, Sp1.
Asunto(s)
Región de Flanqueo 5'/genética , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/genética , Regiones Promotoras Genéticas/genética , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Elementos de Respuesta/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Regulación de la Expresión Génica/fisiología , Hepatocitos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption (TR4(-/-)). The number of TR4(-/-) pups generated by the mating of TR4(+/-) mice is well under that predicted by the normal Mendelian ratio, and TR4(-/-) mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4(-/-) females show defects in reproduction and maternal behavior, with pups of TR4(-/-) dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.