RESUMEN
Thermogenic adipose tissue, consisting of brown and beige fat, regulates nutrient utilization and energy metabolism. Human brown fat is relatively scarce and decreases with obesity and aging. Hence, inducing thermogenic differentiation of white fat offers an attractive way to enhance whole-body metabolic capacity. Here, we show the role of endothelin 3 (EDN3) and endothelin receptor type B (EDNRB) in promoting the browning of white adipose tissue (WAT). EDNRB overexpression stimulates thermogenic differentiation of human white preadipocytes through cAMP-EPAC1-ERK activation. In mice, cold induces the expression of EDN3 and EDNRB in WAT. Deletion of EDNRB in adipose progenitor cells impairs cold-induced beige adipocyte formation in WAT, leading to excessive weight gain, glucose intolerance, and insulin resistance upon high-fat feeding. Injection of EDN3 into WAT promotes browning and improved whole-body glucose metabolism. The findings shed light on the mechanism of WAT browning and offer potential therapeutics for obesity and metabolic disorders.
Asunto(s)
Tejido Adiposo Blanco , Diferenciación Celular , Endotelina-3 , Receptor de Endotelina B , Transducción de Señal , Termogénesis , Animales , Tejido Adiposo Blanco/metabolismo , Termogénesis/genética , Humanos , Ratones , Receptor de Endotelina B/metabolismo , Receptor de Endotelina B/genética , Endotelina-3/metabolismo , Endotelina-3/genética , Masculino , Obesidad/metabolismo , Obesidad/genética , Ratones Endogámicos C57BL , Dieta Alta en Grasa , Resistencia a la Insulina , Adipocitos Blancos/metabolismo , Ratones Noqueados , Adipocitos Beige/metabolismo , Tejido Adiposo Pardo/metabolismo , Intolerancia a la Glucosa/metabolismo , FríoRESUMEN
PURPOSE: Changes in the activity of endothelins and their receptors may promote neoplastic processes. They can be caused by epigenetic modifications and modulators, but little is known about endothelin-3 (EDN3), particularly in endometrial cancer. The aim of the study was to determine the expression profile of endothelin family and their interactions with miRNAs, and to assess the degree of EDN3 methylation. METHODS: The study enrolled 45 patients with endometrioid endometrial cancer and 30 patients without neoplastic changes. The expression profile of endothelins and their receptors was determined with mRNA microarrays and RT-qPCR. The miRNA prediction was based on the miRNA microarray experiment and the mirDB tool. The degree of EDN3 methylation was assessed by MSP. RESULTS: EDN1 and EDNRA were overexpressed regardless of endometrial cancer grade, which may be due to the lack of regulatory effect of miR-130a-3p and miR-485-3p, respectively. In addition, EDN3 and EDNRB were significantly downregulated. CONCLUSION: The endothelial axis is disturbed in endometrioid endometrial cancer. The observed silencing of EDN3 activity may be mainly due to DNA methylation.
Asunto(s)
Carcinoma Endometrioide , Neoplasias Endometriales , MicroARNs , Femenino , Humanos , Endotelina-3/genética , Endotelina-3/metabolismo , Endotelinas/genética , Endotelinas/metabolismo , MicroARNs/genética , Receptor de Endotelina A/genética , Neoplasias Endometriales/genética , Carcinoma Endometrioide/genética , Regulación Neoplásica de la Expresión Génica , Endotelina-1/genética , Endotelina-1/metabolismoRESUMEN
The development of the enteric nervous system (ENS) is highly modulated by the synchronized interaction between the enteric neural crest cells (ENCCs) and the neural stem cell niche comprising the gut microenvironment. Genetic defects dysregulating the cellular behaviour(s) of the ENCCs result in incomplete innervation and hence ENS dysfunction. Hirschsprung disease (HSCR) is a rare complex neurocristopathy in which the enteric neural crest-derived cells fail to colonize the distal colon. In addition to ENS defects, increasing evidence suggests that HSCR patients may have intrinsic defects in the niche impairing the extracellular matrix (ECM)-cell interaction and/or dysregulating the cellular niche factors necessary for controlling stem cell behaviour. The niche defects in patients may compromise the regenerative capacity of the stem cell-based therapy and advocate for drug- and niche-based therapies as complementary therapeutic strategies to alleviate/enhance niche-cell interaction. Here, we provide a summary of the current understandings of the role of the enteric neural stem cell niche in modulating the development of the ENS and in the pathogenesis of HSCR. Deciphering the contribution of the niche to HSCR may provide important implications to the development of regenerative medicine for HSCR.
Asunto(s)
Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/metabolismo , Enfermedad de Hirschsprung/genética , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Nicho de Células Madre , Animales , Biomarcadores , Diferenciación Celular , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Endotelina-3/metabolismo , Predisposición Genética a la Enfermedad , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Enfermedad de Hirschsprung/diagnóstico , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/terapia , Humanos , Cresta Neural/citología , Cresta Neural/metabolismo , Receptor de Endotelina B/metabolismo , Medicina Regenerativa , Transducción de SeñalRESUMEN
Endothelins induce many biological responses, and they are composed of three peptides: ET-1, ET-2, and ET-3. Reports have indicated that ET-1 regulates cell proliferation, adipogenesis, and other cell responses and that ET-3 stimulates the growth of gastrointestinal epithelial cells and melanocytes. However, the signalling pathways of ET3 that mediate the growth of fat cells are still unclear. Using 3T3-L1 white preadipocytes, we found that ET-3 induced increases in both cell number and BrdU incorporation. Pretreatment with an ETAR antagonist (but not an ETBR antagonist) blocked the ET-3-induced increases in both cell number and BrdU incorporation. Additionally, BQ610 suppressed the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3 proteins, and pretreatment with specific inhibitors of AMPK, JNK/c-JUN, or JAK/STAT3 prevented the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3, respectively. Neither p38 MAPK inhibitor nor PKC inhibitor altered the effects of ET-3 on cell growth. These data suggest that ET-3 stimulates preadipocyte growth through the ETAR, AMPK, JNK/c-JUN, and STAT3 pathways. Moreover, ET-3 did not alter HIB1B brown preadipocyte and D12 beige preadipocyte growth, suggesting a preadipocyte type-dependent effect. The results of this study may help explain how endothelin mediates fat cell activity and fat cell-associated diseases.
Asunto(s)
Adipocitos/citología , Endotelina-3/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Proliferación Celular , Endotelina-3/antagonistas & inhibidores , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Several studies on papillary thyroid cancer (PTC) have been performed. However, the effects of endothelin 3 (EDN3) and microRNA (miR)27a3p on PTC cells has yet to be investigated, to the best of the authors' knowledge. The present study aimed to explore the biological functions of EDN3 and miR27a3p in PTC cells. Bioinformatics analysis was conducted to identify possible key genes and miRs involved in PTC progression. Western blot analysis and reverse transcriptionquantitative (RTq) PCR were employed to confirm the key genes or miRs expressed in PTC cells. Cytological methods were used to detect cell viability, proliferation, apoptosis and migration and luciferase reporter assay was performed to confirm the relationship between END3 and miR27a3p. After analyzing the results of gene microarray analyses and RTqPCR, EDN3 with low expression was identified as the key gene associated with PTC progression. It was also found that EDN3 overexpression in PTC cells impaired cell viability, proliferation and migration but promoted cell apoptosis. In addition, the findings revealed that miR27a3p could relieve the inhibitory influence of EDN3 on PTC cells by binding to EDN3 mRNA 3' untranslated region (UTR), thereby suppressing EDN3 expression. Overall, the results of the present study demonstrated that by binding to EDN3 mRNA 3'UTR, miR27a3p could attenuate the inhibitory function of EDN3 in the tumorigenesis of PTC cells.
Asunto(s)
Carcinogénesis/metabolismo , Endotelina-3/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Neoplásico/metabolismo , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/metabolismo , Adulto , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Endotelina-3/genética , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , ARN Neoplásico/genética , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
The enteric nervous system (ENS) constitutes the largest part of the peripheral nervous system. In recent years, ENS development and its neurogenetic capacity in homeostasis and allostasishave gained increasing attention. Developmentally, the neural precursors of the ENS are mainly derived from vagal and sacral neural crest cell portions. Furthermore, Schwann cell precursors, as well as endodermal pancreatic progenitors, participate in ENS formation. Neural precursorsenherite three subpopulations: a bipotent neuron-glia, a neuronal-fated and a glial-fated subpopulation. Typically, enteric neural precursors migrate along the entire bowel to the anal end, chemoattracted by glial cell-derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) molecules. During migration, a fraction undergoes differentiation into neurons and glial cells. Differentiation is regulated by bone morphogenetic proteins (BMP), Hedgehog and Notch signalling. The fully formed adult ENS may react to injury and damage with neurogenesis and gliogenesis. Nevertheless, the origin of differentiating cells is currently under debate. Putative candidates are an embryonic-like enteric neural progenitor population, Schwann cell precursors and transdifferentiating glial cells. These cells can be isolated and propagated in culture as adult ENS progenitors and may be used for cell transplantation therapies for treating enteric aganglionosis in Chagas and Hirschsprung's diseases.
Asunto(s)
Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/crecimiento & desarrollo , Cresta Neural/metabolismo , Neurogénesis , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Endotelina-3/metabolismo , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/patología , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/crecimiento & desarrollo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Cresta Neural/citología , Cresta Neural/crecimiento & desarrollo , Neurogénesis/genética , Neuroglía/citología , Neuronas/citología , Receptores Notch/metabolismo , Células de Schwann/metabolismo , Transducción de Señal/genéticaRESUMEN
Prurigo nodularis is a highly pruritic and hyperplastic chronic dermatosis with unknown pathogenesis. Many pruritogenic mediators, including nerve growth factor, interleukin (IL)-31, thymic stromal lymphopoietin, and endothelin-1, are implicated in chronic itch and inflammation. This study investigated the mRNA levels and immunoreactivity of the nerve growth factor, IL-31, thymic stromal lymphopoietin, and endothelin axes in both lesional and perilesional skin in prurigo nodularis by using quantitative real-time PCR and immunohistochemistry studies. The nerve growth factor high-affinity receptor tyrosine kinase receptor A was upregulated while the low affinity receptor p75 neurotrophin receptor was downregulated in prurigo nodularis lesions. Downregulated expression of IL-31/IL-31 receptor A and endothelin-3/endothelin receptor B and upregulation of thymic stromal lymphopoietin receptor were found in prurigo nodularis lesions. Aberrant expression of nerve growth factor, IL-31, thymic stromal lymphopoietin and endothelin axes was found in prurigo nodularis lesions, especially in the epidermis, indicating the importance of keratinocytes in prurigo nodularis pathogenesis.
Asunto(s)
Epidermis/metabolismo , Queratinocitos/metabolismo , Prurigo/genética , Prurigo/metabolismo , Adulto , Citocinas/genética , Citocinas/metabolismo , Regulación hacia Abajo , Endotelina-3/genética , Endotelina-3/metabolismo , Femenino , Expresión Génica , Humanos , Hipersensibilidad/complicaciones , Hipersensibilidad/genética , Hipersensibilidad/metabolismo , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Persona de Mediana Edad , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Prurigo/complicaciones , ARN Mensajero/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Regulación hacia Arriba , Linfopoyetina del Estroma TímicoRESUMEN
Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.
Asunto(s)
Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Endotelina-3/metabolismo , Endotelinas/química , Endotelinas/metabolismo , Endotelinas/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Receptor de Endotelina B/agonistas , Factor de Crecimiento Transformador alfa/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)-a known melanogenic factor of tetrapods-as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates.
Asunto(s)
Cromatóforos/fisiología , Endotelina-3/metabolismo , Pigmentación/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Proliferación Celular , Embrión no Mamífero , Endotelina-3/genética , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Animales , Fenotipo , Transducción de Señal/genética , Piel/citología , Especificidad de la Especie , Proteínas de Pez Cebra/genéticaRESUMEN
The endothelin system is a vertebrate-specific innovation with important roles in regulating the cardiovascular system and renal and pulmonary processes, as well as the development of the vertebrate-specific neural crest cell population and its derivatives. This system is comprised of three structurally similar 21-amino acid peptides that bind and activate two G-protein coupled receptors. In 1994, knockouts of the Edn3 and Ednrb genes revealed their crucial function during development of the enteric nervous system and melanocytes, two neural-crest derivatives. Since then, human and mouse genetics, combined with cellular and developmental studies, have helped to unravel the role of this signaling pathway during development and adulthood. In this review, we will summarize the known functions of the EDN3/EDNRB pathway during neural crest development, with a specific focus on recent scientific advances, and the enteric nervous system in normal and pathological conditions.
Asunto(s)
Endotelina-3/fisiología , Cresta Neural/metabolismo , Receptor de Endotelina B/fisiología , Animales , Evolución Biológica , Tipificación del Cuerpo/fisiología , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Endotelina-3/metabolismo , Endotelinas/metabolismo , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/fisiología , Humanos , Melanocitos/metabolismo , Cresta Neural/embriología , Cresta Neural/fisiología , Tubo Neural , Neurogénesis , Receptores de Endotelina/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Vertebrados/embriologíaRESUMEN
We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.
Asunto(s)
Endotelina-3/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor de Endotelina B/metabolismo , Factor de Células Madre/metabolismo , Aterosclerosis/patología , Línea Celular Tumoral , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Motilidad Gastrointestinal , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/patología , Tumores del Estroma Gastrointestinal/fisiopatología , Homeostasis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inmunohistoquímica , Melanoma/patología , Plexo Mientérico/metabolismo , Invasividad Neoplásica , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Piel/metabolismo , Luz Solar , Factores de Tiempo , Regulación hacia Arriba/genética , VasodilataciónRESUMEN
We have recently demonstrated that endothelin (ET) is functionally coupled to Nax , a Na+ concentration-sensitive Na+ channel for lactate release via ET receptor type B (ETB R) and is involved in peripheral nerve regeneration in a sciatic nerve transection-regeneration mouse model. Nax is known to interact directly with Na+ /K+ -ATPase, leading to lactate production in the brain. To investigate the role of Na+ /K+ -ATPase in peripheral nerve regeneration, in this study, we applied ouabain, a Na+ /K+ -ATPase inhibitor, to the cut site for 4 weeks with an osmotic pump. While functional recovery and nerve reinnervation to the toe started at 5 weeks after axotomy and were completed by 7 weeks, ouabain delayed them by 2 weeks. The delay by ouabain was improved by lactate, and its effect was blocked by α-cyano-4-hydroxy-cinnamic acid (CIN), a broad monocarboxylate transporter (MCT) inhibitor. In primary cultures of dorsal root ganglia, neurite outgrowth of neurons and lactate release into the culture medium was inhibited by ouabain. Conversely, lactate enhanced the neurite outgrowth, which was blocked by CIN, but not by AR-C155858, a MCT1/2-selective inhibitor. ET-1 and ET-3 increased neurite outgrowth of neurons, which was attenuated by an ETB R antagonist, ouabain and 2 protein kinase C inhibitors. Taken together with the finding that ETB R was expressed in Schwann cells, these results demonstrate that ET enhanced neurite outgrowth of neurons mediated by Na+ /K+ -ATPase via ETB R in Schwann cells. This study suggests that Na+ /K+ -ATPase coupled to the ET-ETB R system plays a critical role in peripheral nerve regeneration via lactate signalling.
Asunto(s)
Ácido Láctico/metabolismo , Regeneración Nerviosa/fisiología , Receptor de Endotelina B/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Células Cultivadas , Antagonistas de los Receptores de la Endotelina B/farmacología , Endotelina-1/metabolismo , Endotelina-3/metabolismo , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Masculino , Ratones Endogámicos C57BL/metabolismo , Ratones Transgénicos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Proyección Neuronal/fisiología , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Células de Schwann/patología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
BACKGROUND & AIMS: Maintenance and differentiation of progenitor cells in the developing enteric nervous system are controlled by molecules such as the signaling protein endothelin 3 (EDN3), its receptor (the endothelin receptor type B [EDNRB]), and the transcription factors SRY-box 10 (SOX10) and zinc finger E-box binding homeobox 2 (ZEB2). We used enteric progenitor cell (EPC) cultures and mice to study the roles of these proteins in enteric neurogenesis and their cross regulation. METHODS: We performed studies in mice with a Zeb2 loss-of-function mutation (Zeb2Δ) and mice carrying a spontaneous recessive mutation that prevents conversion of EDN3 to its active form (Edn3ls). EPC cultures issued from embryos that expressed only wild-type Zeb2 (Zeb2+/+ EPCs) or were heterozygous for the mutation (Zeb2Δ/+ EPCs) were exposed to EDN3; we analyzed the effects on cell differentiation using immunocytochemistry. In parallel, Edn3ls mice were crossed with Zeb2Δ/+mice; intestinal tissues were collected from embryos for immunohistochemical analyses. We investigated regulation of the EDNRB gene in transactivation and chromatin immunoprecipitation assays; results were validated in functional rescue experiments using transgenes expression in EPCs from retroviral vectors. RESULTS: Zeb2Δ/+ EPCs had increased neuronal differentiation compared to Zeb2+/+ cells. When exposed to EDN3, Zeb2+/+ EPCs continued expression of ZEB2 but did not undergo any neuronal differentiation. Incubation of Zeb2Δ/+ EPCs with EDN3, on the other hand, resulted in only partial inhibition of neuronal differentiation. This indicated that 2 copies of Zeb2 are required for EDN3 to prevent neuronal differentiation. Mice with combined mutations in Zeb2 and Edn3 (double mutants) had more severe enteric anomalies and increased neuronal differentiation compared to mice with mutations in either gene alone. The transcription factors SOX10 and ZEB2 directly activated the EDNRB promoter. Overexpression of EDNRB in Zeb2Δ/+ EPCs restored inhibition of neuronal differentiation, similar to incubation of Zeb2+/+ EPCs with EDN3. CONCLUSIONS: In studies of cultured EPCs and mice, we found that control of differentiation of mouse enteric nervous system progenitor cells by EDN3 requires regulation of Ednrb expression by SOX10 and ZEB2.
Asunto(s)
Diferenciación Celular/genética , Endotelina-3/genética , Sistema Nervioso Entérico/embriología , Proteínas de Homeodominio/genética , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Receptor de Endotelina B/metabolismo , Proteínas Represoras/genética , Factores de Transcripción SOXE/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Endotelina-3/metabolismo , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Enfermedad de Hirschsprung , Proteínas de Homeodominio/metabolismo , Inmunoquímica , Ratones , Mutación , Células-Madre Neurales/citología , Reacción en Cadena de la Polimerasa , Proteínas Represoras/metabolismo , Células Madre , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
Endothelin-3 (EDN3) and ß1-integrins are required for the colonization of the embryonic gut by enteric neural crest cells (ENCCs) to form the enteric nervous system (ENS). ß1-integrin-null ENCCs exhibit migratory defects in a region of the gut enriched in EDN3 and in specific extracellular matrix (ECM) proteins. We investigated the putative role of EDN3 on ENCC adhesion properties and its functional interaction with ß1-integrins during ENS development. We show that EDN3 stimulates ENCC adhesion to various ECM components in vitro. It induces rapid changes in ENCC shape and protrusion dynamics favouring sustained growth and stabilization of lamellipodia, a process coincident with the increase in the number of focal adhesions and activated ß1-integrins. In vivo studies and ex-vivo live imaging revealed that double mutants for Itgb1 and Edn3 displayed a more severe enteric phenotype than either of the single mutants demonstrated by alteration of the ENS network due to severe migratory defects of mutant ENCCs taking place early during the ENS development. Altogether, our results highlight the interplay between the EDN3 and ß1-integrin signalling pathways during ENS ontogenesis and the role of EDN3 in ENCC adhesion.
Asunto(s)
Adhesión Celular , Endotelina-3/metabolismo , Sistema Nervioso Entérico/embriología , Integrina beta1/metabolismo , Animales , Movimiento Celular/fisiología , Cruzamientos Genéticos , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Adhesiones Focales/metabolismo , Genotipo , Mucosa Intestinal/metabolismo , Intestinos/embriología , Masculino , Ratones , Mutación , Cresta Neural/citología , Fenotipo , Seudópodos/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: This study aimed to explore the molecular mechanism of cervical cancer (CC) by integrated bioinformatic analyses of gene expression and methylation profiles. METHODS: The gene expression and methylation microarrays in CC samples and normal controls were respectively downloaded from the GEO database. After screening the differentially expressed genes (DEGs) with Limma package and the CC-related methylation sites with CpGassoc package in R language, DEGs with CC-related methylation sites were identified from the intersection of the above two groups of results with 50kb upstream and downstream of a gene as the gene region. Then GO enrichment was performed by GenCLIP2.0 software. Sequentially, analysis of metabolic sub-pathways with pathogenic risk was predicted by iSubpathwayMiner package in R language. RESULTS: A total of 1357 DEGs including 721 up-regulated and 636 down-regulated, as well as 666 CC-related methylation sites were screened out. After being analyzed, 26 DEGs with 35 CC-related methylation sites were identified. EDN3 and EDNRB were significantly involved in a function cluster in GO terms of vein smooth muscle contraction, vascular smooth muscle contraction and phasic smooth muscle contraction. LHX2 and PAX6 were significantly involved in a function cluster in GO terms of telencephalon regionalization and forebrain regionalization. ACOX3, CYP39A1 and DPYS were significantly enriched in 25 sub-pathways of 6 major pathways. CONCLUSIONS: EDN3 and EDNRB might play important roles in the molecular mechanism of CC, and LHX2, ACOX3, CYP39A1 and DPYS might be susceptibility genes and potential risk markers in CC.
Asunto(s)
Cuello del Útero/metabolismo , Metilación de ADN , Endotelina-3/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Receptor de Endotelina B/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Adulto , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , China/epidemiología , Biología Computacional , Bases de Datos Genéticas , Endotelina-3/genética , Epigénesis Genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Prosencéfalo/metabolismo , Receptor de Endotelina B/genética , Riesgo , Telencéfalo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias del Cuello Uterino/epidemiologíaRESUMEN
Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.
Asunto(s)
Endotelina-1/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Regulación Alostérica , Sitio Alostérico , Membrana Celular/metabolismo , Cristalografía por Rayos X , Endotelina-1/química , Endotelina-1/farmacología , Endotelina-3/química , Endotelina-3/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
Decline in oxygen availability experienced under hypobaric hypoxia (HH) mediates imbalance in lung fluid clearance and is a causative agent of acute lung injury. Here, we investigate the pathological events behind acute HH mediated lung injury and assess the therapeutic efficacy of nanocurcumin in its amelioration. We assess the protective efficacy of nanotized curcumin (nanocurcumin) in ameliorating HH induced lung injury and compare to curcumin. Rats exposed to acute HH (6, 12, 24, 48 and 72 h) were subjected to histopathology, blood-gas analysis and clinical biochemistry, cytokine response and redox damage. HH induced lung injury was analysed using markers of lung injury due to pulmonary vasoconstriction (ET-1/2/3 and endothelin receptors A and B) and trans-vascular fluid balance mediator (Na+/K+ ATPase). The protective efficacy of nanocurcumin was analysed by examination of Akt/Erk signalling cascade by western blot. HH induced lung injury was associated with discrete changes in blood analytes, differential circulatory cytokine response and severe pulmonary redox damages. Up-regulation of ET-1/2/3 and its receptors along with down-regulation of Na+/K+ ATPase confirmed defective pulmonary fluid clearance which promoted edema formation. Nanocurcumin treatment prevented lung edema formation and restored expression levels of ET-1/2/3 and its receptors while restoring the blood analytes, circulatory cytokines and pulmonary redox status better than curcumin. Modulation in Akt/Erk signalling pathway in rat lungs under HH confirmed the protective efficacy of nanocurcumin.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Curcumina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipoxia/tratamiento farmacológico , Nanoestructuras/uso terapéutico , Sustancias Protectoras/farmacología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Endotelina-1/genética , Endotelina-1/metabolismo , Endotelina-2/genética , Endotelina-2/metabolismo , Endotelina-3/genética , Endotelina-3/metabolismo , Hipoxia/complicaciones , Hipoxia/genética , Hipoxia/patología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nanoestructuras/química , Oxidación-Reducción/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Endothelin (EDN) is a possible regulating factor of oviductal motility, which is important for the transport of gametes and embryo. To clarify the factors that control the secretion of EDN in the bovine oviduct, the expression of EDNs, EDN-converting enzymes (ECEs) and EDN receptors (EDNRs) were investigated. All isoforms of EDN (EDN1-3), ECE (ECE1 and ECE2) and EDNR (EDNRA and EDNRB) were immunolocalised in the epithelial cells of the ampulla and the isthmus. EDNRs were also immunolocalised in smooth-muscle cells. The mRNA expression of EDN2 and ECE2 was higher in cultured ampullary oviductal epithelial cells than in isthmic cells. The expression of EDN1, EDN2 and ECE2 in the ampullary tissue was highest on the day of ovulation. Oestradiol-17ß increased EDN2 and ECE1 expression, while progesterone increased only ECE1 expression in cultured ampullary epithelial cells. These results indicate that EDNs are produced by epithelial cells and their target site is smooth-muscle and epithelial cells, and suggest that ovarian steroids are regulators of endothelin synthesis in ampullary oviductal epithelial cells.
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Endotelina-1/metabolismo , Endotelina-2/metabolismo , Enzimas Convertidoras de Endotelina/metabolismo , Trompas Uterinas/fisiología , Membrana Mucosa/metabolismo , Músculo Liso/metabolismo , Receptor de Endotelina A/metabolismo , Mataderos , Animales , Animales Endogámicos , Bovinos , Células Cultivadas , Endotelina-1/genética , Endotelina-2/genética , Endotelina-3/genética , Endotelina-3/metabolismo , Enzimas Convertidoras de Endotelina/genética , Trompas Uterinas/citología , Trompas Uterinas/enzimología , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica/veterinaria , Isoenzimas/genética , Isoenzimas/metabolismo , Membrana Mucosa/citología , Membrana Mucosa/enzimología , Músculo Liso/citología , Músculo Liso/enzimología , Especificidad de Órganos , Ovulación/metabolismo , ARN Mensajero/metabolismo , Receptor de Endotelina A/agonistas , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/metabolismo , Transducción de SeñalRESUMEN
Na(x), which is preferentially expressed in the glial cells of sensory circumventricular organs in the brain, is a sodium channel that is poorly homologous to voltage-gated sodium channels. We previously reported that Na(x) is a sodium concentration ([Na(+)])-sensitive, but not a voltage-sensitive channel that is critically involved in body-fluid homeostasis. Na(x)-knockout mice do not stop ingesting salt even when dehydrated and transiently develop hypernatremia. [Na(+)] in body fluids is strictly controlled at 135 to 145 mM in mammals. Although the set point must be within this range, Na(x) was shown to have a threshold value of ~150 mM for extracellular [Na(+)] ([Na(+)]o) for activation in vitro. Therefore, the [Na(+)]o dependency of Na(x) in vivo is presumably modified by an as yet unidentified mechanism. We recently demonstrated that the [Na(+)]o dependency of Na(x) in the subfornical organ was adjusted to the physiological range by endothelin-3. Pharmacological experiments revealed that endothelin receptor B signaling was involved in this modulation of Na(x) gating through protein kinase C and ERK1/2 activation. In addition, we identified a case of essential hypernatremia caused by autoimmunity to Na(x). Occurrence of a ganglioneuroma composed of Schwann-like cells that robustly expressed Na(x) was likely to induce the autoimmune response in this patient. An intravenous injection of the immunoglobulin fraction of the patient's serum, which contained anti-Na(x) antibodies, into mice reproduced the patient's symptoms. This review provides an overview of the physiological functions of Na(x) by summarizing our recent studies.
Asunto(s)
Líquidos Corporales/metabolismo , Encéfalo/metabolismo , Órganos Circunventriculares/fisiología , Canales de Sodio/fisiología , Sodio/metabolismo , Animales , Órganos Circunventriculares/metabolismo , Endotelina-3/metabolismo , Homeostasis , Humanos , Hipernatremia/metabolismo , Ratones , Ratones Noqueados , Neuroglía/metabolismo , Neuronas/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismoRESUMEN
Lipid bilayers, such as the plasma membrane and nuclear envelope, serve as effective cellular barriers to ions and macromolecules, thus allowing regulated access to subcellular compartments including the cytoplasm and nucleus, respectively. Of course, these barriers are semipermeable and a wide variety of proteins including transporters, ion exchangers, pumps, and ion channels are required to permit access as well as establish and maintain molecular and ionic gradients across membranes. However, some experimental designs, such as specifically targeting intracellular receptors, require the administration of membrane-impermeable molecules directly into live cells. The microinjection technique described in this chapter is an efficient, technically simple, and reliable approach that can be used to introduce macromolecules into intracellular compartments while maintaining the integrity of the plasma membrane itself.