RESUMEN
During the development of diabetes ß-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1ß, which in vitro induce ß-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced ß-cell apoptosis, we evaluated the protective potential of CRFR activation in ß-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in ß-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.
Asunto(s)
Apoptosis/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Citocinas/efectos adversos , Citoprotección/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Receptores de Hormona Liberadora de Corticotropina/agonistas , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Secretoras de Insulina/fisiología , Interleucina-1beta/efectos adversos , Ratones , Ratas , Factor de Necrosis Tumoral alfa/efectos adversosRESUMEN
Corticotropin-releasing factor-binding protein (CRF-BP) is considered a key determinant for CRF receptor (CRF-R) activation by CRF and several related peptides. Earlier studies have shown that the CRF system is highly conserved in gene structures throughout evolution, yet little is known about the evolutionary conservation of its biological functions. Therefore, we address the functional properties of CRF-BP and CRF-Rs in a teleost fish (common carp; Cyprinus carpio L.). We report the finding of two similar, yet distinct, genes for both CRF-R1 and CRF-R2 in this species. The four receptors are differentially responsive to CRF, urotensin-I (UI), sauvagine, and urocortin-2 (Ucn-2) and -3 (Ucn-3) as shown by luciferase assays. In vitro, carp CRF-BP inhibits CRF- and UI-mediated activation of the newfound CRF-Rs, but its potency to do so varies between receptor and peptide ligand. This is the first paper to establish the functionality and physiological interplay between CRF-BP, CRF-Rs and CRF-family peptides in a teleostean species.
Asunto(s)
Carpas/metabolismo , Proteínas Portadoras/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Urotensinas/metabolismo , Proteínas Anfibias/metabolismo , Animales , AMP Cíclico/farmacología , Células HEK293 , Humanos , Luciferasas/metabolismo , Hormonas Peptídicas/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Hormona Liberadora de Corticotropina/genética , Proteínas Recombinantes/metabolismo , Urocortinas/metabolismoRESUMEN
Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2ß as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2ß-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2ß expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2ß product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2ß were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2ß membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.
Asunto(s)
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Mamíferos/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Proteínas Anfibias/metabolismo , Animales , Western Blotting , Membrana Celular/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Medios de Cultivo/farmacología , Detergentes/farmacología , Vectores Genéticos , Humanos , Cinética , Ligandos , Ratones , Fragmentos de Péptidos/metabolismo , Hormonas Peptídicas/metabolismo , Unión Proteica/efectos de los fármacos , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Receptores de Hormona Liberadora de Corticotropina/química , Proteínas Recombinantes de Fusión/metabolismo , SolubilidadRESUMEN
OBJECTIVE: Ghrelin is known to regulate appetite control and cellular metabolism. The corticotropin-releasing factor (CRF) family is also known to regulate energy balance. In this study, the links between ghrelin and the CRF family in C2C12 cells, a mouse myoblast cell line was investigated. DESIGN AND METHODS: C2C12 cells were treated with ghrelin in the presence or absence of CRF receptor antagonists and then subjected to different metabolic analyses. RESULTS: Ghrelin enhanced glucose uptake by C2C12 cells, induced GLUT4 translocation to the cell surface and decreased RBP4 expression. A CRF-R2 selective antagonist, anti-sauvagine-30, blocked ghrelin-induced glucose uptake, Ghrelin upregulated CRF-R2 but not CRF-R1 levels. Moreover, ghrelin-treated C2C12 cells displayed a cAMP and pERK activation in response to Ucn3, a CRF-R2 specific ligand, but not in response to CRF or stressin, CRF-R1 specific ligands. Ghrelin also induced UCP2 and UCP3 expression, which were blocked by anti- sauvagine-30. Ghrelin did not induce fatty acids uptake by C2C12 cells or ACC expression. Even though C2C12 cells clearly exhibited responses to ghrelin, the known ghrelin receptor, GHSR1a, was not detectable in C2C12 cells. CONCLUSION: The results suggest that, ghrelin plays a role in regulating muscle glucose and, raise the possibility that suppression of the CRF-R2 pathway might provide benefits in high ghrelin states.
Asunto(s)
Regulación de la Expresión Génica , Ghrelina/metabolismo , Glucosa/metabolismo , Mioblastos/metabolismo , Receptores de Hormona Liberadora de Corticotropina/agonistas , Receptores de Ghrelina/metabolismo , Transducción de Señal , Animales , Anticuerpos Bloqueadores/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 4/metabolismo , Canales Iónicos/agonistas , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , Proteínas Mitocondriales/agonistas , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mioblastos/efectos de los fármacos , Mioblastos/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Pirimidinas/farmacología , Pirroles/farmacología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores de Ghrelina/genética , Proteínas Plasmáticas de Unión al Retinol/antagonistas & inhibidores , Proteínas Plasmáticas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Desacopladora 2 , Proteína Desacopladora 3 , Urocortinas/metabolismoRESUMEN
Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet ß-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of ß-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 ß-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Isocitratos/metabolismo , Ácido Pirúvico/metabolismo , Canales de Potasio Shab/biosíntesis , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Masculino , Modelos Biológicos , Péptidos/farmacología , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/genética , Venenos de Araña/farmacologíaRESUMEN
Mouse (m) and human (h) urocortin 2 (Ucn 2) were identified by molecular cloning strategies and the primary sequence of their mature forms postulated by analogy to closely related members of the corticotropin-releasing factor (CRF) neuropeptide family. Because of the paucity of Ucn 2 proteins in native tissues, skin, muscle, and pancreatic cell lines were transduced with lentiviral constructs and secretion media were used to isolate and characterize Ucn 2 products and study processing. Primary structures were assigned using a combination of Edman degradation sequencing and mass spectrometry. For mUcn 2, transduced cells secreted a 39 amino acid peptide and the glycosylated prohormone lacking signal peptide; both forms were C-terminally amidated and highly potent to activate the type 2 CRF receptor. Chromatographic profiles of murine tissue extracts were consistent with cleavage of mUcn 2 prohormone to a peptidic form. By contrast to mUcn 2, mammalian cell lines transduced with hUcn 2 constructs secreted significant amounts of an 88 amino acid glycosylated hUcn 2 prohormone but were unable to further process this molecule. Similarly, WM-266-4 melanoma cells that express endogenous hUcn 2 secreted only the glycosylated prohormone lacking the signal peptide and unmodified at the C terminus. Although not amidated, hUcn 2 prohormone purified from overexpressing lines activated CRF receptor 2. Hypoxia and glycosylation, paradigms that might influence secretion or processing of gene products, did not significantly impact hUcn 2 prohormone cleavage. Our findings identify probable Ucn 2 processing products and should expedite the characterization of these proteins in mammalian tissues.
Asunto(s)
Hormona Liberadora de Corticotropina , Procesamiento Proteico-Postraduccional , Urocortinas , Secuencia de Aminoácidos , Animales , Hipoxia de la Célula , Células Cultivadas , Hormona Liberadora de Corticotropina/química , Hormona Liberadora de Corticotropina/metabolismo , Glicosilación , Humanos , Ratones , Datos de Secuencia Molecular , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Transducción Genética , Urocortinas/química , Urocortinas/metabolismoRESUMEN
The central nucleus of the amygdala (CeA) is an important integrative site for the reinforcing effects of drugs of abuse, such as ethanol. Activation of corticotropin-releasing factor type 1 (CRF1) receptors in the CeA plays a critical role in the development of ethanol dependence, but these neurons remain uncharacterized. Using CRF1:GFP reporter mice and a combined electrophysiological/immunohistochemical approach, we found that CRF1 neurons exhibit an α1 GABA(A) receptor subunit-mediated tonic conductance that is driven by action potential-dependent GABA release. In contrast, unlabeled CeA neurons displayed a δ subunit-mediated tonic conductance that is enhanced by ethanol. Ethanol increased the firing discharge of CRF1 neurons and decreased the firing discharge of unlabeled CeA neurons. Retrograde tracing studies indicate that CeA CRF1 neurons project into the bed nucleus of the stria terminalis. Together, these data demonstrate subunit-specific tonic signaling and provide mechanistic insight into the specific effects of ethanol on CeA microcircuitry.
Asunto(s)
Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiología , Etanol/farmacología , Subunidades de Proteína/fisiología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Receptores de GABA-A/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Masculino , Ratones , Ratones Transgénicos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We have previously demonstrated that viral-mediated overexpression of corticotropin-releasing factor (CRF) within the central nucleus of the amygdala (CeA) reproduces many of the behavioral and endocrine consequences of chronic stress. The present experiment sought to determine whether administration of the selective serotonin reuptake inhibitor (SSRI) escitalopram reverses the adverse effects of CeA CRF overexpression. In a 2×2 design, adult male rats received bilateral infusions of a control lentivirus or a lentivirus in which a portion of the CRF promoter is used to drive increased expression of CRF peptide. Four weeks later, rats were then implanted with an Alzet minipump to deliver vehicle or 10mg/kg/day escitalopram for a 4-week period of time. The defensive withdrawal (DW) test of anxiety and the sucrose-preference test (SPT) of anhedonia were performed both before and after pump implantation. Additional post-implant behavioral tests included the elevated plus maze (EPM) and social interaction (SI) test. Following completion of behavioral testing, the dexamethasone/CRF test was performed to assess HPA axis reactivity. Brains were collected and expression of HPA axis-relevant transcripts were measured using in situ hybridization. Amygdalar CRF overexpression increased anxiety-like behavior in the DW test at week eight, which was only partially prevented by escitalopram. In both CRF-overexpressing and control groups, escitalopram decreased hippocampal CRF expression while increasing hypothalamic and hippocampal expression of the glucocorticoid receptor (GR). These gene expression changes were associated with a significant decrease in HPA axis reactivity in rats treated with escitalopram. Interestingly, escitalopram increased the rate of weight gain only in rats overexpressing CRF. Overall these data support our hypothesis that amygdalar CRF is critical in anxiety-like behavior; because the antidepressant was unable to reverse behavioral manifestations of CeA CRF-OE. This may be a potential animal model to study treatment-resistant psychopathologies.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Citalopram/farmacología , Hormona Liberadora de Corticotropina/metabolismo , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Amígdala del Cerebelo/efectos de los fármacos , Animales , Ansiedad/genética , Ansiedad/metabolismo , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Hormona Liberadora de Corticotropina/genética , Dexametasona , Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Masculino , Pruebas de Función Adreno-Hipofisaria/métodos , Sistema Hipófiso-Suprarrenal/metabolismo , Ratas , Receptores de Glucocorticoides/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3(+) beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3(+) hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3(+) alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Urocortinas/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula , Células Madre Embrionarias/citología , Expresión Génica , Células Secretoras de Glucagón/citología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica/métodos , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Transactivadores/genética , Transactivadores/metabolismo , Urocortinas/genéticaRESUMEN
BACKGROUND: The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation. METHODS: Transgenic mice overexpressing the highly specific CRFR2 ligand urocortin 3 (UCN3OE) were analyzed for stress-related behaviors and hypothalamic-pituitary-adrenal axis responses. Responses to 5-HT receptor agonist challenge were assessed by local cerebral glucose utilization, while 5-HT and 5-hydroxyindoleacetic acid content were quantified in limbic brain regions. RESULTS: Mice overexpressing urocortin 3 exhibited increased stress-related behaviors under basal conditions and impaired retention of spatial memory compared with control mice. Following acute stress, unlike control mice, they exhibited no further increase in these stress-related behaviors and showed an attenuated adrenocorticotropic hormone response. 5-HT and 5-hydroxyindoleacetic acid content of limbic nuclei were differentially regulated by stress in UCN3OE mice as compared with control mice. Responses to 5-HT type 1A receptor challenge were significantly and specifically reduced in UCN3OE mice. The distribution pattern of local cerebral glucose utilization and 5-HT type 1A receptor messenger RNA expression levels suggested this effect was mediated in the raphé nuclei. CONCLUSIONS: Chronic activation of CRFR2 promotes an anxiety-like state, yet with attenuated behavioral and hypothalamic-pituitary-adrenal axis responses to stress. This is reminiscent of stress-related atypical psychiatric syndromes such as posttraumatic stress disorder, chronic fatigue, and chronic pain states. This new understanding indicates CRFR2 antagonism as a potential novel therapeutic target for such disorders.
Asunto(s)
Ansiedad/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Agonistas del Receptor de Serotonina 5-HT1/metabolismo , Urocortinas/genética , Análisis de Varianza , Animales , Ansiedad/genética , Encéfalo/metabolismo , Cromatografía Liquida , Corticosterona/metabolismo , Ácido Hidroxiindolacético/análisis , Hibridación in Situ , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Serotonina 5-HT1A/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Serotonina/análisis , Estrés Fisiológico , Estrés Psicológico , Urocortinas/metabolismoRESUMEN
Exposure and/or sensitivity to stress have been implicated as conferring risk for development of Alzheimer's disease (AD). Although the basis for such a link remains unclear, we previously reported differential involvement of corticotropin-releasing factor receptor (CRFR) 1 and 2 in acute stress-induced tau phosphorylation (tau-P) and solubility in the hippocampus. Here we examined the role of CRFRs in tau-P induced by repeated stress and the structural manifestations of altered tau solubility. Robust tau-P responses were seen in WT and CRFR2 null mice exposed to repeated stress, which were sustained at even 24 h after the final stress exposure. A portion of phosphorylated tau in these mice was sequestered in detergent-soluble cellular fractions. In contrast, CRFR1 and CRFR double-KO mice did not exhibit repeated stress-induced alterations in tau-P or solubility. Similarly, treatment with CRFR1 antagonist attenuated repeated stress-induced tau-P. Using histochemical approaches in a transgenic CRFR1 reporter mouse line, we found substantial overlap between hippocampal CRFR1 expression and cells positive for phosphorylated tau after exposure to repeated stress. Ultrastructural analysis of negatively stained extracts from WT and CRFR2 null mice identified globular aggregates that displayed positive immunogold labeling for tau-P, as well as conformational changes in tau (MC1) seen in early AD. Given that repeated stress exposure results in chronic increases in hippocampal tau-P and its sequestration in an insoluble (and potentially prepathogenic) form, our data may define a link between stress and an AD-related pathogenic mechanism.
Asunto(s)
Receptores de Hormona Liberadora de Corticotropina/metabolismo , Estrés Psicológico , Proteínas tau/metabolismo , Animales , Western Blotting , Giro Dentado , Detergentes/química , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Inmunoelectrónica , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Pirroles/farmacología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/genética , Solubilidad , Proteínas tau/química , Proteínas tau/ultraestructuraRESUMEN
The transforming growth factor-beta (TGF-ß) superfamily of ligands have been recognized as important signals in vertebrate embryonic development from the blastula stage to adulthood. In addition to roles in early development, TGF-ß superfamily ligands, and particularly activin family ligands, are involved in specification, differentiation, and proliferation of multiple organ systems, including the pancreas. More recently, research has suggested that activin family ligands, binding proteins, receptors, and Smad signal transducers and modulators are involved in regulating adult pancreatic function and maintaining pancreatic islet homeostasis in the adult. This article will focus on outlining common themes in activin family regulation of embryonic pancreatic development and adult pancreatic homeostasis, particularly in activin family involvement in setting and maintaining populations of islet cells such as ß-cells.
Asunto(s)
Activinas/fisiología , Islotes Pancreáticos/fisiología , Receptores de Activinas/metabolismo , Activinas/metabolismo , Animales , Tipificación del Cuerpo , Metabolismo Energético , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas/citología , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Proteínas de la Superfamilia TGF-beta/metabolismo , Proteínas de la Superfamilia TGF-beta/fisiologíaRESUMEN
Activins are multifunctional proteins and members of the TGF-ß superfamily. Activins are expressed locally in most tissues and, analogous to the actions of other members of this large family of pleiotropic factors, play prominent roles in the regulation of diverse biological processes in both differentiated and embryonic stem cells. They have an essential role in maintaining tissue homeostasis in the adult and are known to contribute to the developmental programs in the embryo. Activins are further implicated in the growth and metastasis of tumor cells. Through distinct modes of action, inhibins and follistatins function as antagonists of activin and several other TGF-ß family members, including a subset of BMPs/GDFs, and modulate cellular responses and the signaling cascades downstream of these ligands. In the pituitary, the activin pathway is known to regulate key aspects of gonadotrope functions and also exert effects on other pituitary cell types. As in other tissues, activin is produced locally by pituitary cells and acts locally by exerting cell-type specific actions on gonadotropes. These local actions of activin on gonadotropes are modulated by the autocrine/paracrine actions of locally secreted follistatin and by the feedback actions of gonadal inhibin. Knowledge about the mechanism of activin, inhibin and follistatin actions is providing information about their importance for pituitary function as well as their contribution to the pathophysiology of pituitary adenomas. The aim of this review is to highlight recent findings and summarize the evidence that supports the important functions of activin, inhibin and follistatin in the pituitary.
Asunto(s)
Activinas/fisiología , Folistatina/fisiología , Gonadotrofos/metabolismo , Inhibinas/fisiología , Activinas/metabolismo , Animales , Folistatina/metabolismo , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/fisiología , Humanos , Inhibinas/metabolismo , Hipófisis/citología , Hipófisis/metabolismo , Transducción de Señal , Proteínas de la Superfamilia TGF-beta/metabolismo , Proteínas de la Superfamilia TGF-beta/fisiologíaRESUMEN
Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-ß ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-ß1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of the 78 kDa glucose regulated protein (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-ß and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , Ratones , Modelos Biológicos , Neoplasias/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Células Madre/citologíaRESUMEN
The pituitary gland plays a prominent role in the control of many physiological processes. This control is achieved through the actions and interactions of hormones and growth factors that are produced and secreted by the endocrine cell types and the non-endocrine constituents that collectively and functionally define this complex organ. The five endocrine cell types of the anterior lobe of the pituitary, somatotropes, lactotropes, corticotropes, thyrotropes and gonadotropes, are defined by their primary product, growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH) and follicle stimulating hormone (FSH)/luteinizing hormone (LH). They are further distinguishable by the presence of cell surface receptors that display high affinity and selectivity for specific hypothalamic hormones and couple to appropriate downstream signaling pathways involved in the control of cell type specific responses, including the release and/or synthesis of pituitary hormones. Central control of the pituitary via the hypothalamus is further fine-tuned by the positive or negative actions of peripheral feedback signals and of a variety of factors that originate from sources within the pituitary. The focus of this review is the latter category of intrinsic factors that exert local control. Special emphasis is given to the TGF-ß family of growth factors, in particular activin effects on the gonadotrope population, because a considerable body of evidence supports their contribution to the local modulation of the embryonic and postnatal pituitary as well as pituitary pathogenesis. A number of other substances, including members of the cytokine and FGF families, VEGF, IGF1, PACAP, Ghrelin, adenosine and nitric oxide have also been shown or implicated to function as autocrine/paracrine factors, though, definitive proof remains lacking in some cases. The ever-growing list of putative autocrine/paracrine factors of the pituitary nevertheless has highlighted the complexity of the local network and its impact on pituitary functions.
RESUMEN
Capturing the right ligand at the right spot: a well-balanced system for non-natural amino acid mutagenesis allows the ligand binding sites of a class II G-protein coupled receptor to be mapped and distinct binding domains to be identified for different ligands in the native environment of mammalian cells.
Asunto(s)
Aminoácidos/química , Ligandos , Receptores Acoplados a Proteínas G/química , Animales , Sitios de Unión , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Etiquetas de Fotoafinidad , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Growth factors are proteins secreted by a number of cell types that are capable of modulating cellular growth, proliferation and cellular differentiation. It is well accepted that uterine cellular events such as proliferation and differentiation are regulated by sex steroids and their actions in target tissues are mediated by local production of growth factors acting through paracrine and/or autocrine mechanisms. Myometrial mass is ultimately modified in pregnancy as well as in tumour conditions such as leiomyoma and leiomyosarcoma. Leiomyomas, also known as fibroids, are benign tumours of the uterus, considered to be one of the most frequent causes of infertility in reproductive years in women. METHODS: For this review, we searched the database MEDLINE and Google Scholar for articles with content related to growth factors acting on myometrium; the findings are hereby reviewed and discussed. RESULTS: Different growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and TGF-ß perform actions in myometrium and in leiomyomas. In addition to these growth factors, activin and myostatin have been recently identified in myometrium and leiomyoma. CONCLUSIONS: Growth factors play an important role in the mechanisms involved in myometrial patho-physiology.
Asunto(s)
Sustancias de Crecimiento/fisiología , Leiomioma/etiología , Leiomioma/fisiopatología , Miometrio/fisiopatología , Neoplasias Uterinas/etiología , Neoplasias Uterinas/fisiopatología , Femenino , Hormonas Esteroides Gonadales/fisiología , Humanos , Modelos Biológicos , Embarazo , Transducción de SeñalRESUMEN
Forkhead box L2 (FoxL2) is required for ovarian development and differentiation. FoxL2 is also expressed in the pituitary where it has been implicated in the development and regulation of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. Here, we show that FoxL2 is not required for the specification of gonadotropes; the pituitaries of Foxl2 mutant mice contain normal numbers of gonadotropes that express glycoprotein α subunit and LHß. Whereas the specification of gonadotropes and all other hormonal cell types is normal in the pituitaries of Foxl2 mutant animals, FSHß levels are severely impaired in both male and female animals, suggesting that FoxL2 is required for normal Fshb expression. The size of the pituitary is reduced in proportion to the smaller body size of Foxl2 mutants, with a concomitant increase in the pituitary cellular density. In primary pituitary cultures, activin induces FSH secretion and Fshb mRNA expression in cells from wild-type mice. In cells from Foxl2 mutant mice, however, FSH secretion is not detected, and activin is unable to drive Fshb expression, suggesting that the mechanism of activin-dependent activation of Fshb transcription is impaired. However, a small number of gonadotropes in the ventromedial region of the pituitaries from Foxl2 mutant mice maintain FSHß expression, suggesting that a FoxL2- and activin-independent mechanism can drive Fshb transcription. These data indicate that, in addition to its role in the ovary, FoxL2 function in the pituitary is required for normal expression of FSH.
Asunto(s)
Hormona Folículo Estimulante de Subunidad beta/genética , Factores de Transcripción Forkhead/metabolismo , Hipófisis/metabolismo , Hipófisis/patología , Receptores de Activinas/metabolismo , Activinas/metabolismo , Animales , Recuento de Células , Células Cultivadas , Femenino , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Proteína Forkhead Box L2 , Regulación de la Expresión Génica , Gonadotrofos/metabolismo , Hormona Luteinizante/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Smad/metabolismo , Transcripción GenéticaRESUMEN
G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2ß are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.
Asunto(s)
Sistema Libre de Células/metabolismo , Fructanos/metabolismo , Expresión Génica , Polímeros/metabolismo , Receptores Acoplados a Proteínas G/genética , Detergentes , Fructanos/química , Humanos , Ligandos , Resonancia Magnética Nuclear Biomolecular , Polímeros/química , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays and calcium mobilization measurements, we further demonstrate the functionality of these differentially expressed receptors. Using luciferase reporter assays we show a differential activation of CRFR promoters during myogenic differentiation. Transfections with different fragments of the 5'-flanking region of the mCRFR2ß gene fused to a luciferase reporter gene show a promoter-dependent expression of the reporter gene and reveal the importance of the myocyte enhancer factor 2 consensus sequence located at the 3'-proximal region of CRFR2ß promoter. Furthermore, we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat diet and chronic variable stress conditions. Performing a whole-genome expression microarray analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are elevated in insulin-resistant states. In correlation with the SM CRFR2ß levels, the SM retinol-binding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable stress conditions. The current findings further position the SM CRFR2 pathways as a relevant physiological system that may affect the known reciprocal relationship between psychological and physiological challenges and the metabolic syndrome.