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BACKGROUND AND PURPOSE: Although more common than in the general population, seizures are an atypical manifestation of multiple sclerosis (MS) and their pathophysiology is not well understood. This study aims to examine the prevalence, clinical characteristics, brain imaging findings and course of epilepsy, presenting in patients with MS. METHODS: Observational retrospective study of MS patients evaluated at a single MS reference center in Buenos Aires, Argentina, between 2011 and 2022, focusing on those who developed epilepsy (EMS). Clinical, demographic, and prognostic factors were evaluated and compared to a control group of non-epileptic MS patients (NEMS). To analyze specific epilepsy characteristics, a second control group of patients with non-lesional focal epilepsy (FNLE) was also included. RESULTS: Twenty-five patients (18 women), were diagnosed with epilepsy, corresponding to a prevalence of 1.95%. Comparison of brain imaging characteristics between EMS and NEMS patients showed brain atrophy (32% vs 6.1%, p<0.01), as well as cortical (26% vs 4%, p=0.03) and juxtacortical lesions (84% vs 55%, p=0.05), were more frequent in EMS patients. However, after multivariate analysis, cortical atrophy was the only variable linked to a significant increase in risk of epilepsy (OR 24, 95%CI=2.3-200, p<0.01). No significant differences in clinical characteristics, disease activity, disability levels, response to disease modified treatment (DMT) or lack of DMT efficacy were observed between MS patients with or without epilepsy. Most patients received anti-seizure medication (ASM), and seizure control was better in EMS than in FNLE patients (92% vs 58%, p=0.022) with no differences found in drug resistance. We did not find predictors of seizure recurrence in the population studied. CONCLUSION: We observed a lower prevalence of epilepsy in this group of MS patients, compared to other reported cohorts. Although epilepsy seems to have a benign course in MS patients, cortical atrophy appears to be an important contributor to the development of secondary epilepsy in MS patients. Further investigations will be necessary to identify risk factors or biomarkers predicting increased epilepsy risk in MS.
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Epilepsia , Esclerosis Múltiple , Humanos , Femenino , Estudios de Casos y Controles , Estudios Retrospectivos , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/diagnóstico por imagen , Esclerosis Múltiple/epidemiología , Epilepsia/tratamiento farmacológico , Epilepsia/epidemiología , Epilepsia/etiología , Pronóstico , Atrofia/epidemiología , Atrofia/complicaciones , Convulsiones/tratamiento farmacológico , Anticonvulsivantes/uso terapéuticoRESUMEN
The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.
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Receptores Acoplados a Proteínas G , Ácido Succínico , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Hipoxia , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Succinatos , Ácido Succínico/metabolismoRESUMEN
Invasive fungal infections may complicate infection by SARS-CoV-2 and increase morbidity and mortality. A 59-year-old man with multiple medical comorbidities was transferred to our hospital for worsening hypoxic respiratory failure due to COVID-19 and received high-dose corticosteroids and 2 doses of cyclophosphamide. He was diagnosed with pulmonary aspergillosis and cryptococcosis by culture of a bronchoalveolar lavage sample. This patient's secondary infections were likely due to treatment with immunosuppressants, his comorbidities, and his prolonged critical illness.
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OBJECTIVE: Most contemporary data concerning the frequency and causes of multiple sclerosis (MS) misdiagnosis are from North America and Europe with different healthcare system structure and resources than countries in Latin America. We sought to determine the frequency, and potential contributors to MS misdiagnosis in patients evaluated at an MS referral center in Argentina. METHODS: The study was a retrospective medical record review. We included patients evaluated at the MS Clinic at Fleni between April 2013 and March 2021. Diagnoses prior to consultation, final diagnoses after consultation, demographic, clinical and paraclinical data, and treatment were extracted and classified. RESULTS: Seven hundred thirty-six patients were identified. Five hundred seventy-two presented with an established diagnosis of MS and after evaluation, misdiagnosis was identified in 89 (16%). Women were at 83% greater risk of misdiagnosis (p = 0.034). The most frequent alternative diagnoses were cerebrovascular disease, radiological isolated syndrome (RIS), and headache. Seventy-four (83%) of misdiagnosed patients presented with a syndrome atypical for demyelination, 62 (70%) had an atypical brain magnetic resonance imaging (MRI), and 54 (61%) were prescribed disease-modifying therapy. CONCLUSION: Sixteen percent of patients with established MS were subsequently found to have been misdiagnosed. Women were at higher risk for misdiagnosis. Expert application of the McDonald criteria may prevent misdiagnosis and its associated morbidity and healthcare system cost.
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Esclerosis Múltiple , Argentina/epidemiología , Errores Diagnósticos , Femenino , Humanos , América Latina/epidemiología , Imagen por Resonancia Magnética , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/epidemiología , Derivación y Consulta , Estudios RetrospectivosRESUMEN
The global coronavirus disease 2019 pandemic has raised significant concerns of developing rapid, broad strategies to protect the vulnerable population and prevent morbidity and mortality. However, even with an aggressive approach, controlling the pandemic has been challenging, with concerns of emerging variants that likely escape vaccines, nonadherence of social distancing/preventive measures by the public, and challenges in rapid implementation of a global vaccination program that involves mass production, distribution, and execution. In this review, we revisit the utilization of attenuated vaccinations, such as the oral polio vaccine, which are safe, easy to administer, and likely provide cross-protection against respiratory pathogens. We discuss the rationale and data supporting its use and detail description of available vaccines that could be repurposed for curtailing the pandemic.
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This report presents a case of spontaneous fungal peritonitis (SFP) in a patient with cardiogenic ascites. Physicians need to be aware of this life-threatening condition because early suspicion of SFP can improve survival.
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[This corrects the article DOI: 10.1038/celldisc.2016.17.].
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Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.
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Preterm birth (PTB) is firmly linked to inflammation regardless of the presence of infection. Proinflammatory cytokines, including IL-1ß, are produced in gestational tissues and can locally upregulate uterine activation proteins. Premature activation of the uterus by inflammation may lead to PTB, and IL-1 has been identified as a key inducer of this condition. However, all currently available IL-1 inhibitors are large molecules that exhibit competitive antagonism properties by inhibiting all IL-1R signaling, including transcription factor NF-κB, which conveys important physiological roles. We hereby demonstrate the efficacy of a small noncompetitive (all-d peptide) IL-1R-biased ligand, termed rytvela (labeled 101.10) in delaying IL-1ß-, TLR2-, and TLR4-induced PTB in mice. The 101.10 acts without significant inhibition of NF-κB, and instead selectively inhibits IL-1R downstream stress-associated protein kinases/transcription factor c-jun and Rho GTPase/Rho-associated coiled-coil-containing protein kinase signaling pathways. The 101.10 is effective at decreasing proinflammatory and/or prolabor genes in myometrium tissue and circulating leukocytes in all PTB models independently of NF-κB, undermining NF-κB role in preterm labor. In this work, biased signaling modulation of IL-1R by 101.10 uncovers a novel strategy to prevent PTB without inhibiting NF-κB.
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Inflamación/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Péptidos/farmacología , Nacimiento Prematuro/prevención & control , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Línea Celular , Femenino , Interleucina-1beta/inmunología , Ratones , Miometrio/metabolismo , FN-kappa B/metabolismo , Embarazo , Receptores de Interleucina-1/antagonistas & inhibidores , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Útero/inmunología , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rß1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rß1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rß-IL-12Rß2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.
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Antiinflamatorios/farmacología , Inflamación/prevención & control , Oligopéptidos/farmacología , Receptores de Interleucina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antiinflamatorios/química , Diseño Asistido por Computadora , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Sudunidad beta 1 del Receptor de Interleucina-12/genética , Sudunidad beta 1 del Receptor de Interleucina-12/metabolismo , Células Jurkat , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Fosforilación , Receptores de Interleucina/química , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Factor de Transcripción STAT3/metabolismo , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Neurons have an important role in retinal vascular development. Here we show that the G protein-coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.
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Neovascularización Fisiológica , Neuronas/metabolismo , Receptor PAR-2/metabolismo , Transporte Activo de Núcleo Celular , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Animales , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica/genética , Regiones Promotoras Genéticas , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Células Ganglionares de la Retina/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismo , Nexinas de Clasificación/metabolismo , Factor de Transcripción Sp1/metabolismo , Fracciones Subcelulares/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Prompt post-hypoxia-ischemia (HI) revascularization has been suggested to improve outcome in adults and newborn subjects. Other than hypoxia-inducible factor, sensors of metabolic demand remain largely unknown. During HI, anaerobic respiration is arrested resulting in accumulation of carbohydrate metabolic intermediates. As such succinate readily increases, exerting its biological effects via a specific receptor, G-protein-coupled receptor (GPR) 91. We postulate that succinate/GPR91 enhances post-HI vascularization and reduces infarct size in a model of newborn HI brain injury. APPROACH AND RESULTS: The Rice-Vannucci model of neonatal HI was used. Succinate was measured by mass spectrometry, and microvascular density was evaluated by quantification of lectin-stained cryosection. Gene expression was evaluated by real-time polymerase chain reaction. Succinate levels rapidly increased in the penumbral region of brain infarcts. GPR91 was foremost localized not only in neurons but also in astrocytes. Microvascular density increased at 96 hours after injury in wild-type animals; it was diminished in GPR91-null mice leading to an increased infarct size. Stimulation with succinate led to an increase in growth factors implicated in angiogenesis only in wild-type mice. To explain the mode of action of succinate/GPR91, we investigated the role of prostaglandin E2-prostaglandin E receptor 4, previously proposed in neural angiogenesis. Succinate-induced vascular endothelial growth factor expression was abrogated by a cyclooxygenase inhibitor and a selective prostaglandin E receptor 4 antagonist. This antagonist also abolished succinate-induced neovascularization. CONCLUSIONS: We uncover a dominant metabolic sensor responsible for post-HI neurovascular adaptation, notably succinate/GPR91, acting via prostaglandin E2-prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery.
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Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/efectos de los fármacos , Infarto Cerebral/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Receptores Acoplados a Proteínas G/agonistas , Ácido Succínico/farmacología , Proteínas Angiogénicas/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Línea Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Infarto Cerebral/etiología , Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Infarto Cerebral/fisiopatología , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Hipoxia-Isquemia Encefálica/etiología , Hipoxia-Isquemia Encefálica/genética , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Hipoxia-Isquemia Encefálica/fisiopatología , Inyecciones Intraventriculares , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/metabolismo , Antagonistas de Prostaglandina/farmacología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/efectos de los fármacos , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Succínico/administración & dosificación , Ácido Succínico/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.
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Lesión Renal Aguda/tratamiento farmacológico , Riñón/efectos de los fármacos , Riñón/fisiología , Oligopéptidos/uso terapéutico , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Recuperación de la Función/efectos de los fármacos , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Animales , Cisplatino/efectos adversos , AMP Cíclico/biosíntesis , Modelos Animales de Enfermedad , Perros , Femenino , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Tasa de Filtración Glomerular/efectos de los fármacos , Células HEK293 , Hemo-Oxigenasa 1/biosíntesis , Humanos , Interleucina-6/biosíntesis , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Ratas , Ratas Sprague-Dawley , Flujo Plasmático Renal/fisiología , Vena Safena/efectos de los fármacos , Vena Safena/patología , Porcinos/fisiologíaRESUMEN
SUMO conjugation has emerged as a dynamic process in regulating protein function. Here we identify estrogen receptor ß (ERß) to be a new target of SUMO-1. ERß SUMO-1 modification occurs on a unique nonconsensus sumoylation motif which becomes fully competent upon phosphorylation of its contained serine residue, which provides the essential negative charge for sumoylation. This process is further regulated by phosphorylation of additional adjacent serine residues by glycogen synthase kinase 3ß (GSK3ß), which maximizes ERß sumoylation in response to hormone. SUMO-1 attachment prevents ERß degradation by competing with ubiquitin at the same acceptor site and dictates ERß transcriptional inhibition by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. These findings uncovered a novel phosphorylated sumoylation motif (pSuM), which consists of the sequence ψKXS (where ψ represents a large hydrophobic residue) and which is connected to a GSK3-activated extension that functions as a SUMO enhancer. This extended pSuM offers a valuable signature to predict SUMO substrates under protein kinase regulation.
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Receptor beta de Estrógeno/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteína SUMO-1/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Receptor beta de Estrógeno/química , Receptor beta de Estrógeno/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , SumoilaciónRESUMEN
From classical gland-based endocrinology to nuclear hormone receptor biology, tremendous progress has been made in our understanding of hormone responses underlying cellular communication. Estrogen elicits a myriad of biological processes in reproductive and peripheral target tissues through its interaction with the estrogen receptors ERalpha and ERbeta. However, our knowledge of estrogen-dependent and independent action has mainly focused on ERalpha, leaving the role of ERbeta obscure. This review discusses our current understanding of ERbeta function and the emerging role of intracellular signals that act upon and achieve estrogen-like effects through phosphorylation of ERbeta protein. Improving our understanding of how cellular determinants impact estrogen receptor actions will likely lead to treatment strategies for related endocrine diseases affecting women's health.
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Receptor beta de Estrógeno/metabolismo , Proteínas Quinasas/metabolismo , Animales , Receptor beta de Estrógeno/fisiología , Humanos , Modelos Biológicos , Fosforilación , Receptores de Quimiocina/metabolismo , Receptores de Quimiocina/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Induction of estrogen-regulated gene transcription by estrogen receptors ERalpha and ERbeta plays an important role in breast cancer development and growth. High expression of the chemokine receptor CXCR4 and its ligand CXCL12/stromal cell-derived factor 1 (SDF-1) has also been correlated with aggressive breast tumor phenotypes. Here, we describe a positive regulatory loop between the CXCR4/SDF-1 signaling pathway and ER transcriptional competence in human breast cancer cells. Treatment of breast carcinoma MCF-7 cells with SDF-1 increased ER transcriptional activity and expression of ER target genes, including SDF-1 itself. These effects were blocked by the antiestrogen ICI-182780 and by CXCR4 silencing and, conversely, estrogen-induced gene expression and growth of MCF-7 cells were impaired on CXCR4 inhibition. Both ERalpha and ERbeta were activated by SDF-1 in the presence of CXCR4 and by overexpression of a constitutively active CXCR4, indicating that CXCR4 signals to both receptors. In particular, ERbeta was able to translate the effects of SDF-1 on its own expression, as well as enhance activator protein 1 (AP-1) containing genes cyclin D1 and c-Myc in the presence of tamoxifen. This correlated with an increased ERbeta occupancy of responsive promoters at both estrogen-responsive and AP-1 elements. Ser-87, a conserved mitogen-activated protein kinase site in ERbeta, was highly phosphorylated by SDF-1, revealing an essential role of the AF-1 domain in response to CXCR4 activation. These results identify a complete autocrine loop between the CXCR4/SDF-1 and ERalpha/ERbeta signaling pathways that dictates ER-dependent gene expression and growth of breast cancer cells.
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Quimiocina CXCL12/genética , Receptores CXCR4/genética , Receptores de Estrógenos/genética , Transducción de Señal/genética , Sitios de Unión/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/genética , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología , Factores de Tiempo , Factor de Transcripción AP-1/metabolismoRESUMEN
The ubiquitin-proteasome pathway has been recognized as an important regulator in the hormonal response by estrogen receptor (ER) alpha, but its impact on ERbeta function is poorly characterized. In the current study, we investigated the role of the ubiquitin-proteasome pathway in regulating ERbeta activity and identified regulatory sites within the activation function (AF)-1 domain that modulate ERbeta ubiquitination and nuclear dynamics in a hormone-independent manner. Although both ERalpha and ERbeta were dependent on proteasome function for their maximal response to estrogen, they were regulated differently by proteasome inhibition in the absence of hormone, an effect shown to be dependent on their respective AF-1 domain. Given the role of AF-1 phosphorylation to regulate ER activity, we found that sequential substitutions of specific serine residues contained in MAPK consensus sites conferred transcriptional activation of ERbeta in a proteasome-dependent manner through reduced ubiquitination and enhanced accumulation of mutant receptors. Specifically, serines 94 and 106 within ERbeta AF-1 domain were found to modulate subnuclear mobility of the receptor to transit between inactive clusters and a more mobile state in a proteasome-dependent manner. In addition, cellular levels of ERbeta were regulated through these sites by facilitating the recruitment of the ubiquitin ligase E6-associated protein in a phosphorylation-dependent manner. These findings suggest a role for ERbeta AF-1 in contributing to the activation-degradation cycling of the receptor through a functional clustering of phosphorylated serine residues that cooperate in generating signals to the ubiquitin-proteasome pathway.
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Núcleo Celular/metabolismo , Receptor beta de Estrógeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , Secuencia de Consenso , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Immunoblotting , Inmunoprecipitación , Leupeptinas/farmacología , Ratones , Modelos Biológicos , Mutación , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , Serina/genética , Serina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The hormonal response of estrogen receptors (ER) alpha and ERbeta is controlled by a number of cofactors, including the general transcriptional coactivator CREB-binding protein (CBP). Growing evidence suggests that specific kinase signaling events also modulate the formation and activity of the ER coactivation complex. Here we show that ERbeta activity and target gene expression are decreased upon activation of ErbB2/ErbB3 receptors despite the presence of CBP. This inhibition of ERbeta involved activation of the phosphatidylinositol 3-kinase/Akt pathway, abrogating the potential of CBP to facilitate ERbeta response to estrogen. Such reduced activity was associated with an impaired ability of ERbeta to recruit CBP upon activation of Akt. Mutation of serine 255, an Akt consensus site contained in the hinge region of ERbeta, prevented the release of CBP and rendered ERbeta transcriptionally more responsive to CBP coactivation, suggesting that Ser-255 may serve as a regulatory site to restrain ERbeta activity in Akt-activated cells. In contrast, we found that CBP intrinsic activity was increased by Akt through threonine 1872, a consensus site for Akt in the cysteine- and histidine-rich 3 domain of CBP, indicating that such enhanced transcriptional potential of CBP did not serve to activate ERbeta. Interestingly, nuclear receptors sharing a conserved Akt consensus site with ERbeta also exhibit a reduced ability to be coactivated by CBP, whereas others missing that site were able to benefit from the activation of CBP by Akt. These results therefore outline a regulatory mechanism by which the phosphatidylinositol 3-kinase/Akt pathway may discriminate nuclear receptor response through coactivator transcriptional competence.
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Proteína de Unión a CREB/metabolismo , Receptor beta de Estrógeno/metabolismo , Regulación de la Expresión Génica , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Proteína de Unión a CREB/genética , Línea Celular , Activación Enzimática , Receptor beta de Estrógeno/genética , Regulación de la Expresión Génica/genética , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Mutación Puntual , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Deregulated signaling of ErbB2 receptor tyrosine kinase is often associated with hormone resistance in estrogen receptor alpha (ERalpha)-positive breast cancers, establishing a relationship between ErbB2 and ERalpha pathways. Although ERalpha and ERbeta are expressed in many breast cancer cells, the response of ERbeta to ErbB2 signaling is less well defined. In the present study, we demonstrate that ERbeta activity can be modulated by ErbB2 signaling in ER-expressing breast cancer cells. The estrogen-dependent transcriptional activity of ERbeta was altered in a manner similar to ERalpha by either activation of ErbB2/ErbB3 signaling by growth factor heregulin beta or expression of a constitutively active mutant of ErbB2. However, as opposed to ERalpha, the p38 MAPK pathway was found to be involved in liganded ERbeta repression activity by ErbB2 signaling and in regulating estrogen-responsive promoter occupancy by ERbeta. The repression in ERbeta response to hormone was dependent upon its AF-1 domain which includes serines 106 and 124, two phosphorylation target sites for Erk that we previously showed to be involved in SRC-1 recruitment to ERbeta. Substitution of these two serines by aspartic acid residues abolished the repression of ERbeta by activated ErbB2/ErbB3. Moreover, expression of SRC-1 also relieved the inhibition of ERbeta in heregulin-treated cells. Our study demonstrates a functional coupling between ERbeta and ErbB receptors and outlines the differential role of the AF-1 region in the regulation of the estrogen-dependent cell growth and activity of both estrogen receptors in response to growth factor signaling.