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1.
Front Immunol ; 15: 1364774, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38629075

RESUMEN

Allergic asthma has emerged as a prevalent allergic disease worldwide, affecting most prominently both young individuals and lower-income populations in developing and developed countries. To devise effective and curative immunotherapy, it is crucial to comprehend the intricate nature of this condition, characterized by an immune response imbalance that favors a proinflammatory profile orchestrated by diverse subsets of immune cells. Although the involvement of Natural Killer T (NKT) cells in asthma pathology is frequently implied, their specific contributions to disease onset and progression remain incompletely understood. Given their remarkable ability to modulate the immune response through the rapid secretion of various cytokines, NKT cells represent a promising target for the development of effective immunotherapy against allergic asthma. This review provides a comprehensive summary of the current understanding of NKT cells in the context of allergic asthma, along with novel therapeutic approaches that leverage the functional response of these cells.


Asunto(s)
Asma , Hipersensibilidad , Células T Asesinas Naturales , Humanos , Hipersensibilidad/terapia , Citocinas , Inmunoterapia
2.
Free Radic Biol Med ; 211: 24-34, 2024 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-38043868

RESUMEN

The intricate relationship between calcium (Ca2+) homeostasis and mitochondrial function is crucial for cellular metabolic adaptation in tumor cells. Ca2+-initiated signaling maintains mitochondrial respiratory capacity and ATP synthesis, influencing critical cellular processes in cancer development. Previous studies by our group have shown that the homocysteine-inducible ER Protein with Ubiquitin-Like Domain 1 (HERPUD1) regulates inositol 1,4,5-trisphosphate receptor (ITPR3) levels and intracellular Ca2+ signals in tumor cells. This study explores the role of HERPUD1 in regulating mitochondrial function and tumor cell migration by controlling ITPR3-dependent Ca2+ signals. We found HERPUD1 levels correlated with mitochondrial function in tumor cells, with HERPUD1 deficiency leading to enhanced mitochondrial activity. HERPUD1 knockdown increased intracellular Ca2+ release and mitochondrial Ca2+ influx, which was prevented using the ITPR3 antagonist xestospongin C or the Ca2+ chelator BAPTA-AM. Furthermore, HERPUD1 expression reduced tumor cell migration by controlling ITPR3-mediated Ca2+ signals. HERPUD1-deficient cells exhibited increased migratory capacity, which was attenuated by treatment with xestospongin C or BAPTA-AM. Additionally, HERPUD1 deficiency led to reactive oxygen species-dependent activation of paxillin and FAK proteins, which are associated with enhanced cell migration. Our findings highlight the pivotal role of HERPUD1 in regulating mitochondrial function and cell migration by controlling intracellular Ca2+ signals mediated by ITPR3. Understanding the interplay between HERPUD1 and mitochondrial Ca2+ regulation provides insights into potential therapeutic targets for cancer treatment and other pathologies involving altered energy metabolism.


Asunto(s)
Calcio , Neoplasias , Humanos , Calcio/metabolismo , Señalización del Calcio/fisiología , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Factores de Transcripción/metabolismo
3.
Expert Opin Ther Targets ; 27(3): 207-223, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36880349

RESUMEN

INTRODUCTION: The vascular cell adhesion molecule (VCAM-1) is a transmembrane sialoglycoprotein detected in activated endothelial and vascular smooth muscle cells involved in the adhesion and transmigration of inflammatory cells into damaged tissue. Widely used as a pro-inflammatory marker, its potential role as a targeting molecule has not been thoroughly explored. AREAS COVERED: We discuss the current evidence supporting the potential targeting of VCAM-1 in atherosclerosis, diabetes, hypertension and ischemia/reperfusion injury. EXPERT OPINION: There is emerging evidence that VCAM-1 is more than a biomarker and may be a promising therapeutic target for vascular diseases. While there are neutralizing antibodies that allow preclinical research, the development of pharmacological tools to activate or inhibit this protein are required to thoroughly assess its therapeutic potential.


Asunto(s)
Aterosclerosis , Daño por Reperfusión , Humanos , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Endotelio Vascular
4.
Front Neurosci ; 12: 470, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30057523

RESUMEN

Age is the main risk factor for the onset of neurodegenerative diseases. A decline of mitochondrial function has been observed in several age-dependent neurodegenerative diseases and may be a major contributing factor in their progression. Recent findings have shown that mitochondrial fitness is tightly regulated by Ca2+ signals, which are altered long before the onset of measurable histopathology hallmarks or cognitive deficits in several neurodegenerative diseases including Alzheimer's disease (AD), the most frequent cause of dementia. The transfer of Ca2+ from the endoplasmic reticulum (ER) to the mitochondria, facilitated by the presence of mitochondria-associated membranes (MAMs), is essential for several physiological mitochondrial functions such as respiration. Ca2+ transfer to mitochondria must be finely regulated because excess Ca2+ will disturb oxidative phosphorylation (OXPHOS), thereby increasing the generation of reactive oxygen species (ROS) that leads to cellular damage observed in both aging and neurodegenerative diseases. In addition, excess Ca2+ and ROS trigger the opening of the mitochondrial transition pore mPTP, leading to loss of mitochondrial function and cell death. mPTP opening probably increases with age and its activity has been associated with several neurodegenerative diseases. As Ca2+ seems to be the initiator of the mitochondrial failure that contributes to the synaptic deficit observed during aging and neurodegeneration, in this review, we aim to look at current evidence for mitochondrial dysfunction caused by Ca2+ miscommunication in neuronal models of neurodegenerative disorders related to aging, with special emphasis on AD.

6.
Cell Rep ; 14(10): 2313-24, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26947070

RESUMEN

In the absence of low-level ER-to-mitochondrial Ca(2+) transfer, ATP levels fall, and AMPK-dependent, mTOR-independent autophagy is induced as an essential survival mechanism in many cell types. Here, we demonstrate that tumorigenic cancer cell lines, transformed primary human fibroblasts, and tumors in vivo respond similarly but that autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared. Cancer cell death is due to compromised bioenergetics that can be rescued with metabolic substrates or nucleotides and caused by necrosis associated with mitotic catastrophe during their proliferation. Our findings reveal an unexpected dependency on constitutive Ca(2+) transfer to mitochondria for viability of tumorigenic cells and suggest that mitochondrial Ca(2+) addiction is a feature of cancer cells.


Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Acetilcisteína/farmacología , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Compuestos Macrocíclicos/farmacología , Microscopía por Video , Oxazoles/farmacología , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo
9.
J Neurosci ; 34(20): 6910-23, 2014 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-24828645

RESUMEN

Exaggerated intracellular Ca(2+) signaling is a robust proximal phenotype observed in cells expressing familial Alzheimer's disease (FAD)-causing mutant presenilins (PSs). The mechanisms that underlie this phenotype are controversial and their in vivo relevance for AD pathogenesis is unknown. Here, we used a genetic approach to identify the mechanisms involved and to evaluate their role in the etiology of AD in two FAD mouse models. Genetic reduction of the type 1 inositol trisphosphate receptor (InsP3R1) by 50% normalized exaggerated Ca(2+) signaling observed in cortical and hippocampal neurons in both animal models. In PS1M146V knock-in mice, reduced InsP3R1 expression restored normal ryanodine receptor and cAMP response element-binding protein (CREB)-dependent gene expression and rescued aberrant hippocampal long-term potentiation (LTP). In 3xTg mice, reduced InsP3R1 expression profoundly attenuated amyloid ß accumulation and tau hyperphosphorylation and rescued hippocampal LTP and memory deficits. These results indicate that exaggerated Ca(2+) signaling, which is associated with FAD PS, is mediated by InsP3R and contributes to disease pathogenesis in vivo. Targeting the InsP3 signaling pathway could be considered a potential therapeutic strategy for patients harboring mutations in PS linked to AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio/genética , Corteza Cerebral/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/genética , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Potenciación a Largo Plazo/genética , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Presenilina-1/genética , Presenilina-1/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
10.
Nat Cell Biol ; 14(12): 1336-43, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23178883

RESUMEN

Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.


Asunto(s)
Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Células COS , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/genética
11.
Cell ; 151(3): 630-44, 2012 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-23101630

RESUMEN

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Supervivencia Celular , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Apoptosis , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo
12.
Proc Natl Acad Sci U S A ; 108(32): 13293-8, 2011 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-21784978

RESUMEN

Mutations in presenilins (PS) account for most early-onset familial Alzheimer's disease (FAD). Accumulating evidence suggests that disrupted Ca(2+) signaling may play a proximal role in FAD specifically, and Alzheimer's disease (AD) more generally, but its links to the pathogenesis of AD are obscure. Here we demonstrate that expression of FAD mutant PS constitutively activates the transcription factor cAMP response element binding protein (CREB) and CREB target gene expression in cultured neuronal cells and AD mouse models. Constitutive CREB activation was associated with and dependent on constitutive activation of Ca(2+)/CaM kinase kinase ß and CaM kinase IV (CaMKIV). Depletion of endoplasmic reticulum Ca(2+) stores or plasma membrane phosphatidylinositol-bisphosphate and pharmacologic inhibition or knockdown of the expression of the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel each abolished FAD PS-associated constitutive CaMKIV and CREB phosphorylation. CREB and CaMKIV phosphorylation and CREB target gene expression, including nitric oxide synthase and c-fos, were enhanced in brains of M146V-KI and 3xTg-AD mice expressing FAD mutant PS1 knocked into the mouse locus. FAD mutant PS-expressing cells demonstrated enhanced cell death and sensitivity to Aß toxicity, which were normalized by interfering with the InsP(3)R-CAMKIV-CREB pathway. Thus, constitutive CREB phosphorylation by exaggerated InsP(3)R Ca(2+) signaling in FAD PS-expressing cells may represent a signaling pathway involved in the pathogenesis of AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Muerte Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Fosforilación/efectos de los fármacos , Transcripción Genética/efectos de los fármacos
13.
Antioxid Redox Signal ; 14(7): 1225-35, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20701429

RESUMEN

Familial Alzheimer's disease (FAD) is caused by mutations in amyloid precursor protein and presenilins (PS1, PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca(2+)) homeostasis by proximal mechanisms independent of amyloid production by dramatically enhancing gating of the inositol trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel by a gain-of-function effect that mirrors genetics of FAD and is independent of secretase activity. Electrophysiological recordings of InsP(3)R in FAD patient B cells, cortical neurons of asymptomatic PS1-AD mice, and other cells revealed they have higher occupancy in a high open probability burst mode, resulting in enhanced Ca(2+) signaling. Exaggerated Ca(2+) signaling through this mechanism results in enhanced generation of reactive oxygen species, believed to be an important component in AD pathogenesis. Exaggerated Ca(2+) signaling through InsP(3)R-PS interaction is a disease specific and robust proximal mechanism in AD that may contribute to the pathology of AD by enhanced generation of reactive oxygen species.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedad de Alzheimer/patología , Animales , Linfocitos B/patología , Linfocitos B/fisiología , Línea Celular , Humanos , Peróxido de Hidrógeno/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Activación del Canal Iónico , Potenciales de la Membrana , Ratones , Mutación , Presenilina-1/genética , Presenilina-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
14.
Cell ; 142(2): 270-83, 2010 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-20655468

RESUMEN

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.


Asunto(s)
Linfocitos B/metabolismo , Señalización del Calcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Animales , Autofagia , Calcio/metabolismo , Línea Celular , Pollos , Técnicas de Inactivación de Genes
15.
Neuron ; 58(6): 871-83, 2008 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-18579078

RESUMEN

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio/fisiología , Calcio/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Activación del Canal Iónico/fisiología , Presenilinas/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Calcio/fisiología , Señalización del Calcio/genética , Células Cultivadas , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Spodoptera
16.
Cell Calcium ; 41(5): 491-502, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17074386

RESUMEN

Hydrogen peroxide, which stimulates ERK phosphorylation and synaptic plasticity in hippocampal neurons, has also been shown to stimulate calcium release in muscle cells by promoting ryanodine receptor redox modification (S-glutathionylation). We report here that exposure of N2a cells or rat hippocampal neurons in culture to 200 microM H2O2 elicited calcium signals, increased ryanodine receptor S-glutathionylation, and enhanced both ERK and CREB phosphorylation. In mouse hippocampal slices, H2O2 (1 microM) also stimulated ERK and CREB phosphorylation. Preincubation with ryanodine (50 microM) largely prevented the effects of H2O2 on calcium signals and ERK/CREB phosphorylation. In N2a cells, the ERK kinase inhibitor U0126 suppressed ERK phosphorylation and abolished the stimulation of CREB phosphorylation produced by H2O2, suggesting that H2O2 enhanced CREB phosphorylation via ERK activation. In N2a cells in calcium-free media, 200 microM H2O2 stimulated ERK and CREB phosphorylation, while preincubation with thapsigargin prevented these enhancements. These combined results strongly suggest that H2O2 promotes ryanodine receptors redox modification; the resulting calcium release signals, by enhancing ERK activity, would increase CREB phosphorylation. We propose that ryanodine receptor stimulation by activity-generated redox species produces calcium release signals that may contribute significantly to hippocampal synaptic plasticity, including plasticity that requires long-lasting ERK-dependent CREB phosphorylation.


Asunto(s)
Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipocampo/citología , Peróxido de Hidrógeno/farmacología , Neuronas/enzimología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo
17.
J Biol Chem ; 279(37): 39122-31, 2004 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-15262987

RESUMEN

Membrane depolarization of skeletal muscle cells induces slow inositol trisphosphate-mediated calcium signals that regulate the activity of transcription factors such as the cAMP-response element-binding protein (CREB), jun, and fos. Here we investigated whether such signals regulate CREB phosphorylation via protein kinase C (PKC)-dependent pathways. Western blot analysis revealed the presence of seven isoforms (PKCalpha, -betaI, -betaII, -delta, -epsilon, -, and -zeta) in rat primary myotubes. The PKC inhibitors bisindolymaleimide I and Gö6976, blocked CREB phosphorylation. Chronic exposure to phorbol ester triggered complete down-regulation of several isoforms, but reduced PKCalpha levels to only 40%, and did not prevent CREB phosphorylation upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKCalpha translocation to the nucleus following depolarization, which was blocked by 2-amino-ethoxydiphenyl borate, an inositol trisphosphate receptor inhibitor, and by the phospholipase C inhibitor U73122. In C2C12 cells, which expressed PKCalpha,-epsilon, and -zeta, CREB phosphorylation also depended on PKCalpha. These results strongly implicate nuclear PKCalpha translocation in CREB phosphorylation induced by skeletal muscle membrane depolarization.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Músculo Esquelético/metabolismo , Transporte Activo de Núcleo Celular , Animales , Animales Recién Nacidos , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Estrenos/farmacología , Inmunohistoquímica , Inositol 1,4,5-Trifosfato/química , Ratones , Microscopía Confocal , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ésteres del Forbol/farmacología , Fosforilación , Potasio/química , Pruebas de Precipitina , Isoformas de Proteínas , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo
18.
Endocrinology ; 144(8): 3586-97, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12865341

RESUMEN

Involvement of intracellular Ca(2+) and ERK1/2 phosphorylation in the fast nongenomic effects of androgens in myotubes was investigated. Testosterone or nandrolone produced fast (<1 min) and transient increases in intracellular Ca(2+) with an oscillatory pattern. Calcium signals were slightly reduced in Ca(2+)-free medium, but lack of oscillations was evident. Signals were blocked by U-73122 and xestospongin B, inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway. Furthermore, IP(3) increased transiently 2- to 3-fold 45 sec after hormone addition. Cyproterone neither affected the fast Ca(2+) signal nor the increase in IP(3). Calcium increases could also be induced by the impermeant testosterone conjugated to BSA, and the effect of testosterone was abolished in cells incubated with guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. Stimulation of myotubes with testosterone, nandrolone, or testosterone conjugated to BSA increased immunodetectable phosphorylation of ERK1/2 within 5 min, and this effect was not inhibited by cyproterone. Phosphorylation was blocked by the use of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester, U-73122, and xestospongin B as well as by dominant negative Ras, MAPK kinase (MEK), or the MEK inhibitor PD-98059. In addition, guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin blocked ERK1/2 phosphorylation. These results are consistent with a fast effect of testosterone, involving a G protein-linked receptor at the plasma membrane, IP(3)-mediated Ca(2+) signal, and the Ras/MEK/ERK pathway in muscle cells.


Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Proteínas de Unión al GTP/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Esquelético/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/fisiología , Testosterona/farmacología , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Animales , Animales Recién Nacidos , Quelantes/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Acetato de Ciproterona/farmacología , Expresión Génica/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Nandrolona/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Transfección , Proteínas ras/metabolismo
19.
Am J Physiol Cell Physiol ; 284(6): C1438-47, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12529240

RESUMEN

The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate (IP(3)) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c-fos, c-jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the IP(3) inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells.


Asunto(s)
Calcio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces , Potenciales de la Membrana/fisiología , Músculo Esquelético/fisiología , Transducción de Señal/fisiología , Animales , Cafeína/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN , Proteína 1 de la Respuesta de Crecimiento Precoz , Inhibidores Enzimáticos/metabolismo , Genes fos , Genes jun , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Fosforilación , Potasio/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción , Activación Transcripcional
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