RESUMEN
BACKGROUND: In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial. METHODS: EAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35-55. Glibenclamide (10 µg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET. RESULTS: Administering glibenclamide beginning 24 days after MOG35-55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2. CONCLUSIONS: SUR1-TRPM4 may be a druggable target for disease modification in MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Gliburida/administración & dosificación , Esclerosis Múltiple/metabolismo , Receptores de Sulfonilureas/biosíntesis , Canales Catiónicos TRPM/biosíntesis , Adulto , Anciano , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Gliburida/metabolismo , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Esclerosis Múltiple/patología , Resultado del TratamientoRESUMEN
BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), deletion of transient receptor potential melastatin 4 (Trpm4) and administration of glibenclamide were found to ameliorate disease progression, prompting speculation that glibenclamide acts by directly inhibiting Trpm4. We hypothesized that in EAE, Trpm4 upregulation is accompanied by upregulation of sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which are highly sensitive to glibenclamide, and that Sur1-Trpm4 channels are required for EAE progression. METHODS: EAE was induced in wild-type (WT) and Abcc8-/- mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Lumbar spinal cords were examined by immunohistochemistry, immuno-Förster resonance energy transfer (immunoFRET), and co-immunoprecipitation for Sur1-Trpm4. WT/EAE mice were administered with the Sur1 inhibitor, glibenclamide, beginning on post-induction day 10. Mice were evaluated for clinical function, inflammatory cells and cytokines, axonal preservation, and white matter damage. RESULTS: Sur1-Trpm4 channels were upregulated in EAE, predominantly in astrocytes. The clinical course and severity of EAE were significantly ameliorated in glibenclamide-treated WT/EAE and in Abcc8-/-/EAE mice. At 30 days, the lumbar spinal cords of glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice showed significantly fewer invading immune cells, including leukocytes (CD45), T cells (CD3), B cells (CD20) and macrophages/microglia (CD11b), and fewer cells expressing pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17). In both glibenclamide-treated WT/EAE and Abcc8-/-/EAE mice, the reduced inflammatory burden correlated with better preservation of myelin, better preservation of axons, and more numerous mature and precursor oligodendrocytes. CONCLUSIONS: Sur-Trpm4 channels are newly upregulated in EAE and may represent a novel target for disease-modifying therapy in multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Receptores de Sulfonilureas/antagonistas & inhibidores , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Axones/patología , Femenino , Silenciador del Gen , Gliburida/uso terapéutico , Hipoglucemiantes/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vaina de Mielina/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos , Médula Espinal/patología , Receptores de Sulfonilureas/genéticaRESUMEN
Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and may be responsible for important complications of SAH. Signaling by Toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NFκB) in microglia plays a critical role in neuronal damage after SAH. Three molecules derived from erythrocyte breakdown have been postulated to be endogenous TLR4 ligands: methemoglobin (metHgb), heme and hemin. However, poor water solubility of heme and hemin, and lipopolysaccharide (LPS) contamination have confounded our understanding of these molecules as endogenous TLR4 ligands. We used a 5-step process to obtain highly purified LPS-free metHgb, as confirmed by Fourier Transform Ion Cyclotron Resonance mass spectrometry and by the Limulus amebocyte lysate assay. Using this preparation, we show that metHgb is a TLR4 ligand at physiologically relevant concentrations. metHgb caused time- and dose-dependent secretion of the proinflammatory cytokine, tumor necrosis factor α (TNFα), from microglial and macrophage cell lines, with secretion inhibited by siRNA directed against TLR4, by the TLR4-specific inhibitors, Rs-LPS and TAK-242, and by anti-CD14 antibodies. Injection of purified LPS-free metHgb into the rat subarachnoid space induced microglial activation and TNFα upregulation. Together, our findings support the hypothesis that, following SAH, metHgb in the subarachnoid space can promote widespread TLR4-mediated neuroinflammation.
Asunto(s)
Metahemoglobina/farmacología , Receptor Toll-Like 4/metabolismo , Animales , Bovinos , Línea Celular , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inflamación/etiología , Ligandos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metahemoglobina/química , Metahemoglobina/aislamiento & purificación , Ratones , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Hemorragia Subaracnoidea/complicaciones , Hemorragia Subaracnoidea/patología , Sulfonamidas/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The sulfonylurea receptor 1 (Sur1)-NC(Ca-ATP) channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC(Ca-ATP) channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K(ATP) channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Canales Catiónicos TRPM/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Células COS , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Diazóxido/farmacología , Transferencia Resonante de Energía de Fluorescencia , Gliburida/farmacología , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Ratas , Receptores de Droga/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Receptores de SulfonilureasRESUMEN
N-glycosylation is important for the function and regulation of ion channels. We examined the role of N-glycosylation of transient receptor potential melastatin (Trpm) 4b, a membrane glycoprotein that regulates calcium influx. Trpm4b was expressed in vivo in all rat tissues examined. In each tissue, Trpm4b had a different molecular mass, between â¼129 and â¼141 kDa, but all reverted to â¼120 kDa following treatment with peptide:N-glycosidase F, consistent with N-glycosylation being the principal form of post-translational modification of Trpm4b in vivo. In six stable isogenic cell lines that express different levels of Trpm4b, two forms were found, high mannose, core-glycosylated and complex, highly glycosylated (HG), with HG-Trpm4b comprising 85% of the total Trpm4b expressed. For both forms, surface expression was directly proportional to the total Trpm4b expressed. Complex N-glycosylation doubled the percentage of Trpm4b at the surface, compared with high mannose N-glycosylation. Mutation of the single N-glycosylation consensus sequence at Asn-988 (Trpm4b-N988Q), located near the pore-forming loop between transmembrane helices 5 and 6, prevented glycosylation, but did not prevent surface expression, impair formation of functional membrane channels, or alter channel conductance. In transfection experiments, the time courses for appearance of HG-Trpm4b and Trpm4b-N988Q on the surface were similar. In experiments with cycloheximide inhibition of protein synthesis, the time course for disappearance of HG-Trpm4b from the surface was much slower than that for Trpm4b-N988Q. We conclude that N-glycosylation is not required for surface expression or channel function, but that complex N-glycosylation plays a crucial role in stabilizing surface expression of Trpm4b.
Asunto(s)
Membrana Celular/metabolismo , Canales Catiónicos TRPM/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Señalización del Calcio , Chlorocebus aethiops , Glicosilación , Manosa/química , Ratones , Datos de Secuencia Molecular , Mutación Missense , Estabilidad Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/genéticaRESUMEN
BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) can leave patients with memory impairments that may not recover fully. Molecular mechanisms are poorly understood, and no treatment is available. The sulfonylurea receptor 1-transient receptor potential melastatin 4 (Sur1-Trpm4) channel plays an important role in acute central nervous system injury. We evaluated upregulation of Sur1-Trpm4 in humans with SAH and, in rat models of SAH, we examined Sur1-Trpm4 upregulation, its role in barrier dysfunction and neuroinflammation, and its consequences on spatial learning. METHODS: We used Förster resonance energy transfer to detect coassociated Sur1 and Trpm4 in human autopsy brains with SAH. We studied rat models of SAH involving filament puncture of the internal carotid artery or injection of blood into the subarachnoid space of the entorhinal cortex. In rats, we used Förster resonance energy transfer and coimmunoprecipitation to detect coassociated Sur1 and Trpm4, we measured immunoglobulin G extravasation and tumor necrosis α overexpression as measures of barrier dysfunction and neuroinflammation, and we assessed spatial learning and memory on days 7 to 19. RESULTS: Sur1-Trpm4 channels were upregulated in humans and rats with SAH. In rats, inhibiting Sur1 using antisense or the selective Sur1 inhibitor glibenclamide reduced SAH-induced immunoglobulin G extravasation and tumor necrosis α overexpression. In models with entorhinal SAH, rats treated with glibenclamide for 7 days after SAH exhibited better platform search strategies and better performance on incremental and rapid spatial learning than vehicle-treated controls. CONCLUSIONS: Sur1-Trpm4 channels are upregulated in humans and rats with SAH. Channel inhibition with glibenclamide may reduce neuroinflammation and the severity of cognitive deficits after SAH.
Asunto(s)
Trastornos del Conocimiento/metabolismo , Encefalitis/metabolismo , Hemorragia Subaracnoidea/metabolismo , Receptores de Sulfonilureas/antagonistas & inhibidores , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/fisiopatología , Encefalitis/genética , Encefalitis/fisiopatología , Gliburida/farmacología , Humanos , Aprendizaje por Laberinto/efectos de los fármacos , Ratas , Hemorragia Subaracnoidea/genética , Hemorragia Subaracnoidea/fisiopatología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Females have been understudied in pre-clinical and clinical traumatic brain injury (TBI), despite distinct biology and worse clinical outcomes versus males. Sulfonylurea receptor 1 (SUR1) inhibition has shown promising results in predominantly male TBI. A phase II trial is ongoing. We investigated whether SUR1 inhibition effects on contusional TBI differ by sex given that this may inform clinical trial design and/or interpretation. We studied the moderating effects of sex on post-injury brain tissue loss in 142 male and female ATP-binding cassette transporter subfamily C member 8 (Abcc8) wild-type, heterozygote, and knockout mice (12-15 weeks). Monkey fibroblast-like cells and mouse brain endothelium-derived cells were used for in vitro studies. Mice were injured with controlled cortical impact and euthanized 21 days post-injury to assess contusion, brain, and hemisphere volumes (vs. genotype- and sex-matched naïves). Abcc8 knockout mice had smaller contusion volumes (p = 0.012) and larger normalized contralateral (right) hemisphere volumes (nRHV; p = 0.03) after injury versus wild type. This was moderated by sex: Contusions were smaller (p = 0.020), nRHV was higher (p = 0.001), and there was less global atrophy (p = 0.003) in male, but not female, knockout versus wild-type mice after TBI. Less atrophy occurred in males for each copy of Abcc8 lost (p = 0.023-0.002, all outcomes). In vitro, sex-determining region Y (SRY) stimulated Abcc8 promoter activity and increased Abcc8 expression. Loss of Abcc8 strongly protected against post-traumatic cerebral atrophy in male, but not female, mice. This may partly be mediated by SRY on the Y-chromosome. Sex differences may have important implications for ongoing and future trials of SUR1 blockade.
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Lesiones Traumáticas del Encéfalo/patología , Receptores de Sulfonilureas/fisiología , Animales , Atrofia , Lesiones Traumáticas del Encéfalo/etiología , Lesiones Traumáticas del Encéfalo/metabolismo , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores Sexuales , Proteína de la Región Y Determinante del Sexo/fisiologíaRESUMEN
Limited, and underutilized, therapeutic options for acute stroke require new approaches to treatment. One such potential approach involves better understanding of innate immune response to brain injury such as acute focal cerebral ischemia. This includes understanding the temporal profile, and specificity, of Toll-like receptor 4 (TLR4) signaling in brain cell types, such as astrocytes, following focal cerebral ischemia. This study evaluated TLR4 signaling, and downstream mediators, in astrocytes, during acute and chronic phases post transient middle cerebral artery occlusion (MCAO). We also determined whether high mobility group box 1 (HMGB1), an endogenous TLR4 ligand, was sufficient to induce TLR4 signaling activation in astrocytes in vivo and in vitro. We injected HMGB1 into normal cortex, in vivo, and stimulated cultured astrocytes with HMGB1, in vitro, and determined TLR4, and downstream mediator, expression by immunohistochemistry. We found that expression of TLR4, and downstream mediators, such as inducible nitric oxide synthase (iNOS), occurs in penumbral astrocytes in acute and chronic phases after focal cerebral ischemia, but was undetectable in cortical astrocytes in the contralateral hemisphere. In addition, cortical injection of recombinant HMGB1 led to a trend towards an almost 2-fold increase in TLR4 expression in astrocytes surrounding the injection site. Consistent with these results, in vitro stimulation of the DI TNC1 astrocyte cell line, with recombinant HMGB1, led to increased TLR4 and iNOS message levels. These findings suggest that HMGB1, an endogenous TLR4 ligand, is an important physiological ligand for TLR4 signaling activation, in penumbral astrocytes, following acute and chronic ischemia and HMGB1 amplifies TLR4 signaling in astrocytes.
RESUMEN
The transcriptional activator TonEBP is a central regulator of osmolality in the renal medulla and whole body water homeostasis. In order to understand the regulation of TonEBP in the renal medulla, we examined MDCK cells, a kidney-derived epithelial cell line, under conditions mimicking the renal medulla. Moderate changes in ambient tonicity, which was tolerated without prior adaptation, displayed lasting effects on TonEBP in bidirectional manner-stimulated by hypertonicity and inhibited by hypotonicity. TonEBP expression was further enhanced by extreme hypertonicity observed in the inner medullae of antidiuretic animals. Urea stimulated TonEBP expression and promoted cellular proliferation under the conditions of extreme hypertonicity. On the other hand, the TonEBP activity was negatively modulated under these conditions presumably to temper the highly abundant TonEBP. We conclude that urea is critical to the cellular adaptation to extreme hypertonicity and the high level of TonEBP expression in the inner medulla.
Asunto(s)
Adaptación Fisiológica/fisiología , Médula Renal/metabolismo , Factores de Transcripción NFATC/biosíntesis , Urea/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Betaína/metabolismo , Northern Blotting , Western Blotting , Línea Celular , Perros , Células Epiteliales/metabolismo , Proteínas de Choque Térmico/metabolismo , Soluciones Hipertónicas , Soluciones Hipotónicas , Inmunohistoquímica , Médula Renal/citología , Concentración OsmolarRESUMEN
BACKGROUND: Hemorrhagic transformation is a major complication of ischemic stroke, is linked to matrix metalloproteinase-9 (MMP-9), and is exacerbated by tissue plasminogen activator (tPA). Cerebral ischemia/reperfusion is characterized by SUR1-TRPM4 (sulfonylurea receptor 1-transient receptor potential melastatin 4) channel upregulation in microvascular endothelium. In humans and rodents with cerebral ischemia/reperfusion (I/R), the SUR1 antagonist, glibenclamide, reduces hemorrhagic transformation and plasma MMP-9, but the mechanism is unknown. We hypothesized that tPA induces protease activated receptor 1 (PAR1)-mediated, Ca2+-dependent phasic secretion of MMP-9 from activated brain endothelium, and that SUR1-TRPM4 is required for this process. METHODS: Cerebral I/R, of 2 and 4 hours duration, respectively, was obtained using conventional middle cerebral artery occlusion. Immunolabeling was used to quantify p65 nuclear translocation. Murine and human brain endothelial cells (BEC) were studied in vitro, without and with NF-κB activation, using immunoblot, zymography and ELISA, patch clamp electrophysiology, and calcium imaging. Genetic and pharmacological manipulations were used to identify signaling pathways. RESULTS: Cerebral I/R caused prominent nuclear translocation of p65 in microvascular endothelium. NF-κB-activation of BEC caused de novo expression of SUR1-TRPM4 channels. In NF-κB-activated BEC: (i) tPA caused opening of SUR1-TRPM4 channels in a plasmin-, PAR1-, TRPC3- and Ca2+-dependent manner; (ii) tPA caused PAR1-dependent secretion of MMP-9; (iii) tonic secretion of MMP-9 by activated BEC was not influenced by SUR1 inhibition; (iv) phasic secretion of MMP-9 induced by tPA or the PAR1-agonist, TFLLR, required functional SUR1-TRPM4 channels, with inhibition of SUR1 decreasing tPA-induced MMP-9 secretion. CONCLUSIONS: tPA induces PAR1-mediated, SUR1-TRPM4-dependent, phasic secretion of MMP-9 from activated brain endothelium.
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Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fibrinolíticos/farmacología , Activador de Tejido Plasminógeno/farmacología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Calcio/metabolismo , Línea Celular , Células Endoteliales/metabolismo , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Microvasos/patología , FN-kappa B/metabolismo , Ratas Wistar , Receptores de Sulfonilureas/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
The TonE-binding protein (TonEBP) is a transcriptional activator in the Rel family that includes NFkappaB and NFAT. TonEBP is critical for the development and function of the renal medulla, which is a major regulator of water homeostasis. TonEBP is also implicated in diabetic nephropathy and inflammation. Established methods for biochemical and histochemical detection and functional analysis of TonEBP, including identification of novel TonEBP target genes, are described for those who are interested in investigating function and regulation of TonEBP.
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Diferenciación Celular/fisiología , Factores de Transcripción NFATC/fisiología , Secuencia de Aminoácidos , Animales , Ensayo de Cambio de Movilidad Electroforética , Humanos , Soluciones Hipertónicas/farmacología , Médula Renal/fisiología , Datos de Secuencia Molecular , Factores de Transcripción NFATC/análisis , Factores de Transcripción NFATC/inmunología , Interferencia de ARNRESUMEN
The nonselective monovalent cation channel transient receptor potential melastatin 4 (Trpm4) is transcriptionally upregulated in neural and vascular cells in animal models of brain infarction. It associates with sulfonylurea receptor 1 (Sur1) to form Sur1-Trpm4 channels, which have critical roles in cytotoxic edema, cell death, blood-brain barrier breakdown, and vasogenic edema. We examined Trpm4 expression in postmortem brain specimens from 15 patients who died within the first 31 days of the onset of focal cerebral ischemia. We found increased Trpm4 protein expression in all cases using immunohistochemistry; transcriptional upregulation was confirmed using in situ hybridization of Trpm4 messenger RNA. Transient receptor potential melastatin 4 colocalized and coassociated with Sur1 within ischemic endothelial cells and neurons. Coexpression of Sur1 and Trpm4 in necrotic endothelial cells was also associated with vasogenic edema indicated by upregulated perivascular tumor necrosis factor, extravasation of serum immunoglobulin G, and associated inflammation. Upregulated Trpm4 protein was present up to 1 month after the onset of cerebral ischemia. In a rat model of middle cerebral artery occlusion stroke, pharmacologic channel blockade by glibenclamide, a selective inhibitor of sulfonylurea receptor, mitigated perivascular tumor necrosis factor labeling. Thus, upregulated Sur1-Trpm4 channels and associated blood-brain barrier disruption and cerebral edema suggest that pharmacologic targeting of this channel may represent a promising therapeutic strategy for the clinical management of patients with cerebral ischemia.
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Infarto Cerebral/metabolismo , Receptores de Sulfonilureas/biosíntesis , Canales Catiónicos TRPM/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Cerebral ischemia causes increased transcription of sulfonylurea receptor 1 (SUR1), which forms SUR1-regulated NC(Ca-ATP) channels linked to cerebral edema. We tested the hypothesis that hypoxia is an initial signal that stimulates transcription of Abcc8, the gene encoding SUR1, via activation of hypoxia-inducible factor 1 (HIF1). In the brain microvascular endothelial cells, hypoxia increased SUR1 abundance and expression of functional SUR1-regulated NC(Ca-ATP) channels. Luciferase reporter activity driven by the Abcc8 promoter was increased by hypoxia and by coexpression of HIF1α. Surprisingly, a series of luciferase reporter assays studying the Abcc8 promoter revealed that binding sites for specificity protein 1 (Sp1), but not for HIF, were required for stimulation of Abcc8 transcription by HIF1α. Luciferase reporter assays studying Sp1 promoters of three species, and chromatin immunoprecipitation analysis in rats after cerebral ischemia, indicated that HIF binds to HIF-binding sites on the Sp1 promoter to stimulate transcription of the Sp1 gene. We conclude that sequential activation of two transcription factors, HIF and Sp1, is required to stimulate transcription of Abcc8 following cerebral ischemia. Sequential gene activation in cerebral ischemia provides a plausible molecular explanation for the prolonged treatment window observed for inhibition of the end-target gene product, SUR1, by glibenclamide.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga/genética , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Sitios de Unión , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Gliburida/farmacología , Gliburida/uso terapéutico , Células Hep G2 , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Factor 1 Inducible por Hipoxia/genética , Hipoxia-Isquemia Encefálica/genética , Hipoxia-Isquemia Encefálica/prevención & control , Inmunohistoquímica , Canales KATP/genética , Canales KATP/metabolismo , Luciferasas/genética , Masculino , Microvasos/citología , Microvasos/metabolismo , Técnicas de Placa-Clamp , Plásmidos , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Wistar , Receptores de Droga/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Receptores de Sulfonilureas , TransfecciónRESUMEN
The interstitium of the renal medulla is hypertonic, imposing deleterious effects on local cells. At the same time, the hypertonicity provides osmotic gradient for water reabsorption and is a local signal for tissue-specific gene expression and differentiation of the renal medulla, which is a critical organ for water homeostasis.
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Riñón/fisiología , Presión Osmótica/fisiología , Estrés Fisiológico/fisiología , Equilibrio Hidroelectrolítico/fisiología , Animales , HumanosRESUMEN
TonEBP is a Rel domain-containing transcription factor implicated in adaptive immunity, viral replication, and cancer. In the mammalian kidney, TonEBP is a central regulator of water homeostasis. Animals deficient in TonEBP suffer from life-threatening dehydration due to renal water loss. Ambient tonicity (effective osmolality) is the prominent signal for TonEBP in a bidirectional manner; TonEBP activity decreases in hypotonicity, whereas it increases in hypertonicity. Here we found that TonEBP displayed nuclear export in response to hypotonicity and nuclear import in response to hypertonicity. The nuclear export of TonEBP was not mediated by the nuclear export receptor CRM1 or discrete nuclear export signal. In contrast, a dominant nuclear localization signal (NLS) was found in a small region of 16 amino acid residues. When short peptides containing the NLS were fused to constitutively cytoplasmic proteins, the fusion proteins displayed tonicity-dependent nucleocytoplasmic trafficking like TonEBP. Thus, tonicity-dependent activation of the NLS is crucial in the nucleocytoplasmic trafficking of TonEBP. The novel NLS is present only in the vertebrates, indicating that it developed late in evolution.
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Núcleo Celular/fisiología , Factores de Transcripción NFATC/fisiología , Transducción de Señal/fisiología , Vertebrados/fisiología , Animales , Células COS , División Celular , Chlorocebus aethiops , Haplorrinos , Cinética , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFATC/genéticaRESUMEN
Axin, a negative regulator of Wnt, forms a complex with glycogen synthase kinase 3beta, beta-catenin, and adenomatous polyposis coli and promotes GSK3beta-dependent phosphorylation of beta-catenin, thereby stimulating degradation of the beta-catenin. An essential step in that process is the phosphorylation of Axin. Examination of Axin's amino acid sequence revealed it to contain six arginine-X-leucine (RXL) sequences, the cyclin-dependent kinase 2 (CDK2) binding motif, and 10 CDK2 consensus phosphorylation sequences. We also found that cyclin A/CDK2 phosphorylates Axin, thereby enhancing its association with beta-catenin. This suggests that cyclin A/CDK2 is a negative regulator of beta-catenin-mediated signal transduction, which exerts its effects through phosphorylation of Axin.
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Quinasas CDC2-CDC28/química , Quinasas CDC2-CDC28/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Animales , Proteína Axina , Sitios de Unión , Células COS , Catálisis , Chlorocebus aethiops , Quinasa 2 Dependiente de la Ciclina , Humanos , Fosforilación , Unión Proteica , Ratas , beta CateninaRESUMEN
By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.