RESUMEN
The electrogenic Na+ /HCO3- cotransporter (NBCe1) in astrocytes is crucial in regulation of acid-base homeostasis in the brain. Since many pathophysiological conditions in the brain have been associated with pH shifts we exposed primary mouse cortical and hippocampal astrocytes to prolonged low or high extracellular pH (pHo ) at constant extracellular bicarbonate concentration and investigated activation of astrocytes and regulation of NBCe1 by immunoblotting, biotinylation of surface proteins, and intracellular H+ recordings. High pHo at constant extracellular bicarbonate caused upregulation of NBCe1 protein, surface expression and activity via upregulation of the astrocytic activation markers signal transducer and activator of transcription 3 (STAT3) signaling and glial fibrillary acidic protein expression. High pHo -induced increased NBCe1 protein expression was prevented in astrocytes from Stat3flox/flox ::GfapCre/+ mice. In vitro, basal and high pHo -induced increased NBCe1 functional expression was impaired following inhibition of STAT3 phosphorylation. These results provide a novel regulation mode of NBCe1 protein and activity, highlight the importance of astrocyte reactivity on regulation of NBCe1 and implicate roles for NBCe1 in altering/modulating extracellular pH during development as well as of the microenvironment at sites of brain injuries and other pathophysiological conditions.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/citología , Factor de Transcripción STAT3/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Astrocitos/efectos de los fármacos , Bicarbonatos/farmacología , Biomarcadores/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Espacio Extracelular/metabolismo , Hipocampo/citología , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF-ß) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT-PCR, immunoblotting, immunofluorescence, intracellular H(+ ) recording using the H(+ ) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF-ß signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF-ß receptor-mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4-aminopyridine (4AP)-dependent NBCe1 regulation requires TGF-ß. TGF-ß increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild-type astrocytes but not in cortical astrocytes from Slc4a4-deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF-ß signaling. The data show for the first time that NBCe1 is a direct target of TGF-ß/Smad4 signaling. Through activation of the canonical pathway TGF-ß acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.
Asunto(s)
Astrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Transducción de Señal/fisiología , Simportadores de Sodio-Bicarbonato/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , 4-Aminopiridina/farmacología , Familia de Aldehído Deshidrogenasa 1 , Animales , Benzamidas/farmacología , Células Cultivadas , Corteza Cerebral/citología , Antiportadores de Cloruro-Bicarbonato/farmacología , Dioxoles/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/citología , Concentración de Iones de Hidrógeno , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bloqueadores de los Canales de Potasio/farmacología , Retinal-Deshidrogenasa/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad4/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Simportadores de Sodio-Bicarbonato/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) is expressed in many cell types and is a major regulator of intracellular, and extracellular pH. In astrocytes, membrane depolarization leads to intracellular alkalinization through the activation of NBCe1. However, the molecular mechanisms regulating functional expression of NBCe1 in astrocytes are largely unknown. Astrocytes also express voltage-dependent K(+) channels that are activated after depolarization and are sensitive to the K(+) blocker 4-aminopyridine (4AP). Using acute hippocampal slices and primary hippocampal and cortical astrocyte cultures, we have investigated the role of 4AP for the regulation of NBCe1 and elucidated the underlying signaling pathways by quantitative RT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, and intracellular H(+) recording using the H(+) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein. The results show significant upregulation of NBCe1 transcript, protein, and surface expression after the application of 4AP in both hippocampal slices and astrocyte cultures, effects that were suppressed after the inhibition of c-jun N-terminal kinase (JNK), proto-oncogene tyrosine-protein kinase Src, and Src/extracellular-signal-regulated kinases signaling. In the presence of 4AP, the rate and amplitude of intracellular H(+) changes upon challenging NBCe1 increased in wild-type astrocytes but not in cortical astrocytes from NBCe1-deficient mice. 4AP-dependent effects were suppressed after the inhibition of JNK and Src signaling. Our results demonstrate that transcriptional regulation and targeting of NBCe1, as well as functional operation of NBCe1, may occur through multiple signaling pathways.