RESUMEN
The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker ßIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for ßIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Ergotioneína/metabolismo , Neuronas/fisiología , Proteínas de Transporte de Catión Orgánico/metabolismo , Células Madre/fisiología , Animales , Antioxidantes/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carnitina/genética , Carnitina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Ergotioneína/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Neuronas/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismo , Simportadores , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [(3)H]ERGO in several brain regions of octn1(-/-) mice was much lower than that in wild-type mice, whereas extracellular marker [(14)C]mannitol exhibited similar distribution in the two strains. The [(3)H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and ßIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [(3)H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, ßIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [(3)H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development.