RESUMEN

On incubation with epinephrine, PC12 cells stably expressing alpha2-adrenergic receptor (alpha2-AR) undergo morphological and biochemical changes characteristic of neuron-like differentiation. The present study shows that alpha2-AR stimulation increases the phosphorylation of the transcription factor cAMP-response element-binding protein (CREB), the activity of a CRE-reporter plasmid and the expression of cyclin D1 with subtype-dependent efficiency (alpha2A approximately alpha2C >> alpha2B). The effects of epinephrine were mimicked by cell exposure to forskolin or to exogenous arachidonic acid (AA) and they were abrogated by prior treatment with the inhibitor of phospholipase C (PLC) (U73122) or the inhibitor of cytochrome P450-dependent epoxygenase, ketoconazole. On the other hand, treatment of the cells with epinephrine caused activation of protein kinase A (PKA), which was fully abolished by ketoconazole. Inhibition of PKA activity with H89 or ketoconazole abolished the effects of epinephrine on CREB, suggesting that activation of the cAMP/PKA pathway by AA epoxy-derivatives is responsible for CREB activation by alpha2-ARs. The effects of epinephrine were unaffected by LY294002. Furthermore, treatment with staurosporine, tyrphostin AG1478, PP1 or PD98059 did not change the extent of CREB phosphorylation but enhanced its transcriptional activity. Altogether, our results demonstrate that, in PC12 cells, the alpha2-AR subtypes cause phosphorylation and activation of CREB through a pathway involving stimulation of PLC, AA release, generation of epoxygenase derivative and increase of PKA activity. They also suggest attenuation of CREB transcriptional activity by mitogen-activated protein kinase, protein kinase C and Src kinases.

Asunto(s)

Ácido Araquidónico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Animales , Ácido Araquidónico/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Epinefrina/metabolismo , Epinefrina/farmacología , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología

RESUMEN

Alpha2-adrenergic receptors have been reported to induce subtype-specific neuronal differentiation in vitro, but the signaling mechanisms that mediate this effect have not been characterized. In the present study we found that stimulated alpha2-ARs induce delayed transactivation of TrkA in PC12 cells. The transactivation of TrkA was sensitive to the PP1 inhibitor of the Src family kinases and required prior transactivation of the EGF receptor. Moreover, alpha2-adrenergic receptors induced sustained activation of MAPK and Akt. The sustained activation of Akt, but not of MAPK, was subtype-specific and correlated with the neuronal differentiation of PC12 cells, with the order alpha2A

Asunto(s)

Receptor trkA/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transducción de Señal , Animales , Ciclo Celular , Diferenciación Celular , Receptores ErbB/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Células PC12 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptores Adrenérgicos alfa 2/genética , Transfección

RESUMEN

In the present study we sought to investigate the signal transduction mechanisms that underlie the alpha2-adrenergic receptor (AR)-induced, subtype-specific neuronal differentiation of PC12 cells. Alpha2-ARs induced NF-kappaB transcriptional activity and p21(waf-1) gene transcription in the same subtype-specific manner (alpha2A

Asunto(s)

FN-kappa B/metabolismo , Receptores Adrenérgicos alfa 2/fisiología , Animales , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Humanos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , FN-kappa B/genética , Células PC12 , Fosfatidilinositol 3-Quinasas/fisiología , Ratas , Transducción de Señal , Transcripción Genética

RESUMEN

Previous study carried out on PC12 cells expressing each alpha(2)-adrenergic receptor subtype individually (PC12/alpha(2A), /alpha(2B) or /alpha(2C)) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each alpha(2)-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA(2) inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by alpha(2)-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/alpha(2) induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which alpha(2)-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.

Asunto(s)

Ácido Araquidónico/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasas de la Matriz/metabolismo , Oxidorreductasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Animales , Antifúngicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Epinefrina/metabolismo , Humanos , Cetoconazol/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Quinacrina/metabolismo , Ratas , Receptor trkA/metabolismo , Receptores Adrenérgicos alfa 2/genética , Activación Transcripcional , Familia-src Quinasas/metabolismo