Alpha2-adrenergic receptors activate cyclic AMP-response element-binding protein through arachidonic acid metabolism and protein kinase A in a subtype-specific manner.
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.
Department of Pharmacology, School of Medicine, University of Patras, Rio Patras, Greece.
Georgios
G
Karkoulias
Orthodoxia
O
Mastrogianni
Panagiotis
P
Papathanasopoulos
Hervé
H
Paris
Christodoulos
C
Flordellis
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism* / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004837:Epinephrine / Q000378:metabolism / Q000494:pharmacology; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D018341:Receptors, Adrenergic, alpha-2; D019938:Cyclin D1; D005576:Colforsin; D016718:Arachidonic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D004837:Epinephrine