title: The cAMP pathway regulates both transcription and activity of the paired homeobox transcription factor Phox2a required for development of neural crest-derived and central nervous system-derived catecholaminergic neurons.
abstract: Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, bone morphogenetic protein 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor Phox2a. In NC cultures inhibition of cAMP-response element-binding protein (CREB)-mediated transcription by expression of dominant-negative CREB suppresses Phox2a transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREB(DIEDML), despite inducing Phox2a transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREB(DIEDML)-mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent protein kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-10 nm okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated Phox2a dephosphorylation, cAMP-dependent Phox2a DNA binding in vitro, and cAMP- and Phox2a-dependent dopamine-beta-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent Phox2a dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both Phox2a transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development.
authors.affiliation: Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47906, USA.
authors.forename: Sigeng
authors.initials: S
authors.lastname: Chen
authors.forename: Ming
authors.initials: M
authors.lastname: Ji
authors.forename: Maryline
authors.initials: M
authors.lastname: Paris
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Hullinger
authors.forename: Ourania M
authors.initials: OM
authors.lastname: Andrisani
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology / Q000502:physiology; D002395:Catecholamines / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology*; D002642:Chick Embryo; D003370:Coturnix / Q000196:embryology; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D015870:Gene Expression; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009432:Neural Crest / Q000166:cytology*; D009474:Neurons / Q000502:physiology*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013564:Sympathetic Nervous System / Q000166:cytology; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D002395:Catecholamines; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D007546:Isoquinolines; D012333:RNA, Messenger; D013449:Sulfonamides; D016212:Transforming Growth Factor beta; D019319:Okadaic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine