title: Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway.
abstract: The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca2+-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca2+ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIdelta by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIdelta and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.
authors.affiliation: Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan. yobanno@gifu-u.ac.jp
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Banno
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nemoto
authors.forename: Masashi
authors.initials: M
authors.lastname: Murakami
authors.forename: Masashi
authors.initials: M
authors.lastname: Kimura
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Ueno
authors.forename: Kenji
authors.initials: K
authors.lastname: Ohguchi
authors.forename: Akira
authors.initials: A
authors.lastname: Hara
authors.forename: Yukio
authors.initials: Y
authors.lastname: Okano
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
authors.forename: Minoru
authors.initials: M
authors.lastname: Onozuka
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019922:GAP-43 Protein / Q000096:biosynthesis / Q000235:genetics; D016501:Neurites / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology; D016716:PC12 Cells; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016704:Synapsins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D019922:GAP-43 Protein; D009467:Neuromuscular Depolarizing Agents; D016704:Synapsins; D014157:Transcription Factors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C105606:phospholipase D2; D010739:Phospholipase D