Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway.
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.
Banno
Yoshiko
Y
Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan. yobanno@gifu-u.ac.jp
Nemoto
Satoshi
S
Murakami
Masashi
M
Kimura
Masashi
M
Ueno
Yoshihito
Y
Ohguchi
Kenji
K
Hara
Akira
A
Okano
Yukio
Y
Kitade
Yukio
Y
Onozuka
Minoru
M
Murate
Takashi
T
Nozawa
Yoshinori
Y
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*
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