Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway |
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Authors: | Banno Yoshiko Nemoto Satoshi Murakami Masashi Kimura Masashi Ueno Yoshihito Ohguchi Kenji Hara Akira Okano Yukio Kitade Yukio Onozuka Minoru Murate Takashi Nozawa Yoshinori |
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Affiliation: | Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan; Department of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan; Nagoya University School of Health Sciences, Nagoya, Japan; Department of Molecular Pathobiochemistry, Gifu University Graduate School of Medicine, Gifu, Japan; Faculty of Engineering, Gifu University, Gifu, Japan; Gifu International Institute of Biotechnology, Kakamigahara, Japan; Department of Physiology and Neuroscience, Kanagawa Dental College, Yokosuka, Japan |
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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 CaMKIIδ 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 CaMKIIδ 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. |
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Keywords: | depolarization differentiation growth-associated protein-43 phospholipase D2 signal transduction synapsin I |
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