Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway
Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway
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DOI：
10.1111/j.1471-4159.2007.05085.x
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发表时间：
2008-03-01
影响因子：
4.7
通讯作者：
Nozawa, Yoshinori
中科院分区：
文献类型：
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作者：
Banno, Yoshiko;Nemoto, Satoshi;Nozawa, Yoshinori
本研究探讨磷脂酶D2（PLD 2）在大鼠嗜铬细胞瘤（PC 12）细胞去极化诱导的神经突起生长和生长相关蛋白-43（GAP-43）和突触素I表达的调节中的作用。50 mmol/L KCl去极化可促进PC 12细胞突起的生长，增加GAP-43和synapsin I的mRNA和蛋白表达。这些增加被抑制的钙离子-钙调蛋白依赖性蛋白激酶II（CaMKII），PLD，或丝裂原活化蛋白激酶激酶（MEK）的抑制。通过小干扰RNA（siRNA）敲低PLD 2抑制去极化诱导的神经突生长，以及GAP-43和突触蛋白I表达的增加。去极化引起Ca 2+升高，从而激活各种信号传导酶和cAMP反应元件结合蛋白（CREB）。通过siRNA沉默CaMKII δ阻断KCl诱导的富含脯氨酸的蛋白酪氨酸激酶2（Pyk 2）、Src激酶和细胞外信号调节激酶（ERK）的磷酸化。Src或MEK的抑制消除了ERK和CREB的磷酸化。此外，Pyk 2，ERK和CREB的磷酸化被PLD抑制剂，1-丁醇和PLD 2 siRNA转染抑制，而野生型PLD 2的过表达增强。去极化诱导的PLD 2激活被CaMKII和Src抑制剂抑制，但不被MEK或蛋白激酶A抑制剂抑制。这些结果表明，去极化诱导的PLD 2激活的信号通路位于CaMKII δ和Src的下游，Pyk 2（Y881）和ERK/CREB的上游，但不依赖于蛋白激酶A。这是首次证明PLD 2激活参与了细胞凋亡过程中GAP-43和突触蛋白I的表达。
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 CaMKII delta 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 overexpression 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 CaMKII delta 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