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 Ca 2+ -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 Ca 2+ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIId 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 CaMKIId 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.

Increased Expression of Calcium/Calmodulin-Dependent Protein Kinase Type II Subunit Delta after Rat Traumatic Brain Injury

“…Important functions of CaMKII include the control of neurotransmitter release and the mediation of stimulus-induced gene expression (Moreno-Delgado et al 2009;Shen et al 2010). CaMKIIδ, by the very nature of its core function as a kinase, is a multifunctional signaling protein that regulates numerous physiological processes including cellular differentiation (Donai et al 2000;Ju et al 2004;Banno et al 2008;Han et al 2011;Mouton-Liger et al 2011), ion channel function (Wagner et al 2011), arrhythmogenesis during atrial fibrillation (Tobimatsu and Fujisawa 1989;Qin et al 2011), modulating inflammatory and proliferative responses (Currie et al 2011), vascular smooth muscle proliferation (House et al 2007;House and Singer 2008;Li et al 2011), heart failure (Zhang et al 2004;Maier 2005;Rokita et al 2011), and pathological processes such as apoptosis induced by ischemia/reperfusion injury in cardiomyoctyes (Ma et al 2009;Wang et al 2010). Knowledge of the participation of CaMKIIδ in cell physiology and apoptotic pathways in central nervous system is limited.…”

Molecular Mechanisms of N-Formyl-Methionyl-Leucyl-Phenylalanine-Induced Superoxide Generation and Degranulation in Mouse Neutrophils: Phospholipase D Is Dispensable

“…In contrast, PLD2 has a constitutively high activity in the presence of PI4,5P 2 and is insensitive to PLD1 activators (28,32). PLD2 has been implicated in various cellular events, such as antiapoptosis (38), endocytic recycling (39), endocytosis (40), differentiation of PC12 cells (41), actin-based membrane dynamics (42), and L1-dependent neurite outgrowth (43). Conclusions about some of these PLD functions are based on observations from experiments using primary alcohols, such as ethanol (EtOH) and 1-butanol.…”

Expression and Role of T-type Calcium Channels during Neuroendocrine Differentiation

“…Another study demon-strated that depolarizationinduced calcium entry promotes NE differentiation of PC12 cells. This NE differentiation characterized by neurite formation, GAP-43 and synapsin I overexpression, requires the activation of phospholipase D2 -ErK-CREB pathway and Src phosphorylation by CaMKinase II [99]. Such a pathway is similar to the one demonstrated in hippocampal neurons, where NCAM (neural cell adhesion molecules) induced neurite formation is dependent on the T-type calcium channel [100].…”