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 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.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Phospholipase D2 activation by p38 MAP kinase is involved in neurite outgrowth

p38 mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth. However, the underlying molecular mechanism(s) remains unclear. Here, we demonstrate that phospholipase D2 (PLD2) mediates p38 signaling in neurite outgrowth. Stimulation of rat pheochromocytoma PC12 cells with nerve growth factor activated PLD2 and augmented neurite outgrowth, both of which were inhibited by pharmacological suppression of p38. Overexpression of constitutively active MAP kinase kinase 6 (MKK6-CA) activated coexpressed PLD2 in PC12 and mouse neuroblastoma N1E-115 cells. Overexpression of wild-type PLD2 in these cells strongly augmented the neurite outgrowth induced by MKK6-CA, whereas lipase-deficient PLD2 suppressed it. These findings provide evidence that PLD2 functions as a downstream molecule of p38 in the neurite outgrowth signaling cascade.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.     

Immunosuppressant FK506 induces neurite outgrowth in PC12 mutant cells with impaired NGF-promoted neuritogenesis via a novel MAP kinase signaling pathway

We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

The neurite outgrowth multiadaptor RhoGAP, NOMA-GAP, regulates neurite extension through SHP2 and Cdc42

Neuronal differentiation involves the formation and extension of neuronal processes. We have identified a novel regulator of neurite formation and extension, the neurite outgrowth multiadaptor, NOMA-GAP, which belongs to a new family of multiadaptor proteins with RhoGAP activity. We show that NOMA-GAP is essential for NGF-stimulated neuronal differentiation and for the regulation of the ERK5 MAP kinase and the Cdc42 signaling pathways downstream of NGF. NOMA-GAP binds directly to the NGF receptor, TrkA, and becomes tyrosine phosphorylated upon receptor activation, thus enabling recruitment and activation of the tyrosine phosphatase SHP2. Recruitment of SHP2 is required for the stimulation of neuronal process extension and for sustained activation of ERK5 downstream of NOMA-GAP. In addition, we show that NOMA-GAP promotes neurite outgrowth by tempering activation of the Cdc42/PAK signaling pathway in response to NGF. NOMA-GAP, through its dual function as a multiadaptor and RhoGAP protein, thus plays an essential role downstream of NGF in promoting neurite outgrowth and extension.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Artepillin C Derived from Propolis Induces Neurite Outgrowth in PC12m3 Cells via ERK and p38 MAPK Pathways

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 μM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system

The rat pheochromocytoma PC12 cell line has been an invaluable model system for studying neuritogenesis. Nerve growth factor (NGF) elicits multiple aspects of neurite outgrowth in PC12 cells. It is therefore difficult to dissect and assign an individual signaling pathway to each stage of neuritogenesis. We have recently reported the isolation of a variant PC12 cell line, PC12-N1 (N1), which spontaneously extends neuritic processes and exhibits an increased sensitivity to NGF. Here, we show that, under different culture conditions, the cells display three distinct phases of neuritogenesis consisting of neurite initiation, rapid neurite elongation, and a maturation process characterized by the thickening of neurites and increase in cell soma sizes. We demonstrate that signaling through ERK, but not p38 or JNK, is required for the spontaneous neurite initiation and extension. Treatment with low concentrations of NGF induces rapid neurite elongation without affecting neurite branching and cell soma sizes. Such a rapid neurite outgrowth can be blocked by the inhibition of ERK, but not JNK, activities. In the presence of higher concentrations of NGF, the N1 cells undergo further differentiation with many characteristics of mature neurons in culture, e.g. larger cell soma and numerous branches/connections. This process can be completely blocked by inhibiting ERK or JNK activities using specific inhibitors. These results suggest that ERK and JNK signals play different roles in neuritogenesis, and that JNK activity is essential in the late stages of neuritogenesis. Furthermore, our results demonstrate that signaling dosage is important in the activation of a specific pathway, leading to distinctive biological outcomes.