Effect of NGF on the Subcellular Localization of Group IIA Secretory Phospholipase A2 (GIIA) in PC12 Cells: Role in Neuritogenesis

Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.     

On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A₂ (sPLA₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA₂s have the same fold as Atx. The first three sPLA₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA₂ and hPDI is however different from that of the other sPLA₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA₂s confirmed the model-based expectation that GV sPLA₂ was a more effective inhibitor than GX sPLA₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA₂s in relation to their action intracellularly.     

A possible involvement of cytoplasmic Ca2+ in sodium dependency of neurite outgrowth of rat pheochromocytoma PC12 cells

The effects of extracellular Na⁺, K⁺ and Cl^? on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na⁺ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60–150 mM Na⁺, whereas it decreased significantly at lower concentrations of Na⁺. Na⁺ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast, neurite outgrowth was independent of K⁺ in the range 5–106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl^? replaced by NO^?₃, SO^2?₄, or 2-hydroxyethanesulfonate. Thus, extracellular Na⁺ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca²⁺ concentration monitored by a fluorescent dye sensitive to Ca²⁺.     

Low Molecular Weight Phospholipases A2 in Mammalian Brain and Neural Cells: Roles in Functions and Dysfunctions

Several “low molecular weight” or “secretory” phospholipases A₂ isoforms may be expressed in mammalian neural cells. Indeed, mRNAs for GIB, GIIA, GIIE, GIII, GV, GX, and GXII were detected in brain tissues despite different levels. However, only the presence of GIB, GIIA, and GV proteins has been clearly demonstrated in neural cells or in the nervous tissue. Although the roles of GIB and GV in the nervous tissue are still elusive, there is evidence to support the involvement of GIIA in physiological and pathological events, including neurotransmission, long-term potentiation, and neuritogenesis. The neurotoxic effects of an increase in GIIA may be envisaged under pathological conditions associated with the activation of astrocytes during inflammation or through activation of neurons and enzymes due to the stimulation of the NMDA glutamate receptor. In the past, elevation of GIIA expression in many acute and chronic neurological diseases is well known. Although each neurodegenerative disease has a separate etiology, many share similar neurochemical common processes, such as excitotoxicity, oxidative stress, and mitochondrial dysfunction, phenomena where GIIA play an important role.     

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.     

Synergistic effect of immobilized laminin and nerve growth factor on PC12 neurite outgrowth

Immobilized extracellular matrix proteins and neurotrophins have been extensively studied to enhance neuronal adhesion and proliferation on surfaces for applications in nerve tissue engineering and neuroprosthetic devices. This article describes how the coimmobilization of laminin, an extracellular matrix protein and nerve growth factor (NGF), a neurotrophin can enhance neurite outgrowth observed separately with each type of molecule. In the absence of immobilized NGF, PC12 neurite outgrowth is influenced strongly by the presence of NGF in solution and unaffected by significant increases in laminin surface density (18.7–93.5 ng/mm²). However, when both laminin and NGF are immobilized together, the surface density of laminin is an important factor in determining whether or not the neurite outgrowth‐promoting effect of NGF can be obtained. PC12 neurite outgrowth on surfaces with coimmobilized laminin and NGF with surface densities of 27.6 ng/mm² and 1.4 ng/mm², respectively, are similar to that observed on surfaces with immobilized laminin and dissolved NGF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

The neurite-initiating effect of microbial extracellular glycolipids in PC12 cells

The effects of several kinds of microbial extracellular glycolipids on neurite initiation in PC12 cells were examined. Addition of mannosylerythritol lipid-A (MEL-A), MEL-B, and sophorose lipid (SL) to PC12 cells caused significant neurite outgrowth. Other glycolipids, such as polyol lipid (PL), rhamnose lipid (RL), succinoyl trehalose lipid-A (STL-A) and STL-B caused no neurite-initiation. MEL-A increased acetylcholine esterase (AChE) activity to an extent similar to nerve growth factor (NGF). However, MEL-A induced one or two long neurites from the cell body, while NGF induced many neurites. In addition, MEL-A-induced differentiation was transient, and after 48 h, percentage of cells with neurites started to decrease in contrast to neurons induced by NGF, which occurred in a time-dependent manner. MEL-A could induce neurite outgrowth after treatment of PC12 cells with an anti-NGF receptor antibody that obstructed NGF action. These results indicate that MEL-A and NGF induce differentiation of PC12 cells through different mechanisms. This revised version was published online in August 2006 with corrections to the Cover Date.     

Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.     

The A2A adenosine receptor rescues neuritogenesis impaired by p53 blockage via KIF2A,a kinesin family member

The A_2A adenosine receptor (A_2AR) is a G‐protein–coupled receptor. We previously reported that the C terminus of the A_2AR binds to translin‐associated protein X (TRAX) and modulates nerve growth factor (NGF)‐evoked neurite outgrowth in PC12 cells. Herein, we show that neuritogenesis of primary hippocampal neurons requires p53 because blockage of p53 suppressed neurite outgrowth. The impaired neuritogenesis caused by p53 blockage was rescued by activation of the A_2AR (designated the A_2A rescue effect) in a TRAX‐dependent manner. Importantly, suppression of a TRAX‐interacting protein (kinesin heavy chain member 2A, KIF2A) inhibited the A_2A rescue effect, whereas overexpression of KIF2A caused a rescue effect. Expression of a KIF2A fragment (KIF2A₅₁₄), which disturbed the interaction between KIF2A and TRAX, blocked the rescue effect. Transient colocalization of TRAX and KIF2A was detected in the nucleus of PC12 cells upon NGF treatment. These data suggest that functional interaction between KIF2A and TRAX is critical for the A_2A rescue effect. Moreover, p53 blockage during NGF treatment prevented the redistribution of KIF2A from the nucleus to the cytoplasmic region. Expression of a nuclear‐retained KIF2A variant (NLS‐KIF2A) did not rescue the impaired neurite outgrowth as did the wild‐type KIF2A. Therefore, redistribution of KIF2A to the cytoplasmic fraction is a prerequisite for neurite outgrowth. Collectively, we demonstrate that KIF2A functions downstream of p53 to mediate neuritogenesis of primary hippocampal neurons and PC12 cells. Stimulation of the A_2AR rescued neuritogenesis impaired by p53 blockage via an interaction between TRAX and KIF2A. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 604–621, 2010     

Gicerin/CD146 is involved in neurite extension of NGF-treated PC12 cells

Gicerin/CD146 is a cell adhesion molecule, which belongs to the immunoglobulin (Ig) superfamily. We have reported that it has a homophilic binding activity, which participates in the neurite extension from embryonic neurons. To elucidate how gicerin is involved in the neurite extension mechanism, we employed PC12 cells, which expresses gicerin/CD146. PC12 cells extend longer neurites by nerve growth factor (NGF) on gicerin substrate than on without gicerin substrate, which indicates that gicerin participates in neurite extension by NGF. We also found that the expression of gicerin in PC12 cells is induced by NGF. Over-expression of gicerin also promotes neurite extension by gicerin-gicerin homophilic interaction. These findings suggested that increase of gicerin expression by NGF promotes the gicerin-gicerin homophilic interaction resulting in the neurite extension.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

Regulation of PC12 cell survival and differentiation by the new P2Y-like receptor GPR17

The P2Y-like receptor GPR17 has been reported to respond to both uracil nucleotides and cysteinyl-leukotrienes (cysLTs), such as UDP-glucose and LTD₄. Our previous data suggest a potential role for GPR17 in regulation of both cell viability and differentiation state of central nervous system cells. On this basis, in the present paper we investigated the effect of GPR17 receptor ligands on PC12 cell viability, following induction of morphological differentiation by nerve growth factor (NGF). In addition, the role of GPR17 ligands, either alone or in combination with growth factors, on the degree of PC12 cell differentiation was investigated. GPR17, which was not basally expressed in undifferentiated PC12 cells, was specifically induced by a 10 day-treatment with NGF, suggesting a role in the control of neuronal specification. Both UDP-glucose and LTD₄, agonists at the nucleotide and cysLT GPR17 binding sites, respectively, induced a significant pro-survival effect on PC12 cells after priming with NGF. By in vitro silencing experiments with specific small interfering RNAs and by using receptor antagonists, we confirmed that the agonist effects are indeed mediated by the selective activation of GPR17. We also demonstrated that GPR17 agonists act, both alone and synergistically with NGF, to promote neurite outgrowth in PC12 cells. In addition, GPR17 ligands were able to confer an NGF-like activity to the epidermal growth factor (EGF), that, under these experimental conditions, also promoted cell differentiation and neurite elongation.Finally, we show that GPR17 ligands activate the intracellular phosphorylation of both ERK 1/2 and p38 kinases, that have been identified as important signalling pathways for neurotrophins in PC12 cells.Our results establish GPR17 as a neurotrophic regulator for neuronal-like cells and suggest a possible interplay between endogenous uracil derivatives, cysLTs and NGF in the signalling pathways involved in neuronal survival and differentiation. They also represent the first direct demonstration, in a native system, that GPR17 can indeed be activated by uracil nucleotides and cysLTs, in line with what previously demonstrated in recombinant expression systems.     

Differential action of nerve growth factor, cyclic AMP and neurotropin on PC12h cells

Nerve growth factor (NGF) induced the activities of acetylcholinesterase (AChE) and Na+,K+-ATPase concomitant with neurite outgrowth in PC12h cells, while dibutyryl cyclic AMP (DBcAMP) caused the induction of AChE activity and neurite outgrowth but not Na+,K+-ATPase activity. A nonproteinaceous extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin) induced neurite outgrowth and cell surface change similar to NGF without affecting AChE activity. The results suggest that NGF, DBcAMP and Neurotropin act on PC12h cells through different mechanisms.     

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.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor-stimulated PC12 cells

The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP₃) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)–induced neurite outgrowth. To further explore the mechanism, we visualized PIP₃, phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP₃ upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain–containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP₃ accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue–depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP₃ coordinately works with the PI3-kinase–mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP₃ accumulation to maintain proper neurite outgrowth.