Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase

1996 ◽  
Vol 109 (2) ◽  
pp. 289-300 ◽  
Author(s):  
T.R. Jackson ◽  
I.J. Blader ◽  
L.P. Hammonds-Odie ◽  
C.R. Burga ◽  
F. Cooke ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Specific Inhibitor ◽  
Morphological Differentiation ◽  
Sympathetic Neuron ◽  
Neuronal Morphology ◽  
Regulatory Subunit ◽  
Membrane Reorganization ◽  
Phosphoinositide 3 Kinase

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.

scholarly journals The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.

1986 ◽  
Vol 102 (3) ◽  
pp. 821-829 ◽  
Author(s):  
C Richter-Landsberg ◽  
B Jastorff
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Morphological Differentiation ◽  
Intracellular Camp ◽  
Nerve Growth ◽  
Process Formation ◽  
Dependent Protein

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.

scholarly journals Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase.

1994 ◽  
Vol 269 (29) ◽  
pp. 18961-18967
Author(s):  
K. Kimura ◽  
S. Hattori ◽  
Y. Kabuyama ◽  
Y. Shizawa ◽  
J. Takayanagi ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Specific Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells

2000 ◽  
Vol 113 (15) ◽  
pp. 2705-2713
Author(s):  
K. Sawada ◽  
Y. Konishi ◽  
M. Tominaga ◽  
Y. Watanabe ◽  
J. Hirano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurite Outgrowth ◽  
Neuronal Differentiation ◽  
Pc12 Cells ◽  
Mammalian Cells ◽  
Homeobox Gene ◽  
S Phase ◽  
Bhlh Transcription Factor ◽  
Spemann Organizer

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis

1993 ◽  
Vol 106 (2) ◽  
pp. 611-626 ◽  
Author(s):  
K.K. Teng ◽  
I.S. Georgieff ◽  
J.M. Aletta ◽  
J. Nunez ◽  
M.L. Shelanski ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Neurite Outgrowth ◽  
Pc12 Cell ◽  
Cell Types ◽  
Neuronal Morphology ◽  
Cytoskeletal Proteins ◽  
Parental Line ◽  
Pheochromocytoma Cells ◽  
Tau Expression

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

Phospholipase D1 mediates bFGF-induced Bcl-2 expression leading to neurite outgrowth in H19-7 cells

2011 ◽  
Vol 441 (1) ◽  
pp. 407-416 ◽  
Author(s):  
Sung Nyo Yoon ◽  
Kang Sik Kim ◽  
Ju Hwan Cho ◽  
Weina Ma ◽  
Hye-Jin Choi ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Dominant Negative ◽  
Signal Transducer ◽  
Downstream Effector ◽  
Phospholipase D1 ◽  
Protein Kinase Cα ◽  
Phosphoinositide 3 Kinase ◽  
Interfering Rna ◽  
Transcription 3

The purpose of the present study was to investigate the role of PLD (phospholipase D) in bFGF (basic fibroblast growth factor)-induced Bcl-2 expression and to examine whether overexpressed Bcl-2 influences neurite outgrowth in immortalized hippocampal progenitor cells (H19-7 cells). We found that Bcl-2 expression was maximally induced by bFGF within 24 h, and that this effect was reduced by inhibiting PLD1 expression with PLD1 small interfering RNA or by overexpressing DN (dominant-negative)-PLD1, whereas PLD1 overexpression markedly induced Bcl-2 expression. bFGF treatment activated Ras, Src, PI3K (phosphoinositide 3-kinase), PLCγ (phospholipase Cγ) and PKCα (protein kinase Cα). Among these molecules, Src and PKCα were not required for Bcl-2 expression. PLD activity was decreased by Ras, PI3K or PLCγ inhibitor, suggesting that PLD1 activation occurred through Ras, PI3K or PLCγ. We found that Ras was the most upstream molecule among these proteins, followed by the PI3K/PLCγ pathway, indicating that bFGF-induced PLD activation took place through the Ras/PI3K/PLCγ pathway. Furthermore, PLD1 was required for activation of JNK (c-Jun N-terminal kinase), which led to activation of STAT3 (signal transducer and activator of transcription 3) and finally Bcl-2 expression. When Bcl-2 was overexpressed, neurite outgrowth was stimulated along with induction of neurotrophic factors such as brain-derived neurotrophic factor and neurotrophin 4/5. In conclusion, PLD1 acts as a downstream effector of bFGF/Ras/PI3K/PLCγ signalling and regulates Bcl-2 expression through JNK/STAT3, which leads to neurite outgrowth in H19-7 cells.

scholarly journals Role of phosphoinositide 3-kinase in the autophagic death of serum-deprived PC12 cells

APOPTOSIS ◽  
2005 ◽  
Vol 10 (5) ◽  
pp. 1031-1041 ◽  
Author(s):  
A. Guillon-Munos ◽  
M. X. P. van Bemmelen ◽  
P. G. H. Clarke
Keyword(s):  
Pc12 Cells ◽  
Phosphoinositide 3 Kinase

scholarly journals Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Junko Shibato ◽  
Fumiko Takenoya ◽  
Takahiro Hirabayashi ◽  
Ai Kimura ◽  
Michio Yamashita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Molecular Mechanism ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Two Dimensional Gel Electrophoresis ◽  
Downstream Signaling ◽  
Mediator Protein ◽  
Gsk 3Β ◽  
Signaling Components

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10−7 M, 10−8 M, and 10−9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10−7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

The Pivotal Role of Intracellular Calcium in Oxaliplatin-Induced Inhibition of Neurite Outgrowth but Not Cell Death in Differentiated PC12 Cells

2011 ◽  
Vol 24 (11) ◽  
pp. 1845-1852 ◽  
Author(s):  
Miki Takeshita ◽  
Yoshiko Banno ◽  
Mitsuhiro Nakamura ◽  
Mayuko Otsuka ◽  
Hitomi Teramachi ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracellular Calcium ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Pivotal Role ◽  
Differentiated Pc12 Cells

scholarly journals Secretory phospholipases A2 induce neurite outgrowth in PC12 cells

2003 ◽  
Vol 376 (3) ◽  
pp. 655-666 ◽  
Author(s):  
Satoru NAKASHIMA ◽  
Yutaka IKENO ◽  
Tatsuya YOKOYAMA ◽  
Masakazu KUWANA ◽  
Angelo BOLCHI ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Streptomyces Coelicolor ◽  
Dependent Manner ◽  
Group V ◽  
Phospholipases A2 ◽  
Fatty Acid Release ◽  
Activity Dependent

sPLA2s (secretory phospholipases A2) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn-2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA2s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA2 activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA2 family, bee venom sPLA2, also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA2s, but not by group IB and IIA sPLA2s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca2+ channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca2+- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA2s in the nervous system.

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