GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.

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Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3644 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3644-3653 ◽

Cited By ~ 79

Author(s):

R R Vaillancourt ◽

L E Heasley ◽

J Zamarripa ◽

B Storey ◽

M Valius ◽

...

Keyword(s):

Cell Differentiation ◽

Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Pc12 Cell ◽

Growth Factor Receptor ◽

Receptor Activation ◽

Mitogen Activated Protein ◽

Map Kinase Pathway ◽

Kinase Pathway

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

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AMPN1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nem146 ◽

2010 ◽

Vol 7 (1) ◽

pp. 63-68 ◽

Cited By ~ 17

Author(s):

Noriko Hattori ◽

Hiroshi Nomoto ◽

Hidefumi Fukumitsu ◽

Satoshi Mishima ◽

Shoei Furukawa

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Royal Jelly ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Unique Compound ◽

Kinase A

Earlier we identified adenosine monophosphate (AMP)N1-oxide as a unique compound of royal jelly (RJ) that induces neurite outgrowth (neuritegenesis) from cultured rat pheochromocytoma PC12 cells via the adenosine A2Areceptor. Now, we found that AMPN1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK) but also that of cAMP/calcium-response element-binding protein (CREB) in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMPN1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA) by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMPN1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMPN1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMPN1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2Areceptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

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Nerve growth factor and fibroblast growth factor regulate neurite outgrowth and gene expression in PC12 cells via both protein kinase C- and cAMP-independent mechanisms.

The Journal of Cell Biology ◽

10.1083/jcb.110.4.1333 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1333-1339 ◽

Cited By ~ 81

Author(s):

D H Damon ◽

P A D'Amore ◽

J A Wagner

Keyword(s):

Protein Kinase C ◽

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Neurite Outgrowth ◽

Fibroblast Growth Factor ◽

Pc12 Cells ◽

Pc12 Cell ◽

Fibroblast Growth ◽

Kinase C

Nerve growth factor (NGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) promote the survival and differentiation of a variety of peripheral and central neurons. The signal transduction mechanisms that mediate the actions of these factors in neuronal cells are not well understood. We examined the effect of a deficiency in protein kinase C (PKC) and/or cAMP second messenger systems on the actions of NGF, aFGF, and bFGF in the pheochromocytoma (PC12) cell line. Activation of PKC was not required for NGF, aFGF, and bFGF to maximally induce ornithine decarboxylase (ODC), transcription of the early response genes, d2 and d5, or neurite outgrowth. In a PC12 cell mutant that is deficient in cAMP responsiveness (A126-1B2), all three growth factors maximally induced the transcription of d5 and neurite outgrowth, but aFGF and bFGF did not induce significant increases in ODC. NGF and aFGF maximally induced the transcription of d2 in A126-1B2 cells, but bFGF-induced d2 transcription was attenuated. NGF, aFGF, and bFGF maximally induced neurite outgrowth and d5 transcription in A126 cells that were made deficient in PKC. The d2 transcriptional response was substantially reduced in cells deficient in both PKC and cAMP responsiveness. These observations lead us to conclude that (a) cAMP- and PKC-dependent events are, at least in part, causally linked to NGF, aFGF, and bFGF induction of both ODC and transcription of d2 and may control functionally redundant pathways; (b) NGF, aFGF, and bFGF can elicit neurite outgrowth and increase transcription of d2 and d5 in PC12 cells via mechanisms that are independent of both PKC and cAMP; (c) NGF, aFGF, and bFGF can induce ODC in the absence of PKC; and (d) aFGF and bFGF require cAMP responsiveness to induce ODC in PC12 cells.

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E2F4 Actively Promotes the Initiation and Maintenance of Nerve Growth Factor-Induced Cell Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6048 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6048-6056 ◽

Cited By ~ 44

Author(s):

Stephan P. Persengiev ◽

Ivanela I. Kondova ◽

Daniel L. Kilpatrick

Keyword(s):

Cell Differentiation ◽

Nerve Growth Factor ◽

Growth Factor ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Pc12 Cell ◽

Growth Regulation ◽

Critical Role ◽

Terminal Differentiation ◽

Nerve Growth

ABSTRACT E2F transcription factors play a critical role in cell cycle progression through the regulation of genes required for G1/S transition. They are also thought to be important for growth arrest; however, their potential role in the cell differentiation process has not been previously examined. Here, we demonstrate that E2F4 is highly upregulated following the neuronal differentiation of PC12 cells with nerve growth factor (NGF), while E2F1, E2F3, and E2F5 are downregulated. Immunoprecipitation and subcellular fractionation studies demonstrated that both the nuclear localization of E2F4 and its association with the Rb family member p130 increased following neuronal differentiation. The forced expression of E2F4 markedly enhanced the rate of PC12 cell differentiation induced by NGF and also greatly lowered the rate at which cells lost their neuronal phenotype following NGF removal. Importantly, this effect occurred in the absence of any significant change in the growth regulation of PC12 cells by NGF. Further, the downregulation of E2F4 expression with antisense oligodeoxynucleotides inhibited NGF-induced neurite outgrowth, indicating an important role for this factor during PC12 cell differentiation. Finally, E2F4 expression was found to increase dramatically in the developing rat cerebral cortex and cerebellum, as neuroblasts became postmitotic and initiated terminal differentiation. These findings demonstrate that, in addition to its effects on cell proliferation, E2F4 actively promotes the neuronal differentiation of PC12 cells as well as the retention of this state. Further, this effect is independent of alterations in cell growth and may involve interactions between E2F4 and the neuronal differentiation program itself. E2F4 may be an important participant in the terminal differentiation of neuroblasts.

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Specific activation of the p38 mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein-2.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)46537-9 ◽

1999 ◽

Vol 274 (47) ◽

pp. 33826

Author(s):

Shoji Iwasaki ◽

Makoto Iguchi ◽

Kazushi Watanabe ◽

Rika Hoshino ◽

Masafumi Tsujimoto ◽

...

Keyword(s):

Protein Kinase ◽

Neurite Outgrowth ◽

Signaling Pathway ◽

Pc12 Cells ◽

Mitogen Activated Protein Kinase ◽

Bone Morphogenetic Protein 2 ◽

Morphogenetic Protein ◽

Mitogen Activated Protein ◽

Protein Kinase Signaling ◽

Kinase Signaling Pathway

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Protein kinase B is involved in Nogo-66 inhibiting neurite outgrowth in PC12 cells

Neuroreport ◽

10.1097/wnr.0b013e32834a58e8 ◽

2011 ◽

Vol 22 (15) ◽

pp. 733-738 ◽

Cited By ~ 12

Author(s):

Haijun Wang ◽

Jianying Shen ◽

Nanxiang Xiong ◽

Hongyang Zhao ◽

Yan Chen

Keyword(s):

Protein Kinase ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Protein Kinase B

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Protein kinase- and staurosporine-dependent induction of neurite outgrowth and plasminogen activator activity in PC12 cells

Biochemical Pharmacology ◽

10.1016/s0006-2952(96)00472-8 ◽

1996 ◽

Vol 52 (9) ◽

pp. 1399-1405 ◽

Cited By ~ 8

Author(s):

Pierre Leprince ◽

Catherine Bonvoisin ◽

Bernard Rogister ◽

Cécile Mazy-Servais ◽

Gustave Moonen

Keyword(s):

Protein Kinase ◽

Neurite Outgrowth ◽

Plasminogen Activator ◽

Pc12 Cells ◽

Plasminogen Activator Activity

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Constitutively Active Protein Kinase A Qualitatively Mimics the Effects of Follicle-Stimulating Hormone on Granulosa Cell Differentiation

Molecular Endocrinology ◽

10.1210/me.2008-0103 ◽

2008 ◽

Vol 22 (8) ◽

pp. 1842-1852 ◽

Cited By ~ 42

Author(s):

Rosalba Escamilla-Hernandez ◽

Lynda Little-Ihrig ◽

Kyle E. Orwig ◽

Junming Yue ◽

Uma Chandran ◽

...

Keyword(s):

Cell Differentiation ◽

Protein Kinase ◽

Protein Kinase A ◽

Granulosa Cell ◽

Follicle Stimulating Hormone ◽

Active Protein ◽

Kinase A ◽

Constitutively Active ◽

Stimulating Hormone

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The Insulin Receptor: A Potential Target of Amarogentin Isolated from Gentiana rigescens Franch That Induces Neurogenesis in PC12 Cells

Biomedicines ◽

10.3390/biomedicines9050581 ◽

2021 ◽

Vol 9 (5) ◽

pp. 581

Author(s):

Lihong Cheng ◽

Hiroyuki Osada ◽

Tianyan Xing ◽

Minoru Yoshida ◽

Lan Xiang ◽

...

Keyword(s):

Protein Kinase ◽

Insulin Receptor ◽

Signaling Pathways ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Mitogen Activated Protein Kinase ◽

Potential Target ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Gentiana Rigescens

Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.

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