Essential Role for Phospholipase D2 Activation Downstream of ERK MAP Kinase in Nerve Growth Factor-stimulated Neurite Outgrowth from PC12 Cells

ABSTRACT Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.

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Different Outcomes of Unliganded and Liganded Estrogen Receptor-α on Neurite Outgrowth in PC12 Cells

Endocrinology ◽

10.1210/en.2008-0449 ◽

2008 ◽

Vol 150 (1) ◽

pp. 200-211 ◽

Cited By ~ 17

Author(s):

Yohann Mérot ◽

François Ferrière ◽

Luc Gailhouste ◽

Guillaume Huet ◽

Frédéric Percevault ◽

...

Keyword(s):

Estrogen Receptor ◽

Nerve Growth Factor ◽

Growth Factor ◽

Signaling Pathways ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Estrogen Receptor Α ◽

Akt Signaling ◽

Induced Response ◽

Nerve Growth

A precise description of the mechanisms by which estrogen receptor-α (ERα) exerts its influences on cellular growth and differentiation is still pending. Here, we report that the differentiation of PC12 cells is profoundly affected by ERα. Importantly, depending upon its binding to 17β-estradiol (17βE2), ERα is found to exert different effects on pathways involved in nerve growth factor (NGF) signaling. Indeed, upon its stable expression in PC12 cells, unliganded ERα is able to partially inhibit the neurite outgrowth induced by NGF. This process involves a repression of MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways, which leads to a negative regulation of markers of neuronal differentiation such as VGF and NFLc. This repressive action of unliganded ERα is mediated by its D domain and does not involve its transactivation and DNA-binding domains, thereby suggesting that direct transcriptional activity of ERα is not required. In contrast with this repressive action occurring in the absence of 17βE2, the expression of ERα in PC12 cells allows 17βE2 to potentiate the NGF-induced neurite outgrowth. Importantly, 17βE2 has no impact on NGF-induced activity of MAPK and Akt signaling pathways. The mechanisms engaged by liganded ERα are thus unlikely to rely on an antagonism of the inhibition mediated by the unliganded ERα. Furthermore, 17βE2 enhances NGF-induced response of VGF and NFLc neuronal markers in PC12 clones expressing ERα. This stimulatory effect of 17βE2 requires the transactivation functions of ERα and its D domain, suggesting that an estrogen-responsive element-independent transcriptional mechanism is potentially relevant for the neuritogenic properties of 17βE2 in ERα-expressing PC12 cells. In the absence of its ligand, ERα partially inhibits the nerve growth factor-induced neurite outgrowth of PC12 cells, whereas, once liganded, it enhances differentiation.

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Overexpression of HuD, but Not of Its Truncated Form HuD I+II, Promotes GAP-43 Gene Expression and Neurite Outgrowth in PC12 Cells in the Absence of Nerve Growth Factor

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2000.0751103.x ◽

2002 ◽

Vol 75 (3) ◽

pp. 1103-1114 ◽

Cited By ~ 62

Author(s):

Kim D. Anderson ◽

Melissa A. Morin ◽

Andrea Beckel-Mitchener ◽

Charlotte D. Mobarak ◽

Rachael L. Neve ◽

...

Keyword(s):

Gene Expression ◽

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Truncated Form ◽

Nerve Growth

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Nitric Oxide Synthase Inhibitors Modulate Nerve-Growth-Factor Mediated Activation of Akt

ISRN Cell Biology ◽

10.5402/2012/847974 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Cheryl L. Cragg ◽

Janet C. MacKinnon ◽

Bettina E. Kalisch

Keyword(s):

Nitric Oxide ◽

Nerve Growth Factor ◽

Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Cell Lines ◽

Nerve Growth ◽

Akt Phosphorylation ◽

Map Kinase Pathway ◽

Kinase Pathway

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

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