Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

doi:10.1016/0006-8993(96)00449-0

Brain Research

Volume 730, Issues 1–2, 19 August 1996, Pages 212-222

https://doi.org/10.1016/0006-8993(96)00449-0 Get rights and content

Abstract

We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC12D, a subline whose phenotype resembles that of the parent PC12 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites and their survival can be induced by NGF in enucleated PC12D cells, (ii) individual neurites of a single ‘giant cell’, produced by cell fusion of PC12D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might be based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF.

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Cited by (7)

NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth
2007, Neurochemistry International
Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3β are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3β inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3β pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3β and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3β. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3β, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.
Activation of mitogen-activated protein kinases is not required for the extension of neurites from PC12D cells triggered by nerve growth factor
1998, Brain Research
Numerous studies with PC12 cells have suggested that the mitogen-activated protein (MAP) kinase pathway might play a major role in the neuronal differentiation that is induced by nerve growth factor (NGF). Cells of the PC12D subline extend neurites within several hours in response to NGF in the presence of inhibitors of the synthesis of RNA and protein. We examined the effects of a specific inhibitor 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD98059) of the MAP kinase kinase (MEK)/MAP kinase pathway on the NGF-induced outgrowth of neurites in PC12D cells. The increase in MAP kinase activity in response to NGF was reduced by 80% upon treatment of PC12D cells with 50 μM PD98059, whereas the NGF-dependent formation of ruffles and the subsequent outgrowth of neurites were not blocked by PD98059 at this concentration. The outgrowth of neurites from conventional PC12 cells by NGF was suppressed by the addition of 50 μM PD98059 as reported by Pang et al. [L. Pang, T. Sawada, J. Stuart,S.J. Decker, A.R. Saltiel, Inhibition of MAP kinase kinase blocks the differentiation of PC12 cells induced by nerve growth factor, J. Biol. Chem. 270 (1995) 13585–13588]. In contrast, the rapid regeneration of neurites from PC12 cells primed with NGF, was not altered in the presence of the same dose of the inhibitor of MEK. It appeared, therefore, that the activation of the MAP kinase pathway was not necessarily required for the NGF-dependent extension of neurites. When PC12D cells were transfected with the dominant inhibitory Ha-ras Asn-17 gene, the induction of the mutant Ras protein led the suppression of the rapid outgrowth of neurites in response to NGF but not to dibutyryl cyclic AMP (dbcAMP). The result implies a direct involvement of Ras protein in the NGF-induced signal transduction that lead to the formation of neurites in PC12D cells. We can conclude that the activation of MAP kinase and selective gene expression are required for the differentiation of conventional PC12 cells to sympathetic neuron-like cells and that activation of Ras protein and, subsequently, of a MAP kinase-independent pathway might be involved in the extension of neurites from PC12D cells or in the regeneration of neurites from primed PC12 cells in response to NGF.
Development of microarray device for functional evaluation of PC12D cell axonal extension ability
2014, Journal of Micro/Nanolithography, MEMS, and MOEMS
NGF signaling in PC12 cells: The cooperation of p75<sup>NTR</sup> with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade
2013, Biology Open
Parallels between tooth development and repair: Conserved molecular mechanisms following carious and dental injury
2004, Journal of Dental Research
Expression of the urokinase plasminogen activator receptor is transiently required during "priming" of PC12 cells in nerve growth factor-directed cellular differentiation
2001, Journal of Neuroscience Research

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Research reportRe-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

Abstract

Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth

J. Cell Biol.

PC12 cells express juvenile microtubule-associated proteins during nerve growth factor-induced neurite out-growth

J. Cell Biol.

Different neurotrophins are expressed and act in a developmental sequence to promote the survival of embryonic sensory neurons

Development

Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor

Development of sympathetic neurons in compartmentized cultures. I. Local control of neurite growth by nerve growth factor

Dev. Biol.

Development of sympathetic neurons in compartmentized cultures. II. Local control of neurite survival by nerve growth factor

Dev. Biol.

Neutrophin receptors: a window into neuronal differentiation

Neuron

Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells

Cell

The mitogen-activated protein kinase signal transduction pathway

J. Biol. Chem.

Axon guidance and the patterning of neuronal projections in vertebrates

Science

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

J. Cell Biol.

Novel form of growth cone motility involving site-directed actin filament assembly

Nature

The nerve growth factor: biochemistry, synthesis, and mechanism of action

Annu. Rev. Neurosci.

Characterization of the turning response of dorsal root neurites toward nerve growth factor

J. Cell Biol.

Similarities and differences in the way neutrophins interact with the Trk receptors in neuronal and nonneuronal cells

Neuron

Neuritic growth from a new subline of PC12 pheochromocytoma cells: cyclic AMP mimics the action of nerve growth factor

J. Neurosci. Res.

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules

J. Cell Biol.

Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength

J. Neurosci.

The nerve growth factor: thirty-five years later

EMBO J.

Research report
Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells