scholarly journals Changes in the organization of the neuritic cytoskeleton during nerve growth factor-activated differentiation of PC12 cells: a serial electron microscopic study of the development and control of neurite shape.

1986 ◽  
Vol 103 (3) ◽  
pp. 895-906 ◽  
Author(s):  
J R Jacobs ◽  
J K Stevens
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Structural Changes ◽  
Shape Change ◽  
Sympathetic Neurons ◽  
Computer Assisted ◽  
Electron Microscopic ◽  
Nerve Growth ◽  
Major Transitions

After exposure to nerve growth factor, PC12 cells differentiate within a period of only a few days into cholinergic sympathetic neurons. Using computer-assisted three-dimensional serial electron microscopic reconstruction, we describe the progressive cytoskeletal and structural changes of PC12 neurites at different stages in their differentiation. Developmental changes in these neurites can be characterized by two major transitions. First, microtubules (MTs), which define the longitudinal axis of the neurite, increase in number leading to a more cylindrical and uniform neurite shape. Second, there are major changes in the relative numbers of other organelle types, which reflect the functional specialization of the neurite. These changes do not in themselves seriously affect shape change of the neurite during development, however the presence of these organelles and their associated obligatory volumes (volumes surrounding organelle) account for well over 50% of the neurite's volume at all stages of development. The MT-MT distances and obligatory volumes associated with the organelles remain constant throughout development. Thus, we can conclude that many of the observed changes seen in developing PC12 neurites are due simply to the production of a greater number of MTs in the cell, and that many of the other important parameters that can be measured and contribute to neurite shape remain constant during development.

scholarly journals Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons.

1992 ◽  
Vol 119 (6) ◽  
pp. 1669-1680 ◽  
Author(s):  
P W Mesner ◽  
T R Winters ◽  
S H Green
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Programmed Cell Death ◽  
Pc12 Cells ◽  
Sympathetic Neurons ◽  
Nerve Growth ◽  
Neuronal Pc12 Cells ◽  
New Protein

Previous studies have shown that in neuronal cells the developmental phenomenon of programmed cell death is an active process, requiring synthesis of both RNA and protein. This presumably reflects a requirement for novel gene products to effect cell death. It is shown here that the death of nerve growth factor-deprived neuronal PC12 cells occurs at the same rate as that of rat sympathetic neurons and, like rat sympathetic neurons, involves new transcription and translation. In nerve growth factor-deprived neuronal PC12 cells, a decline in metabolic activity, assessed by uptake of [3H]2-deoxyglucose, precedes the decline in cell number, assessed by counts of trypan blue-excluding cells. Both declines are prevented by actinomycin D and anisomycin. In contrast, the death of nonneuronal (chromaffin-like) PC12 cells is not inhibited by transcription or translation inhibitors and thus does not require new protein synthesis. DNA fragmentation by internucleosomal cleavage does not appear to be a consistent or significant aspect of cell death in sympathetic neurons, neuronal PC12 cells, or nonneuronal PC12 cells, notwithstanding that the putative nuclease inhibitor aurintricarboxylic acid protects sympathetic neurons, as well as neuronal and nonneuronal PC12 cells, from death induced by trophic factor removal. Both phenotypic classes of PC12 cells respond to aurintricarboxylic acid with similar dose-response characteristics. Our results indicate that programmed cell death in neuronal PC12 cells, but not in nonneuronal PC12 cells, resembles programmed cell death in sympathetic neurons in significant mechanistic aspects: time course, role of new protein synthesis, and lack of a significant degree of DNA fragmentation.

scholarly journals Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation.

1992 ◽  
Vol 12 (3) ◽  
pp. 1239-1247 ◽  
Author(s):  
R M Frederickson ◽  
W E Mushynski ◽  
N Sonenberg
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Translation Initiation ◽  
Sympathetic Neurons ◽  
Translation Initiation Factor ◽  
Tumor Promoter ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Nerve Growth

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.

scholarly journals Lithium ion inhibits nerve growth factor-induced neurite outgrowth and phosphorylation of nerve growth factor-modulated microtubule-associated proteins.

1985 ◽  
Vol 101 (3) ◽  
pp. 862-870 ◽  
Author(s):  
D E Burstein ◽  
P J Seeley ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Sympathetic Neurons ◽  
Lithium Ion ◽  
Newborn Mouse ◽  
Nerve Growth ◽  
Tumor Promotor ◽  
Associated Proteins

LiCl (2.5-20 mM) reversibly suppressed nerve growth factor (NGF)-induced neurite outgrowth by cultured rat PC 12 pheochromocytoma cells. Similar concentrations of LiCl also reversibly blocked NGF-dependent regeneration of neurites by PC12 cells that had been primed by long-term pre-exposure to NGF and by cultured newborn mouse sympathetic neurons. In contrast, transcription-dependent responses of PC12 cells to NGF such as priming and induction of the NGF-inducible large external glycoprotein, occurred despite the presence of Li+. SDS PAGE analysis of total cellular phosphoproteins (labeled by 2-h exposure to 32P-orthophosphate) from neurite-bearing primed PC12 cells revealed that Li+ reversibly inhibited the phosphorylation of a band of Mr 64,000 that was barely detectable in NGF-untreated PC12 cells. However, Li+ did not appear to affect the labeling of other phosphoproteins in either NGF-primed or untreated PC12 cultures, nor did it affect the rapid increase in phosphorylation of several proteins that occurs when NGF is first added to unprimed cultures. Several criteria indicated that the NGF-inducible phosphoprotein of Mr 64,000 is a microtubule-associated protein (MAP). Of the NGF-inducible phosphorylated MAPs that have been detected in PC12 cells (Mr 64,000, 72,000, 80,000, and 320,000), several (Mr 64,000, 72,000, and 80,000) were found to be substantially less phosphorylated in the presence of Li+. Neither a phorbol ester tumor promotor nor permeant cAMP analogs reversed the inhibitory effects of Li+ on neurite outgrowth or on phosphorylation of the component of Mr 64,000. Microtubules are a major and required constituent of neurites, and MAPs may regulate the assembly and stability of neuritic microtubules. The observation that Li+ selectively inhibits NGF-induced neurite outgrowth and MAP phosphorylation suggests a possible causal relationship between these two events.

Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation

1992 ◽  
Vol 12 (3) ◽  
pp. 1239-1247
Author(s):  
R M Frederickson ◽  
W E Mushynski ◽  
N Sonenberg
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Translation Initiation ◽  
Sympathetic Neurons ◽  
Translation Initiation Factor ◽  
Tumor Promoter ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Nerve Growth

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.

scholarly journals Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+and Calmodulin

2000 ◽  
Vol 20 (6) ◽  
pp. 1931-1946 ◽  
Author(s):  
Joaquim Egea ◽  
Carme Espinet ◽  
Rosa M. Soler ◽  
Sandra Peiró ◽  
Nativitat Rocamora ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Sympathetic Neurons ◽  
Moderate Increase ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Kinase Pathway

ABSTRACT Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.

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