The mode of action of nerve growth factor in PC12 cells

This review deals with the mechanism of nerve growth factor action. In view of the many and diversified effects of this growth factor, and since it could utilize different mechanism(s) in distinct types of cells, we have confined our analysis to the best characterized and more extensively studied target, the clonal cell line PC12. When exposed to NGF in vitro, these neoplastic cells recapitulate the last major steps of neuronal differentiation, i.e., the commitment to become a neuron and the acquisition of the neuronal phenotype. This is characterized by electrically excitable neurites, a display of a highly organized cytoskeleton, and the specific chemical and molecular neuronal properties. These effects are elicited upon the interaction of NGF with a receptor whose gene has been cloned and whose kinetic properties are now relatively well characterized. It is not yet clear, on the contrary, if and which of the several potential second messengers (cAMP, Ca, or phosphoinositides) that undergo marked fluctuations following NGF binding, transduce and amplify the NGF message. Among both the early and late effects of NGF is the modulation of expression of several genes. Some of the products of these genes are mainly restricted to nerve cells and others appear to play a crucial role in regulating the proper assembly of cytoskeletal elements. It is hypothesized that this complex array of chemical, molecular, and ultrastructural changes is triggered by NGF, not through activation of a single pathway, but more likely via combinatorial processes whereby several intracellular signals interplay before the irreversible commitment of becoming a neuron is undertaken.     

Effect of nerve growth factor on lesioned PC12 cells.

The protecting effect of nerve growth factor (NGF) from hydrogen peroxide was studied on PC12 cells conditioned at 1 mM hydrogen peroxide with NGF and without NGF in comparison with cells treated with neither hydrogen peroxide nor NGF. NGF treatment of PC12 cells increased significantly the activity of catalase representing induction of free radical detoxifying mechanisms. The protection effect of NGF was reflected also on enhanced activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the cells.     

Hidden receptors for nerve growth factor in PC12 cells

The binding of nerve growth factor (NGF) to its receptors in PC12 cells was studied in two experimental conditions: (a) cell fixation with paraformaldehyde followed by permeabilization of the plasma membrane with methanol and (b) metabolic poisoning of living cells with sodium azide. Paraformaldehyde fixation of PC12 cells causes a 60-70% reduction of NGF binding capacity; the original binding capacity is restored following permeabilization with methanol. A kinetic analysis of NGF binding under these conditions reveals a single homogeneous population of receptors at variance with experiments performed in living cells where two kinetically distinct types of NGF receptors were demonstrated [Landreth, G. E. and Shooter, E. M. (1980) Proc. Natl Acad. Sci. USA, 77, 4751-4755; Schechter, A. L. and Bothwell, M. A. (1981) Cell, 24, 867-874]. Our results suggest that a proportion of the NGF receptors in PC12 cells is hidden, i.e. not available for binding to the ligand, and in a dynamic equilibrium with exposed receptors. The existence of hidden receptors is confirmed by treatment of PC12 cells with sodium azide, which causes a 50% reduction in NGF binding capacity and protection from trypsin digestion of the remaining pool of hidden receptors. The latter become exposed at the cell surface following removal of sodium azide. Our data provide an interpretation for the as yet unsatisfactorily explained data on NGF receptors.     

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characteristics of nerve growth factor receptors on PC12 cells

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.     

Differential action of nerve growth factor, cyclic AMP and neurotropin on PC12h cells

Nerve growth factor (NGF) induced the activities of acetylcholinesterase (AChE) and Na+,K+-ATPase concomitant with neurite outgrowth in PC12h cells, while dibutyryl cyclic AMP (DBcAMP) caused the induction of AChE activity and neurite outgrowth but not Na+,K+-ATPase activity. A nonproteinaceous extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin) induced neurite outgrowth and cell surface change similar to NGF without affecting AChE activity. The results suggest that NGF, DBcAMP and Neurotropin act on PC12h cells through different mechanisms.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

Protein tyrosine phosphatase activation during nerve growth factor-induced neuronal differentiation of PC12 cells.

We have studied the role of protein tyrosine phosphatases (PTPases) during neuronal differentiation of PC12 cells. Nerve growth factor (NGF), a well-characterized differentiating agent for these cells, led to a decrease in DNA synthesis within 24 h. This was accompanied by a 2- to 3-fold increase in the activity of PTPases, measured as the dephosphorylation of polyacidic or polybasic substrates phosphorylated on tyrosine. PTPase activation was independent of cell density and proportional to NGF concentration, with a half-maximal effect occurring at 0.35 nM. High-performance liquid chromatography size exclusion chromatography revealed that PTPases with molecular masses of 550, 300, and 60 kilodaltons were activated in response to NGF. Additional studies showed that the presence of NGF made PC12 cells refractory to the mitogenic effect of epidermal growth factor. Our data indicate that NGF-induced neuronal differentiation and growth arrest in PC12 cells are associated with activation of several PTPases. We speculate that PTPase activation in response to NGF may inhibit the mitogenic actions of other growth factors.     

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.     

Relationship among types of nerve growth factor receptors on PC12 cells

We analyzed the kinetics and thermodynamics of 125I-nerve growth factor (125I-NGF) binding to NGF-receptor on PC12 cells. We used conditions of pseudo-first order kinetics and techniques to quantitate internalized complexes, "slow" or high affinity binding complexes, and cell surface "fast" or low affinity complexes. Two possible models were examined: binding to two independent receptors at the cell surface (i.e. high and low affinity forms of NGF-receptor) and a model for consecutive formation of fast, low affinity binding followed by slow, high affinity binding or internalization. Our data are consistent with the consecutive model only. The rates of association and dissociation of NGF with slow, high affinity sites and internalized, acid wash-resistant sites are indistinguishable from each other. We also analyzed, in detail, the two assays primarily used to distinguish slow binding complexes from internalized complexes. Scatchard analysis of total binding and dissociation of pre-equilibrated 125I-NGF in the presence of unlabeled NGF at high concentration (cold wash). Neither of these assays shows any evidence that the slow, high affinity binding step is different from internalization of the 125I-NGF-receptor complex. Based on this analysis, there are only two detectable forms of NGF-receptor on PC12 cells: complexes on the surface of the cells with a binding affinity of 0.5 nM at 37 degrees C and complexes internalized by the cells. Furthermore, the data are consistent with a model in which NGF-receptor is internalized constitutively and independently of occupancy by NGF. We also examined the fate of internalized 125I-NGF. In the first 60 min after contact with PC12 cells, no degradation of 125I-NGF was observed. Moreover, a significant amount of 125I-NGF recirculates to the cell surface and is released as intact, Mr = 13,000 NGF. The cells were also stimulated by NGF in a primary neurite outgrowth assay with an ED50 of 2-16 pM under conditions of low initial cell numbers in a large extracellular volume of NGF-containing medium. Thus, low level occupancy of the cell surface receptors, Kd = 0.5 nM, for several days is sufficient to stimulate neurite outgrowth. This indicates the presence of spare NGF-receptors on the surface PC12 cells.     

The effects of nerve growth factor on polyamine metabolism in PC12 cells

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.     

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.     

Internalization of nerve growth factor by PC12 cells. A description of cellular pools

Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.     

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.     

The phosphorylation and activation of B-raf in PC12 cells stimulated by nerve growth factor.

Treatment of PC12 cells with nerve growth factor does not alter the levels of B-raf mRNA, but does induce rapid phosphorylation of B-raf proteins. Phosphorylation was observed after 1.5 min and reached a maximum by 10-15 min. B-raf protein was phosphorylated almost exclusively on serine residues; no tyrosine phosphorylation was detected. Nerve growth factor-induced phosphorylation was not affected by depletion of protein kinase C or by removal of extracellular calcium but was inhibited by K-252a. Concomitant with the increase in serine phosphorylation, nerve growth factor treatment also increased the serine/threonine kinase activity of B-raf protein within 1-2 min.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.