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)     

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.     

Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells.

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the regulation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity were assessed in rat pheochromocytoma (PC12) cells. Both NGF and EGF induced a rapid activation of PtdIns 3-kinase as assessed by a dramatic rise in growth factor-induced PtdIns 3-kinase activity found in antiphosphotyrosine immunoprecipitates. The intracellular levels of two of the lipid products of PtdIns 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), also rose dramatically, exhibiting time courses very similar to the appearance of PtdIns 3-kinase in immunoprecipitates. The activation of PtdIns 3-kinase is, therefore, a common event in the signal transduction processes elicited by growth factors stimulating distinct cellular end points in PC12 cells, namely the NGF-induced neuronal differentiation and EGF-stimulated mitogenesis. Thus the intracellular products of this enzyme may function in early biochemical events that are common components of the pathways controlling both differentiation and proliferation.     

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.     

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.     

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.     

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.     

Binding, sequestration, and processing of epidermal growth factor and nerve growth factor by PC12 cells

The rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well-characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4 degrees C. At 37 degrees C both ligands are "sequestered," but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reaction appears to compete favorably with NGF sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.     

Treatment of PC12 cells by nerve growth factor,dexamethasone, and forskolin

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.     

Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.     

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.     

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.     

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.     

Calcium-dependent activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells by nerve growth factor

Glycogen phosphorylase in PC12 cells exists in two forms analogous to those found in brain and muscle. The active phosphorylated form of the enzyme, phosphorylase-a, represents about 20-30% of total glycogen phosphorylase in these cells. Incubation of PC12 cells with 100 ng 7S nerve growth factor/ml increased phosphorylase-a within minutes. In contrast to nerve growth factor, insulin (6 ng/ml) and epidermal growth factor (6 ng/ml) decreased phosphorylase-a. Activation of phosphorylase-a by nerve growth factor was not accompanied by increases in cyclic AMP; however, removal of extracellular Ca2+ or incubation of cells with calcium channel blockers inhibited activation of glycogen phosphorylase by nerve growth factor.