Functional receptors for nerve growth factor on Ewing's sarcoma and Wilm's tumor cells

Quantification of changes in levels of c-fos RNA was used as an indicator of the presence of functional responses to nerve growth factor in several human non-neuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC-EW1 showed greatly increased levels (10-20-fold) of c-fos RNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c-fos RNA expression. In IARC-EW1 cells, this induction was analyzed for kinetics, dose-response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high-affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism of action of NGF.     

Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.     

PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor

Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.     

Cell-cell interactions modulate the responsiveness of PC12 cells to nerve growth factor

The growth of PC12 cells on a collagen substratum or on monolayers of several non-neuronal cell types was studied by measuring nerve growth factor (NGF)-dependent increases in the expression of a 150 X 10(3) (Mr) neurofilament protein subunit and the membrane glycoprotein Thy-1. Both responses were found to be greatly suppressed in cultures of fibroblasts as compared to the C2 and G8-1 muscle cell lines and the C6 glioma cell line. This suppression was associated with an inhibition of NGF-dependent neuritic outgrowth from PC12 cells grown on fibroblast monolayers. There was no evidence that fibroblasts secrete soluble molecules that directly inhibit these responses or neutralize NGF. In addition, there was no difference in the neurofilament protein response from PC12 cells that had been treated with NGF prior to coculture, and the now primed PC12 cells readily extended axons over fibroblast monolayers. These data demonstrate that cell-cell and/or cell-matrix interactions can modulate biochemical responses to NGF and suggest that responsiveness of neuronal cells to environmental cues is not immutable. Control of the latter may be at the level of expression of receptor molecules for cell-surface- or matrix-associated macromolecules and a similar mechanism operating during development could play a role in growth cone guidance.     

Cell cycle dependent growth factor regulation of gene expression

The expression of the proto-oncogenes c-fos and c-myc is a rapid response of G0-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c-fos and c-myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and platelet-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c-myc expression was PDGF-dependent and plasma-independent, and PDGF was required for maintenance of elevated c-myc levels during G1 transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been "primed" by exposure to PDGF during S phase.     

Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocampal cells by two criteria: rapid induction of c-fos and neurotrophic responses. The selective induction of c-fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CA1 through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.     

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

Expression of Nerve Growth Factor and Nerve Growth Factor Receptor Genes in Human Tissues and in Prostatic Adenocarcinoma Cell Lines

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.     

K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.

The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.     

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.     

Effect of nerve growth factor and fibroblast growth factor on SCG10 and c-fos expression and neurite outgrowth in protein kinase C-depleted PC12 cells

The role of protein kinase C (PKC) in mediating nerve growth factor (NGF) or basic fibroblast growth factor (bFGF)-stimulated SCG10 and c-fos expression as well as neurite outgrowth was studied in PC12 cells. Activators of PKC such as phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol mimicked the stimulatory effect of NGF and bFGF on SCG10 mRNA levels. Induction involved a protein synthesis-dependent mechanism and was maximal within 12-24 h of exposure. Chronic treatment of the cells with PMA for up to 8 days resulted in a substantial decrease (approximately 90%) in total PKC activity in the continued presence of PMA. PKC depletion did not affect NGF- or bFGF-stimulated SCG10 mRNA induction and bFGF-stimulated c-fos mRNA induction. However, NGF-stimulated c-fos mRNA induction was attenuated. In addition, induction of neurite outgrowth was not abolished in PKC-depleted cells. The results imply that PKC is not involved in NGF- and bFGF-stimulated SCG10 mRNA induction and neurite outgrowth. Furthermore, while the effect of bFGF on c-fos mRNA induction is PKC-independent, that of NGF is mediated by PKC-dependent and -independent pathways.     

Pit formation and rapid changes in surface morphology of sympathetic neurons in response to nerve growth factor

Scanning and transmission electron microscope studies were carried out on the rapid cell surface responses of cultured newborn rat sympathetic neurons to nerve growth factor (NGF), a substance that promotes their survival and differentiation. The somas of sympathetic neurons continuously exposed to NGF or deprived of the factor for 4-5 h have a very smooth surface. After readdition of NGF to the latter type of cultures, there is rapidly initiated a transient, sequential change in the cell surface. Microvilli and small ruffles appear within 30 s and are most prominent by 1 min. By 3 min of exposure, the microvilli and ruffles decrease in prominence, and by 7 min the somal surface is again smooth. By 30 s after NGF readdition, as increase in the number of 60- tp 130-nm coated pits is also detectable. This increase reaches a maximum of about threefold from 0.5 to 3 min and then gradually decreases. Alterations in the surface did not occur on the nonneuronal cell types present in the cultures and were not observed in response to another basic protein (cytochrome c) or to physical manipulation. Changes in cell surface architecture induced by NGF in normal sympathetic neurons and, as previously described, in PC12 pheochromocytoma cells indicate that such responses may present or reflect primary events in the mechanism of the factor's action.     

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.     

Distribution of rat embryonal fibroblasts through cell cycle phases in the presence of inhibitors of active oxygen species formation and N-acetylcysteine

Intracellular level of reactive oxygen species (ROS) and distribution of primary rat embryo fibroblast throughout the cell cycle have been studied. Serum-starved cells were activated by the addition of 10% serum to the culture medium in the presence on N-acetyl-cystein (NAC) and ROS-inhibitors, diphenileniodonium (DPI) and rothenone. It has been shown that serum starvation could block the cells at the G1/S boundary. Activation of serum-starved cells by the addition of serum reactivated the cell cycle and caused cell progression into S phase, which was paralleled with the increase in the intracellular level of ROS. Effects of NAC, PAI and rothenone, similar to that of serum starvation, blocked cell progression into S phase and decreased ROS formation due to the action of serum growth factors. The antiproliferative effect of NAC is discussed.     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle

Induction of c-fos mRNA levels associated with the stimulation of growth by fetal bovine serum following quiescence was examined in three cell types following brief (24 h) serum starvation. Starved NIH-3T3 and HeLa S3 cells experienced c-fos mRNA induction 20-30 min after addition of serum. In contrast, Swiss-3T3 cells expressed c-fos constitutively following serum starvation. The pattern of oncogene expression coincided with the level of quiescence of each cell line prior to induction. Serum inductions of c-fos expression was dependent upon the response of each cell line to serum starvation, c-fos expression was also examined in HeLa S3 cells that had been separated into sequential cell cycle phases by centrifugal elutriation, c-fos expression peaked during the earliest part of the synchronous G1 phase. The amount of c-fos mRNA measured was approximately twice that found during other cell cycle phases. This suggests that, in addition to its role during the transition from quiescence, the c-fos gene product may play a regulatory role during the earliest part of G1 phase of the continuous cell cycle.