Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.     

Gicerin/CD146 is involved in neurite extension of NGF-treated PC12 cells

Gicerin/CD146 is a cell adhesion molecule, which belongs to the immunoglobulin (Ig) superfamily. We have reported that it has a homophilic binding activity, which participates in the neurite extension from embryonic neurons. To elucidate how gicerin is involved in the neurite extension mechanism, we employed PC12 cells, which expresses gicerin/CD146. PC12 cells extend longer neurites by nerve growth factor (NGF) on gicerin substrate than on without gicerin substrate, which indicates that gicerin participates in neurite extension by NGF. We also found that the expression of gicerin in PC12 cells is induced by NGF. Over-expression of gicerin also promotes neurite extension by gicerin-gicerin homophilic interaction. These findings suggested that increase of gicerin expression by NGF promotes the gicerin-gicerin homophilic interaction resulting in the neurite extension.     

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.     

Sustained signaling by phospholipase C-gamma mediates nerve growth factor-triggered gene expression

In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-gamma (PLC-gamma) binding site, but not the Shc binding site, for NGF-triggered induction of PN1. In concordance with results with Trk mutants, drug-mediated inhibition of PLC-gamma activity also blocks PN1 induction by NGF. Examination of the kinetics of TrkA autophosphorylation indicates that triggered signaling does not result from sustained activation and autophosphorylation of the TrkA receptor kinase, whose phosphorylation state declines rapidly after NGF removal. Rather, TrkA triggers an unexpectedly prolonged phosphorylation and activation of PLC-gamma signaling that is sustained for up to 2 h. Prevention of the elevation of intracellular Ca2+ levels using BAPTA-AM results in a block of PN1 induction by NGF. Sustained signaling by PLC-gamma provides a means for differential neuronal gene induction after transient exposure to NGF.     

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.     

Nuclear factor kappaB/p49 is a negative regulatory factor in nerve growth factor-induced choline acetyltransferase promoter activity in PC12 cells

Anovel nuclear factor kappaB (NF-kappaB) binding site has been identified within the promoter region of the mouse gene encoding choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine and has been implicated in the cognitive deficits associated with aging and Alzheimer's disease. This binding site, which is located within the nerve growth factor (NGF)-responsive enhancer element, was recognized by the NF-kappaB protein p49 but not p65 or p50. p49 from both basal forebrain and PC12 nuclear extracts interacted with this specific sequence in electrophoretic mobility shift assays. Mutation of the NF-kappaB site caused an increase in NGF-induced promoter activation, whereas overexpression of p49 in NGF-differentiated PC12 cells caused a decrease in endogenous ChAT enzyme activity and a decrease in promoter activity that was specifically mediated through this NF-kappaB binding site. Treatment of PC12 cells with NGF resulted in a drastic reduction in nuclear p49 binding to the ChAT NF-kappaB site after 24 h, but nuclear p49 levels were not altered, suggesting that late NGF-mediated events prevent binding of p49 to the ChAT promoter by an unknown mechanism other than nuclear translocation. Decreased ChAT expression and increased NF-kappaB activity in the brain are associated with aging and Alzheimer's disease. These data indicate that p49 is a negative regulator of ChAT expression and suggest a possible mechanism for aging-associated declines in cholinergic function.     

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.     

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.     

Glia maturation factor beta regulates the growth of N18 neuroblastoma cells

Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the brain. The effect of bovine GMF-beta on neurons was tested on the neuroblastoma line N18 and the pheochromocytoma line PC12. GMF-beta inhibited the proliferation of N18 cells and promoted their neurite outgrowth, with an increase in neurofilament protein, but had no effect on PC12 cells. This was in contrast to nerve growth factor (NGF) which regulated PC12 but not N18. Acidic fibroblast growth factor (FGF), on the other hand, had a weak effect on PC12 but none on N18. Antisera against GMF-beta and NGF neutralized the biological activity of the corresponding growth factors but showed no cross-neutralization. Fluorescence visualization revealed the binding of GMF-beta to N18 cells but not to PC12 cells; the opposite was true with NGF.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.     

Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.     

A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor

Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF-receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins.     

A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells

A nerve growth factor (NGF) receptor interactive monoclonal antibody (192-IgG) which enhances beta-NGF binding to PC12 cells has been produced. The hybridoma clone was obtained by fusing Sp2/0- Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside solubilized proteins from isolated PC12 cell plasma membranes. The antibody is an IgG, which does not bind beta-NGF. It binds to the same number of sites on PC12 cells at low temperature as does beta-NGF. The 192-IgG increases the apparent affinity of beta-NGF binding to fast receptors on PC12 cells at low temperature by a factor of 2.5- to 4-fold and enhances the photoactivatable cross-linking of beta-NGF to the same receptor while decreasing the cross-linking of beta-NGF to the slow NGF receptor. At 37 degrees C 192-IgG partially inhibits the regeneration of neurites from primed PC12 cells. The 192-IgG also reduces the rate of appearance of binding to slow NGF receptors and increases the proportion of beta-NGF bound to fast receptors at 37 degrees C. These results implicate the slow receptor as the mediator of the biological response. This antibody provides a tool for examining steps in the mechanism of action of beta-NGF after binding to the receptor.     

The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines.

The trk tyrosine kinase proto-oncogene product gp140prototrk binds nerve growth factor (NGF) and is rapidly and selectively activated by this neurotrophic factor. To determine whether gp140prototrk is involved in transducing a functional NGF signal, PC12 cell mutants (PC12nnr) deficient in high affinity NGF binding and unresponsive to NGF were used. Northern analysis revealed that these mutant cells have greatly reduced levels of trk expression. PC12nnr cultures were transiently transfected with expression vectors encoding the full-length rat trk cDNA and assessed for responsiveness to NGF. Expression of exogenous trk rescued the capacity for NGF-promoted neurite outgrowth, cellular hypertrophy, and serum-free survival by these cells. These results indicate that gp140prototrk is necessary for functional NGF signal transduction.