Dexamethasone blocks nerve growth factor induction of nerve growth factor receptor mRNA in PC12 cells.

Glucocorticoids and nerve growth factor (NGF) have been shown to have antagonistic effects on chromaffin cells in vivo. Here we determined the effect of the synthetic glucocorticoid, dexamethasone, on levels of mRNA for the nerve growth factor receptor (NGFR) in rat PC12 pheochromocytoma cells. Following administration of dexamethasone (1 microM) there is a decline in NGFR mRNA expression. More importantly, administration of dexamethasone appears to block the NGF-mediated induction of NGFR when both agents are administered simultaneously. These data support the hypothesis that glucocorticoids and NGF act in opposition in determination of the phenotype of chromaffin cells.     

Cytoskeletal elements regulate the distribution of nerve growth factor receptors in PC12 cells.

Nerve growth factor receptor (NGFR)-like immunoreactivity (IR) was studied in PC12 cells treated for 96 hr with NGF (40 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin (IgG) conjugated with colloidal gold. PC12 cells exhibited occasional gold label (positive NGFR-IR) on all surfaces. Cells treated with colcemid (0.05 micrograms/ml) or cytochalasin D (2 micrograms/ml), which limit microtubule (MT) and microfilament (MF) formation, respectively, displayed an increased NGFR-IR in terms of gold labeling. NGFR-IR was also seen on taxol (0.85 micrograms/ml)-exposed cells, an agent that promotes MT assembly. Cells treated simultaneously with cytochalasin D and taxol had a dramatically augmented NGFR-IR on their surfaces, which exceeded levels obtained with either agent alone. Prominent NGFR-IR was localized frequently in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes, in both treated and untreated cells. The results suggest that a large number of NGFRs (positive NGFR-IR) in PC12 cells are cryptic and not available for ligand binding. Changes in cytoskeletal organization that may affect mobility of integral membrane proteins can modulate the distribution of NGFR-IR on neuronal surfaces.     

Differentiation of PC12 cells with K-ras: comparison with nerve growth factor.

The cell line PC12, derived from a rat pheochromocytoma, has served as a model for studies on the mechanism of action of nerve growth factor, as well as for the exploration of neuronal differentiation in general. When treated with nanomolar concentrations of nerve growth factor, these neoplastic chromaffin-like cells stop dividing and acquire, for all intents and purposes, the phenotype of mature sympathetic neurons. This phenotype is characterized by the extensive outgrowth of electrically excitable neurites, the ability to form functional synapses, and the acquisition of a number of biochemical markers. Treatment of PC12 cells with retroviral vectors encoding the K-ras, the N-ras, or the v-src oncogenes also produces a marked morphological differentiation very similar to that seen upon treatment with nerve growth factor. Treated cells stop dividing and develop an extensive network of neurites. It has recently been shown that PC12 cells differentiated with v-src, while resembling, morphologically, those treated with nerve growth factor, differ substantially in the biochemical characteristics normally associated with nerve growth factor-induced differentiation. Cells infected with K-ras also develop a neurite network similar to that seen after treatment with nerve growth factor. In addition, such cells develop tetanus toxin-binding sites and saxitoxin-binding sites, as do cells treated with nerve growth factor. Decreases in the binding of epidermal growth factor and in the activity of calpain also occur and these, as well, are characteristic of nerve growth factor-treated cells. But the adhesive properties of cells infected with K-ras are different than those of nerve growth factor-treated cells, and the former do not show an increase in the NILE glycoprotein. Finally, K-252a, an inhibitor of the actions of nerve growth factor on PC12 cells, has no effect on the neurite outgrowth produced by infection with K-ras. Thus, many of the key markers of nerve growth factor-induced differentiation of PC12 cells also appear upon differentiation with K-ras, but there are, nevertheless, some crucial differences in the properties of these two sets of cells.     

Decreased levels of nerve growth factor receptor on dexamethasone-treated PC12 cells

Treatment of PC12 cells with dexamethasone leads, in a period of days, to a 60% decrease in the binding of (125I)nerve growth factor. The decrease was maximal after 3 days of treatment with 1 microM dexamethasone, but some decrease was seen after 6 hr and at concentrations as low as 10 nM. The effect was specific for the glucocorticosteroids. Scatchard plots confirmed the overall loss of nerve growth factor binding, and studies with trypsin digestion and Triton X-100 extraction indicated that the decrease in binding was largely due to a decrease in the number of low-affinity receptors. Nerve growth factor-induced changes, such as the induction of ornithine decarboxylase and the generation of neurites, were inhibited, but only minimally, in dexamethasone-treated cells.     

Nerve growth factor regulates the subcellular localization of the nerve growth factor-inducible protein PC4 in PC12 cells

The immediate early gene (IEG) PC4, which encodes a protein related to γ interferon, is activated at the onset of the neuronal differentiation induced by nerve growth factor (NGF) in PC12 cells. With an antibody raised to a bacterial β gal-PC4 fusion protein, the PC4 protein is detected as an immunoreactive molecular species of 49 kDa, whose synthesis is rapidly induced by NGF in parallel with the induction of its mRNA. Immunofluorescence, electron microscopy and subfractionation studies indicate that the PC4 immunoreactivity is localized in the cytoplasm of PC12 cells, where it is increased transiently by NGF within 3 hr of treatment. In addition, the PC4 immunoreactivity presents an NGF-dependent pattern of intracellular localization. In fact, within 3 hr after addition of NGF, PC4 is also significantly expressed on the inner face of the plasma membrane, to which it is physically associated. After longer NGF treatment, PC4 disappears from the plasma membrane and appears in the nucleus, with reduced cytoplasmic expression. Localization in the nucleus is reversed by removal of NGF and closely parallels changes in the state of differentiation of the cell. The existence within the PC4 protein of a consensus sequence for the addition of myristic acid and of a putative sequence for the nuclear localization suggests possible mechanisms for the NGF-dependent redistribution. For an NGF-inducible IEG product, such growth factor-dependent localization of PC4 is a novel type of regulation in the pathways from the NGF receptor to the adjacent membrane proteins and to the nucleus. © 1994 Wiley-Liss, Inc.     

Ethanol enhances growth factor-induced neurite formation in PC12 cells.

Ethanol can injure the nervous system by disturbing the growth of neural processes. PC12 cells, which form neurites in response to nerve growth factor (NGF), fibroblast growth factor (FGF), and cAMP analogues, were used to study mechanisms by which ethanol alters process outgrowth. Ethanol potentiated NGF-induced neurite outgrowth in cells cultured on different substrata and in serum-containing or defined medium. Ethanol did not increase NGF receptor binding or internalization of NGF. Neurite outgrowth induced by basic FGF was also increased by ethanol but outgrowth induced by forskolin was not. Ethanol potentiated NGF-induced expression of Thy-1, but not of neural cell adhesion molecule (N-CAM), indicating that some, but not all actions of NGF are enhanced by ethanol. In some brain regions, chronic exposure to ethanol increases the growth of dendrites. This has been explained as a compensatory response of surviving neurons to the loss of neighboring cells, and not as a direct effect of ethanol. The present findings suggest that, in some cells, ethanol directly promotes growth factor-mediated neurite formation. This could harm the nervous system by disturbing the balanced development and organization of synapses.     

Induction of neurofilament triplet proteins in PC12 cells by nerve growth factor

The localization of neurofilament triplet proteins in PC12 cells grown in the absence of (PC12−) or maintained in the presence of (PC12+) nerve growth factor (NGF) was studied using indirect immunofluorescence and monospecific, immunosorbent purified antibodies to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins. The intensity of immunofluorescent staining of the triplet protein was always greater in PC12+ compared with PC12− cells. Neuritic staining was seen in PC12+ cells with all 3 monospecific antibodies to neurofilament proteins. However, the perikaryal distribution of each of the neurofilament proteins differed in both PC12+ and PC12− ells. Monospecific antibodies to P68 protein yielded a ‘ball-like’ cytoplasmic staining pattern whereas monospecific antibodies to P150 protein stained in a stippled pattern. Monospecific antibodies to P200 on the other hand diffusely stained the perikaryal cytoplasm with very faint but detectable foci of f ‘ball-like’ configurations and stippling. Electron microscopic study of PC12+ and PC12− cells revealed intermediate filaments in the cell bodies of both as well as in the processes of the former. ‘Ball-like’ clusters of such filaments were rarely seen. However, these filaments lacked the three-dimensional organization typical of intact neurofilaments.It is concluded that PC12 cells contain dissociated or incompletely assembled immunoreactive neurofilament triplet proteins and that these proteins can be induced by NGF. The PC12 cells are therefore an attractive model system not only for studies of neuronal differentiation but also for studies of neurofilament metabolism and disorders thereof.     

Differentiation of PC12 cells with v-src: comparison with nerve growth factor

The PC12 rat pheochromocytoma cell line is used extensively as a model to study neuronal differentiation. These cells resemble adrenal chromaffin cells, differentiating both morphologically and biochemically when cultured in the presence of dexamethasone, but develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. Expression of the protein product of the v-src oncogene in PC12 cells also induces neurite outgrowth similar to that resulting from nerve growth factor treatment (Alema et al: Nature 316:557-559, 1985). It is thus possible that c-src or a src-like tyrosine kinase participates in the signal transduction pathway by which nerve growth factor acts on PC12 cells. In this study a temperature-sensitive v-src gene has been introduced into PC12 cells. When cultures of these src-transformed cells are switched from the nonpermissive (40 degrees C) to the permissive (37 degrees C) temperature they elaborate neurites. The differentiation induced by src has been compared with that induced by nerve growth factor by determining whether src-transformed PC12 cells at 37 degrees C exhibit the same biochemical alterations as those induced in PC12 cells treated with nerve growth factor. Neurite extension at 37 degrees C in v-src-transformed cells, like NGF-induced differentiation, is accompanied by an increase in the nerve growth factor-inducible large external (NILE) protein. However, neurite extension in v-src-transformed cells is not blocked by the protein kinase inhibitor K-252a, which completely blocks NGF-induced neurite extension. Likewise, EGF receptor down-regulation and the development of saxitoxin and tetanus toxin binding sites are either much reduced or completely absent in src-differentiated compared with NGF-differentiated PC12 cells.     

The low-affinity nerve growth factor receptor p75NGFR mediates death of PC12 cells after nerve growth factor withdrawal

We have investigated the role of the low-affinity nerve growth factor (NGF) receptor p75^NGFR in determining the death of neuronally differentiated PC12 cells after withdrawal of NGF. A range of high and low p75^NGFR-expressing cells were obtained by a combination of fluorescence activated cell sorting (FACS) and stable transfection with a p75^NGFR expression vector. Cells were readily differentiated to a neuronal phenotype irrespective of the level of p75^NGFR expression. However, the rate and extent of neuronal death following NGF deprivation were extremely sensitive to the level of p75^NGFR expression. The highest expressing cells died most rapidly. Cells selected for very low levels of p75^NGFR expression exhibited resistance to NGF withdrawal, and remained as viable, differentiated neurons, with minimal cell death, for at least 5 days in the absence of NGF. Antisense oligonucleotides against p75^NGFR were shown to down-regulate p75^NGFR in PC12 cells and, further, to significantly enhance survival in the absence of NGF. These results consolidate and generalize our previous findings that p75^NGFR induces cell death in postnatal sensory neurons in the absence of NGF. The ability to induce cell death in the absence of NGF appears to be a more general role of p75^NGFR in differentiated neurons, and an important new paradigm for the mechanism of NGF-dependent survival. © 1996 Wiley-Liss, Inc.     

Mechanisms whereby nerve growth factor increases diacylglycerol levels in differentiating PC12 cells

We previously showed indirectly that the increase in diacylglycerol (DAG) levels caused by exposing differentiating PC12 cells to nerve growth factor (NGF) must derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [3H]phosphatidylinositol (PI). To explore further the biochemical mechanisms of this increase, we measured, in PC12 cells, DAG synthesis from glycerol or various fatty acids; its liberation from phosphatidylcholine (PC); and the activities of various enzymes involved in DAG production and metabolism. Among cells exposed to NGF (0-116 h), the labeling of DAG from [3H]glycerol peaked earlier than that of [3H]PC, and the specific radioactivity of [3H]glycerol-labeled DAG was much higher than those of the [3H]phospholipids, indicating that [3H]DAG synthesis precedes [3H]phospholipid synthesis. NGF treatment also increased (by 50-330%) the incorporation of monounsaturated ([3H]oleic acid) and polyunsaturated ([14C]linoleic acid or [3H]arachidonic acid) fatty acids into DAG, and, by 15-70%, into PC. NGF treatment increased the activities of long chain acyl-CoA synthetases (LCASs), including oleoyl-CoA synthetase and arachidonoyl-CoA synthetase, by 150-580% over control, but cholinephosphotransferase activity rose by only 60%, suggesting that the synthesis of DAG in the cells was increased to a greater extent than its utilization. NGF did not promote the breakdown of newly formed [3H]PC to [3H]DAG, nor did it consistently affect the activities of phospholipase C or D. NGF did increase phospholipase A2 activity, however the hydrolysis catalyzed by this enzyme does not liberate DAG. Hence the major source of the increased DAG levels in PC12 cells exposed to NGF appears to be enhanced de novo DAG synthesis, probably initiated by the activation of LCASs, rather than the breakdown of PC or PI.     

Effect of nerve growth factor on the synthesis of amino acids in PC12 cells

Radioactive short-chain fatty acids preferentially label glutamine relative to glutamate in brain due to compartmentation of glutamine and glutamate. To determine whether this phenomenon occurs in a single cell culture model, we examined the effect of fatty acid chain length on the synthesis as well as pool size of selected amino acids in rat pheochromocytoma PC12 cells, a cell culture model of the large glutamate compartment in neurons. Intracellular 14C-amino acids were quantitated by HPLC, and the incorporation of [U-14C]-glucose, [1-14C]-butyrate, [1-14C]-octanoate, and [1-14C]-palmitate into five amino acids was measured in native and NGF-treated PC12 cells. NGF pretreatment decreased the intracellular concentration of amino acids as did addition of fatty acids but these effects were not additive. Specific activities of amino acids in native cells labelled by 14C-octanoate were 1,300 DPM/nmol, 490 DPM/nmol, 200 DPM/nmol, and 110 DPM/nmol for glutamate, aspartate, glutamine, and serine, respectively. No radioactivity was detected in alanine. Similar specific activities were noted when 14C-butyrate was the precursor; however, there was at least 5-fold less if 14C-palmitate was the precursor. Pretreatment of cells with NGF decreased the specific activity of amino acids by 25-65%. Specific activities of amino acids synthesized from 14C-glucose decreased in the following order: glutamate, 1,640 DPM/nmol; aspartate, 1,210 DPM/nmol; alanine, 580 DPM/nmol; glutamine, 275 DPM/nmol; and serine, 80 DPM/nmol for native cells. NGF pretreatment decreased the specific activities of glutamate and glutamine, but not of the other 3 amino acids. The preferred precursor for glutamate synthesis in native PC12 cells was glucose followed by octanoate, butyrate and palmitate (16:6:3:1).     

PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells.

The uptake of divalent cations and the intracellular concentration of calcium in PC12 cells were studied by flow cytometric analysis using the calcium-sensitive dye, Fluo-3, under a variety of conditions. In particular the actions of nerve growth factor were analyzed. The data show that nerve growth factor stimulates the uptake of divalent cations and increases the intracellular calcium levels of cells attached to collagen-coated plates. The data further indicate that nerve growth factor-dependent increases in the uptake of divalent cations become less pronounced as the intracellular concentration of calcium increases. Intracellular calcium levels increase upon detachment of the cells from the plates and also with increasing cell density. Studies on the uptake of 45calcium confirmed the influence of intracellular calcium levels on nerve growth factor-stimulated calcium uptake. Thus, the effect of nerve growth factor on the uptake of divalent cations is dependent on the calcium levels in the cells, perhaps explaining why previous studies in this field have provided inconsistent results.     

Neuronatin mRNA in PC12 cells: downregulation by nerve growth factor

Neuronatin was recently cloned from neonatal rat brain (Biochem. Biophys. Res. Commun., 201 (1994) 1227–1234). In subsequent studies, we noted that neuronatin mRNA was brain-specific and that there were two alternatively spliced forms, α and β (Brain Res., 690 (1995) 92–98). Furthermore, on sequencing the human neuronatin gene, it was determined that the α-form was encoded by three exons, and the β-form was encoded by the first and third exons only (Genomics, 33 (1996) 292–297). The middle exon was spliced out in the β-form. The human neuronatin gene is located in single copy on chromosome 20q11.2-12 (Brain Res., 723 (1996) 8–22). These studies called for an understanding of the function of this gene. Therefore, we studied the expression of neuronatin in PC12 cells, an established model of neuronal growth and differentiation. Neuronatin mRNA expression was found to be abundant in undifferentiated PC12 cells. Treatment with nerve growth factor (NGF), resulting in neuronal differentiation, was associated with a downregulation of neuronatin mRNA expression. Removal of NGF was associated with a return of neuronatin mRNA levels towards baseline. These effects appear to be specific for NGF as they were not seen with transforming growth factor, epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate or dexamethasone. Although, basic fibroblast growth factor also reduced neuronatin mRNA levels, the effect was less pronounced than with NGF. The NGF-induced decrease in neuronatin mRNA occurred even in the presence of protein and RNA syntheses inhibitors. Of the two spliced forms, only the α-form was expressed in PC12 cells. In conclusion, we report the presence of neuronatin mRNA in PC12 cells, and that NGF downregulates its expression. These findings provide a basis for investigating the role of neuronatin in neuronal growth and differentiation.     

Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors (10% of those found in intact cells) are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor, since greater than 90% of cell surface glycoprotein receptors for WGA become associated with Triton X-100-insoluble material at lectin concentrations greater than 33 micrograms/ml. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin.     

Morphological properties and membrane channels of the growth cones induced in PC12 cells by nerve growth factor

Large growth cones were produced in vitro by nerve growth factor (NGF) treatment of multinucleate cells produced by chemical fusion of cells of the neuron-like clone PC12. These endings were studied both at the light microscopic and ultrastructural levels. The activity of ionic channels at growth cones was recorded with intracellular microelectrodes, patch recording of single channels, and whole cone recording from mechanically isolated growth cones. Morphologically, these large growth cones were characterized by the presence of microspikes and filopodia, by the presence of actin demonstrated immunohistochemically, and by the presence of catecholamine fluorescence. At the ultrastructural level they contained a broad spectrum of organelles with a distribution characteristic of neuronal growth cones, including dense core vesicles, abundant smooth membrane cisternae, microtubules, and a filamentous network. The presence of channels capable of generating action potentials was revealed by intracellular microelectrode recording from the growth cone in the presence of locally applied tetraethylammonium (TEA). TEA appeared to block outward current channels that could effectively shunt inward current activated by depolarization. Action potentials elicited by depolarizing current in the presence of TEA could be blocked reversibly by Cd2+, a specific blocker of Ca channels. These action potentials were often followed by a long after-hyperpolarization lasting hundreds of milliseconds. This after-hyperpolarization was similar to that recorded in the cell body of PC12 cells where it appears to be mediated by Ca-activated K current. Single channel recording from outside-out excised patches of membrane from the growth cones perfused with KF revealed the presence of voltage sensitive Na channels, Ca-activated K channels, and K channels resembling delayed rectifier K channels. Macroscopic currents recorded from mechanically isolated growth cones in the "whole cone" configuration showed rapid inward currents at potentials greater than or equal to -40 mV, followed by delayed outward currents at more positive potentials, a finding providing additional evidence for the presence of Na and K channels in growth cones.     

Induction of peptidylglycine α-hydroxylating monooxygenase activity by nerve growth factor in PC12 cells

Pheochromocytoma PC12 cells grown in the presence of nerve growth factor (NGF) undergo marked neuronal differentiation. During this process gene expression is altered, resulting in the activation of genes specific for neuronal properties, including the gene encoding neuropeptide Y (NPY). Here we sought to determine whether NGF also induces the activity of peptidylglycine α-hydroxylating monooxygenase (PHM) (EC1.4.17.3). PHM catalyzes the rate limiting step in the formation of α-amidated NPY from its glycine extended precursor, a posttranslational modification essential for biologic activity. PC12 cells were grown with or without NGF and assayed for PHM activity under optimal conditions. Whole cell extracts, medium and soluble and membrane bound fractions were assayed; total cellular PHM activity was found to be primarily membrane bound (fivefold greater than in soluble) and very little activity was released into the medium. Compared to control cells, PHM activity was increased significantly by NGF by 24 h but not before 4 h exposure. Through kinetic analysis, it was determined that the NGF-induction of PHM was a result of an increase inV _max with no change inK _m. It was found that the glucocorticoid, dexamethasone (DEX), decreased basal PHM activity and prevented its induction by NGF. Cotreatment with DEX for up to 7 d, however, did not dramatically alter the pronounced changes in cell morphology that occurred in response to NGF. These findings indicate that NGF and glucocorticoids exert reciprocal control over the activity of PHM in PC12 cells. As such, the process of differentiation in PC12 cells is a model for studying the mechanisms that coordinate the expression and activity of peptide processing enzymes with the regulation of their substrates and products.