Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Selective Inhibition of Nerve Growth Factor-Stimulated Protein Kinases by K-252a and 5''-S-Methyladenosine in PC12 Cells

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Down-Regulation of c-neu Receptors by Nerve Growth Factor in PC12 Cells

Abstract: A small number of p185^{c- neu} receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185^{c- neu} , in this case on tyrosine residues. Neither heregulin-β1 nor gp30 stimulates the tyrosine phosphorylation of p185^{c- neu} , and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185^{c- neu} receptors. This down-regulation does not occur when PC12nnr5 cells, which lack the high-affinity NGF receptor, p140 ^trk , are treated with NGF.The level of p185^{c- neu} mRNA is not altered by NGF treatment, suggesting that the down-regulation is due to either a translational or a posttranslational alteration.     

Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Receptors for insulin-like growth factor I (IGF-I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-I receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differentiated cells found in S-phase. But although the addition of IGF-I to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-I fails to induce cell proliferation in differentiated PC12 cells.     

Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.     

Nerve Growth Factor Promotes Neurite Regeneration in PC12 Cells by Translational Control

Abstract: When PC12 cells are primed with nerve growth factor (NGF) for periods of ≥1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short-lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.     

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro- trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per- oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.     

Multiple Levels for Regulation of TrkA in PC12 Cells by Nerve Growth Factor

Abstract: TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140 ^trkA was rapidly followed by down-regulation of cell surface and total cellular gp140 ^trkA . At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140 ^trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Production of 1,2-Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.     

Down-Regulation of Cyclin F Levels during Nerve Growth Factor-Induced Differentiation of PC12 Cells

Treatment with nerve growth factor (NGF) produces a marked decrease of cyclin F levels in PC12EY cells. This decrease is prevented by the simultaneous addition of K-252a. A smaller decrease is observed when the cells are treated with fibroblast growth factor, but the addition of epidermal growth factor has no comparable effect. Time course studies show that the decrease in cyclin F precedes the changes produced by NGF in the distribution of cells within the cell cycle. The data suggest that cyclin F is involved in NGF-mediated cell cycle events during the differentiation of PC12EY cells.     

Lithium Stimulation of Membrane-Bound Phospholipase C from PC 12 Cells Exposed to Nerve Growth Factor

LiCl stimulated the formation of inositol monophosphate in PC12 cells that had been exposed to nerve growth factor (NGF) for 4-5 days. Half-maximal accumulation was observed at approximately 8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inositol trisphosphate was half-maximal at approximately 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol 4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at approximately 0.7 mM and the second half-maximal at approximately 15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from approximately 200 to approximately 70 microM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.     

Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.     

Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ～30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [³⁵S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [³⁵S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [³⁵S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor-Differentiated PC12 Cells Induced by Chemical Hypoxia

Abstract: Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca²⁺ in growth cones and cell bodies. Hypoxia increased [Ca²⁺]_i and exaggerated its response to K⁺ depolarization in both parts of the cells. [Ca²⁺]_i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K⁺ or hypoxia. Ca²⁺-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca²⁺]_i following K⁺ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker ω-conotoxin GVIA (ω-CgTX) diminished K⁺-induced changes in [Ca²⁺]_i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω-CgTX diminished K⁺-induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω-CgTX diminished the [Ca²⁺]_i response to K⁺ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca²⁺ entry with K⁺ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca²⁺ regulation in the growth cone and cell body of the same cell.     

Trans Fatty Acids Enhanced Beta-Amyloid Induced Oxidative Stress in Nerve Growth Factor Differentiated PC12 Cells

The effects of trans fatty acids, elaidic acid (trans-9, C18:1) and linoelaidic acid (trans-9, trans-12 C18:2), at 20 or 40 μM in nerve growth factor differentiated PC12 cells with or without beta-amyloid peptide (Aβ) were examined. Elaidic acid treatment alone did not affect cell viability and oxidative injury associated markers (P > 0.05). However, co-treatments of elaidic acid and Aβ led to more reduction in mitochondrial membrane potential (MMP) and Na⁺-K⁺-ATPase activity, and more increase in DNA fragmentation and 8-hydroxydeoxyguanosine (8-OHdG) production than Aβ treatment alone (P < 0.05). Linoelaidic acid alone exhibited apoptotic and oxidative effects in cells via decreasing MMP and Na⁺-K⁺-ATPase activity, increasing reactive oxygen species (ROS) level, lowering glutathione content and glutathione peroxidase (GPX) activity (P < 0.05). The co-treatments of linoelaidic acid with Aβ further enhanced oxidative damage via enhancing the generation of ROS, nitrite oxide and 8-OHdG, elevating caspase-3, caspase-8 and nitric oxide synthase activities, as well as declining GPX, catalase and superoxide dismutase activities (P < 0.05). These results suggested that the interaction of linoelaidic acid and Aβ promoted oxidative stress and impaired mitochondrial functions in neuronal cells.