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.     

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.     

Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells

Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.     

The quantitative bioassay of nerve growth factor: use of frozen ‘primed’ PC12 pheochromocytoma cells

A quantitative bioassay for nerve growth factor (NGF) has been previously described which employs PC13 rat pheochromocytoma cells. The basis of this assay is that rapid neurite regeneration by PC12 cells pretreated ('primed') with NGF for at least a week is quantitatively related to the amount of NGF present in the culture medium. The present experiments demonstrate that NGF-pretreated PC12 cells retain their priming for over a year when appropriately frozen and stored and that such frozen cells can be employed for the quantitative bioassay of NGF. The major advantage of this assay is that large numbers of cells can be primed at a time (in suspension culture) and stored in aliquots until future use. The newly described bioassay employs the frozen primed cells essentially as reagents and avoids the need to have freshly primed cultures on hand.     

Endocytosis of nerve growth factor by ‘differentiated’ PC12 cells studied by quantitative ultrastructural autoradiography

The endocytosis of [¹²⁵I]nerve growth factor (NGF) by rat pheochromocytoma cells (PC12 line), previously exposed to the growth factor (‘differentiated’ or ‘primed’ cells), was studied by ultrastructural quantitative autoradiography. Cells previously grown in the presence of NGF were incubated at 37 °C with [¹²⁵I]NGF for periods of up to 24 h. Under these culture conditions, PC12 cells have a rich network of neurites.

At the commencement of the experiment, after incubation of cells with [¹²⁵I]NGF for 1 min at room temperature, the plasma membranes of perikarya and processes showed similar levels of labeling by [¹²⁵I]NGF of0.186 ± 0.03grains/μm and0.152 ± 0.013grains/μm respectively.

The density of grains per micron of plasma membrane of perikarya reached a plateau between 15 min to 2 h of incubation of cells at 37 °C with [¹²⁵I]NGF (0.58 ± 0.15grains/μm and0.65 ± 0.18grains/μm, respectively).

The endocytosis of [¹²⁵I]NGF in perikarya of cells incubated for 6 h at 37 °C was studied by the ‘mask’ analysis method of Salpeter et al.²². At this time, the greatest amount of endocytosis was observed, corresponding to 28.4% of total grain counts. The following optimized computed source densities, or relative specific activities± standard errors of measurement (S.E.M.), were obtained: plasma membrane,16.52 ± 0.86; multivesicular bodies,9.58 ± 2.84; endosomes,5.00 ± 0.97; smooth vesicles and tubules,1.66 ± 0.38; lysosomes,1.13 ± 0.20; mitochondria,0.46 ± 0.10; nuclear membranes or envelopes,0.32 ± 0.14; nuclei,0.06 ± 0.01; the Golgi apparatus,0.08 ± 0.06; and other cytoplasmic elements0.07 ± 0.03.

Our findings indicate that smooth vesicles and tubules, endosomes, multivesicular bodies and lysosomes are part of the pathway(s) of endocytosis of NGF, while all other cytoplasmic and nuclear elements, including the nuclear membrane, are not.

The heavy plasma membrane labeling of NGF and the relatively low degree of its endocytosis are consistent with the hypothesis that the NGF action is mediated through plasma membrane activated second messenger(s).     

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.     

Axl和Tyro-3受体表达受神经生长因子调控并参与PC12细胞的分化

目的 Axl和Tyro-3受体在脑内有区域性的分布,但两者在中枢神经系统中的生物学功能尚不明确.本研究旨在探讨Axl和Tyro-3受体在神经元分化中的作用.方法 PC12细胞分别转染CMV-Axl-eGFP、CMV-Tyro-3-eCFP和CMV-eGFP质粒后,给予神经生长因子(nerve growth factor...     

Axl和Tyro-3受体表达受神经生长因子调控并参与PC12细胞的分化(英文)

目的Axl和Tyro-3受体在脑内有区域性的分布,但两者在中枢神经系统中的生物学功能尚不明确。本研究旨在探讨Axl和Tyro-3受体在神经元分化中的作用。方法PC12细胞分别转染CMV-Axl-eGFP、CMV-Tyro-3-eCFP和CMV-eGFP质粒后,给予神经生长因子(nerve growth factor,NGF)诱导,观察绿色荧光蛋白和青色荧光蛋白的表达和分布。结果Axl-eGFP和Tyro-3-eCFP的表达随着NGF作用时间的延长而逐渐上调,并且荧光蛋白在细胞内的定位也发生变化。作为对照组,CMV-eGFP转染的PC12细胞并没有出现此变化。此外,过表达Axl和Tyro-3能够促进PC12细胞的突起生长。结论Axl和Tyro-3受体的表达受NGF调控,其过表达可能参与了PC12细胞的分化。     

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 dexamethasone on the nerve growth factor-induced increase in the NILE glycoprotein in PC12 cells.

The long-known and well-documented increase in the NILE glycoprotein produced by nerve growth factor in PC12 cells is prevented by simultaneous treatment with dexamethasone. The absence of this surface marker on the fully differentiated cells has been demonstrated by both glucosamine incorporation and immunohistofluorescence.     

Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells

We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC12D, a subline whose phenotype resembles that of the parent PC12 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites and their survival can be induced by NGF in enucleated PC12D cells, (ii) individual neurites of a single ‘giant cell’, produced by cell fusion of PC12D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might be based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF.     

Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells

Immunoblotting analysis and immunofluorescence studies of proteins that react with MAP₁- and MAP₂-specific antibodies in PC12 rat pheochromocytoma cells were carried out. When cells of the PC12D subline of PC12 cells, which rapidly extend neurites in response to NGF or drugs that elevate intracellular levels of cyclic AMP, were examined, they were found to contain a relatively higher level of MAP₁ or of a MAP₁-like protein than conventional PC12 cells. Immunoblotting study showed that levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins increased in PC12D cells, but not in conventional PC12 cells, in response to forskolin. Immunofluorescence studies also revealed increases in levels of MAP₁ and MAP₂ or of MAP₁ or MAP₂-like proteins in conjunction with the outgrowth of neurites from the cells. These results support the hypothesis that the induction of MAPs may be one of the first steps required for outgrowth of neurites from PC12 cells. Furthermore, PC12D cells may contain a sufficiently high level of MAP₁ or MAP₁-like protein to permit the extension of neurites in the absence of the lag period normally required by PC12 cells. The MAP₁ or a MAP₁-like protein was localized in the cell soma and neurites. An increase of MAP₂-specific immunoreactivity in perikarya was observed in the differentiated cells. After immunostaining with a monoclonal antibody that reacted with phosphorylated MAP₁, intense fluorescence was seen in the growth cones of neurites. This observation supports the hypothesis that the phosphorylation of MAP₁ or of a MAP₁-like protein may play a regulatory role in the formation of neurites in growth cones.     

Mechanisms of apoptosis in PC12 cells irreversibly differentiated with nerve growth factor and cyclic AMP

PC12 cells treated with cAMP become irreversibly differentiated and die by apoptosis when deprived of trophic support, instead of dedifferentiating and reentering the cell cycle. To approach the molecular mechanism underlying the cAMP-induced switch from differentiation/proliferation to apoptosis, we compared three sequential markers of a candidate apoptogenic signal transduction pathway (ceramide, free radicals and NF-kappaB), after trophic factor withdrawal in PC12 cells before and after irreversible differentiation. Serum withdrawal increased ceramide and free radical production regardless of the state of differentiation of the cells. It was followed by cell death, however, only in the absence of NGF and/or cAMP, and was no longer required for apoptosis in NGF/cAMP-differentiated cells. NGF and cAMP withdrawal sufficed. NF-kappaB was activated by NGF withdrawal in reversibly differentiated PC12 cells during dedifferentiation and reentry into the cell cycle, whereas in NGF/cAMP-differentiated cells, it was activated, at a late stage of the apoptotic process, concomitantly with cell death. These results show that a serum factor inhibits ceramide-dependent apoptosis upstream of ceramide and free radical production, whereas NGF- and cAMP-dependent mechanisms inhibit apoptosis either downstream or parallel to these events. After terminal differentiation by cAMP, apoptosis appears to be initiated from the second site, consistent with the serum independence of these cells and the absence of ceramide and free radical production when the NGF/cAMP-dependent inhibitions are released. The differential regulation of NF-kappaB appears to be an important step in the switch from mitosis to apoptosis that occurs during irreversible differentiation of PC12 cells by cAMP.     

HMG-CoA reductase inhibitor induces a transient activation of high affinity nerve growth factor receptor, trk, and morphological differentiation with fatal outcome in PC12 cells

The present study was aimed at investigating the possible toxicity of simvastatin on a neuronal cell line, PC12 cells. Simvastatin clearly induced a transient morphological differentiation as evidenced by the occurrence of neurite outgrowth with a transient activation of the high affinity nerve growth factor receptor, Trk, but died at 36 h after its addition. Tyrosine autophosphorylation of the Trk protein also disappeared at 36 h after addition. During the morphological differentiation, NGF mRNA expression was upregulated transiently and returned to the basal level at 36 h after addition of simvastatin. These results suggest that simvastatin is neurotoxic and PC12 cells elicited a protective response, involving a transient activation of a Trk-mediated intracellular signal transduction pathway by an autocrine secretion of NGF, although these responses did not persist against pro-apoptotic signals and resulted in an apoptosis of the PC12 cells.