Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B.

The retinoid-X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR-RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor.

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.

p-Chlorophenylalanine-induced chemical manifestations of phenylketonuria in rats.

p-Chlorophenylalanine, a potent inhibitor of phenylalanine hydroxylase in vivo, has been used to simulate phenylketonuria in rats. This inhibitor, when administered with phenylalanine, produces marked elevation of blood and tissue phenylalanine without an increase in tyrosine. Disproportionate ratios of phenylalanine to tyrosine are characteristic of phenylketonuria in humans. The use of p-chlorophenylalanine permits the production of this characteristics amino acid imbalance in experimental animals.

Ultrastructural changes in rat optic nerve associated with hyperphenylalaninemia induced by para-chlorophenylalanine and phenylalanine.

The morphologic effects of hyperphenylalaninemia induced by treatment with para-chlorophenylalanine (PCP) plus phenylalanine on optic nerve were studied in developing F344 rats. PCP and phenylalanine were infected daily between days 5 and 20 days of life. At 20 days optic nerve of treated animals, as compared with saline-injected controls, showed enhanced neuroglial activity with broad astrocytic septae and debris-laden oligodendrocytes. In specimens obtained long after treatment with PCP and phenylalanine, continuing gliosis with evidence of focally abnormal myelination and axonal degeneration were observed. The results are consistent with a metabolic insult sustained in early development by astrocytes and oligodendrocytes, and are considered in relation to other work in experimental hyperphenylalaninemia and to human phenylketonuria.

Sequence of a rat TIS11 cDNA, an immediate early gene induced by growth factors and phorbol esters.

We report here the nucleotide sequence of a rat TIS11 cDNA, an immediate early gene, induced by nerve growth factor and epidermal growth factor, by 12-O-tetradecanoyl phorbol-13-acetate, and other stimuli in PC12 pheochromocytoma cells. The deduced protein consists of 320 amino acid residues with two tandem repeats of a putative Zn(2+)-finger motif.

Identification of Nsp100 as elongation factor 2 (EF-2).

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.

Regulation of the differentiation of PC12 pheochromocytoma cells.

The PC12 clone, developed from a pheochromocytoma tumor of the rat adrenal medulla, has become a premiere model for the study of neuronal differentiation. When treated in culture with nanomolar concentrations of nerve growth factor, PC12 cells stop dividing, elaborate processes, become electrically excitable, and will make synapses with appropriate muscle cells in culture. The changes induced by nerve growth factor lead to cells that, by any number of criteria, resemble mature sympathetic neurons. These changes are accompanied by a series of biochemical alterations occurring in the membrane, the cytoplasm, and the nucleus of the cell. Some of these events are independent of changes in transcription, while others clearly involve changes in gene expression. A number of the alterations seen in the cells involve increases or decreases in the phosphorylation of key cellular proteins. The information available thus far allows the construction of a hypothesis regarding the biochemical basis of PC12 differentiation.

Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells.

Neural crest cells maintained as primary cultures for 4-7 days demonstrated specific binding of radioiodinated nerve growth factor ([125I]NGF); occasionally, significant binding of ([125I]NGF could be detected in 3-day cultures. Parallel cultures processed for indirect immunofluorescence revealed that the NGF receptor-positive neural crest cells represented a subpopulation of the total cells in culture. Cultures aged 4-7 days demonstrated fluorescent cells which were often grouped as aggregates. Some 3-day cultures contained faintly fluorescent cells. One- and two-day cultures were non-fluorescent. Melanocytes did not appear to bind NGF. Preliminary flow cytofluorometric analysis indicated that ca. 28% of neural crest cells in 5-day cultures possessed specific receptors for NGF.

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells.

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.

The significance of retrograde axonal transport for the accumulation of systemically administered nerve growth factor (NGF) in the rat superior cervical ganglion.

The present study has shown that after intravenous injection of [125I]NGF the time-course of appearance of radioactivity in all organs studied with the exception of sympathetic and sensory ganglia, roughly paralleled that of the blood. The highest levels were reached immediately after injection, after which the radioactivity decayed rapidly within the firsh hour. By contrast, in the superior cervical ganglion there was a small but significant increase within the first hour. After this the radioactivity remained constant for about 4 h and then increased dramatically (7-fold) when the radioactivity in other tissues had declined to very low levels. Measuring the proportion of radioactivity in the plasma which represents immunologically active NGF, we found that within 30 min after injection all the radioactivity represented unchanged [125I]NGF. After this time the proportion of immunologically active NGF decreased gradually and reached a final level of about 10-15%. Evidence that the radioactivity accumulated in the superior cervical ganglion by retrograde axonal transport represents unchanged [125I]NGF was provided by gel electrophoresis. The results are interpreted as follows: the initial small increase in the sympathetic ganglia may result either from [125I]NGF taken up by short collateral fibres within the ganglion or from a direct accumulation of blood-borne [125I]NGF by the cell bodies of the adrenergic neurones. The dramatic increase occurring after 4 h is caused by the moiety of [125I]NGF reaching the cell body by retrograde axonal transport. This interpretation is supported by autoradiographic studies which showed that 1 h after [125I]NGF injection there was only very sparse labelling of the ganglion, whereas 24 h later virtually all the cell bodies were heavily labelled. Moreover, it could be shown that the lag period between intravenous injection and subsequent accumulation of [125I]NGF in the adrenergic cell bodies was considerably shorter after transection of the postganglionic fibres distal to the cell body [the transected fibres were allowed to regenerate for 7 days] resulting in a reduction of the distance between the site of uptake and accumulation.

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence.

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.

The role of phosphorylation in the action of nerve growth factor.

Although there is a great deal of information available about the biological effects of nerve growth factor, the detailed mechanism(s) by which it works is still not known. Some general outlines, however, are becoming clear. It is known that a number of second messenger systems are recruited and there is evidence that this recruitment has functional consequences. There is also increasingly strong evidence that nerve growth factor causes the activation of parallel pathways of phosphorylative cascades leading to changes in the phosphorylation and, consequently, in the function of key proteins throughout the cell. It can be suggested that changes in the phosphorylation of nuclear proteins initiate the alterations in gene expression known to occur after nerve growth factor treatment, which underlie the dramatic changes in the overall biology of nerve growth factor-responsive cells.

Evidence for an indirect effect of nerve growth factor (NGF) on the ADP-ribosylation of a 22 kDa rho-like protein in PC12 cells.

EDIN catalyzes the ADP-ribosylation of a 22 kDa protein, probably related to rho, in permeabilized PC12 cells in a time- and dose-dependent manner. Pre-treatment of the cells with nerve growth factor inhibits this EDIN-catalyzed ADP-ribosylation, both in vitro and in vivo. This inhibition is largely prevented by the addition of K-252a. Nerve growth factor itself causes neither ADP-ribosylation nor phosphorylation of this 22 kDa protein. These results suggest that the ADP-ribosylation of the rho protein is inhibited, albeit indirectly, by nerve growth factor treatment.

Nerve growth factor-induced increase in calcium uptake by PC12 cells.

Treatment of PC12 cells with nerve growth factor (NGF) produces a rapid and transient increase in calcium uptake into the cells. The increased uptake is maximal after 5 minutes of NGF treatment, but after 15 minutes of NGF treatment, no such increase can be observed. The effect of NGF is partially inhibited by blockers of L-type calcium channels. K-252a, an alkaloid-like kinase inhibitor that usually is found to inhibit the actions of NGF on PC12 cells, produces an increase in calcium uptake similar to, but smaller than, that seen with NGF. NGF had no effect on calcium release under these conditions.

Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.