Biomedical subjects
Publications and source records attributed to G Guroff.
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.
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.
Nerve growth factor stimulates the uptake of radioactive calcium into PC12 cells. This stimulation is inhibited by low concentrations of dideoxyforskolin or staurosporine, and by high concentrations of nifedipine or cadmium. On the other hand, neither dideoxyforskolin nor staurosporine inhibited the stimulation of calcium uptake caused by BK-8644 or adenosine triphosphate (ATP). Nickel inhibited only the effect of ATP on calcium uptake, and actually stimulated the effects of either BK-8644 or nerve growth factor. Down-regulation of L-calcium channels by BK-8644 blocked the subsequent stimulation of calcium uptake by this agent, but not the stimulation by nerve growth factor. Conversely, pre-treatment of the cells with nerve growth factor inhibited the subsequent stimulation of calcium uptake by nerve growth factor, but not the stimulation by BK-8644. The effects of BK-8644 and nerve growth factor on calcium uptake were additive, as were the effects of nerve growth factor and ATP. Phosphatase 2A inhibited the effect of nerve growth factor on calcium uptake, but did not influence the action of BK-8644. On the other hand, calcineurin inhibited the effect of BK-8644 on calcium uptake, but potentiated the action of nerve growth factor. Calmidazolium or fluphenazine also inhibited the effect of nerve growth factor on calcium uptake, but okadaic acid stimulated it. A comparison of the effects of these inhibitors on the actions of various calcium channel agonists shows that the channels on which the action of nerve growth factor is exerted are different than either the L-type calcium channels or the ATP-activated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.
Treatment of PC12 cells with nerve growth factor does not alter the levels of B-raf mRNA, but does induce rapid phosphorylation of B-raf proteins. Phosphorylation was observed after 1.5 min and reached a maximum by 10-15 min. B-raf protein was phosphorylated almost exclusively on serine residues; no tyrosine phosphorylation was detected. Nerve growth factor-induced phosphorylation was not affected by depletion of protein kinase C or by removal of extracellular calcium but was inhibited by K-252a. Concomitant with the increase in serine phosphorylation, nerve growth factor treatment also increased the serine/threonine kinase activity of B-raf protein within 1-2 min.
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.
PC12 cells contain at least three immunologically distinct phospholipase C (PLC) isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of PC12 cells with nerve growth factor (NGF) leads to an increase in the phosphorylation of PLC-gamma, but not of PLC-beta or PLC-delta. This increase can be seen in as little as 1 minute. The increased phosphorylation occurs on both serine and tyrosine residues, with the major increase being in the former. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinases associated with the NGF receptor.
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.
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.
PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. The cells contain catecholamines and secrete them in response to depolarizing stimuli and cholinergic agonists. Treatment of the cells with nerve growth factor produces a number of very rapid changes, including the structural rearrangement of the cell membrane, the generation of a number of different second messengers, and the phosphorylation of several proteins. The present studies show that nerve growth factor treatment increases the release of dopamine and norepinephrine from the cells within a few minutes and does so independently of its effect on their metabolism. The experiments indicate that this effect on nerve growth factor is dependent on the presence of extracellular calcium and can be blocked by calcium channel antagonists. K-252a, an alkaloid-like material, usually found to inhibit the actions of nerve growth factor on PC12 cells, also increases the release of catecholamines under these conditions.
Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1:1 to 20:1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60-80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using 125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2-3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these "primed" cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.
The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.
PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. Treatment with nerve growth factor causes the cells to differentiate. One of the hallmarks of this differentiation is the generation of neurites. PC12 cells contain both calpain I and calpain II; about 90% of the total calpain activity is due to calpain II. Treatment of the cells with nerve growth factor causes a time-dependent decrease in calpain activity, more than 50% being lost over a 5-day period. Both the decrease in calpain activity and the growth of neurites are reversible upon the removal of nerve growth factor from the cultures. Agents other than nerve growth factor that cause neurite outgrowth, such as fibroblast growth factor and dibutyryl cyclic AMP, also cause a decrease in calpain activity. Calpain levels, as detected with immunoblotting or immunohistochemistry, show no decrease. Removal of calpastatin, the endogenous inhibitor of the calpains, by phenyl-Sepharose chromatography increases the calpain activity of extracts from both control and nerve growth factor-treated cells and brings the activity in the extracts from treated cells up to the activity in those from controls. Calpastatin-containing fractions from extracts of nerve growth factor-treated cells inhibit more calpain activity than do comparable fractions from control cells. These studies suggest that nerve growth factor causes a decrease in the activity of calpain in morphologically differentiating PC12 cells by causing an increase in the activity of calpastatin.
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.
PC12 cells were manipulated in such a way as to permit the study of differentiation-specific responses independently from proliferative responses. Cells were starved for serum then exposed to nerve growth factor (NGF) or serum. Following addition of serum, cells incorporated thymidine in a synchronous manner. Subsequent to the wave of DNA synthesis, the cell number increased approximately two-fold. Addition of NGF to serum-starved cultures had no measurable effect on either parameter. Neurite outgrowth was more rapid and extensive and appearance of Na+ channels, measured as saxitoxin binding sites, more rapid than when NGF was added to exponentially-growing cells. Epidermal growth factor receptors were heterologously down-regulated by NGF with similar kinetics under both conditions. Induction of the proto-oncogene c-fos by NGF was also greater in the serum-starved cells than in exponentially-growing cultures. These results indicated that serum starvation resulted in synchronisation of the cultures and that NGF action may be cell cycle-specific. Analysis of the cellular response to NGF at different times during the cell cycle showed that c-fos was induced in the G1 phase but not in S or G2. Fluorescence-activated cell sorter analysis demonstrated that addition of NGF to exponentially-growing cells, resulted in their accumulation in a G1-like state. With regard to the study of the mechanism of NGF action, these results illustrate that measurements of NGF effects on specific components in the signal transduction pathway may be confounded by the use of exponentially-growing cultures.
K-252a, a kinase inhibitor isolated from the culture broth of Nocardiopsis sp., selectively inhibits, in a dose- and time-dependent fashion, the increased transcription of the protooncogene c-fos induced by nerve growth factor in PC12 cells. Induction of c-fos by epidermal growth factor, A23187, dBcAMP, or TPA in the same cells is not affected. Pretreatment with K-252a for 30 min results in a complete inhibition of the nerve growth factor-induced increase in intracellular calcium. Increases in intracellular calcium induced by carbachol or by high K+ are not altered. K-252a derivatives selective for the inhibition of various known kinases were used to inhibit the nerve growth factor-dependent induction of c-fos mRNA, the nerve growth factor-dependent increase in intracellular calcium levels, and the nerve growth factor-dependent outgrowth of neurites. K-252a is the most effective inhibitor of all three of these actions of nerve growth factor. The possible mechanisms by which K-252a acts on PC12 cells are considered in the light of the characteristics of the inhibitions seen here.
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.