©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Okadaic Acid Increases Nerve Growth Factor Secretion, mRNA Stability, and Gene Transcription in Primary Cultures of Cortical Astrocytes (*)

(Received for publication, June 20, 1994; and in revised form, January 3, 1995)

Sergey P. Pshenichkin Bradley C. Wise (§)

From the Fidia-Georgetown Institute for the Neurosciences and the Department of Pharmacology, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF) in response to cytokines, growth factors, and activators of protein kinases. To further implicate a protein phosphorylation mechanism in the regulation of NGF expression, astrocytes were treated with okadaic acid and calyculin A, inhibitors of phosphoprotein phosphatases 1 and 2A. Okadaic acid dramatically increased both NGF mRNA content (50-fold) and NGF secretion (100-fold) in astrocytes, while calyculin A, which has a spectrum of phosphatase inhibitory activity different from okadaic acid, failed to augment NGF expression. The increased mRNA accumulation was due mainly to an increase (4-fold) in the half-life of the NGF mRNA following 9 or 24 h of treatment. Nuclear run-on assays indicated that okadaic acid also activated NGF gene transcription, which was preceded by an induction of c-fos and c-jun gene transcription. The induction of NGF expression by okadaic acid appeared independent from protein kinase C activity because down-regulating protein kinase C activity failed to decrease the okadaic acid stimulation. In contrast, interleukin-1beta acted synergistically with okadaic acid to stimulate NGF secretion. The results indicate that okadaic acid profoundly stimulates NGF expression in astrocytes mainly by enhancing NGF mRNA stability and suggest important roles for phosphoprotein phosphatases in regulating NGF production.


INTRODUCTION

Nerve growth factor (NGF), (^1)a member of the growing family of neurotrophins, promotes the survival and differentiation of various types of neurons(1) . The factors comprising this neurotrophin family, which includes brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5, share about 50% homology in amino acid and nucleotide sequences, exhibit specific expression patterns in different brain regions, and act on different neuronal targets(2) . The promotion of neuron survival by neurotrophins suggests that increasing tissue neurotrophin levels might be beneficial in certain chronic and progressive neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease(3) .

Primary cultures of glial cells and glial-derived cell lines synthesize and secrete NGF(4, 5, 6, 7, 8) . In C6 astrocytoma cells, NGF expression is regulated by beta-adrenergic receptor agonists(4, 9) , which increase NGF mRNA content through a cAMP-dependent mechanism involving immediate early gene (IEG) induction(10) . In primary cultures of rat astrocytes, glial cell growth factors, and cytokines, including basic fibroblast growth factor(6, 11, 12) , interleukin-1beta (IL-1)(6, 8, 11, 13) , and transforming growth factor beta1 (14) are very potent and efficacious activators of NGF gene expression and NGF secretion. In contrast, glucocorticoids inhibit basal and stimulated NGF production in astrocytes (15, 16) and other nonneuronal cells(17) . The action of IL-1 is of interest because it can activate both NGF gene transcription (16, 18) and NGF mRNA stabilization(11, 18) , although the intracellular signals mediating these actions in astrocytes are unknown. We have recently provided evidence excluding a role of protein kinase C (PKC) in the mediation of IL-1 effects on NGF gene expression in astrocytes, but our results did implicate a role for other protein phosphorylation systems(16) . In contrast to NGF, astroglial brain-derived neurotrophic factor expression is not increased by cytokines and growth factors that increase NGF, but brain-derived neurotrophic factor mRNA is markedly increased by forskolin, ionomycin, and norepinephrine(19) . The presence of multiple and different promoters in the brain-derived neurotrophic factor gene (20) compared with the NGF gene (21, 22) may underlie the differential regulation of these neurotrophins.

Okadaic acid (OA) is a polyether fatty acid isolated from marine sponges that has been shown to be a potent tumor promoter(23) . Instead of activating PKC like the phorbol ester tumor promoters, OA specifically inhibits phosphoprotein phosphatases 1 and 2A leading to an increase in the phosphorylation state of many cellular proteins (23) . Interestingly, OA treatment of fibroblasts mimicked the effects of IL-1 on protein phosphorylation(24) , suggesting that one cellular action of IL-1 might be to inhibit phosphoprotein phosphatase activity. OA has also been found to increase NGF mRNA content in mixed glial-neuronal hippocampal cell cultures similar to IL-1(25) . In the present study, we examined the mechanism responsible for the induction of NGF expression by OA in primary cultures of cortical astrocytes. Our results indicate that OA stimulates both NGF gene transcription and NGF mRNA stabilization. Moreover, the action of OA does not require basal or activated PKC activity and may be mediated by induction of IEGs.


EXPERIMENTAL PROCEDURES

Cell Culture and Treatment

Primary cultures of cortical astroglial cells were prepared from newborn (postnatal day 2) Sprague-Dawley rats (Zivic-Miller Laboratories, Inc.) as described(6) . After careful removal of meninges, the cortices were dissociated by passage through 75-µm sterile Nitex screens (Tetko) into 10 ml of culture medium. The medium consisted of Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1), 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). Cells were plated onto 100-mm tissue culture dishes (Nunc) (16 times 10^6 cells/dish) or 24-well plates (Nunc) (0.4 times 10^6 cells/15 mm well). Cells were grown for 8-10 days (80-90% confluent) at 37 °C in a water-saturated air environment containing 6% CO(2). Culture medium was changed every 3 days of cultivation.

Cells were treated under serum-free conditions (Dulbecco's modified Eagle's medium/Ham's F-12 medium plus antibiotics) with OA (Life Technologies, Inc.), IL-1 (Boehringer Mannheim), TPA, staurosporine, (Sigma), or the respective vehicle solutions (control cells). Concentrations and times of treatment are indicated in the text and figure legends. For NGF determinations, culture medium was collected and immediately frozen, and NGF was extracted from cells (in 100-mm dishes) as described earlier(6) . For RNA determination, cells in 100-mm dishes were incubated for 3 h in serum-free medium to equilibrate the cells and then treated with the various agents for the times indicated in the text and figure legends. For mRNA stability studies, cells were pretreated for different times with or without OA (20 nM) followed by the addition of actinomycin D (10 µg/ml). Cells were harvested for RNA isolation at various times after actinomycin D addition.

NGF Enzyme-linked Immunoassay

The NGF enzyme-linked immunoassay (NGF-EIA) was performed as described previously (6) using purified monoclonal anti-NGF antibodies unconjugated and conjugated to beta-galactosidase (Boehringer Mannheim). Chlorophenol red-beta-galactopyranoside was used as the enzyme substrate, and standard curves were generated using mouse NGF.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated by the guanidine isothiocyanate method as described previously (11) . RNA (40 µg) was fractionated on a 1.1% agarose, 6% formaldehyde gel, transferred to a nylon membrane (Schleicher and Schuell), and hybridized at 65 °C for 24 h with an NGF P-labeled cRNA probe (specific activity of 8-9 times 10^8 cpm/µg)(11, 16) . The NGF cRNA probe was prepared by transcription with SP6 polymerase (Life Technologies, Inc.) of a pGEM-3Z vector containing a 543-base pair cDNA encoding mouse beta-NGF (9, 11) . Following hybridization, blots were washed with 0.1 times SSC, 0.1% SDS at 65 °C. Hybridization of RNA blots to P-labeled, randomly primed c-fos and c-jun cDNA probes was performed at 42 °C for 24 h(16) . Following exposure of blots to Hyperfilm-MP (Amersham Corp.) with intensifying screens for 1-2 days at -70 °C, the radioactivity was removed by washing, and the blots rehybridized at 45 °C with a P-labeled nick-translated p1B15 cDNA probe that hybridizes to mRNA encoding the structural protein cyclophilin. The amount of NGF mRNA was expressed in arbitrary units, defined as the ratio between the densitometric area of the NGF mRNA hybridization and that of the cyclophilin mRNA hybridization.

Nuclear Run-on Transcription Assays

Nuclei were prepared from 5 times 10^7 astroglial cells and incubated at 37 °C for 40 min with 100 µCi each of [P]CTP and [P]GTP (Amersham Corp.) to label the in vitro synthesized RNA transcripts as described previously(16) . After two deoxyribonuclease (30 µg/ml) and proteinase K (0.15 mg/ml) digestions (each for 30 min at 37 °C), nascent labeled RNA was extracted 3 times with phenol/chloroform and precipitated with ethanol. The labeled RNA transcripts were hybridized at 45 °C for 4 days to plasmids containing the NGF, p1B15, c-fos, and c-jun cDNA inserts(16) , which had been linearized with EcoRI, denatured, and slot blotted (about 5 µg) onto nitrocellulose paper. Blots were also prepared with the parent pGEM-3Z plasmid without the cDNA inserts to control for nonspecific hybridization. After hybridization, the filters were washed twice (60 min each) at 45 °C with buffer A (10 mM Tris/Cl, pH 7.5, 0.3 M NaCl, 2 mM EDTA) containing 0.1% SDS, washed twice (1 min each) at 45 °C with buffer A, and then incubated with RNase A (10 µg/ml) and RNase T1 (2 µg/ml) for 30 min at 37 °C. The blots were washed 2 times (60 min each time) at 45 °C with buffer A plus 0.1% SDS followed by exposure to Hyperfilm-MP with intensifying screens for 3-6 days at -70 °C. Transcriptional rates were expressed as the ratio between the density of the NGF and cyclophilin hybridizations after correction for nonspecific background hybridization.

Other Methods

Protein determinations were performed with the protein assay kit from Bio-Rad using -globulin as a standard. One-way analysis of variance followed by the multiple comparison Neuman-Keuls test was used for statistical analysis of the data.


RESULTS

Induction of NGF Expression by OA

OA was used to examine the role of protein dephosphorylation pathways in the regulation of NGF expression. Primary cultures of astrocytes were treated with OA (30 nM), and, at various times of treatment, total cellular RNA was isolated. Northern blot analysis showed that OA elicited a dramatic induction of NGF mRNA content, whereas it had only small effects on the levels of p1B15 mRNA, which was used as a reference for selective quantification of NGF mRNA content (Fig. 1). The results of several such experiments are summarized in Fig. 2, where it is evident that the increase in NGF mRNA peaked between 18 and 24 h of OA treatment and was maintained for up to 48 h before declining by about 60% at 72 h. Removal of OA after 24 h of treatment by washing the cells led to a decline of NGF mRNA over a period of 5 h (Fig. 2), indicating the reversibility of OA's effects. OA also increased cell content of NGF by about 12-fold following 48 h of incubation with 30 nM OA, while NGF accumulation in the medium was stimulated up to 100-fold over basal levels of secretion after 72 h of OA treatment (Fig. 2). NGF secreted into the medium was biologically active because conditioned medium from OA-treated astrocytes stimulated neurite outgrowth of PC-12 cells (data not shown).


Figure 1: Induction of NGF mRNA in astrocytes treated with OA. Astrocytes were treated with OA (30 nM) for the indicated times. Total RNA was extracted, electrophoresed in a 1.1% agarose, 6% formaldehyde gel, transferred to nylon membranes, and hybridized with an NGF cRNA probe followed by hybridization with a p1B15 cDNA probe as described under ``Experimental Procedures.'' The positions of the NGF and p1B15 mRNAs detected in a representative autoradiograph following a 2-day exposure are indicated.




Figure 2: Time course of NGF expression in astrocytes stimulated with OA. Astrocytes were treated with OA (30 nM) for the indicated times. After 24 h of treatment, some dishes were washed with fresh medium to remove OA and then incubated for the times indicated (dottedline). Total RNA was extracted, and NGF mRNA content was determined by Northern blot hybridization as described under ``Experimental Procedures.'' NGF content in the cells and culture medium was determined by the NGF-EIA. The data are expressed as percent of respective control (vehicle-treated) values measured at each time point and are the means ± S.E. of three independent experiments.



The dose-dependent effect of OA on NGF secretion (during 24 h) is shown in Fig. 3. OA was effective over a narrow range of concentrations with maximal activation seen at 30 nM OA. Identical concentration-dependent effects of OA were seen on NGF mRNA accumulation. The efficacy of OA was limited by its toxicity, which became apparent at concentrations greater than 50 nM. OA, at these higher concentrations, induced morphological changes (observed by phase contrast microscopy), manifested as rounding and shrinking of cells, and detachment of cells from the culture dish. These changes were associated with DNA fragmentation into oligonucleosomal fragments (^2)(assessed by agarose gel electrophoresis) characteristic of apoptosis. The potent induction of NGF expression by OA was not related to these cellular changes because staurosporine (see below) or dexamethasone^2 inhibited the induction of NGF by OA but failed to block the morphological changes and DNA fragmentation induced by OA. Treatment of cells with different concentrations of calyculin A, an inhibitor with a different spectrum of phosphatase inhibitory activity compared to OA(23) , failed to induce NGF secretion (Fig. 3), but induced similar cell morphological changes as did OA.


Figure 3: Dose-dependent effect of OA and calyculin A on NGF secretion from astrocytes. Astrocytes were treated with OA or calyculin A at the indicated concentrations for 24 h. NGF content in the culture medium was measured by the NGF-EIA and expressed as pg of NGF/mg of cell protein. Values are the means ± S.E. of four independent determinations each assayed in duplicate. Similar results were seen in two additional experiments.



Involvement of Protein Kinases in the Induction of NGF by OA

Astrocytes were treated with 100 nM staurosporine, a protein kinase inhibitor that blocks PKC- and non-PKC-dependent secretion of NGF(16) . Staurosporine significantly blocked the increase in NGF mRNA, cellular NGF, and secreted NGF induced by OA (Fig. 4), suggesting that a basal level of protein phosphorylation is necessary for the action of OA. Because NGF expression in nonneuronal cells is enhanced by PKC activation(16, 26, 27, 28) , we examined the role of PKC in the induction of NGF by OA. Down-regulating PKC activity by prolonged treatment (24 h) with TPA failed to affect OA's action (Fig. 5). Under control conditions (i.e. minus TPA pretreatment), TPA and OA treatment increased NGF secretion (during 24 h) by 700 and 2100 pg/mg of protein, respectively, above control levels (Fig. 5, leftcolumns). Following pretreatment with TPA for 24 h, the addition of fresh TPA failed to further stimulate NGF secretion compared with pretreatment control, demonstrating a reduction of PKC activity (Fig. 5, rightcolumns). OA stimulated NGF accumulation by about 2200 pg/mg of protein under these conditions of PKC down-regulation (Fig. 5, rightcolumns). Similar results were seen when NGF mRNA content was estimated under identical conditions (data not shown). To further support the noninvolvement of PKC in OA's action, astrocytes were treated simultaneously with maximally active concentrations of TPA (100 nM) and OA (30 nM) for 24 h. NGF secretion during this time was 108 ± 6 pg/mg of protein (n = 4 determinations) in control cells, 945 ± 20 in TPA-treated cells, 3000 ± 280 in OA-treated cells, and 5200 ± 420 in TPA- and OA-treated cells. This additive action of the two agents and the results of the PKC down-regulation experiments demonstrate that OA affects protein phosphorylation pathways different from those regulated by PKC in enhancing NGF expression.


Figure 4: Staurosporine inhibition of OA stimulation of NGF expression in astrocytes. Astrocytes were treated with OA (20 nM) in the absence and presence of staurosporine (100 nM) for 24 h. NGF mRNA content was estimated by Northern blot hybridization, and NGF content in cells and culture medium were determined by the NGF-EIA. The data are expressed as percent of respective control (vehicle-treated) values and are the means ± S.E. of three independent determinations. *, p < 0.05 compared with control;**, p < 0.05 compared with OA only treatment; analysis of variance and Neuman-Keuls test.




Figure 5: Effect of TPA pretreatment on OA- and TPA-stimulated NGF secretion. Astrocytes were pretreated with (rightcolumns) or without (leftcolumns) TPA (100 nM) for 24 h. The medium was then changed, fresh TPA (100 nM) or OA (20 nM) was added, and cells were incubated for 24 h. The amount of NGF secreted into the medium was measured, and the values are the means ± S.E. of four determinations each assayed in duplicate. Similar results were found in three additional experiments. *, p < 0.05 compared with respective controls; analysis of variance and Newman-Keuls test.



Based on our previous studies showing that IL-1 stimulates NGF expression in a PKC-independent manner(16) , a possible interaction between OA and IL-1 was investigated. Astrocytes were treated simultaneously with IL-1 (at the maximal concentration of 10 units/ml) and with 3 or 30 nM OA for 24 h. IL-1 by itself gave about a 5-fold increase in NGF secretion, while in the presence of an ineffective concentration of OA (3 nM, see Fig. 3) NGF secretion was increased by about 22-fold (Fig. 6). Treatment with IL-1 and a maximal concentration of OA (30 nM, see Fig. 3) failed to significantly increase NGF accumulation in the medium above that produced by OA alone (Fig. 6). These results indicate a synergism and nonadditivity in the action of OA and IL-1, suggesting that these two agents act via a common mechanism to induce NGF expression.


Figure 6: IL-1 potentiates OA stimulation of NGF secretion from astrocytes. Astrocytes were treated for 24 h with the indicated concentrations of OA in the absence or presence of IL-1 (10 units/ml). NGF content in the culture medium was measured by the NGF-EIA, and values are the means ± S.E. of four determinations each assayed in duplicate. This experiment was replicated three times with similar results. *, p < 0.05 compared with respective no IL-1 treatment controls; analysis of variance and Newman-Keuls test.



Effect of OA on NGF mRNA Stability and Gene Transcription

The elevation of NGF mRNA content by OA may be a result of mRNA stabilization and/or gene transcription. To examine NGF mRNA stability, we estimated the half-life of the mRNA following inhibition of gene transcription with actinomycin D. Astrocytes were treated with OA for 9 or 26 h prior to the addition of actinomycin D. Total RNA was isolated from cells at different times and analyzed by Northern blot hybridization with the NGF cRNA probe (Fig. 7). The half-life (t) of the NGF mRNA under control conditions was 26 ± 3 min (n = 5 experiments), while OA treatment of astrocytes for 26 h increased the t to 110 ± 22 min (n = 5 experiments). OA treatment of cells for 9 h produced comparable increases in the NGF mRNA half-life as seen after 26 h of treatment (Fig. 7).


Figure 7: NGF mRNA half-life in astrocytes treated with OA. Astrocytes were treated with vehicle (control) or OA (20 nM) for 9 or 26 h before the addition of actinomycin D (10 µg/ml) to inhibit gene transcription. RNA was isolated from the cells at the indicated times after addition of actinomycin D and processed for NGF and p1B15 mRNA determinations by Northern blot hybridization. The data, expressed as percent of the initial NGF mRNA content before actinomycin D addition, is from a representative experiment, and the mean NGF mRNA half-lives determined from several such experiments are given in the text.



To establish whether the induction of NGF mRNA by OA was due to an increased rate of gene transcription in addition to mRNA stabilization, nuclear run-on assays were performed with nuclei from control and OA-treated astrocytes. Radiolabeled nascent RNA transcripts were hybridized to NGF, cyclophilin (p1B15), and pGEM-3Z cDNAs immobilized on nitrocellulose paper (Fig. 8). Compared with the slight increase in p1B15 gene transcription after 9 and 26 h of OA treatment, in three separate experiments, OA stimulated by 1.5 ± 0.2-fold the NGF gene transcriptional rate after 26 h of treatment (Fig. 8).


Figure 8: OA stimulation of NGF and IEG gene transcription. Astrocytes were treated with OA (20 nM) for the indicated times. Nuclei were prepared and subjected to transcriptional run-on assays as described under ``Experimental Procedures.'' Radiolabeled nascent RNA transcripts were purified and hybridized to NGF, p1B15, pGEM-3Z, c-fos, or c-jun cDNAs immobilized on nitrocellulose paper, as indicated. The parent plasmid pGEM-3Z was used to control for nonspecific hybridization. The results shown are from a representative experiment (exposure time was 10 days) demonstrating the induction of NGF, c-fos, and c-jun gene transcription by OA. The mean rates of NGF, c-fos, and c-jun gene transcription from three such experiments are given in the text.



Stimulation of c-fos and c-jun Gene Transcription and mRNA Accumulation by OA

Since the NGF gene contains a functional AP-1 transcriptional regulatory element(21, 22) , induction of the IEGs c-fos and c-jun by OA was examined to elucidate a possible involvement of these genes in increasing the NGF gene transcriptional rate. Nuclear run-on experiments showed that OA maximally stimulated c-fos and c-jun gene transcription after 3 h of treatment (Fig. 8). The relative increase in c-fos and c-jun transcription, compared with that of p1B15, was 1.6 ± 0.2-fold and 1.8 ± 0.4-fold (n = 3), respectively. Measurement of the mRNA content for these IEGs reflected the results of the transcription assays. OA treatment induced c-fos and c-jun mRNA content with maximal increases seen between 3 and 6 h of treatment (Fig. 9). It is significant to note that c-fos mRNA content remained elevated above control levels even after 24 h of treatment (Fig. 9), indicating a prolonged expression of this mRNA similar to that seen with NGF mRNA.


Figure 9: Induction of c-fos and c-jun mRNA by OA in astrocytes. Cells were treated with OA (20 nM) for the indicated times. Following treatment, total RNA was isolated, electrophoresed, and blotted as described under ``Experimental Procedures.'' The individual blots were hybridized with P-labeled cDNA probes against c-fos, c-jun, and p1B15 as indicated. Shown is an autoradiograph from a representative experiment. Similar results were seen in two additional experiments.




DISCUSSION

Previous studies have implicated PKC and cAMP-dependent protein phosphorylation mechanisms in the regulation of NGF expression in primary cultures of astrocytes(16, 25, 27, 28) , astroglioma cells(4, 9, 10) , and fibroblasts(26) . We now show that OA, but not calyculin A, dramatically increased steady state NGF mRNA levels and NGF production in primary cultures of astrocytes. Because OA inhibits phosphoprotein phosphatase 2A with greater potency than protein phosphatase 1 and because calyculin A inhibits protein phosphatase 1 about 100-fold better than OA(23) , the difference in action of these two inhibitors on NGF expression may be a result of protein phosphatase 2A inhibition by OA. However, there is insufficient evidence at present to conclusively state that protein phosphatase 2A inhibition is responsible for the stimulation of NGF expression in astrocytes. The action of OA did require a basal level of protein phosphorylation activity because staurosporine, a protein kinase inhibitor, significantly blocked OA's stimulatory effects on NGF expression. Moreover, the effects of OA were observed under conditions of PKC down-regulation, indicating that PKC activity was not required in the mechanism of action of OA. Because IL-1 acted synergistically with OA, it is apparent that the signal transduction processes activated by IL-1 converge with the inhibition of phosphoprotein phosphatases by OA to augment NGF expression. As suggested by Guy et al.(24) , IL-1 may inactivate a protein phosphatase similar to the action of OA, or it may stimulate, through activation of protein kinases, the phosphorylation of proteins. By either mechanism, OA and IL-1 treatment would lead to a net increase in the phosphorylation state of proteins critical for the activation of NGF gene expression in astrocytes.

One cellular mechanism, although not the major one, by which OA increases NGF expression in astrocytes is by activation of NGF gene transcription (Fig. 8). Indirect evidence indicates that the induction of NGF gene expression by activators of protein kinases is mediated by an induction of IEGs, such as c-fos and c-jun(10, 16, 25) . The protein products of IEGs form a functional transcription complex that binds to an AP-1 regulatory DNA element in the first intron of the NGF gene and thereby activates its transcription(21, 22) . OA has been shown to stimulate c-fos gene transcription as well as c-fos mRNA stabilization in NIH3T3 cells(29) . Our results show that OA increased IEG transcription and IEG mRNA content ( Fig. 8and Fig. 9) in astrocytes. It is significant to note that c-fos mRNA content induced by OA was prolonged such that by 24 h after treatment, c-fos mRNA was still about 5-fold higher than control levels (Fig. 9), an effect due probably to a stabilization of the c-fos mRNA by OA(29) . This late elevation in c-fos mRNA content coincided with the induction of NGF gene transcription by OA. These results are consistent with the notion that IEG activation is involved in NGF gene transcription stimulated by OA, as well as by IL-1 and TPA as reported in other studies(16, 25) . However, since there is a time delay between the maximal induction by OA of c-fos and c-jun compared with NGF, other cellular influences may be controlling NGF gene transcription. For example, the strong repressor element in the NGF promoter (21) may overide the AP-1 stimulatory element and, therefore, may need to be inactivated prior to a stimulation of NGF gene transcription in astrocytes.

The most prominent action of OA on astroglial NGF gene expression was to increase the half-life of the rather unstable NGF mRNA. In control astrocytes, NGF mRNA declined following actinomycin D inhibition of gene transcription with an apparent half-life of about 30 min as seen in other studies(11, 16, 18) . OA treatment for 9 or 26 h increased the NGF mRNA half-life by about 4-fold (Fig. 7), and, in preliminary studies, this stabilization of NGF mRNA was seen as early as 3 h of OA treatment. The short half-life of the NGF mRNA is similar to cytokine and proto-oncogene mRNAs, which are rapidly degraded and, hence, transiently expressed(30, 31) . A common feature of the mRNAs coding for cytokines and proto-oncogenes is the presence of an AU-rich region including repetitive AUUUA sequences in the 3`-untranslated region (UTR) of their mRNAs, which have been implicated as determinants of the destabilization of these mRNA species(30, 31) . Computer sequence analysis of the 3`-UTR of the NGF mRNA from different species (rat, mouse, guinea pig, and humans) indicates a high degree of sequence homology in an AU-rich region of the 3`-UTR and the presence of one to two AUUUA sequences within this region. This RNA domain may serve an important functional role in NGF mRNA regulation, perhaps acting as a determinant of its stability. The binding of proteins to nucleotide sequences in the 3`-UTR of various mRNA species has led to the hypothesis that trans-acting protein factors may serve to regulate the rate of cytoplasmic mRNA degradation(31, 32, 33) . We have identified by Northwestern blotting procedures a protein that binds to an RNA probe encompassing the AU-rich region of the NGF mRNA 3`-UTR and which is induced in astrocytes by OA treatment under identical conditions that increase NGF mRNA half-life. (^3)This protein, by binding to the destabilizing AU-rich region of the NGF mRNA 3`-UTR, might slow the rate of degradation of the mRNA.

The results in this report demonstrate a dual action of OA on NGF expression in astrocytes; an early and prolonged stabilization of the NGF mRNA followed by an activation of NGF gene transcription possibly mediated by an induction of IEGs. Both actions lead to dramatic increases in NGF mRNA content followed by NGF secretion in astrocytes. Glial cell production of NGF following neuronal injury or neurodegeneration might limit the extent of neuronal loss and promote regenerative processes to restore neuronal function(6, 7, 12) . Of major future interest would be to see whether similar increases in NGF production by OA occurs in vivo, and if so, whether such increases would promote neuronal survival in animal models of neurodegeneration.


FOOTNOTES

*
This work was supported in part by National Institutes of Mental Health Grant MH45181. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, D. C. 20007. Tel.: 202-687-1270; Fax: 202-687-6437.

(^1)
The abbreviations used are: NGF, nerve growth factor; IEG, immediate early gene; IL-1, interleukin-1beta; PKC, protein kinase C; OA, okadaic acid; NGF-EIA, NGF enzyme-linked immunoassay; TPA, 12-O-tetradecanoylphorbol-13-acetate; UTR, untranslated region.

(^2)
S. P. Pshenichkin and B. C. Wise, manuscript in preparation.

(^3)
B. Tang and B. C. Wise, manuscript in preparation.


REFERENCES

  1. Levi-Montalcini, R. (1987) Science 237, 1154-1162 [Medline] [Order article via Infotrieve]
  2. Thoenen, H. (1991) Trends Neurosci. 14, 165-170 [CrossRef][Medline] [Order article via Infotrieve]
  3. Springer, J. E. (1993) Exp. Neurol. 124, 2-4 [CrossRef][Medline] [Order article via Infotrieve]
  4. Schwartz, J. P., Chuang, D. M., and Costa, E. (1977) Brain Res. 137, 369-375 [Medline] [Order article via Infotrieve]
  5. Furukawa, S., Furukawa, Y., Satoyoshi, E., and Hayashi, K. (1987) Biochem. Biophys. Res. Commun. 142, 395-402 [Medline] [Order article via Infotrieve]
  6. Carman-Krzan, M., Vigé, X., and Wise, B. C. (1991) J. Neurochem. 56, 636-643 [Medline] [Order article via Infotrieve]
  7. Lu, B., Yokoyama, M., Dreyfus, C. F., and Black, I. B. (1991) J. Neurosci. 11, 318-326 [Abstract]
  8. Spranger, M., Lindholm, D., Bandtlow, C., Heumann, R., Gnahn, H., Näher-Noé, M., and Thoenen, H. (1990) Eur. J. Neurosci. 2, 69-76 [Medline] [Order article via Infotrieve]
  9. Dal Toso, R., De Bernardi, M. A., Brooker, G., Costa, E., and Mocchetti, I. (1988) J. Pharmacol. Exp. Ther. 246, 1190-1193 [Abstract]
  10. Mocchetti, I., De Bernardi, M. A., Szekely, A. M., Alho, H., Brooker, G., and Costa, E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3891-3895 [Abstract]
  11. Vigé, X., Costa, E., and Wise, B. C. (1991) Mol. Pharmacol. 40, 186-192 [Abstract]
  12. Yoshida, K., and Gage, F. H. (1991) Brain Res. 538, 118-126 [Medline] [Order article via Infotrieve]
  13. Friedman, W. J., Larkfors, L., Ayer-Lelievre, C., Ebendal, T., Olson, L., and Persson, H. (1990) J. Neurosci. Res. 27, 374-382 [Medline] [Order article via Infotrieve]
  14. Lindholm, D., Hengerer, B., Zafra, F., and Thoenen, H. (1990) Neuroreport 1, 9-12 [Medline] [Order article via Infotrieve]
  15. Lindholm, D., Castrén, E., Hengerer, B., Zafra, F., Berninger, B., and Thoenen, H. (1992) Eur. J. Neurosci. 4, 404-410 [Medline] [Order article via Infotrieve]
  16. Pshenichkin, S. P., Szekely, A. M., and Wise, B. C. (1994) J. Neurochem. 63, 419-428 [Medline] [Order article via Infotrieve]
  17. D'Mello, S. R., and Heinrich, G. (1991) J. Neurochem. 57, 1570-1576 [Medline] [Order article via Infotrieve]
  18. Lindholm, D., Heumann, R., Hengerer, B., and Thoenen, H. (1988) J. Biol. Chem. 263, 16348-16351 [Abstract/Free Full Text]
  19. Zafra, F., Lindholm, D., Castrén, E., Hartikka, J., and Thoenen, H. (1992) J. Neurosci. 12, 4793-4799 [Abstract]
  20. Timmusk, T., Palm, K., Metsis, M., Reintam, T., Paalme, V., Saarma, M., and Persson, H. (1993) Neuron 10, 475-489 [Medline] [Order article via Infotrieve]
  21. D'Mello, S. R., and Heinrich, G. (1991) Mol. Brain Res. 11, 255-264 [Medline] [Order article via Infotrieve]
  22. Hengerer, B., Lindholm, D., Heumann, R., Ruther, U., Wagner, E. F., and Thoenen, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3899-3903 [Abstract]
  23. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. (1990) Trends Biochem. 15, 98-102 [CrossRef][Medline] [Order article via Infotrieve]
  24. Guy, G. R., Cao, X., Chua, S. P., and Tan, Y. H. (1992) J. Biol. Chem. 267, 1846-1852 [Abstract/Free Full Text]
  25. Friedman, W. J., Altiok, N., Fredholm, B. B., and Persson, H. (1992) J. Neurosci. Res. 33, 37-46 [Medline] [Order article via Infotrieve]
  26. D'Mello, S., and Heinrich, G. (1990) J. Neurochem. 55, 718-721 [Medline] [Order article via Infotrieve]
  27. Neveu, I., Jehan, F., Houlgatte, R., Wion, D., and Brachet, P. (1992) Brain Res. 570, 316-322 [Medline] [Order article via Infotrieve]
  28. Carman-Krzan, M., and Wise, B. C. (1993) J. Neurosci. Res. 34, 225-232 [Medline] [Order article via Infotrieve]
  29. Schonthal, A., Tsukitani, Y., and Feramisco, J. R. (1991) Oncogene 6, 423-430 [Medline] [Order article via Infotrieve]
  30. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667 [Medline] [Order article via Infotrieve]
  31. Schiavi, S. C., Belasco, J. G., and Greenberg, M. E. (1992) Biochim. Biophys. Acta 1114, 95-106 [CrossRef][Medline] [Order article via Infotrieve]
  32. Malter, J. S. (1989) Science 246, 664-666 [Medline] [Order article via Infotrieve]
  33. Klausner, R. D., and Harford, J. B. (1989) Science 246, 870-872 [Medline] [Order article via Infotrieve]

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