(Received for publication, June 20, 1994; and in revised form, January 3, 1995)
From the
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-1
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.
Nerve growth factor (NGF), ()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 -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-1
(IL-1)(6, 8, 11, 13) , and
transforming growth factor
1 (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.
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.
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 ()(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
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.
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.
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.
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.
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. ()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.