Rapid attenuation of AP-1 transcriptional factors associated with nitric oxide (NO)-mediated neuronal cell death.

Stimulation of glutamate receptors causes several intracellular reactions including activation of activator protein-1 (AP-1) production and nitric oxide (NO) generation. Exposing mouse cerebellar granule cells to N-methyl-D-aspartate or kainate (KA) in culture induced an increase of AP-1 DNA binding activity that was blocked by further addition of sodium nitroprusside (SNP), a typical NO donor. Immunoblotting using anti-c-Fos antiserum revealed the specific attenuation of AP-1, although total protein synthesis was not affected. Since the level of c-fos mRNA expression stimulated by KA remained constant even after exposure to SNP, the AP-1 attenuation can be post-transcriptionally induced. SNP did not affect the Ca2+ influx into the cells stimulated by KA. The involvement of NO in the AP-1 attenuation was supported by the fact that potassium ferrocyanide (K4Fe(CN)6), an analogue of SNP but devoid of NO, failed to inhibit the AP-1 DNA binding activity stimulated by KA. SNP alone induced neuronal cell death, which was blocked by the simultaneous addition of antioxidants, superoxide dismutase and catalase, and an NO scavenger, suggesting a direct role of peroxynitrite in the cell death. In good agreement with these effects, the AP-1 attenuation by SNP was also blocked by antioxidants. These results indicated that post-transcriptional attenuation of AP-1 is involved in the early processes of NO-mediated neuronal cell death.

The stimulation of glutamate receptors elicits an extracellular Ca 2ϩ influx into neurons through NMDA 1 receptors or voltage-dependent calcium channels (VDCCs) (1). The increased cytosolic Ca 2ϩ concentration resulting from the stimulated Ca 2ϩ influx into neurons successively activates a variety of cellular reactions, including the activation of Ca 2ϩ -dependent protein kinases (2) and the induction of immediate early genes such as c-fos and zif/268 (3,4). The transduced calcium signals also enhance the activity of nitric oxide synthase through Ca 2ϩ /calmodulin (5), resulting in the increased pro-duction of NO. Synthesized NO then diffuses out to neighboring cells where it activates soluble guanylate cyclase that can produce cGMP (6). NO may be involved in long term potentiation (7) and long term depression (8) in the central nervous system. During ischemic brain injury, however, NO may be responsible for neuronal cell death (9), which can be achieved by the cytosolic Ca 2ϩ overload evoked via overstimulating glutamate receptors. Thus, NO seems to have dual functions as an intercellular messenger for synaptic plasticity and, if in excess, as a neurodestructive effector of neuronal injury. The mechanisms proposed for NO-mediated neurotoxicity include inactivation of the mitochondrial respiratory chain (10), S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (11), inhibition of cis-aconitase (12), activation of poly(ADP-ribose) synthase, and DNA damage (13), most of which can be mediated by the formation of nitrosocompounds by cellular components. However, it remains uncertain which cellular mechanisms are exactly responsible for the NO-mediated neurotoxicity. To elucidate these mechanisms, intracellular reactions that can be induced with NO-mediated neuronal cell death must be identified.
Activator protein-1 (AP-1) mainly consists of c-Fos and c-Jun, which are the members of the basic leucine zipper (bZIP) family, and it mediates immediate-early cellular responses by controlling the transcription of a variety of genes carrying AP-1 recognition sequences termed 12-O-tetradecanoylphorbol-13acetate (TPA)-responsive element (TRE) (14). The AP-1 DNA binding activity can be modulated by the reduction-oxidation (redox) of a single conserved cysteine residue in the DNAbinding domains of c-Fos and c-Jun (15,16). NO from sodium nitroprusside (SNP), an NO donor, may directly modulate the AP-1 DNA binding activity in vitro (17). In addition, reactive oxygen species including superoxide anions also affect c-fos mRNA expression and AP-1 DNA binding activity in cultured cells (18,19). Thus, AP-1 DNA binding activity seems to be sensitive to changes in the redox state of cells and, hence, provides a useful molecular tool for investigating the mechanisms responsible for NO-mediated neuronal cell death.
Although brain ischemia may cause neuronal injury via a production of reactive oxygen species or NO in vivo (20,21), NO-mediated neuronal cell death can be reproduced in primary cultures of neurons, which can be driven by the formation of peroxynitrite from NO and superoxide anion (22). For further understanding of the molecular mechanisms of NO-mediated neuronal cell death, we investigated the effect of NO on the AP-1 DNA binding activity using primary cultures of cerebellar granule cells. Stimulation of cerebellar granule cells with NMDA and KA, which causes the Ca 2ϩ influx into the cells through NMDA receptors and VDCCs, respectively, leads to the activation of AP-1 DNA binding activity resulting from an increase in c-fos mRNA expression (23,24). In this study, we * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom all correspondence should be addressed. found that NO, released from an NO donor, rapidly causes the post-transcriptional attenuation of AP-1, followed by NO-mediated neuronal cell death.
Cell Culture and Drug Stimulation-Primary cultures of cerebellar granule cells were prepared from 7-day-old mice (ICR) as described (23). In brief, cells were seeded on plastic culture dishes coated with 5 g/ml poly-L-lysine (Pharmacia Biotech Inc.) and incubated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 25 mM KCl, and 60 g/ml kanamycin sulfate (Wako Purechemicals Co.). Within 30 -48 h after seeding, the proliferation of glial cells was prevented by replacing the medium with that containing 10 M cytosine arabinoside. We used cells cultured for 6 -7 days.
For drug stimulation, the cells were washed twice with Locke's solution containing 10 mM Hepes-NaOH (pH 7.35), 154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO 3 , 2.3 mM CaCl 2 and 5.6 mM glucose, and incubated in the same solution for 1 h. The glutamate receptor agonist, NMDA or KA, was added at the indicated concentrations to stimulate the cells. Cytoplasmic RNAs and nuclear mini-extracts were prepared 1 h after drug stimulation. The samples for measurement of lactate dehydrogenase (LDH) were prepared at the indicated time after drug stimulation. SNP was added 10 min before NMDA and KA stimulation.
45 Ca 2ϩ Uptake-Details of procedure for 45 Ca 2ϩ uptake were previously described by Ohtani et al. (24). Cells were washed three times with Locke's solution containing 1.3 mM CaCl 2 , and incubated for 10 min. NMDA or KA was added with 1 Ci of 45 CaCl 2 (Amersham) to the culture dishes, and the cells were incubated for another 10 min. After removing the incubation solution, the cells were washed four times with Ca 2ϩ -and glucose-free Locke's solution containing 2 mM EGTA, then solubilized with 0.5 M NaOH. The amount of radioactivity incorporated by the cells was measured using a liquid scintillation counter. About 2 mg of protein was recovered from each culture dish (data not shown).
Northern Blot Analysis-After removing the incubation solution, cells were scraped off with a rubber policeman with 1 ml of Trisbuffered saline containing 25 mM Tris-HCl (pH 7.4), 130 mM NaCl, and 5 mM KCl. Cytoplasmic RNAs were extracted as described by Wilkinson et al. (25). Aliquots of RNAs (4.5-10 g) were separated by an electrophoresis on denaturing formaldehyde gels and transferred onto a nylon membrane (Hybond N, Amersham). The membrane was prehybridized for 2 h at 42°C in a hybridization solution containing 5 ϫ SSPE (750 mM NaCl, 50 mM NaH 2 PO 4 , 5 mM EDTA), 50% formamide, 5 ϫ Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 20 g/ml heatdenatured salmon sperm DNA. Thereafter, heat-denatured 32 P-labeled DNA probes were added, and the membrane was hybridized at 42°C overnight. The membrane was washed twice with 2 ϫ SSPE and 0.1% SDS for 10 min at 60°C, once with 1 ϫ SSPE and 0.1% SDS for 30 min at 60°C, and finally twice with 0.1% SSPE and 0.1% SDS for 15 min at room temperature. After autoradiography, the level of mRNA expression was quantified using an imaging scanner (BAS 2000, Fuji).
Preparation of Nuclear Mini-extracts and the Gel Mobility Assay-The procedures for extracting nuclear mini-extracts and the conditions for the gel mobility assay were as described (23). The synthetic 20-base pair oligonucleotides containing TRE (5Ј-GATTCGTGACTCAGCA-CAGG-3Ј) was end-labeled with [ 32 P]dCTP(Amersham) and used as a DNA probe to detect AP-1 DNA binding activity. DNA-protein complexes were separated on a 4% polyacrylamide gel in 1 ϫ TAE (6.7 mM Tris-HCl (pH 7.5), 3.3 mM sodium acetate, 2.5% glycerol, and 0.1 mM

FIG. 1. The inhibitory effect of SNP on the increase in TRE binding activity induced by NMDA, KA, or TPA and its reversibility.
The culture medium was replaced with Locke's solution and the cells were incubated for 1 h. A, SNP was added to the Locke's solution at the indicated concentrations 10 min before 100 M NMDA, 100 M KA, or 100 ng/ml TPA. One hour later, nuclear mini-extracts were prepared and their gel mobility was assayed. B, the cells were incubated for 1 h with 100 M SNP (lanes 3-5). After washing with Locke's solution without SNP and incubating for another 1 h, 100 M KA was added (lane 4) or not (lane 3) and the cells were incubated for 1 h before preparing nuclear mini-extracts. SNP was added again 10 min before KA (lane 5). When the pretreatment of cells with SNP was omitted, the cells were continuously incubated without SNP for 2 h before KA stimulation was added (lane 2) or not (lane 1). The relative levels of TRE binding activities are shown as -fold increase compared with that of the control. Cont. and pre mean the unstimulated control and the pretreatment of cells with SNP, respectively. T means the bands formed by the DNA-protein complexes on TRE. F means free TRE-probe. Bars represent the mean Ϯ S.D. obtained from two to three experiments. EDTA). After electrophoresis, the gel was dried and visualized by autoradiography. Radioactivity in the bands resolved by gel electrophoresis were quantified using the imaging scanner.
Immunoblotting Analysis-Nuclear mini-extracts (30 g each) were denatured in 1 ϫ sample buffer (10 mM Tris-HCl (pH 6.8), 1% SDS, 1% ␤-mercaptoethanol, 20% glycerol) for 5 min at 95°C and then separated by 10% SDS-polyacrylamide gel electrophoresis at 30 mA. For preparing total cellular extracts, cells were lysed with 100 l of lysis buffer containing 62.5 mM Tris-HCl (pH 6.2), 2% Nonidet P-40, 9 M urea, 5% ␤-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. Aliquots (60 g of protein) of total cellular extracts were used for analysis. Protein concentration was quantified by the method of Schaffner and Weismann (26). Sample proteins were transferred onto nylon membranes (Millipore) and blocked with 5% skim milk, 1% bovine serum albumin, and 0.02% antifoam A (Sigma) in phosphate-buffered saline. The membrane was incubated with rabbit anti-c-Fos antibody (Santa Cruz Biotechnology) overnight at 4°C, then with goat biotinylated anti-rabbit IgG antibody (Oncogene Science) for 2 h at 37°C. Thereafter, c-Fos was finally detected following the ABC procedure (Elite ABC kit, Vectastain). Labeled proteins were electrophoretically separated by 8% SDSpolyacrylamide gel electrophoresis, and the radioactivity levels in the lanes were quantified using the Imaging scanner.

Measurement of [ 35 S]Methionine Incorporation-Total
Assay of Cytotoxicity-Cytotoxicity was assessed by measuring LDH release into the extracellular fluid after drug stimulation. Samples were prepared and enzymatic activity was measured as described by Murphy et al. (27). After drug stimulation for the indicated periods, the incubation solution was transferred into fresh tubes and stored at 4°C until use. After removing any remaining solution, the cells were scraped from the plates, lysed with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.5% Triton X-100, and kept in an ice bath for 15 min. After centrifugation for 5 min at 700 ϫ g, the supernatant of the cell lysate was stored at 4°C until use. The LDH activities in the incubation solution and in the supernatant were spectrophotometrically measured at 340 nm. The percent of LDH release was defined as the value of LDH activity included in the incubation solution divided by the additive values included in both the incubation solution and the supernatant. SNP was about 4-fold higher cytotoxic in Locke's solution than in the culture medium of Dulbecco's modified Eagle's medium containing 10% fetal calf serum (data not shown).

Effect of SNP on TRE Binding
Activity-To investigate whether or not NO modulates AP-1 DNA binding activity, we examined the effect of the NO donor, SNP, on the increase of TRE binding activity induced by stimulating cultured cerebellar granule cells with NMDA, KA, and TPA, a potent activator for protein kinase C (23,24). As shown in Fig. 1A, NMDA, KA, and TPA in Locke's solution increased the TRE binding activities (lanes 2, 5, and 8) but further addition of SNP inhibited these induced increases in a dose-dependent manner (lanes 3, 4, 6, 7, 9, and 10). When the KA was administered after exposure to SNP for 1 h (Fig. 1B, lane 4), the TRE binding activity increased in a manner similar to that obtained when KA was administered alone (lane 2). When KA was omitted after the exposure to SNP, the level of TRE binding activity remained constant (lane 3). In addition, SNP was effective in inhibiting the increase in TRE binding activity stimulated by KA even after the exposure to SNP (lane 5). These results indicated that the inhibitory effect of SNP on the increase in TRE binding activity was reversible and, moreover, that exposing cells to SNP for 1 h does not irreversibly damage the cells.
Attenuation of AP-1 by SNP-We investigated whether the amounts of AP-1 in nuclear extracts decreased in accordance with the inhibitory effects of SNP on the TRE binding activity, by means of immunoblotting using anti-c-Fos antiserum (Fig.  2, A and B). The immunoblots showed that c-Fos accumulation was induced by NMDA and KA ( Fig. 2A, lanes 2 and 5, respectively), but inhibited by SNP (lanes 3, 4, 6, and 7). In addition, the inhibition of c-Fos protein accumulation by SNP was also observed with the total cellular extracts prepared from the cells treated with NMDA or KA (Fig. 2B). This attenuation of c-Fos proteins induced by SNP does not seem to be caused by a nonspecific inhibition of total protein synthesis, because the intensity of other nonspecific protein bands detected by cross- reacting with anti-c-Fos antiserum remained unchanged (Fig.  2, A and B). Furthermore, SNP at concentrations of 100 and 200 M did not alter the rate of protein synthesis (Fig. 2C, lanes  1-3), measured by the incorporation of [ 35  Effect of SNP on c-fos mRNA Expression-To further investigate whether or not the attenuation of c-Fos proteins corresponds to a decrease in the amount of c-fos mRNA in the cells, we examined the effect of SNP on the increase in c-fos mRNA expression induced by NMDA, KA, or TPA by means of Northern blotting. As shown in Fig. 3A, SNP inhibited the increase of c-fos mRNA expression induced by NMDA in a dose-dependent manner. In contrast, SNP did not change the level of c-fos mRNA expression stimulated by KA (Fig. 3B) or TPA (Fig. 3C). Even a higher concentration (1 mM) of SNP did not change the level of c-fos mRNA expression increased by KA (data not shown). SNP alone did not affect the c-fos mRNA expression, and ␤-actin mRNA expression in the cells was not affected by NMDA, KA, TPA, or SNP. Overall, SNP inhibited both the accumulation of c-Fos proteins and the c-fos mRNA expression induced by NMDA, whereas SNP inhibited the accumulation of c-Fos proteins but did not affect the c-fos mRNA expression induced by KA or TPA (Figs. 2 and 3).
Effect of SNP on 45 Ca 2ϩ Uptake-Since the activation of TRE binding activities and c-fos mRNA expression induced by NMDA or KA depends upon the extracellular Ca 2ϩ influx through NMDA receptors or VDCCs (24), we examined the effect of SNP on the Ca 2ϩ influx into the cells. As shown in Fig.  4A, stimulating cells with NMDA or KA induced the increase of 45 Ca 2ϩ uptake by the cells, and the addition of 100 M SNP decreased the 45 Ca 2ϩ uptake stimulated by NMDA to the level of the control, but did not affect that stimulated by KA. In addition, SNP inhibited the NMDA-but not the KA-evoked 45 Ca 2ϩ uptake in a dose-dependent manner (Fig. 4B). Potassium ferrocyanide (K 4 Fe(CN) 6 ), a chemical analogue of SNP (Na 2 Fe(CN) 5 NO), mimicked the effects of SNP on the 45 Ca 2ϩ uptake by the cells, in which K 4 Fe(CN) 6 inhibited the NMDAevoked 45 Ca 2ϩ uptake but not that by KA (Fig. 5A). K 4 Fe(CN) 6 Does Not Decrease the TRE Binding Activity Stimulated by KA-As shown in Fig. 5B (lanes 2-4), the increase in TRE binding activity induced by NMDA was also inhibited by K 4 Fe(CN) 6 as was observed with SNP ( Fig. 1A). In contrast, however, K 4 Fe(CN) 6 did not inhibit the increase in TRE binding activity induced by KA (Fig. 5B, lanes 5-7), whereas SNP did (Fig. 1A). Since the main difference in the chemical structures of SNP and K 4 Fe(CN) 6 is NO, we examined whether or not NO could be released from SNP in culture. We monitored the levels of intracellular cGMP after incubating cells with SNP or K 4 Fe(CN) 6 and found that cGMP accumulated with increasing concentrations of SNP in a time-and dose-dependent manner but not with those of K 4 Fe(CN) 6 (data not shown), suggesting that NO is released from SNP in culture and that it affects the cGMP production in the cells.
Cytotoxic Effect of SNP-Since NO can be largely involved in neuronal cell death (9, 13, 20 -22, 28), we examined whether or not SNP causes the death of cerebellar granule cells. As shown in Fig. 6A, exposing cells to SNP for 1 h elicited only a small increase in LDH release, whereas that for 4 h significantly increased it. SNP induced a high level of LDH release regardless of KA. The LDH release detected at 4 h increased in a dose-dependent manner, starting at 50 M and reaching a plateau at 500 M SNP (data not shown). K 4 Fe(CN) 6 did not cause LDH release (Fig. 6A). The addition of SOD, a superoxide anion scavenger, significantly reduced the LDH release in- duced by SNP, whereas catalase did not (Fig. 6B). The simultaneous addition of SOD and catalase reduced the level of LDH release but not completely (Fig. 6B). The addition of SOD and catalase alone had no effect on the LDH release (Fig. 6B). Furthermore, the addition of carboxy-PTIO, an NO scavenger (29), also reduced the LDH release induced by SNP in a dosedependent manner (Fig. 6C). The simultaneous addition of carboxy-PTIO with SOD and catalase reduced the LDH release almost to that of the control. Carboxy-PTIO alone had no effect on the LDH release from the cells.
Recovery of TRE Binding Activity by SOD and Catalase-We investigated the relationship between the SNP-mediated inhibition of the TRE binding activity stimulated by KA (Fig. 1A) and the SNP-mediated cytotoxicity (Fig. 6). Although a slight recovery of TRE binding activity from the SNP-mediated inhibition was detected after adding SOD (Fig. 7, compare lane 4  with lane 3), the simultaneous addition of SOD and catalase markedly recovered the TRE binding activity to the same level as that obtained by KA stimulation (Fig. 7, compare lane 6 with lane 2). SOD and catalase alone had no effect on the TRE binding activity (lanes 7 and 8). When KA stimulation was omitted, SNP did not significantly decrease TRE binding activity and subsequent addition of SOD and catalase did not affect the level of TRE binding activity (lanes 9 -11).

DISCUSSION
In this study, we found that SNP, a potent NO donor, effectively inhibited the increases in TRE binding activity induced by NMDA or KA in primary cultures of mouse cerebellar granule cells (Fig. 1A). Corresponding to this inhibitory effect of SNP, the amounts of c-Fos proteins in the nuclear extracts decreased when the cells were incubated with SNP ( Fig. 2A). These results indicated that the attenuation of AP-1 was induced in the cells incubated with SNP. However, the mode of this AP-1 attenuation by SNP differed in cells stimulated with NMDA or KA. Since the increase in TRE binding activity stimulated by NMDA and KA is dependent upon the Ca 2ϩ influx into neurons through NMDA receptors and VDCCs, respec- tively (24), the AP-1 attenuation in the cells stimulated by NMDA was assumed to be largely due to the blockade of Ca 2ϩ influx through the NMDA receptors by SNP (Fig. 4). In contrast, SNP did not affect the Ca 2ϩ influx evoked via KA stimulation (Fig. 4) although it induced AP-1 attenuation (Figs. 1A  and 2A). Since the increase in the c-fos mRNA expression induced by KA was not affected by SNP (Fig. 3B), the AP-1 attenuation is assumed to be post-transcriptionally induced. In support of this notion, the increase in TRE binding activity induced by TPA was also inhibited by SNP (Fig. 1A), while the c-fos mRNA expression stimulated by TPA was not affected (Fig. 3C).
Potassium ferrocyanide (K 4 Fe(CN) 6 ) mimicked the effects of SNP on the Ca 2ϩ influx when the cells were incubated with NMDA or KA, where it blocked the NMDA-but not the KAinduced Ca 2ϩ influx (Figs. 4 and 5A). The blockade of the NMDA-induced Ca 2ϩ influx by SNP and K 4 Fe(CN) 6 is assumed to be due to the ferrocyanide portion of these molecules, which has been suggested by Costa et al. (30). Since SNP did not block the Ca 2ϩ influx evoked via Bay K8644 (data not shown), a potent agonist for VDCCs, the ferrocyanide portion of these molecules appears to be unable to block the Ca 2ϩ influx through VDCCs, accounting for the failure of SNP and K 4 Fe(CN) 6 to block the KA-induced Ca 2ϩ influx that is caused through VDCCs (24). Potassium ferricyanide (K 3 Fe(CN) 6 ) did not block the increase in 45 Ca 2ϩ uptake stimulated by NMDA or KA (data not shown).
The possible involvement of NO in the AP-1 attenuation by SNP was supported by the failure of K 4 Fe(CN) 6 to inhibit the increase in TRE binding activity induced by KA (Fig. 5B). The release of NO from SNP and the action of NO on the cells were confirmed by the accumulation of intracellular cGMP in the cells incubated with SNP (data not shown), which can be mediated by guanylate cyclase stimulated by NO (6). However, the AP-1 attenuation observed with the KA stimulation is independent of cGMP signals because 8-Br-cGMP had no effect on the NMDA-and KA-induced increases in TRE binding activity (data not shown). Since ferrocyanide ions do not pass through the cytoplasmic membrane (30), it seems reasonable that NO released from SNP can act on the cells, leading to AP-1 attenuation, directly or indirectly.
SNP requires reduction before NO is released (31). Actually, only a small signal of NO release from SNP was detected in the culturing medium, which was indirectly examined by the formation of nitrite, while the addition of dithiothreitol, a reducing agent, with SNP dramatically enhanced its formation (data not shown). Therefore, it can be speculated that in culturing medium NO could be released from SNP through the interaction with some reducing equivalents of the cells and subsequently incorporated into the cells. Thus, the mode of action of SNP on the cells remains to be further elucidated.
As shown in Fig. 6, SNP potently induced cell death. The cytotoxic effect of SNP can be largely avoided by adding SOD, suggesting that superoxide anion is key to the cell death caused by SNP. Furthermore, the finding that carboxy-PTIO prevented the LDH release (Fig. 6C) revealed the involvement of NO in the SNP cytotoxic effect. The involvement of NO in the SNP-cytotoxic effect was further supported by the failure of K 4 Fe(CN) 6 to induce cell death (Fig. 6A). The simultaneous addition of SOD, catalase, and carboxy-PTIO almost totally prevented SNP-mediated cell death (Fig. 6C). These results indicated that peroxynitrite, a potent oxidant, which can be produced by NO and superoxide anion, is a direct cause of SNP-mediated neuronal cell death rather than NO or superoxide anion alone. However, we do not know exactly how superoxide anion is supplied from the cells.
The inhibitory effect of SNP on the increase in TRE binding activity induced by KA was effectively overcome by SOD and catalase (Fig. 7), suggesting that the AP-1 attenuation is associated with neuronal cell death induced by SNP. If so, AP-1 attenuation should be immediately caused before cell death because SNP inhibited the KA-stimulated TRE binding activity within 1 h (Fig. 1A), whereas significant cytotoxic effects were detected 4 h after incubating cells with SNP (Fig. 6A). Thus, the AP-1 attenuation might be an acute response of cells that is required to promote NO-mediated cell death.
Reduction-oxidation (redox) regulation is one factor that controls AP-1 DNA binding activity (15,16). The conserved cysteine residues in the DNA-binding domain of AP-1 can be modulated by the redox regulation, leading to the control of AP-1 DNA binding activities (15,16). Cysteine residues interact with NO to produce S-nitrosyl compounds through redoxregulatory mechanisms (32,33). On the other hand, proteolytic events involving the cysteine proteases such as interleukin 1␤-converting enzyme (34) are necessary for apoptotic cell death. Several kinds of cellular proteins including poly(ADPribose) polymerase (35,36) are proteolytically degraded in cells undergoing apoptosis. These results support the notion that AP-1 can be attenuated by its proteolytic decomposition, which might be mediated by S-nitrosylation of AP-1.
Although SNP inhibits the total protein synthesis in hepatocytes at concentrations over 500 M (37), we did not detect a decrease in the rate of protein synthesis when SNP was added at a concentration of 100 or 200 M (Fig. 2C). Nevertheless, it seems likely that a selective blockade of c-Fos protein synthesis by SNP is involved in the AP-1 attenuation. Since c-Fos proteins can be rapidly degraded by the ubiquitin-related proteolytic pathway after their syntheses (38), c-Fos proteins should disappear from the cells immediately after inhibiting protein synthesis. Other scenarios in which AP-1 attenuation involving its translocation to the nucleus being blocked by SNP can be excluded, because the amounts of c-Fos proteins decreased in both the nuclear and total cellular extracts (Fig. 2, A and B).
Various insults to the brain such as hypoxic ischemia (HI) and status epilepticus (SE) can cause two types of neuronal cell death; moderate HI and SE lead to apoptosis, whereas more severe HI leads to necrosis (39). While moderate HI and SE induce a prolonged c-fos mRNA expression in brain neurons (39), severe HI rapidly induces c-fos mRNA expression but does not cause the accumulation of c-Fos proteins in the brain (40), suggesting that the attenuation of c-Fos proteins could be posttranscriptionally induced in brain neurons by severe HI. Even in primary cultures of neurons, production of peroxynitrite can elicit not only apoptotic neuronal cell death with moderate exposure, but also necrosis with severe exposure (41). In addition, apoptotic and necrotic cell death can also be induced in cerebellar granule cells incubated with glutamate (42). Thus, it seems likely that the rapid attenuation of AP-1 is one indicator of acute neuronal cell death mediated by NO, which could be caused by ischemic brain injury.