cytokine-induced expression of type ii nitric oxide synthase in astrocytes is downregulated by atp...

GLIA 15:77-82 (1995)

Cytokine-Induced Expression of Type I1 Nitric Oxide Synthase in Astrocytes Is Downregulated by ATP and Glutamate

SEAN MURPHY, HSIN LEE LIN, AND SONG KYU PARK Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52242

KEY WORDS Cytokines, ATP, Glutamate, NFKB, Gene activation

ABSTRACT Combinations of cytokines andor phorbol ester induce expression of Type I1 nitric oxide synthase (NOS) mRNA in astrocyte cultures via protein kinase mediated pathways (Simmons and Murphy: GLIA 11:227,1994; Feinstein et al.: J Neu- rochem 62:811, 1994). Agonists that activate receptors linked to protein kinases did not reproduce this effect of cytokines in astrocytes. On the contrary, ATP and glutamate treatment of astrocytes prior to a combination of interleukin-lp and interferon-y markedly reduced (30-50%) subsequent NOS mRNA expression. The effect was not seen if treatment coincided with or followed cytokine activation, suggesting that ATP and glutamate were not destabilizing NOS mRNA. The effects of ATP and glutamate were additive and could be mimicked by selective receptor agonists, but were insensitive to a specific inhibitor of protein kinase C. The inhibition of cytokine-induced NOS mRNA expression caused by these agents was not the result of interference with the activatiodtranslocation of nuclear factor-& by interleukin-lp. These results suggest that exposure of astrocytes to ATP and glutamate, both of which increase markedly in a variety of neuropathologies, could modulate the subsequent responsiveness of these cells to NOS-inducing stimuli. As such, this may be an important regulatory mechanism in the expression of Type I1 NOS in vivo. 0 1995 Wiley-Liss, Inc.

INTRODUCTION

Transcriptional induction of a Type I1 nitric oxide synthase (NOS) is initiated in rat astrocytes in vitro by bacterial endotoxin or combinations of cytokines (for a review, see Murphy et al., 1993) that activate signalling pathways involving protein kinases (Feinstein et al., 1994; Simmons and Murphy, 1994). The cDNA for this rat astrocyte NOS has been cloned and sequenced (Galea et al., 1994) and an inducible NOS has recently been characterized in a human glioblastoma cell line (Hokari et al., 1994). There is evidence for the expression of Type I1 NOS in astrocytes in vivo, associated with par- ticular neuropathologies (Bo et al., 1994; Endoh et al., 1994; Wallace and Bisland, 1994). Therefore, under- standing how NOS expression is regulated at the transcriptional, translational and post-translational levels is important.

Studies with astrocytes in vitro have revealed that NOS expression can be inhibited at the transcriptional 0 1995 Wiley-Liss, Inc.

level by corticosteroids, anti-inflammatory cytokines, and NO itself (Park et al., 1994a; Simmons and Mur- phy, 1993). At least in vitro, the NOS transcript is quite unstable (Galea et al., 1994; Park and Murphy, 1994) but can be preserved by inhibitors of protein synthesis, suggesting the existence of destabilising factors which can regulate NOS expression. The Type I1 NOS protein displays target residues for phosphorylation by protein kinases (PKs), but subsequent effects on enzyme activity have yet to be investigated in astrocytes.

Receptors on astrocytes for a variety of molecules signal through calcium andor PKs, including those for excitatory amino acids (Pearce et al., 1986), ATP (Pearce et al., 1989), amines (Pearce et al., 1985), and peptides

Received December 20,1994; accepted April 27,1995. Address reprint requests to Sean Murphy, Department of Pharmacology,

Bowen Science Building, University of Iowa, Iowa City, IA 52242.

MURPHY ET AL. 78

(Wilkin and Cholewinski, 1988). It is feasible, therefore, that such signal molecules could regulate NOS expression in any one of a variety of ways, from transcription through post-translational modification. In- deed, Feinstein et al. (1993) have shown that norepinephrine, via the activation of p-adrenergic receptors and increases in cyclic AMP, will blunt the subsequent expression of Type I1 NOS in astrocytes. In macrophages, an elevation of intracellular calcium is reported to initiate Type I1 NOS expression (Raddassi et al., 1994) and Tonetti et al. (1994) have shown that activation of the purinergic PPy receptor augments NOS expression by endotoxin.

Recently, we reported that exposure of astrocytes to ATP and glutamate diminishes the subsequent expression of Type I1 NOS induced by cytokines (Park et al., 1994b). In the present study we have investigated the mechanism underlying their effects.

MATERIALS AND METHODS Materials

Fetal calf serum (FCS), basal medium Eagle (BME), and recombinant rat IFN-.y were obtained from Gibco BRL (Grand Island, NY). Recombinant human IL-10 was purchased from R and D Systems (Minneapolis, MN), the antibody to p65 was from Rockland (Gilberts- ville, PA), bisindolylmaleimide (BIM) was from Calbio- chem (San Diego, CA), and 2-methylthio-ATP (2-MeS) was from Research Biochemicals (Natick, MA). Trisolv was purchased from Biotex (Houston, TX), Formazol from TelTest B (Friendswood, TX), human G3PDH probe from Clontech (Palo Alto, CA), S & S Nytran from Schleicher & Schuell (Keene, NH 03431), and [32Pl- dCTP (3,000 CUmmole) from NEN-DuPont (Boston, MA). Other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell Culture

Primary astrocyte cultures were prepared from the cerebra of neonatal (<48 h) rats as previously described (Murphy, 1990). Cultures were grown in T-150 culture flasks (original seeding density 1.4 x lo6 cells) a t 37°C in humidified 95% air/5% C02 in Eagle's minimal es- sential medium (EMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 33 mM glucose, 2 mM glutamine, and 50 pglml gentamycin until confluent at 17 DIV. At this point they were 90-95% glial fibrillary acidic protein positive and contained approximately 5% microglial cells. Cultures were then passaged by trypsiniza- tion (0.025% trypsin, 0.02% EDTA) and extensive (80 times) trituration through a Pasteur pipette, and re- plated (1:l) in T-75 flasks. This rigorous passaging and high seeding density limited microglial survival and the resulting cultures routinely contained 0.5% or fewer microglia if used within 24 h (70% confluence). These secondary astrocyte cultures were induced with a com-

bination of IFN--y (50 U/ml) and IL-1p (500 pg/ml) for 5 h. Other agents were added at times indicated in the text or in legends to the figures.

Northern Blotting

Total cellular RNA was collected by the Trisolv method (adapted from Chomczynski and Sacchi, 1987), dissolved in Formazol, resolved by electrophoresis in a 0.9% agarose/formaldehyde gel (1 0 pgllane), and transferred to a nylon membrane overnight, which was then U V irradiated to covalently bind the RNA. The membrane was prehybridized for 2 h at 42°C with a solution containing 10 x Denhardt's solution, 100 pg/ml salmon sperm DNA, 50% formamide, 5 X SSPE (1 x SSPE is 0.15 M NaC1, 10 mM NaH2P04, and 1 mM EDTA), and 1% SDS. The probe for inducible NOS mRNA was a mouse macrophage cDNA clone (Xie et al., 1992). SspI and HincII digestion of the pUC19 plasmid containing a fragment of the cloned NOS cDNA yielded a 4 kb insert which was radiolabeled with [32P]-dCTP (2 x lo6 cpml ml) by a random priming kit (Stratagene). Blots were hybridized with 32P-labeled probe overnight a t 42°C in the same solution used for prehybridization. Blots were washed for 15 min with 2 x SSC (1 x SSC is 0.15 M NaCl and 15 mM sodium citrate) a t 65"C, for 30 min with 2 x SSC/O.l% SDS at 65"C, and finally for 10 min with 0.1 x SSC/O.l% SDS at 50°C. Autoradiography was performed by overnight exposure to Kodak X-Omat film at -70°C in the presence of intensifying screens. To control for loading of RNA, the membrane was rehy- bridized with a probe for human G3PDH mRNA. Rela- tive mRNA levels were quantitated by scanning densit- ometry. Comparison between NOS:G3PDH ratios for cells induced with and without prior treatment (% change) were made using a paired t test.

Activation of NF-KB

This was determined by monitoring on immunoblots translocation of the p65 subunit into nuclei harvested one hour after exposure of cells to cytokines. Cells (1 x T- 150) were washed twice in ice-cold phosphate-buffered saline (PBS), then collected in 5 ml PBS and pelleted at 400g for 4 min at 4°C. The pellet was resuspended in lysis buffer (10 mM Tris-HC1 pH 8, 60 mM KC1, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsul- fonyl fluoride, 3 pM pepstatin, 3 pM leupeptin, and 0.2% NP-40). After a 10 min incubation on ice the ly- sates were spun at 500g for 4 min a t 4°C. The superna- tant was then removed and recentrifuged at 13,OOOg for 5 min at 4°C (cytoplasmic extracts). The pelleted nuclei were briefly washed in lysis buffer without NP-40, resuspended in an equal volume of nuclear extract buffer (20 mM Tris-HC1, pH 8, 420 mM NaC1, 1.5 mM MgCl,, 0.2 mM EDTA, and 25% glycerol) and enough 5 M NaCl added to achieve a final salt concentration of 400 mM.

REGULATION OF ASTROCYTE NOS EXPRESSION

2 4- C

79

B

After 10 min incubation at 4°C the nuclei were vortexed briefly and centrifuged at 13,OOOg for 5 min. The super- natant was used as the nuclear extract.

Protein contents of cytosolic and nuclear fractions were estimated, the fractions combined with an equal volume of sample buffer and denatured by boiling for 10 min. Equal amounts of protein (30 pg) were separated in a 8% polyacrylamide/SDS gel and transferred to nitrocellulose membrane. The nitrocellulose was blocked in Tris buffered saline (TBS)/O.l% Tweed1 % non-fat dry milk and incubated overnight in primary antibody (p65, 1:1,000). The nitrocellulose was washed in TBS/ 0.1% Tween and incubated with an alkaline phosphatase conjugated secondary antibody for 2 h, washed again, and developed in an alkaline buffer with nitroblue tet- razolium as substrate.

RESULTS ATP and Glutamate Modulate Cytokine-Induced

NOS mRNA Expression

Neither ATP nor glutamate (100 p,M) evoked the transcriptional induction of NOS, either in IFN-y primed or unprimed astrocytes (see Fig. 1A). On the contrary, application of ATP or glutamate one hour prior to cytokines resulted consistently in a marked decrease in Type I1 NOS mRNA measured 5 h later. Similar experiments with different cell preparations resulted in decreases in NOS mRNA of, on average, 50% for ATP and 30% for glutamate, as compared with non-pretreated, induced controls (Fig. 1B).

Pretreatment for 1 h with a combination of ATP and glutamate (both at 100 pM) resulted in NOS mRNA expression of 35 -+ 5.2% (n = 5; P < 0.051, a value lower than either agonist alone. Presentation of a combination of ATP and glutamate at various times before or after cytokines resulted in differential effects on NOS mRNA expression. As seen in Figure 2 (the results of a single experiment), while ATP and glutamate inhibited the subsequent expression of NOS mRNA when given 1 or 2 h before cytokines, the treatment of cells 4 h before, coincident with, or following, cytokines was ineffective. In fact, the expression of NOS mRNA in cells treated with ATP and glutamate 2 h following cytokines appeared to be somewhat enhanced.

Evidence for Receptor-Mediated Effects of ATP and Glutamate

To reveal if these effects were receptor-mediated, potent agonists at the astrocyte metabotropic glutamate (quisqualate) and P, purinergic receptors (2-MeS) were employed (Bruner and Murphy, 1990; Pearce et al., 1989). As seen in Figure 3A, both analogs caused a decrease in subsequent cytokine-induced NOS mRNA expression measured 5 h later. Similar experiments with different cell preparations resulted in decreases of 30-35% for quisqualate and 2-MeS, as compared with non-pretreated, induced controls (Fig. 3B).

1 2 3 4 5 6 7 8

0 0

*

$? =- 50 0 n m 0 v) 0 ..

= o ATP GLUT

Fig. 1. Glutamate and ATP pretreatment of astrocytes blunts cytokine-induced NOS expression. A A representative northern blot showing in lane 1, macrophage NOS mRNA; 2, uninduced astrocytes; 3, ILlplIFNy-induced astrocytes; 4, induced astrocytes pretreated for 1 h with 100 pM glutamate; 5, induced astrocytes pretreated for 1 h with 100 ELM ATP; 6, IFNy-primed astrocytes; 7, IFNy-primed astrocytes treated with glutamate; 8, IFNy-primed astrocytes treated with ATP. B. Data combined from 4 to 5 experiments showing the effects of ATP and glutamate expressed as % NOS:G3PDH in cytokine-induced cells (*P < 0.05).

To reveal whether these effects ofATP and glutamate were mediated by PKC the cells were first treated with the specific inhibitor bisindolylmaleimide (BIM) at a concentration (0.2 p,M) ten times that of the reported Ki (Toullec et al., 1991). We have shown previously that BIM does not inhibit cytokine-induced NOS expression in astrocytes (Simmons and Murphy, 1994). However, the inhibitory effects of ATP and glutamate on cytokine-induced NOS expression were retained even in the presence of BIM (data not shown).

Integrity of NF-KB Activation by ILlp

To determine whether ATP or glutamate pretreatment interfered with the ability of IL-1p to activate and subsequently translocate NF-KB (Moynagh et al., 19931, we looked for the p65 subunit in fractions of nuclei from cytokine-activated astrocytes (Fig. 4). As expected, p65 was detected in the cytosol (lanes 9 and 14) and in nuclei derived from cells exposed to cytokines (lanes 2-4 and 10, 11) but not in nuclei from uninduced cells (lane 1). Treatment with glutamate and ATP did not promote activation and translocation of p65 (lanes 5

80 MURPHY ET AL.

I 0 m

cn 0 z

n

(3

A

NOS

GSPDH

c - 1 2 3 4 5 6 7 8 9

Fig. 2. Timing of ATP/glutamate treatment produces differential effects. A A northern blot showing in lane 1, macrophage NOS mRNA, 2, uninduced astrocytes; 3, ILlp/IFNy-induced astrocytes; 4, induced astrocytes pretreated for 4 h with 100 yM glutamate and 100 yM ATP; 5, pretreated for 2 h; 6, pretreated for 1 h; 7, simultaneous addition; 8, treated 1 h after cytokines; 9, treated 2 h after cytokines. B: The ratios for NOS:G3PDH derived from this blot. The dashed lines indicate values for induced (upper) and uninduced cells (lower).

and 6) . In addition, neither agent appeared to interfere with the ability of cytohnes subsequently to cause translocation of p65 (lanes 7,8,12,13 vs. 24,10,11) .

DISCUSSION

The results from this study show that astrocytes pretreated with ATP or glutamate, seemingly via the activation of specific receptors, exhibit markedly dimin- ished induction of Type I1 NOS mRNA in response to cytokines. Timing of the presentation of ATP and glutamate suggests that these effects are on transcriptional activation of the NOS gene rather than on mRNA sta- bility, though nuclear run-on experiments will be re- quired to confirm this.

The signalling systems invoked by IFN-y and IL-1p lead to binding of transcription factors to the promoter region of the NOS gene (Kamijo et al., 1994; Xie and Nathan, 1994). The effect of ATP or glutamate receptor activation on subsequent NOS induction could be at the level of IFN-y and/or IL-1 P receptor expression, their

NOS

1 2 3 4 5 6 7 8 91011

50

0 2MeS QUlS

Fig. 3. Analogs mimic the effects of ATP and glutamate. A A representative northern blot showing in lane 1, macrophage NOS mRNA; 2, uninduced astrocytes; 3, ILlp/IFNy-induced astrocytes; 4, uninduced astrocytes pretreated with 100 yM quisqualate; 5 , uninduced astrocytes pretreated with 10 yM 2-MeS; 6, induced astrocytes pretreated with quisqualate; 7, induced astrocytes pretreated with 2-MeS; 8-11, as lanes 4-7. B: Data combined from three experiments showing the combined effects of ATP and glutamate expressed as % NOS:G3PDH in cytokine-induced cells (*P < 0.05).

coupling to transcription factor activation, or the binding of these transcription factors to consensus sequences of the promoter region of the NOS gene. Our observa- tion that ATP/glutamate pretreatment did not affect NF-KB activation and translocation suggests that the IL-1p signalling mechanism is intact. While in these experiments we have not addressed effects on the IFN-y pathway, the fact that addition of agonists 4 h before or together with cytokines had no effect might suggest that neither do they impair IFN-y signalling. It remains to be determined whether activation of glutamate/ATP receptors on astrocytes somehow promotes an alteration in the nuclear environment in which transcription factors bind to the NOS promoter (Stamler, 1994; Xie and Nathan, 1994).

There is other evidence to suggest that cells can be made refractory to cytokine-evoked transcriptional induction of NOS. Activation of PKA blunted endotoxin- induced NOS in both astrocytes (Feinstein et al., 1993) and macrophages (Bulut et al., 1993). Pretreatment of macrophages with low doses of endotoxin and tumor necrosis factor (TNF)a, or with TGFP, suppressed sub-

REGULATION OF ASTROCYTE NOS EXPRESSION 81

- P65

1 2 3 4 5 6 7 8 9

. r c l l e

10 11 Fig. 4. Glutamate and ATP do not interfere with cytokine activation

and translocation of NFKB p65. Two immunoblots showing nuclei (lanes 1-8 and 10-13) from astrocytes treated with in lane 1, nothing; 2 4 , cytokines; 5, glutamate (100 pM); 6, ATP (100 pM); 7, glutamate prior to cytokines; 8, ATP prior to cytokines; 10, cytokines; 11, cytokines; 12,

sequent NOS induction and caused destabilization of NOS mRNA (Bogdan et al., 1993). It is conceivable that ATP and glutamate in our experiments caused the release of cytokines. such as TGFp and TNFa from astrocytes, which then desensitized the cells to inducers of NOS (Xie and Nathan, 1994).

We have observed a similar desensitization in astrocytes and in cerebral endothelial cells following exposure to NO donors, in that they are less responsive subsequently to inducers of NOS (Borgerding and Mur- phy, 1995; Park et al., 1994a). Astrocytes activated with agents such as glutamate and quisqualate have been shown to produce small amounts of NO transiently from a constitutive NOS (Agullo and Garcia, 1992; Murphy et al., 19911, but it is very unlikely that this NO could be responsible for the effects of quisqualate and glutamate seen here. However, mechanisms similar to those associated with ATP and glutamate treatment might also be invoked by NO.

Do the effects of ATP and glutamate that we have observed here in vitro have any relevance in vivo? There is abundant evidence that cells expressing Type I1 NOS (either resident or infiltrating) are to be found in neuropathologies with and without associated inflammation (for a review, see Murphy et al., 1995). Extracellular levels of glutamate and ATP rise significantly under these circumstances and are suggested to contribute to the pathology via excitotoxicity (Dawson et al., 1993) and activation of resident microglia (Kettenmann et al., 1993). We have suggested recently that the feedback inhibition of NOS induction by NO in astrocytes may generate foci of NO-producing cells in and around an area of damage in the CNS, through NO limiting the induction of NOS in surrounding cells (Park et al., 1994a). The desensitizing actions of ATP and glutamate on the induction of NOS in astrocytes may produce the same effect, restricting the recruitment of NO-producing cells. Alternatively, the effects of ATP and glutamate that we have observed might simply serve to delay the production of large and continuous amounts of potentially toxic NO following damage to the nervous system (Merrill and Murphy, 1995).

12 13 14 glutamate prior to cytokines; 13, ATP prior to cytokines. Lanes 9 and 14, astrocyte cytosol as a positive control to show the position of p65. Lanes 1-9 and 10-14 are results from two separate experiments. Non- specific binding to a molecular weight species of <65 kD indicates uniform loading of the gels.

ACKNOWLEDGMENTS

We thank Sherry Kardos for generating the primary astrocyte cultures and Carl Nathan and Qiao-wen Xie for providing the Type I1 NOS cDNA. This work was supported by NIH grant NS29226.

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cytokine-induced expression of type ii nitric oxide synthase in astrocytes is downregulated by atp...

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