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J Biol Chem, Vol. 273, Issue 45, 29754-29763, November 6, 1998

Pyrimidinoceptor-mediated Potentiation of Inducible Nitric-oxide Synthase Induction in J774 Macrophages
ROLE OF INTRACELLULAR CALCIUM^*

Bing-Chang Chen, Chun-Fen Chou^§, and Wan-Wan Lin^¶

From the  Department of Pharmacology, College of Medicine, National Taiwan University and the ^§ Research Center of Immunology, National Yang-Ming University, Taipei, Taiwan

	ABSTRACT

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We have shown that, in murine J774 macrophages, binding of UTP to pyrimidinoceptors stimulates phosphoinositide (PI) breakdown and an increase in [Ca²⁺]_i. In this study, UTP modulation of the expression of inducible nitric-oxide synthase (iNOS) was investigated. Although UTP alone had no effect, stimulation of J774 cells with a combination of UTP (10-300 µM) and LPS (0.1-3 µg/ml) resulted in a potentiated increase in nitrite levels. In parallel, the amount of iNOS protein induced by LPS was also potentiated by UTP treatment. The UTP potentiating effect was attenuated by U73122, suggesting involvement of the downstream signaling pathways of phosphatidylinositide turnover. The tyrosine kinase inhibitor genistein inhibited both the LPS-induced nitrite response and the UTP potentiation. Conversely, two protein kinase C inhibitors, Ro 31-8220 and Go 6976, and a phosphatidylcholine-specific phospholipase C inhibitor, D609, inhibited LPS-stimulated nitrite induction, but did not affect the potentiating effect of UTP, which was also unaffected by pretreatment with phorbol 12-myristate 13-acetate for 8 h. Furthermore, the UTP-induced potentiation was abolished by BAPTA/AM or KN-93 (a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase (CaMK)). Nitrite potentiation and iNOS induction were prominent when UTP was added simultaneously with LPS, with the potentiating effect being lost when UTP was added 3 h after treatment with LPS. Pyrrolidinedithiocarbamate (3-30 µM), an inhibitor of NF-B, caused a concentration-dependent reduction in the nitrite response to LPS and UTP. In electrophoretic mobility shift assays, LPS produced marked activation of NF-B and AP-1, which was potentiated by UTP. LPS-induced degradation of IB- as well as the phosphorylation of IB- were also increased by UTP. Moreover, the UTP-potentiated activation of NF-B and AP-1 and the degradation and phosphorylation of IB- were inhibited by KN-93. Taken together, these data demonstrate that nucleotides, especially UTP, can potentiate the LPS-induced activation of NF-B and AP-1 and of iNOS induction via a CaMK -dependent pathway and suggest that the UTP-dependent up-regulation of iNOS may constitute a novel element in the inflammatory process.

	INTRODUCTION

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NO is a free radical gas mediating intracellular communication in many mammalian organs (1). Three isoforms of nitric-oxide synthase have been identified and cloned (2, 3). The brain (type I) and endothelial (type III) enzymes are constitutively expressed, and their enzymatic activity is regulated by changes in concentrations of free Ca²⁺. The third member of the family is the inducible (type II) nitric-oxide synthase (iNOS),¹ which is expressed in many different cell types and produces high levels of NO. Formation of large amounts of NO is involved in the bactericidal and tumoricidal actions of macrophages.

In macrophages, lipopolysaccharide (LPS), alone or in combination with cytokines such as interferon- (IFN-), interleukin-1, or tumor necrosis factor  (TNF-), has been shown to increase iNOS activity by increasing the transcription of the iNOS gene (4). The extent of iNOS induction depends on the cell type and on the animal species, with the enzyme being more easily induced in rodent cells than in their human counterparts, in which a cooperative interaction between several signals is required for iNOS expression (5). iNOS protein can catalyze the formation of NO from the terminal guanidino nitrogen atom of L-Arg (3). NO generation plays an important role in the host defense response (6).

The regulation of iNOS gene expression is governed by the activity of several transcription factors. Sequence analysis of the murine iNOS promoter has revealed the presence of a number of consensus motifs for transcription factors, including NF-B (nuclear factor-B), AP-1 (activator protein-1), IFN- response element, -activated site, IFN-stimulated response element, TNF--responsive element, Oct, and NF-IL6 (nuclear factor-interleukin-6) (7-11); of these, NF-B is of paramount importance (9, 10). NF-B, named for its ability to recognize a  light chain immunoglobulin gene regulatory element, can participate in the regulation of numerous genes (12). It is a heterodimer composed of two subunits (50 and 65 kDa) belonging to the Rel family of transcriptional activators. In dormant cells, NF-B is normally complexed with a member of the IB proteins, which localize the transcription factor to the cytosol in an inactive state. Stimulation of the cells with ligands, such as LPS, TNF-, or interleukin-1, results in the rapid dissociation of IB and the subsequent entry of active NF-B into the nucleus, where it can interact with DNA (13, 14). Although the signaling mechanisms leading to NF-B activation are not completely understood, it is widely believed that phosphorylation of IB- at Ser-32 or Ser-36, resulting in the degradation of IB-, is pivotal in the activation of NF-B (15, 16).

Extracellular ATP and other purine/pyrimidine nucleotides mediate intracellular signaling via P₂ purinoceptors and induce many biological effects in various cell types. Since 1993, at least eight G protein-coupled P2Y purinoceptor subtypes have been cloned; their signaling cascades have been recently reviewed (17). Recently, two pyrimidinoceptors, designated the P2Y₄ and P2Y₆ subtypes, have been cloned and sequenced (18-20). Pyrimidinoceptors are known to activate phosphoinositide (PI)-specific phospholipase C and phospholipase A₂ and to increase intracellular Ca²⁺. In mouse J774 and RAW 264.7 macrophages, except the described P2X₇ on the macrophage surface, we have previously demonstrated that UTP, acting via pyrimidinoceptors (another P2Y receptor subtype), can stimulate PI breakdown, increase intracellular Ca²⁺ levels, activate phospholipase A₂, and modulate prostaglandin E₁-induced cAMP formation (21-23). Although it has been shown in RAW 264.7 macrophages that extracellular ATP, UTP, and other nucleotides via an unknown P2Y purinoceptor subtype can potentiate LPS-induced NO production (24), the receptor subtype involved and the underlying mechanism are still unclear.

To gain insight into the effect of nucleotides, particularly UTP, the selective agonist on pyrimidinoceptors, on the LPS-mediated induction of iNOS and its possible role in inflammatory diseases, we further examined the intracellular events involved in iNOS induction in macrophages. We report here that UTP via a calcium-dependent pathway potentiates the LPS-mediated activation of transcriptional factors and expression of iNOS in mouse J774 macrophages. This is the first report elucidating the stimulatory mechanism of nucleotides in NO release.

	MATERIALS AND METHODS

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Reagents-- Oligonucleotides were synthesized on a PS 250 CRUACHEM DNA synthesizer, using the cyanoethyl phosphoroamidate method, and purified by gel filtration. The sequences of the double-stranded oligonucleotides used to detect the DNA-binding activities of NF-B and AP-1 are as follows (the binding site is underlined): NF-B, 5'-GATCAGTTGAGGGGACTTTCCCAGGC-3'; and AP-1, 5'-GATCCGCTTGATGACTCAGCCGGAA-3'. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, and streptomycin were obtained from Life Technologies, Inc. [-³²P]ATP (3000 Ci/mmol), [³²P]orthophosphate, horseradish peroxidase-coupled anti-mouse and anti-rabbit antibodies, and the enhanced chemiluminescence detection agent (ECL) were purchased from Amersham Pharmacia Biotech. myo-[³H]Inositol (2 Ci/mmol) was obtained from NEN Life Science Products. Ro 31-8220, KN-93, H-89, and Go 6976 were purchased from Calbiochem. Genistein, 2-methylthio-ATP, and AMP-CPP were from RBI (Natick, MA). FK506 was a kind gift from Fujisawa Pharmaceuticals (Osaka, Japan). U73122, U73343, and D609 were from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). IFN- was purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal antibodies specific for PKC, PKC, PKC, PKCµ, and PKC and rabbit polyclonal anti-iNOS antibody were purchased from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies specific for p65 NF-B, IB-, PKCI, and PKCII were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific IB- antibody was from New England Biolabs Inc. (Beverly, MA). All materials for SDS-PAGE were obtained from Bio-Rad. All other chemicals were obtained from Sigma.

Cell Culture-- The mouse macrophage cell line J774 (obtained from American Type Culture Collection) was cultured, as described previously (23), in DMEM containing 10% fetal bovine serum and antibiotics (100 units/ml penicillin, and 100 µg/ml streptomycin). Cells were seeded onto 24-well plates for the nitrite assay, onto 10-cm dishes for immunoblotting, or onto 60-mm dishes for NF-B and AP-1 activation tests.

Nitrite Production-- Nitrite production, an indicator of NO synthesis, was measured in the supernatants of J774 macrophages. Briefly, the cells were cultured in 24-well plates in 500 µl of culture medium until confluence. To induce iNOS, fresh culture medium containing LPS was added at the concentrations indicated, and nitrite accumulation in the medium was measured 24 h later. Unless otherwise indicated, to assess the effects of various drugs (such as U73122, genistein, Ro 31-8220, and BAPTA/AM), they were added to the cells at the same time as LPS and left in the medium during the 24-h treatment with LPS. Nitrite was measured by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100-µl samples of culture medium. The absorbance at 550 nm (A_550 nm) was measured using a microplate reader, and the nitrite concentration was calculated by comparison with the A_550 nm produced using standard solutions of sodium nitrite in culture medium.

Immunoblot Analysis of iNOS, IB-, and PKC Isoforms-- To quantify iNOS and IB- protein, following 24 h (for iNOS) or 30 min (for IB-) of incubation in the presence of various stimuli, cells were washed twice in ice-cold phosphate-buffered saline and then solubilized in buffer containing 20 mM Tris-HCl, 0.5 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin (pH 7.5). To detect increased phosphorylation of IB-, quiescent cells treated with LPS plus UTP or thapsigargin for 7.5 min were harvested. To assess PKC down-regulation, cells incubated for 8 h with 1 µM PMA were treated similarly. Samples of equal amounts of protein (50-100 µg) were subjected to SDS-PAGE on 7.5% (iNOS), 9% (PKC isoforms), or 12% (IB-) polyacrylamide gels and transferred onto a nitrocellulose membrane, which was then incubated in 150 mM NaCl, 20 mM Tris, and 0.02% Tween (pH 7.4) containing 1% milk; and the iNOS, IB-, or PKC band was visualized by immunoblotting with specific antibodies. Immunoreactivity was detected by ECL following the manufacturer's instructions.

Phosphorylation of IB- and Immunoprecipitations-- J774 cells were starved in medium without serum for 24 h. The medium was removed, replaced with phosphate-free DMEM containing 0.1 mCi/ml [³²P]orthophosphate, and incubated for overnight. Cells were then pretreated with 10 µM KN-93 prior to stimulation with LPS plus UTP or thapsigargin for 7.5 min; lysed in 1 ml of buffer containing 20 mM Tris (pH 7.5), 1 mM MgCl₂, 125 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mM -glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate; and centrifuged. The supernatant was immunoprecipitated with 10 µl of IB--specific polyclonal antibody overnight. The immunoprecipitates were then analyzed by SDS-PAGE followed by autoradiography.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSAs)-- Nuclear extracts from stimulated or non-stimulated macrophages were prepared by cell lysis followed by nuclear lysis; cells were suspended in 30 µl of buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride; vigorously vortexed for 15 s; allowed to stand at 4 °C for 10 min; and centrifuged at 2000 rpm for 2 min. The pelleted nuclei were resuspended in buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride for 20 min on ice, and then the lysates were centrifuged at 15,000 rpm for 2 min. The supernatants containing the solubilized nuclear proteins were stored at 70 °C until used for EMSAs. Binding tests for NF-B and AP-1 were performed as described (25). Briefly, binding reaction mixtures (15 µl) contained 0.25 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 dpm of ³²P-labeled DNA probe in binding buffer consisting of 10 mM Tris (pH 7.5), 1 mM EDTA, 4% Ficoll, 1 mM dithiothreitol, and 75 mM KCl; the binding reaction was started by the addition of cell extracts and continued for 30 min. Samples were analyzed on native 5% polyacrylamide gels. For competition experiments, 50 ng of the labeled oligonucleotide was mixed with 1 µg of unlabeled competitor oligonucleotides prior to protein addition. For supershift experiments, 4 µg of anti-p65 antibody was mixed with the nuclear extract proteins.

Measurement of PI Turnover-- PI hydrolysis was measured by the accumulation of inositol phosphates in the presence of 10 mM LiCl as described previously (21). Confluent cells on 35-mm Petri dishes were labeled with myo-[³H]inositol (2.5 µCi/dish) in growth medium for 24 h, washed with phosphate-buffered saline containing 10 mM LiCl, and incubated at 37 °C for 20 min. After this preincubation, the indicated drugs were added; incubation was continued for another 30 min; and the reaction was terminated by aspiration of the reaction solution and addition of ice-cold methanol. The cells were scraped off, and [³H]inositol phosphate was isolated using an AG 1-X8 column (formate form, 100-200 mesh, Bio-Rad) and elution with 0.2 N ammonium formate and 0.1 N formic acid.

Statistical Evaluation-- Values are expressed as the mean ± S.E. of at least three experiments. Student's t test was used to assess the statistical significance of the differences, with a p value of <0.05 being considered statistically significant.

	RESULTS

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Effect of Nucleotides on LPS-induced Nitrite Production-- Incubation of J774 macrophages for 24 h with LPS (0.01-3 µg/ml) produced a concentration-dependent increase in nitrite levels from the basal value of 8 ± 2 µM (n = 10) to 50 ± 1 µM (n = 5) after treatment with 3 µg/ml LPS (Fig. 1A). UTP (100 µM) alone did not stimulate nitrite formation, but, when added at the same time as LPS and left in the cell supernatants during the 24-h treatment with LPS, it increased the response to 3 µg/ml LPS to 79 ± 4 µM (n = 6). This potentiating response of UTP was also concentration-dependent; as shown in Fig. 1B, stimulation of the cells with a fixed concentration of LPS (1 µg/ml) and increasing concentrations of UTP (3-300 µM) resulted in increased nitrite production, with a 98 ± 1% (n = 5) increase seen with 300 µM UTP. A variety of other nucleotide analogues were tested, and a modest potentiation (~30-40%) was elicited by ATP, 2-methylthio-ATP, and AMP-CPP at 100 µM; 100 µM adenosine had no effect (Table I).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent induction of nitrite in LPS- and LPS/UTP-treated J774 macrophages. A, cells were incubated with increasing concentrations of LPS in the absence () or presence () of 100 µM UTP for 24 h before assaying for nitrite. B, cells were incubated with vehicle (white bar) or 1 µg/ml LPS in the absence (black bar) or presence () of increasing concentrations of UTP for 24 h before assaying for nitrite. The data represent the mean ± S.E. of three experiments performed in duplicate.

Involvement of Phosphoinositide Turnover in the UTP Response-- Our previous study demonstrated the presence, in J774 macrophages, of pyrimidinoceptors coupled to stimulation of PI turnover, increased intracellular Ca²⁺ levels, and membrane translocation of several PKC isoforms (23). To understand the connection between the NO-potentiating effect of UTP and its PI signaling cascades, the PI-specific phospholipase C (PI-PLC) inhibitor U73122 (26) was tested. As shown in Fig. 2, at 3 µM, it inhibited both the UTP-induced inositol phosphate accumulation (decreased from 1269 ± 32% (n = 3) to 647 ± 20% (n = 3) of control) and its nitrite potentiation (decreased by 65 ± 4% (n = 3)). The inactive analogue, U73343 (26), had no effect. In contrast, D609, at a concentration (30 µM) at which it specifically inhibits phosphatidylcholine-specific phospholipase C (PC-PLC) (27), had no significant effect on the UTP-induced inositol phosphate response (Fig. 2B), but attenuated the LPS-induced NO response by 48 ± 5% (n = 3); however, LPS-induced nitrite formation was still markedly potentiated by the addition of UTP (Fig. 2A).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Involvement of PI turnover in UTP-induced nitrite potentiation. A, cells were pretreated with 3 µM U73122 or U73343 or 30 µM D609 for 20 min before the addition of LPS (1 µg/ml) with or without 100 µM UTP. After a 24-h incubation, nitrite levels in the medium were determined. B, cells labeled with myo-[3H]inositol were pretreated with U73122 (3 µM), U73343 (3 µM), or D609 (30 µM) for 20 min before stimulation with 100 µM UTP for 30 min and the measurement of [3H]inositol monophosphate (3H-IP). The data represent the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05 as compared with the control response to LPS (A) or UTP (B) in the absence of drug pretreatment; **, p < 0.05 as compared with the control LPS response without UTP treatment, indicating the potentiating effect of UTP.

Role of PKC Activation in NO Production-- In J774 cells, PKC activation has been implicated in the control of iNOS by the synergistic action of phorbol esters and IFN- (28). Fig. 3 shows the effects of two PKC inhibitors, Ro 31-8220 (29) and Go 6976 (30), on the LPS and LPS/UTP induction of iNOS activity. At concentrations of 100 nM and 1 µM, respectively, Go 6976 and Ro 31-8220 inhibited the response to LPS alone by 40 ± 5% (n = 4) and 60 ± 5% (n = 4), but failed to affect the potentiating response of UTP, with 30 ± 7 µM (n = 3) and 23 ± 5 µM (n = 3) increases in the presence of Go 6976 and Ro 31-8220, respectively, compared with 31 ± 6 µM (n = 5) in the absence of these inhibitors. These results suggest that PKC activation, although an obligatory event in the regulation of iNOS induction by LPS, might not be the main mechanism responsible for the UTP potentiating effect.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of protein kinase inhibitors on LPS- and LPS/UTP-induced nitrite formation. Cells were incubated for 20 min with 50 µM genistein, 100 nM Go 6976, 1 µM Ro 31-8220, or 1 µM H-89 before the addition of LPS with or without 100 µM UTP. After a 24-h incubation, nitrite formation in the medium was determined. The data represent the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05 as compared with the control response (LPS ± UTP) without treatment with protein kinase inhibitors.

The possible involvement in the UTP potentiating response of tyrosine kinase and protein kinase A, both of which are responsible for iNOS induction in LPS- or cytokine-stimulated macrophages (31-33), was tested by treating cells with genistein or H-89. As shown in Fig. 3, 50 µM genistein attenuated the LPS-induced nitrite response by 50 ± 5% (n = 3) and completely blocked the UTP potentiation. In contrast, 1 µM H-89 had no effect on either response.

To confirm that the PKC-dependent pathway was not involved in the UTP potentiating response, J774 cells were treated with 1 µM PMA for 8 h to down-regulate certain PKC isoforms before stimulation with LPS and UTP. Such pretreatment has been reported to cause the loss of immunoreactive PKCII and PKC in J774 macrophages, leading to decreased LPS-induced iNOS gene activation (28). However, we found that it down-regulated not only PKCII and PKC, but also PKC (Fig. 4A), and that the PKCI level was also slightly reduced; the immunoreactivity of other PKC isoforms (, µ, and ), known to be expressed in J774 macrophages and activated by UTP (23), was not significantly reduced (data not shown). Despite the down-regulation of PKC and the decrease in LPS-induced nitrite production by 43 ± 2% (n = 4), the potentiating effect of UTP (100 µM) was not affected (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Involvement of PKC in the LPS-, but not LPS/UTP-, induced nitrite response. A, shown are PKCI, PKCII, PKC, and PKC immunoblots of cells before and after 8 h of treatment with 1 µM PMA. B, cells were either untreated or pretreated with 1 µM PMA for 8 h before the addition of 1 µg/ml LPS alone or in combination with 100 µM UTP. The data represent the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05 as compared with the control response (LPS ± UTP) without PMA pretreatment.

Involvement of Intracellular Calcium in the UTP Response-- The role of intracellular calcium, suggested as a priming signal for iNOS induction in murine peritoneal macrophages (34), was investigated by pretreating cells with BAPTA/AM to block the increase in cytosolic free Ca²⁺ or with thapsigargin, a specific and potent endoplasmic reticulum Ca²⁺-ATPase inhibitor. Fig. 5 shows that 30 nM thapsigargin had a similar effect to 100 µM UTP in increasing LPS-induced nitrite production, although the degree of potentiation was less. When cells were treated with the intracellular Ca²⁺ chelator BAPTA/AM (30 µM) before stimulation with LPS (1 µg/ml) plus UTP (100 µM) or thapsigargin (30 nM), the potentiation of nitrite production by UTP or thapsigargin was completely inhibited; this treatment had no effect on the response seen with LPS alone (Fig. 5A). These results suggest that the increased [Ca²⁺]_i is a crucial factor in the UTP- or thapsigargin-induced potentiation of nitrite production.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Involvement of cytosolic calcium in the potentiation of nitrite production. Cells grown in DMEM were pretreated with vehicle, 30 µM BAPTA/AM, 100 ng/ml FK506, 3 or 10 µM KN-93, or 1 µM Ro 31-8220 for 20 min. They were then stimulated with 1 µg/ml LPS alone or in combination with 100 µM UTP or 30 nM thapsigargin (TG) for 24 h, and nitrite production was measured (A and B). Data are presented as the mean ± S.E. of three experiments. *, p < 0.05 as compared with the control response (LPS, LPS + UTP, or LPS + thapsigargin) without drug pretreatment. In C, the typical iNOS immunoactivity after overnight treatment with the various combinations of reagents is shown. Data in D represent the average from two independent experiments.

A recent study has indicated that the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin can potentiate the PKC-dependent and PKC-independent activation of NF-B in Jurkat T cells and U937 cells by enhancing the inactivation of IB, thereby increasing the nuclear NF-B DNA-binding activity (35, 36). To investigate this possible regulatory mechanism for the UTP and thapsigargin responses, the immunosuppressant FK506, a calcineurin inhibitor, was used and was found to have no effect on the potentiating effects of UTP or thapsigargin (Fig. 5A).

To correlate the increased intracellular Ca²⁺ with Ca²⁺/calmodulin-dependent protein kinase (CaMK), the selective inhibitor KN-93 (37) was used. At concentrations (3 and 10 µM) selective for CaMK inhibition, it inhibited the potentiating effects of UTP and thapsigargin in a concentration-dependent manner, while only slightly, but non-significantly, reducing the LPS response by 14 ± 7% (n = 4) at 10 µM (Fig. 5A). Further confirmation of the involvement of CaMK in the UTP response and of PKC in the LPS response comes from the findings that the co-presence of KN-93 and Ro 31-8220 further decreased LPS/UTP- and LPS/thapsigargin-induced nitrite formation (Fig. 5B).

To ensure that the increase in nitrite activity seen corresponded to increased expression of iNOS, cells were collected at the end of the incubation period, and their iNOS content was assessed by Western blotting. The results showed that UTP (100 µM) and thapsigargin (1 µM) did not themselves induce iNOS, but caused potentiation of the LPS (1 µg/ml) induction effect by 140 ± 22% (n = 3) and 87 ± 25% (n = 3), respectively. Consistent with the nature of nitrite formation, KN-93 (10 µM) also inhibited the UTP- or thapsigargin-induced increase in iNOS protein, but had no effect on the iNOS production induced by LPS alone (Fig. 5, C and D). In agreement with previous findings on the potentiating effect of IFN- on LPS-mediated iNOS induction, 10 units/ml IFN- increased iNOS induction by 341 ± 65% (n = 3) compared with the effect of LPS alone (data not shown).

To understand whether Ca²⁺ acts as a "priming" signal in the synergistic cooperation with LPS in NO production, the effects of UTP and thapsigargin, applied at various times after LPS stimulation, were examined. Fig. 6 shows that neither UTP or thapsigargin had any effect on either nitrite formation (Fig. 6A) or iNOS induction (Fig. 6B) when applied 3 or 6 h after LPS. Since extracellular nucleotides can be hydrolyzed by ectonucleotidase present on the extracellular surface of a variety of cells as we previously reported (38), we wanted to determine whether short-term treatment of macrophages with UTP is enough to potentiate LPS induction of iNOS. As shown in Fig. 6C, a simultaneous 30-min treatment of cells with LPS plus UTP, followed by washing with DMEM and re-addition of LPS, significantly increased the LPS response by 54 ± 3% (n = 3).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Time-dependent potentiation of LPS-induced nitrite production by UTP or thapsigargin. UTP (100 µM) or thapsigargin (TG; 30 nM) was added to the cell cultures at the same time as or 3 or 6 h after LPS (1 µg/ml). Twenty-four hours after LPS addition, nitrite production in the medium (A) and iNOS immunoreactivity (B) were determined. Data are presented as the mean ± S.E. of a single experiment, which was repeated once with similar results. In C, 30 min after LPS ± UTP addition, the cells were washed twice with DMEM and kept in the presence of LPS for 24 h. In another group, the nitrite production in cells treated without washing was compared. *, p < 0.05 as compared with the potentiating effect of UTP or thapsigargin.

Effect of UTP on LPS-induced NF-B Activation-- Since, as previously mentioned, NF-B activation is necessary for iNOS induction, the effect of UTP on the LPS-induced activation of NF-B was studied in order to delineate the basis of the UTP-mediated regulation of NO production. Fig. 7 shows that the nitrite formation produced by either LPS or LPS/UTP was inhibited in a concentration-dependent manner by pyrrolidinedithiocarbamate (PDTC; 3-30 µM), an NF-B inhibitor (39).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Inhibitory effect of PDTC on LPS- and LPS/UTP-induced nitrite production. Cells were either untreated or preincubated with PDTC (3-30 µM) for 20 min before treatment with 1 µg/ml LPS alone or in combination with 100 µM UTP for 24 h. Data are presented as the mean ± S.E. of three experiments. *, p < 0.05 as compared with the control response (LPS ± UTP) without PDTC pretreatment.

NF-B activation was directly evaluated by the nuclear translocation of NF-B and a gel shift DNA binding assay. Treatment of macrophages with 1 µg/ml LPS resulted in the time-dependent activation of NF-B (Fig. 8A). This gel shift assay detected a specific band produced in the presence of LPS that was competed off by an unlabeled probe (Fig. 8A, 20X competitor, compare with third lane (time of 30 min)) or supershifted by co-incubation with a p65-specific antibody (Anti-p65). NF-B activation by LPS was also concentration-dependent (Fig. 8B). Although UTP (100 µM) alone failed to induce NF-B activation in macrophages, it significantly increased LPS-induced NF-B activation by 60 ± 7% (n = 3) (Fig. 8C).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Effects of UTP on NF-B activation and on IB- degradation induced by LPS. A, time-dependent activation of NF-B by LPS. J774 cells were incubated with LPS (1 µg/ml) for 0-120 min. Following incubation, a nuclear extract was prepared, and an EMSA was performed as described under "Materials and Methods." The top band represents NF-B. NS, nonspecific binding. A competition experiment using 20-fold unlabeled NF-B oligonucleotide (20X competitor) and a supershift experiment with 4 µg of anti-p65 antibody performed on the nuclear extract from LPS-stimulated cells for 30 min are also shown. B, concentration-dependent activation of NF-B by LPS. J774 cells were incubated with LPS (0, 0.1, 1, or 3 µg/ml) for 30 min. After incubation, a nuclear extract was prepared, and an EMSA was performed. In C and D, cells were preincubated with vehicle or KN-93 (10 µM) for 20 min and then incubated with UTP (100 µM), LPS (1 µg/ml) or both for an additional 30 min. NF-B activation (C) and IB- degradation (D) were determined by EMSA and immunoblotting, respectively. The typical traces are representative of three experiments with similar results.

Since NF-B activation is usually associated with IB- degradation, the effects of UTP or LPS on IB- levels were also determined. Fig. 8D shows that LPS, but not UTP alone, decreased IB- immunoreactivity to 63 ± 6% within 30 min, whereas a further decrease to 33 ± 3% was seen when co-incubated with UTP. Likewise, 30 nM thapsigargin can also potentiate LPS-induced NF-B activation and IB- degradation (data not shown).

Since the UTP and thapsigargin responses appear to be CaMK-dependent (Fig. 5), this hypothesis was further tested by examining the effect of KN-93 on NF-B activation and IB- degradation and phosphorylation. Fig. 8C shows that, whereas KN-93 (10 µM) slightly increased LPS-induced NF-B activation by 23 ± 3% (n = 3), it abolished the UTP-induced potentiating effect. Fig. 8D shows that, in the presence of 10 µM KN-93, the increased IB- degradation seen with UTP was partially, but significantly, attenuated. To verify the involvement of CaMK-dependent IB- phosphorylation in UTP-potentiated IB- degradation and NF-B activation, we measured the phosphorylation changes of IB-. Immunoblotting using antibody against Ser-32-phosphorylated IB-, as shown in Fig. 9A, revealed that although UTP and thapsigargin alone did not induce serine phosphorylation on IB-, both of them could prominently potentiate LPS-induced IB- phosphorylation, which was attenuated by the presence of KN-93. Supporting this finding, the ³²P-labeled immunoprecipitation assay further demonstrated the presence of a UTP/thapsigargin-elicited and KN-93-sensitive increase in IB- phosphorylation (Fig. 9B).

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Effects of KN-93 on the UTP potentiation of LPS-induced IB- phosphorylation. A, cells were preincubated with vehicle or KN-93 (10 µM) for 20 min and then incubated with UTP (100 µM), thapsigargin (TG; 30 nM), or LPS (1 µg/ml) or various combinations for another 7.5 min. Equal amounts of cell homogenates (50 µg of protein) were resolved by SDS-PAGE, and IB- phosphorylation (P) was measured by immunoblotting. B, cells loaded with [32P]orthophosphate were treated with drugs as indicated, and then IB- was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The typical traces are representative of three experiments with similar results.

Effect of UTP on LPS-induced AP-1 Activation-- The effect of UTP on the activation of AP-1, another factor required for iNOS transcription, was then investigated. Fig. 10 shows that, after 4 h of treatment, either LPS or UTP alone was able to induce AP-1 activation and that the responses were additive. Unexpectedly, treatment with KN-93 (10 µM) alone also caused weak activation of AP-1 (65 ± 10% (n = 3) of the LPS response); however, in the presence of KN-93, the AP-1 response to LPS or UTP was reduced by 57 ± 9% (n = 3) and 39 ± 7% (n = 3), respectively.

View larger version (45K): [in this window] [in a new window]   Fig. 10.   Effects of KN-93 on LPS- and LPS/UTP-induced AP-1 activation. Cells were left untreated or were pretreated with KN-93 (10 µM) for 20 min, followed by incubation with UTP (100 µM), LPS (1 µg/ml), or both for an additional 4 h. AP-1 activation was determined by EMSA as described under "Materials and Methods." The traces are representative of three experiments.

	DISCUSSION

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NO, a diffusible free radical, is known to play many roles in diverse pathological conditions. Once iNOS is induced in monocytes/macrophages, the primary source of iNOS in inflammation, NO release may continue for hours or even days (40). In addition, recent studies have demonstrated the crucial role of NO in macrophage antimicrobial and tumoricidal activity. iNOS activity is mainly controlled at the transcriptional level in response to a wide array of pro-inflammatory cytokines and bacterial cell wall products and through the involvement of signaling pathways involved in host defense against pathogens and tumor cells (3). In this report, we have examined the effects of UTP, both alone and in combination with LPS, on iNOS expression and nitrite production in mouse J774 macrophages and have demonstrated that UTP potentiates LPS-induced nitrite production by ~2-fold and increases iNOS induction by 140%, whereas UTP alone, at concentrations as high as 300 µM, induces neither nitrite production nor iNOS expression.

In parallel with its effect on LPS-induced nitrite formation, UTP also potentiated the induction of iNOS by LPS. Pharmacological analysis revealed a requirement for PI turnover in the UTP response. The inhibitory effect seen with U73122 (a G protein-activated PI-PLC inhibitor), but not with its inactive analogue, U73343 (26), or with D609 (a PC-PLC inhibitor) (27), indicates that UTP-elicited PI breakdown, which occurs via pyrimidinoceptors (21), plays an essential role in NO production.

Two bifurcate signaling cascades involved in stimulus-triggered PI breakdown (inositol 1,4,5-trisphosphate-mediated intracellular Ca²⁺ mobilization and diacylglycerol-mediated PKC activation) have been established (41) and are known to regulate a variety of cellular functions. In our previous study, we demonstrated a UTP-induced Ca²⁺ increase and PKC activation in mouse J774 macrophages (23). The present results demonstrate that intracellular Ca²⁺ is more important than PKC in the UTP-mediated NO response; this is based on the following evidence. First, a potentiating effect on NO induction similar to that produced by UTP is seen with thapsigargin, which elicits an increase in intracellular Ca²⁺ by inhibiting the endoplasmic reticulum Ca²⁺-ATPase. Second, both the UTP- and thapsigargin-induced potentiating effects are abrogated by BAPTA/AM, a cell-permeable Ca²⁺ chelator. Our results also support the observation that, in murine peritoneal macrophages (34), thapsigargin has a marked cooperative effect on LPS-, IFN--, or PMA-induced NO synthesis. Third, the exposure time-dependent synergistic cooperation between LPS and UTP or thapsigargin results in both nitrite production and iNOS expression, and we therefore conclude that the increase in intracellular Ca²⁺ has a crucial priming effect on iNOS expression in mouse J774 macrophages. Fourth, two specific PKC inhibitors, Ro 31-8220 (an inhibitor of all PKC isoforms) and Go 6976 (a selective inhibitor of the "conventional" PKC, PKCI, PKCII, and PKC) (30), have no significant inhibitory effects on the UTP response. Fifth, PMA-mediated down-regulation of certain PKC isoforms does not alter the potentiating effect of UTP. According to the report of Fujihara et al. (28), in J774 macrophages, PKCII is primarily involved in LPS induction of NO formation, although the LPS-triggered signal transduction pathway may involve other PKC isoenzymes and/or different protein kinases in addition to PKCII (28). In our experiments, after 8 h of PMA treatment, a marked decrease in PKCII, PKC, and PKC, with a parallel decrease in NO release, was seen. In contrast, the down-regulation of PKCI was less marked, and PKCµ, PKC, and PKC were resistant to PMA treatment for 24 h (data not shown).

Our present results also confirmed the involvement of PKC in the signal transduction pathway by which LPS induces iNOS gene activation in macrophages, as previously suggested in J774 macrophages (28, 42), RAW 264.7 macrophages (32), and murine peritoneal macrophages (43). Our data show that the LPS-induced NO production was attenuated by PKC inhibition with Ro 31-8220 or Go 6976 and by the PMA-mediated down-regulation of PKC. In addition, diacylglycerol, derived from PC-PLC, is responsible for LPS-mediated PKC activation in J774 macrophages (44) and also participates in the LPS-induced NO synthesis, as shown by the fact that D609, a PC-PLC inhibitor (27), attenuated the LPS response by 48% at a concentration not affecting cell viability.

Although iNOS gene expression is distinctly regulated in response to specific cytokines in different cell types, it has recently been demonstrated that cAMP induces the expression of iNOS in LPS- or cytokine-stimulated macrophages (31, 33). Our work, in which we used an inhibitor, rules out the involvement of this possible mechanism in the direct action of LPS and the potentiating effect of UTP since the protein kinase A inhibitor H-89 (45) cannot prevent LPS-mediated iNOS induction or its potentiation by UTP. Additionally, although release of TNF-, known to be an autocrine factor for iNOS expression, was enhanced by ATP in RAW 264.7 macrophages (46), we did not detect any significant change of TNF- release by UTP in J774 cells (data not shown). Thus, we exclude the possible involvement of TNF- in the regulation of UTP-induced NO potentiation in J774 macrophages.

There is increasing evidence for the presence, in the iNOS gene, of a consensus sequence for NF-B binding (9, 10); this has been shown to be functionally important for the induction of iNOS. Indeed, our data showed that PDTC treatment of J774 cells blocks both LPS- and LPS/UTP-induced nitrite formation, demonstrating the requirement for NF-B activation in this process. The time course study (Fig. 6) showed that the NO potentiation by UTP requires its co-addition with LPS and that even the short-term presence of UTP for 30 min is sufficient to elicit NO increase, suggesting the involvement of enhanced NF-B activation in the UTP response. In correlation with this suggestion, we indeed found that the NF-B activation caused by LPS was increased after UTP co-presence for 30 min (Fig. 8C). Unexpectedly, we also found that the induction of iNOS by LPS and the potentiation of this effect by UTP showed different sensitivity to PDTC (Fig. 7), with the UTP effect being more sensitively inhibited than LPS. To explore this phenomenon, we have tested the effects of PDTC on UTP-induced increases in intracellular Ca²⁺. We found that PDTC at 3 µM can significantly reduce the Ca²⁺ response elicited by 100 µM UTP from an 856 ± 120 nM increase to 440 ± 45 nM (n = 3) (data not shown).

To date, although more than one protein kinase has been shown to be responsible for the enhancement of NF-B DNA binding by a variety of agents, the signaling mechanisms responsible for NF-B activation are still not completely clear. Transfection studies have shown PKC to be a likely candidate for mediating NF-B activation/NO synthesis in RAW 264.7 macrophages in response to phorbol ester, but not to LPS or pro-inflammatory cytokines (47). An in vitro study by Diaz-Meco et al. (48) showed that PKC induces phosphorylation and inactivation of IB-. Protein kinase A-mediated phosphorylation of NF-B is also involved in the inducible and constitutive activation of NF-B (49). Ribosomal S6 protein kinase is reported to be an essential kinase required for the phosphorylation and subsequent degradation of IB- in response to PMA (50). Recently, a new serine/threonine kinase IKK (named CHUK) was shown to be an IB--specific kinase common to receptors of the TNF family and to the interleukin-1 receptor (51, 52). Casein kinase II has also been proposed as a candidate for IB- phosphorylation (53).

Although NF-B activation in T cells has also been shown to be induced by Ca²⁺ ionophores, especially in the presence of PMA (36, 54, 55), to date, only limited studies have demonstrated regulation of iNOS induction by calcium-dependent pathways. Interestingly, the synergistic effect seen here between calcium-dependent (represented by the action of UTP) and LPS-dependent pathways on NF-B activation suggests the involvement of two distinct kinases acting on NF-B. It is still unclear how the Ca²⁺-dependent protein kinase is involved in iNOS expression. However, in this work, we suggest, for the first time, that CaMK activation might play an important role in this downstream Ca²⁺-dependent signaling cascade. This is based on the fact that the selective CaMK inhibitor KN-93, at concentrations selective for the protein kinase (3 and 10 µM), can inhibit the potentiating effects of UTP on LPS-induced NO production, iNOS induction, NF-B activation, IB- degradation and phosphorylation, and AP-1 activation. Similar prevention was also observed for the effects of thapsigargin on NF-B activation and IB- degradation and phosphorylation. Although the CaMK-dependent pathway has been demonstrated in vivo here to participate in IB- phosphorylation, we still need more investigation to understand its direct or indirect action on the IB- molecule. To further confirm the specific action of KN-93 on CaMK, we determined the ineffectiveness of KN-93 (10 µM) on UTP-induced PI turnover and intracellular Ca²⁺ increase (data not shown). In this study, we demonstrated the activation of AP-1 by UTP and KN-93, and these effects are due to their activation of c-Jun N-terminal kinase/stress-activated kinase, which in turn stimulates AP-1 via phosphorylation of the c-Jun component (data not shown).

Regarding the regulatory role of the calcium/calmodulin-activated phosphatase calcineurin in NF-B activation, conflicting results have been obtained. In Jurkat T cells and U937 monocytes, calcineurin has been suggested to act in synergy with PMA to accelerate IB- degradation and to increase NF-B DNA-binding activity (35, 36), whereas it inhibits the PMA/ionomycin-mediated down-regulation of IB- in T cells (56). Our present results rule out any involvement of either of these possible mechanisms in the Ca²⁺-dependent induction of iNOS in J774 macrophages since FK506, an inhibitor of calcineurin, did not inhibit nitrite production (Fig. 5A) or NF-B DNA activity (data not shown).

In conclusion, we have demonstrated the involvement of Ca²⁺/calmodulin-dependent protein kinase in the pyrimidinoceptor-mediated potentiation of iNOS induction in mouse J774 macrophages. This effect is dependent on the activation of NF-B and AP-1. The ability of nucleotides, especially UTP, to modulate iNOS expression via NF-B activation reflects a potentially novel function of nucleotides in host defense, inflammation, and cytotoxic responses.

	FOOTNOTES

^* This work was supported by Grant NSC88-2314-B002-108 from the National Science Council, Taiwan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., Sec. 1., Taipei, Taiwan. Tel.: 886-2-23123456 (ext. 8315); Fax: 886-2-23915297; E-mail: wwl{at}ha.mc.ntu.edu.tw.

The abbreviations used are: iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; IFN, interferon; TNF, tumor necrosis factor; PI, phosphatidylinositide; DMEM, Dulbecco's modified Eagle's medium; AMP-CPP, adenosine 5'-(,-methylenetriphosphate); PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidinedithiocarbamate CaMK, Ca²⁺/calmodulin-dependent protein kinase; PC, phosphatidylcholine, PLC, phospholipase C.

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