Activation of Transcription Factor Nuclear Block TNF-Induced 2 ...

Selective Inhibitors of Cytosolic or Secretory Phospholipase A2 Block TNF-Induced Activation of Transcription Factor Nuclear Factor-kB and Expression of ICAM-1 Liv Thommesen,*† Wenche Sjursen,*‡ Kathrine Gåsvik,*‡ Wenche Hanssen,*§ Ole-Lars Brekke,¶ Lars Skattebøl,i Anne Kristin Holmeide,i Terje Espevik,# Berit Johansen,*§ and Astrid Lægreid1*† TNF signaling mechanisms involved in activation of transcription factor NF-kB were studied in the human keratinocyte cell line HaCaT. We show that TNF-induced activation of NF-kB was inhibited by the well-known selective inhibitors of cytosolic phospholipase A2 (cPLA2): the trifluoromethyl ketone analogue of arachidonic acid (AACOCF3) and methyl arachidonyl fluorophosphate. The trifluoromethyl ketone analogue of eicosapentaenoic acid (EPACOCF3) also suppressed TNF-induced NF-kB activation and inhibited in vitro cPLA2 enzyme activity with a similar potency as AACOCF3. The arachidonyl methyl ketone analogue (AACOCH3) and the eicosapentanoyl analogue (EPACHOHCF3), which both failed to inhibit cPLA2 enzyme activity in vitro, had no effect on TNF-induced NF-kB activation. TNF-induced NF-kB activation was also strongly reduced in cells stimulated in the presence of the secretory PLA2 (sPLA2) inhibitors 12-epi-scalaradial and LY311727. Addition of excess arachidonic acid suppressed the inhibitory effect of 12-epi-scalaradial and LY311727. Moreover, both methyl arachidonyl fluorophosphate and 12epi-scalaradial blocked TNF-mediated enhancement of expression of ICAM-1. Activation of NF-kB by IL-1b was markedly less sensitive to both cPLA2 and sPLA2 inhibitors. The results indicate that both cPLA2 and sPLA2 may be involved in the TNF signal transduction pathway leading to nuclear translocation of NF-kB and to NF-kB-activated gene expression in HaCaT cells. The Journal of Immunology, 1998, 161: 3421–3430.

T

umor necrosis factor is a central mediator of inflammation, as demonstrated by its ability to activate leukocytes, enhance adherence of neutrophils and monocytes to the intercellular matrix, stimulate fibroblast proliferation, and trigger local production of inflammatory cytokines (1). Thus, TNF mediates many of the cellular responses associated with infection, injury, and invasion, including cytotoxicity against malignant cells. TNF is also involved in triggering the lethal effects of septic shock syndrome, cachexia, and other systemic manifestations of disease (2). The transcription factor NF-kB, which plays a central role in TNF-mediated activation of gene expression, belongs to the Rel family of transcription factors and participates in the regulation of genes coding for cytokines, cytokine receptors, MHC Ags, adhesion molecules, as well as viruses (reviewed in Ref. 3). NF-kB proteins are constitutively expressed in the cytoplasm, bound to inhibitor IkB, and are released and translocated to the nucleus upon phosphorylation and degradation of IkB (4 – 6). Two different TNF receptors, TNFR p55 and TNFR p75, can mediate NF-kB activation (7) although in most cells, TNFR p55 is *UNIGEN–Center for Molecular Biology, †Department of Physiology and Biomedical Engineering, ‡Institute of Chemistry, §Institute of Botany, #Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, and iDepartment of Clinical Chemistry, Trondheim Regional Hospital, Trondheim, Norway; and iDepartment of Chemistry, University of Oslo, Oslo, Norway Received for publication May 14, 1997. Accepted for publication May 14, 1998. 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. 1 Address correspondence and reprint requests to Dr. Astrid Lægreid, Department of Physiology and Biomedical Engineering, Norwegian University of Science and Technology, Medisinsk Teknisk Center, N-7005 Trondheim, Norway. E-mail address: [email protected]

Copyright © 1998 by The American Association of Immunologists

the main signaling receptor (7–9). The TNFR p55 signal transduction pathway leading to NF-kB activation has been proposed to involve type C phospholipases, such as phosphatidyl choline-specific phospholipase C (10) and sphingomyelinase (8, 11), as well as serine/threonine protein kinases, such as protein kinase Cz (12), ceramide-activated protein kinase (11), and members of the mitogen-activated protein kinase cascade (13, 14). At present it is not clear how these intracellular components relate to the TNFR p55 pathway, which mediates activation of NF-kB via the TNFR p55 binding proteins TNFR-associated factor-2 (TRAF-2)2 (15, 16) or receptor interacting protein (RIP) (17), followed by activation of NIK kinase (18), which triggers IkB kinase (19, 20) and IkB degradation. TNF induces phospholipase A2 (PLA2) activity in a number of different cell types, including mesangial cells (21), Jurkat cells (8), astrocytes (22), bone-forming cells (23), and intestinal epithelial cells (24). PLA2s catalyze the release of unsaturated fatty acid from the sn-2 position of phospholipids or phosphatidic acid (reviewed in Ref. 25). Cellular PLA2s comprise three Ca21-dependent enzymes: cytosolic (c) group IV PLA2 (26) and two closely related secretory (s) PLA2s, type II PLA2 (27) and the group V PLA2 (28) and Ca21-independent enzymes, iPLA2 (29, 30). The 85-kDa cPLA2 is constitutively expressed in all cells, is highly selective for arachidonic acid in the sn-2 position, and is activated 2 Abbreviations used in this paper: TRAF-2, TNFR-associated factor-2; PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; iPLA2, Ca21-independent phospholipase A2; p75 AS, TNFR p75 antiserum; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; AACOCF3, trifluoromethyl arachidonyl ketone; AACOCH3, arachidonyl methyl ketone analogue; MAFP, methyl arachidonyl fluorophosphate; EPACOCF3, (all-Z)-1,1,1-trifluoro6,9,12,15,18-heneicosapentaen-2-one; EPACHOHCF3, (all-Z)-1,1,1-trifluoro6,9,12,15,18-heneicosapentaen-2-ol; NMR, nuclear magnetic resonance; PE, phycoerythrin; NIK, NFkB-inducing kinase; NDGA, nordihydroguayaretic acid.

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INVOLVEMENT OF PLA2 IN TNF-MEDIATED ACTIVATION OF NF-kB

by phosphorylation at micromolar concentrations of Ca21 (31–33). Type II and V sPLA2s, both 14-kDa enzymes, are active extracellularly at millimolar Ca21 concentrations and show no specificity toward unsaturated fatty acids in the sn-2 position (reviewed in Ref. 34). The two secretory PLA2s may substitute for each other depending on tissue-specific expression, while the group V is mainly expressed in heart (35) and macrophages (36). PLA2 has been suggested to be involved in the signal transduction mechanism mediating TNF-induced cytotoxicity (37–39). PLA2-generated lipid mediators such as unsaturated free fatty acids have been found to activate protein kinase Cz (40), which is suggested to be involved in TNF-mediated activation of NF-kB (41). To examine whether PLA2 could also be involved in TNFmediated NF-kB activation, we studied the human keratinocyte cell line HaCaT, which we have found to express both cPLA2 and sPLA2 and where we have shown that TNF can induce PLA2 activity, measured as the release of arachidonic acid and its metabolites.3 We stimulated HaCaT cells with TNF in the presence of inhibitors with high selectivity against either cPLA2 or sPLA2 and measured 1) NF-kB nuclear translocation by quantitative bandshift assays and 2) NF-kB-mediated gene activation by flow cytometric analysis of ICAM-1 expression. The results indicate that both cPLA2 and sPLA2 may be involved in the TNF signal transduction pathway leading to functional activation of NF-kB.

Materials and Methods Cells and reagents The spontaneously immortalized human skin keratinocyte cell line HaCaT (provided by Prof. N. E. Fusenig, Heidelberg, Germany) was grown in DMEM with 1 g glucose/l (Life Technologies, Paisley, Scotland), supplemented with 5% (v/v) FCS (HyClone, Logan, UT), 0.3 mg/ml L-glutamine (Sigma, St. Louis, MO), 0.1 mg/ml gentamicin (Sigma), and 1 mg/ml fungizone (Life Technologies). Human rTNF (sp. act., 7.6 3 107 U/mg protein) and human rIL-1b (sp. act., 5 3 107 U/mg protein) were supplied by Dr. Refaat Shalaby (Genentech, South San Francisco, CA) and Dr. A. Shaw (Glaxo, Geneva, Switzerland), respectively. The generation and purification of the mAb htr-9 against TNFR p55 (provided by Dr. M. Brockhaus, Hoffmann-La Roche, Basel, Switzerland) has been reported previously (42). TNFR p75 antiserum (p75 AS) was generated by multiple injections of a rabbit with recombinant soluble TNFR p75 (7). Benzamidine (Sigma) was dissolved in 50% ethanol at 0.5 M, PMA (Sigma) was dissolved in 96% ethanol at 1 mg/ml, and PMSF (Sigma) was dissolved in isopropanol at 0.1 M. DTT (Sigma) was dissolved in 0.01 M sodium acetate at 1 M, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) was dissolved in PBS at 5 mg/ml. The reagents were stable for some weeks (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 4°C) or months (PMA, 280°C; benzamidine, 220°C; PMSF, 4°C). Trifluoromethyl arachidonyl ketone (AACOCF3) and arachidonic acid (AACOOH) (20 mM; Biomol, Plymouth Meeting, PA), AACOCH3 (20 mM; provided by Dr. Michael H. Gelb (43)), and 12-epi-scalaradial (4.6 mM; Cascade, Berkshire, U.K.) were dissolved in ethanol. LY311727 (1 or 10 mM; provided by Dr. G. Camejo, Astra Ha¨ssle, Goteborg, Sweden, and Dr. E. Mihelich, Eli Lilly, Indianapolis, IN) (44) was dissolved in ethanol, and methyl arachidonyl fluorophosphate (MAFP; 27 mM; Cayman, Ann Arbor, MI) was dissolved in methyl acetate. Arachidonic acid and arachidonyl analogues were stored under N2 at 280°C.

Synthesis and purification of eicosapentanenoic acid analogues All reactions were performed in a nitrogen atmosphere (45). (All-Z)-1,1,1-trifluoro-6,9,12,15,18-heneicosapentaen-2-one (EPACOCF3). To a solution of (all-Z)-5,8,11,14,17-eicosapentaenoic acid (donated by Pronova Oleochemicals, Sandefjord, Norway; 5 g, 0.017 mol) in anhydrous dichlorometane (250 ml) was added oxalyl chloride (1.7 ml, 0.020 mol). The reaction was stirred at 20°C for 2 to 3 h. Solvent and excess oxalyl 3 W. Sjursen, O. L. Brekke, B. Johansen. Both secretory and cytosolic phospholipase A2 regulate the long-term-induced eicosanoid production in human differentiated keratinocytes. Submitted for publication.

chloride were removed by distillation under reduced pressure. The residue was taken up in dry dichloromethane (250 ml). To this solution trifluoroacetic anhydride (19.7 g, 0.094 mol) and pyridine (9.5 g, 0.120 mol) were added at 0°C. The cooling bath was removed. After 2 h the reaction mixture was poured into water, and the layers were separated. The water phase was extracted with petroleum ether. The combined organic layers were washed with a saturated NaHCO3 solution and water and dried using MgSO4. Evaporation of solvents under reduced pressure gave a crude product that was purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate, 7/3) to give 4.2 g (70%) of EPACOCF3. Spectroscopic data were as follows: infrared, film: 3015, 1790, 1765, 1210, 1145 cm21; 1 H NMR (300 MHz): d 5.3–5.4 (m, 10H), 2.2.7–2.9 (m, 8H), 2.70 (t, 2H), 2.11 (m, 4H), 1.74 (m, 2H), 0.95 (t, 3H, J 5 7.5 Hz); 13C NMR (300 MHz): d 191.7 (q, J 5 35 Hz), 132.4, 130.0, 129.0, 128.7, 128.6, 128.4, 128.3, 128.2, 127.4, 116 (q, J 5 292), 36.0, 26.4, 26.0, 25.9, 22.5, 20.9, 14.4; 19F NMR (200 MHz): d 279.8 (s); mass spectra: 254 (M1), 285, 218, 91, 79. (All-Z)-1,1,1-trifluoro-6,9,12,15,18-heneicosapentaen-2-ol (EPACHOHCF3). Sodium borohydride (160 mg, 4.2 mmol) was added to a stirred solution of EPA (500 mg, 1.4 mmol) in 20 ml of methanol at 0°C. The solution was stirred for 1 h at 20°C. The mixture was poured in water and extracted with ether. The organic layer was dried using MgSO4. Evaporation of solvents under reduced pressure gave a crude product that was purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate, 7/3) to give 400 mg (80%) of EPACHOHCF3. Spectroscopic data were as follows: infrared film: 3420, 3010, 1165, 1130 cm21; 1H NMR (300 MHz): d 5.3– 5.4 (m, 10H), 3.9 (m, 1H), 2.8 to 2.9 (m, 8H), 2.0 to 2.2 (m, 4H), 1.4 to 1.8 (m, 4H), 0.97 (t, 3H, J 5 7.5 Hz); 13C NMR (300 MHz): d 132.4, 129.5, 129.1, 129.0, 128.7, 128.5, 128.4, 128.2, 127.4, 124.2, 70.4 (q, J 5 30 Hz), 29.5, 28.8, 27.1, 26.0, 25.9, 25.3, 20.9, 14.6; 19F NMR (200 MHz): d 280.47 (d, J 5 7Hz); mass spectra: 356 (M1), 327, 287, 260, 220, 133. EPACOCF3 and EPACHOHCF3 (20 mM) were dissolved in ethanol and stored under N2 at 280°C.

Quantitative bandshift assays Cells were seeded out in normal growth medium in six-well plates (1 3 106 cells/well) and cultivated for 3 days (2 days postconfluence) before pretreatment with PLA2 inhibitors in serum-free medium for 1 or 2 h as indicated. Then, TNF or IL-1 was added in a small volume of serum-free medium (,10% of the total volume), and incubation was continued for 1 h in the presence of inhibitors. Preparation of nuclear extracts, native polyacrylamide gel electrophoresis in the presence of 32P-labeled NF-kB oligonucleotide probe (Promega, Madison, WI), and quantitation with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) were conducted as previously described (72). Radioactivity counts were termed PhosphorImager units and were used to compare the relative amounts of radioactivity in bands within one gel. Bandshift assays with 32P-labeled OCT oligonucleotide (Promega) were performed to ensure that a similar amount of nuclear proteins was applied for each sample. All samples were analyzed in at least two bandshift assays.

Flow cytometric analysis of ICAM-1 Cells (;3106) were labeled with 10 ml of phycoerythrin (PE)-conjugated mouse anti-human CD54 (ICAM-1) mAb (PharMingen, San Diego, CA) as previously described (46). Background fluorescence was estimated by adding 50 mg/ml of PE-conjugated anti-human Leu M3 (CD14, Becton Dickinson, Mountain View, CA). After washing twice with 0.1% (w/v) BSA in PBS, cells were analyzed in a FACScan flow cytometer (Becton Dickinson).

Quantitation of arachidonic acid release and eicosanoid production HaCaT cells were seeded out in normal growth medium in 60-mm culture dishes (6 3 105 cells/dish) or in six-well plates (3 3 105 cells/well) and analyzed after cultivation for 3 days (1 day postconfluence). The cells were labeled by addition of [3H]arachidonic acid (Amersham; 1 mCi/ml, in growth medium with 1% (v/v) FCS) 24 h before treatments. [3H]arachidonic acid was removed by washing three times with PBS, and cells were pretreated with inhibitors in medium containing BSA (2 mg/ml) for 1 h before addition of TNF (10 ng/ml) or IL-1 (10 ng/ml; in ,10% of the total volume). Conditioned medium after 1 h TNF or IL-1 treatment was cleared by centrifugation (5 min, 300 3 g) and extracted using Bond Elut C18 octadecyl columns (500 mg; Varian, Harbor City, CA) according to the method of Powell (47) with modifications as previously described (48). The extracted arachidonic acid and its metabolites were quantified by beta scintillation counting using a Packard 1900 CA beta counter (Packard, Meriden, CT).

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Cytosolic PLA2 enzyme activity assay The cPLA2 enzyme activity was measured using [14C]L-3 phosphatidylcholine, 1-stearoyl-2-arachidonyl as substrate as described by Wijkander et al. (49) with previously described modifications.4 The sources of cPLA2 enzyme activity were HaCaT cells stimulated with IL-1 or insect cells overexpressing recombinant human cPLA2 (10 mg cPLA2 protein/106 cells; BacPAK Baculovirus expression system, Clontech, Palo Alto, CA; see Footnote 3). The cytosolic fraction from HaCaT or insect cells was prepared as described (50).

MTT assay Cells were seeded out in 96-well microtiter plates in normal growth medium (1 3 104 or 3 3 104 cells/well) and cultivated for 3 days (1 and 2 days postconfluence, respectively) before pretreatment with inhibitors in serum-free medium for 1 h (12-epi-scalaradial and LY311727) or 2 h (AACOCF3 and EPACOCF3), followed by treatment for 1 h with TNF (10 ng/ml) or IL-1 (10 ng/ml). Conversion of substrate (MTT) was measured as OD at 580 nm after 4 h according to the method of Mosmann (51).

Results

TNF induces rapid activation of NF-kB in HaCaT via TNFR p55 TNF induced strong activation of transcription factor NF-kB in the human keratinocyte cell line HaCaT (Fig. 1, lane 4). Increased amounts of nuclear NF-kB were clearly detectable in cells treated with TNF for 10 min and reached maximum levels after 30 to 40 min. To examine the roles of the two TNF receptors, bandshift analyses were performed after treatment of the cells with agonistic TNFR p55 or p75 Abs. The TNFR p55 mAb htr9 induced a similar response as TNF (Fig. 1, lane 5), indicating that the p55 receptor can mediate activation of NF-kB in HaCaT. However, agonistic TNFR p75 antiserum, which induces activation of NF-kB via TNFR p75 in human adenocarcinoma SW480 and rhabdomyosarcoma KYM-1 cells (7), had no such effect (Fig. 1, lane 6). Thus, HaCaT seems to lack the intracellular components necessary for efficient TNFR p75-mediated NF-kB activation. PMA was also unable to induce activation of NF-kB in HaCaT after treatment for 1 h (Fig. 1, lane 8) or 24 h (data not shown), indicating that activation of PMA-sensitive protein kinases C is not sufficient for nuclear translocation of NF-kB in this cell line. Specific inhibitors of cPLA2 strongly reduce TNF-mediated activation of NF-kB in HaCaT Since we have found that TNF augments the release of arachidonic acid from HaCaT cells, indicating that PLA2 is activated by TNF in this cell line (see Footnote 3), we were interested in examining whether PLA2 could be involved in TNF-mediated activation of NF-kB. We therefore stimulated the cells with TNF in the presence of the selective cPLA2 inhibitory, synthetic arachidonic acid analogues AACOCF3 and MAFP. AACOCF3 inhibits cPLA2-mediated phospholipid hydrolysis by binding tightly and reversibly to the enzyme (43), while MAFP is an irreversible inhibitor of cPLA2 (52). Quantitative NF-kB bandshift analysis of nuclear extracts from cells stimulated in the presence of AACOCF3 or MAFP showed that both inhibitors caused a strong reduction in TNFmediated NF-kB activation (;70% reduction at 2 mM), while the noninhibitory compound AACOCH3 had no effect (Fig. 2, A–C). We then synthesized eicosapentaenoic acid analogue EPACOCF3. Cytosolic PLA2 shows similar affinity for eicosapentaenoic and arachidonic acids (53) and thus would be expected to be inhibited by EPACOCF3 in a similar manner as by AACOCF3. TNF-mediated NF-kB activation was reduced about 40% in cells 4 A. Lægreid, W. Richardson, L. Thommesen, A. E. Medvedev, A. Sundan, and T. Espevik. Comparison of TNFR p75- and TNFR p55-induced activation of NFWB by using inhibitors of intracellular signaling. Submitted for publication.

FIGURE 1. Bandshift analysis of NF-kB in HaCaT cells treated with TNF, agonistic TNFR Abs, and PMA. Nuclear extracts from unstimulated cells (lane 1) or from cells stimulated with TNF, TNFR Abs, and PMA (lanes 2– 8) were analyzed by band shift with 32P-labeled NF-kB oligonucleotide. The main NF-kB-specific band shift (arrowhead) was identified by its displacement with an excess of unlabeled oligonucleotide containing HIV long terminal repeat NF-kB consensus sequences (lane 3). The same band shift was unaffected by the addition of an excess of unlabeled HIV long terminal repeat oligonucleotide containing mutated NF-kB consensus sequences (lane 2). Supershift analysis indicated that the main NF-kB band shift (arrowhead) contained NF-kB p65 and p50 proteins (data not shown).

stimulated with EPACOCF3 (10 mM; Fig. 2D). In the presence of EPACHOHCF3, in which the carbonyl group of EPACOCF3 is reduced to a hydroxyl group, no inhibition of the TNF response was observed. The inhibitory effect of EPACOCF3 or MAFP could not be reduced by excess arachidonic acid. This may indicate that the role of cPLA2 is mediated by a phospholipid hydrolysis product that is different from arachidonic acid, e.g., lysophospholipid. Alternatively, the signaling molecule involved in the TNF pathway may be an arachidonic acid metabolite, and the failure to abolish inhibition may be due to the fact that EPACOCF3 and MAFP may inhibit enzymes involved in the production of this metabolite, as reported for AACOCF3, which can inhibit cyclooxygenase (54). IL-1 also induces PLA2 activity in HaCaT cells (see Footnote 3). However, when we examined the effects of cPLA2 inhibitors on IL-1-mediated NF-kB activation, we found the IL-1 response to be consistently more resistant to these compounds, as AACOCF3 (2 mM), MAFP (4 mM), and EPACOCF3 (10 mM) only reduced IL-1-induced activation of NF-kB by 25, 10, and 20%, respectively (Fig. 3, A–C). Both TNF and IL-1 were used

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FIGURE 2. TNF-mediated activation of NF-kB in the presence of selective cPLA2 inhibitors. Cells were pretreated for 2 h with the indicated concentrations of AACOCF3, AACOCH3, MAFP, or EPACOCF3 before stimulation with TNF (2 ng/ml) for 1 h. Nuclear extracts were prepared and subjected to NF-kB bandshift analysis. A, Autoradiogram of NF-kB and OCT bandshift analysis of nuclear extracts from cells stimulated in the presence of AACOCF3 and PhosphorImager quantitation of specific NF-kB band shifts (mean of triplicate measurements of the same band shift). B, Autoradiogram of NF-kB and OCT bandshift analysis of nuclear extracts from cells stimulated in the presence of AACOCH3 and PhosphorImager quantitation of specific NF-kB band shifts. C and D, PhosphorImager quantitation of specific NF-kB band shifts in nuclear extracts from cells stimulated in the presence of MAFP (C) or EPACOCF3 (D). Similar results were obtained in two other experiments.

at 2 ng/ml, a concentration that induces submaximal activation of NF-kB (at least 10 ng/ml TNF or IL-1 is needed for a maximal response in HaCaT; data not shown). Thus, our results indicate that the cPLA2 inhibitors interfere more potently with the TNF-activated signal transduction pathway leading to activation of NF-kB than with the IL-1 pathway. The relative resistance of the IL-1 response suggests that the fatty acid analogues do not inhibit TNF-mediated NF-kB activation due to a general toxic effect. This was also confirmed in the MTT assay, where no reduction in cell viability was detected after pretreatment for 2 h with 25 mM AACOCF3, MAFP, or EPACOCF3 followed by 1-h treatment with TNF (data not shown).

TNF-induced activation of NF-kB is also blocked by selective inhibitors of sPLA2 In contrast to the ubiquitously expressed cPLA2, sPLA2 expression is restricted. Secretory PLA2 is known to be activated by TNF in intact cells (21–23, 55). Since we have shown that HaCaT cells express sPLA2 mRNA (see Footnote 3), we examined whether the selective sPLA2 inhibitors 12-epi-scalaradial and LY311727 could affect TNF-mediated NF-kB activation. 12-Epi-scalaradial (56), an epi analogue of the marine sponge product scalaradial, is proposed to cause irreversible inhibition of sPLA2 by formation of a Schiff’s base (imine) with a lysine residue on the surface of the enzyme. The sPLA2 enzyme contains a high number of lysine


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FIGURE 3. IL-1-mediated NF-kB activation in the presence of selective cPLA2 inhibitors. Cells were pretreated for 2 h with the indicated concentrations of AACOCF3, MAFP, or EPACOCF3 before stimulation with IL-1 (2 ng/ml) for 1 h. A, Autoradiogram of NF-kB and OCT bandshift analysis of nuclear extracts from cells stimulated in the presence of AACOCF3 and PhosphorImager quantitation of the specific NF-kB band. B and C, PhosphorImager quantitation of specific NF-kB band shifts in nuclear extracts from cells stimulated in the presence of MAFP (B) and EPACOCF3 (C). Similar results were obtained in two other experiments.

residues and is very sensitive to 12-epi-scalaradial. The other sPLA2 inhibitor, LY311727, is a structurally based, designed indole derivative that interacts with the active site of the enzyme in a noncovalent manner (44). Analysis of nuclear extracts from cells stimulated with TNF in the presence of 12-epi-scalaradial showed that TNF-induced activation of NF-kB was inhibited in a dose-dependent manner (55% reduction at 2 mM; Fig. 4A). A similar effect was seen in cells stimulated in the presence of LY311727 (50% reduction at 10 mM; Fig. 4B). The inhibitory effect of both 12-epi-scalaradial (2 mM) and LY311727 (10 mM) was clearly suppressed in the presence of excess (10 mM) arachidonic acid (data not shown). This indicates that inhibition of TNF-induced activation of NF-kB by the compounds is indeed due to their specific effects on sPLA2. Although IL-1-induced NF-kB activation was completely blocked by 5 mM 12-epi-scalaradial, the IL-1 response was consistently more resistant to the sPLA2 inhibitors (,10% inhibition at 2 mM 12-episcalaradial and 25% inhibition at 10 mM LY311727; Fig. 5, A and B). This suggests that, compared with the TNF response, IL-1mediated NF-kB activation is less dependent on components inhibited by these compounds. The MTT assay showed no reduction in HaCaT cell viability after 1-h pretreatment with LY311727 (10 mM) or 12-epi-scalaradial (5 mM), followed by 1-h TNF stimulation (data not shown). In contrast, Marshall et al. found that 12-epi-scalaradial would compromise neutrophil cell viability at concentrations .3 mM (57). This discrepancy may derive from the different cell types studied or, alternatively, may be due to the fact that Marshall et al. treated their cells in HBSS (57) while our experiments were conducted in

DMEM medium and thus in the presence of lysine amino acids, which may exhaust the inhibitor. TNF-mediated up-regulation of ICAM-1 is inhibited by 12-epi-scalaradial and MAFP Since the bandshift analyses indicated that PLA2 inhibitors strongly suppressed the TNF-induced increase in nuclear NF-kB (Figs. 2 and 4), it was of interest to examine whether a similar effect could be observed at the level of NF-kB-regulated gene expression. We therefore studied TNF mediated regulation of ICAM-1 in the presence of PLA2 inhibitors. TNF is known to be one of the main inducers of ICAM-1 expression, and transcription of the ICAM-1 gene is mainly regulated by NF-kB (reviewed in Ref. 58). Flow cytometric analyses showed that untreated HaCaT cells appear as a heterogeneous population displaying varying degrees of ICAM-1 expression (Fig. 6, untreated control). Treatment with TNF shifted the specific ICAM-1 immunofluorescence of the cells to a higher intensity (Fig. 6, TNF treated). In the presence of the sPLA2 inhibitor 12-epi-scalaradial, TNF-mediated enhancement of ICAM-1 expression was reduced in a dose-dependent manner (Fig. 6A). A concentration of 5 mM 12-epi-scalaradial completely blocked up-regulation of ICAM -1 expression, as shown by the fact that the ICAM-1 fluorescence intensity of cells stimulated in the presence of 5 mM 12-epi-scalaradial was similar to that of untreated cells (Fig. 6A). The cPLA2 inhibitor MAFP also strongly suppressed TNF-induced ICAM-1 expression (Fig. 6B). Thus, 12-epi-scalaradial- and MAFP-mediated inhibition of the TNF-induced increase in nuclear NF-kB is paralleled by their inhibition of TNF-induced ICAM-1 up-regulation. These results indicate that the reduced levels of NF-kB measured in nuclear

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FIGURE 4. TNF-mediated NF-kB activation in the presence of selective sPLA2 inhibitors. Cells were pretreated for 1 h with the indicated concentrations of 12-epi-scalaradial or LY311727 before stimulation with TNF (2 ng/ml) for 1 h. A, Autoradiogram of NF-kB and OCT bandshift analysis of nuclear extracts from cells stimulated in the presence of 12-epi-scalaradial and PhosphorImager quantitation of specific NF-kB band shifts. B, PhosphorImager quantitation of specific NF-kB band shifts in nuclear extracts from cells stimulated in the presence of LY311727. Similar results were obtained in two other experiments.

extracts from cells treated with PLA2 inhibitors are not caused by interference of these compounds with NF-kB binding in the bandshift assay, and that 12-epi-scalaradial and MAFP both inhibit the generation of functional NF-kB in intact cells. EPACOCF3 inhibits cPLA2 enzyme activity in vitro EPACOCF3 has not been previously characterized as a PLA2 inhibitor. We therefore analyzed its effect on cPLA2 enzyme activity using 1) cytosol fractions from insect cells overexpressing recom-

FIGURE 5. IL-1-mediated NF-kB activation in the presence of selective sPLA2 inhibitors. Cells were pretreated for 1 h with the indicated concentrations of 12epi-scalaradial before stimulation with IL-1 (2 ng/ml) for 1 h. A, Autoradiogram of NF-kB and OCT bandshift analysis of nuclear extracts from cells stimulated in the presence of 12-epi-scalaradial and PhosphorImager quantitation of specific NF-kB band shifts. B, PhosphorImager quantitation of specific NF-kB band shifts in nuclear extracts from cells stimulated in the presence of LY311727. Similar results were obtained in two other experiments.

binant cPLA2 or 2) cytosol from IL-1-treated HaCaT cells as enzyme sources. Similar to AACOCF3, EPACOCF3 inhibited cPLA2 activity both when analyzed with recombinant enzyme (Fig. 7A) or with HaCaT cytosolic extracts (Fig. 7B). The effect of the noninhibitory analogues EPACHOHCF3 and AACOCH3 was negligible when measured with recombinant enzyme (Fig. 7A), while AACOCH3 showed a weak, but reproducible, inhibition (15%) of cPLA2 enzyme activity in HaCaT cytosol (Fig. 7B). MAFP also reduced cPLA2 enzyme activity (Fig. 7B).


FIGURE 6. Analysis of TNF-induced ICAM-1 expression in the presence of cPLA2 and sPLA2 inhibitors. Cells were pretreated for 1 h with 12-episcalaradial (A) or for 2 h with MAFP before stimulation with TNF (2 ng/ml) for 4 h. Cells were treated with PE-labeled ICAM-1 mAb and analyzed by flow cytometry. Similar results were obtained in two other experiments.

To compare the potencies of EPACOCF3 and AACOCF3, we measured their dose-dependent effects on recombinant cPLA2 activity. As shown in Figure 8, the IC50 values of EPACOCF3 and AACOCF3 were similar and ranged from 2 to 7 mM. Both cPLA2 and sPLA2 inhibitors block TNF- and IL-1-induced release of arachidonic acid Extracellular release of arachidonic acid from intact cells is commonly used to measure PLA2 activity in intact cells (59). One-hour treatment with IL-1 approximately doubled the arachidonic acid release from HaCaT cells, while treatment with TNF resulted in a weaker, but reproducible, increase (;50%; Table I). AACOCF3 strongly inhibited both the TNF- and IL-1-induced increases in arachidonic acid release, while the noninhibitory

FIGURE 7. Effects of inhibitors on in vitro cPLA2 activity. Cytosol fractions from insect cells overexpressing recombinant cPLA2 (diluted 1/750; A) or from HaCaT cells stimulated with IL-1 (B) were incubated at room temperature without (control) or with PLA2 inhibitors (5 mM) 10 min before admixture of substrate and the start of the assay. The cPLA2 enzyme activity is given as a percentage of the control value (activity in the absence of inhibitors). Similar results were obtained in two other experiments.

3427

FIGURE 8. Dose-dependent effects of EPACOCF3 and AACOCF3 on cPLA2 activity. Recombinant cPLA2 enzyme activity was measured after preincubation with EPACOCF3 or AACOCF3 (as described in Materials and Methods). The cPLA2 enzyme activity is given as a percentage of the control value (activity in the absence of inhibitors) and expressed as the mean (SD) of two independent experiments.

arachidonic acid analogue AACOCH3 had no such effect. EPACOCF3 inhibited TNF-induced arachidonic acid release to a similar extent as AACOCF3 (Table I), indicating that the eicosapentenoic acid analogue can inhibit PLA2 activity in intact cells with similar efficiency as the arachidonic acid analogue. The sPLA2 inhibitor 12-epi-scalaradial also strongly inhibited both TNF- and IL-1-induced enhancement of arachidonic acid release (Table I). LY311727 consistently blocked TNF-induced arachidonic acid release with a higher efficiency than 12-epi-scalaradial (Table I). These data indicate that both cPLA2 and sPLA2 may be involved in receptor-mediated release of arachidonic acid in HaCaT cells.

Discussion The data presented in this study indicate that PLA2 is involved in TNF-induced nuclear translocation of transcription factor NF-kB


3428

Table I. Extracellular release of unsaturated fatty acids and metabolites from HaCaT cells treated with TNF or IL-1 in the presence of PLA2 inhibitors a Stimuli Pretreatment

TNF

IL-1

None AACOCF3, 1 mM EPACOCF3, 1 mM AACOCH3, 1 mM LY311727, 1 mM 12-Epi-scalaradial, 5 mM

156 6 38 87 6 27 74 6 22 152 6 29 88 6 41 114 6 42

191 6 49 136 6 25 ND ND ND 120 6 32

a 3 [ H]Arachidonic-labeled HaCaT cells were pretreated for 1 h with PLA2 inhibitors or noninhibitory control, followed by 1-h stimulation with 10 ng/ml TNF or IL-1 in the presence of the compounds. Arachidonic acid and metabolites were measured by b-scintillation counting as described in Materials and Methods. CPM values were calculated by subtraction of background counts and are given as % of unstimulated cells (set to 100%). Results are given as mean of duplicates or triplicates of at least two experiments, 6SD. ND, not determined.

and activation of ICAM-1 gene expression. Our analyses suggest that both cPLA2 and sPLA2 may participate in the TNF p55 receptor signal transduction pathway leading to functional activation of NF-kB in the human keratinocyte cell line HaCaT. These conclusions are based on the observed effects of potent inhibitors with high selectivity against either cPLA2 or sPLA2. Cytosolic and secretory PLA2 show no structural homology, exhibit different substrate specificity and substrate recognition, and employ different catalytic mechanisms (60, 61). These differences provide the basis for the potency and selectivity of the inhibitors that comprise compounds directed toward the active site of sPLA2 (LY311727), high sensitivity for lysine modifications (12-epi-scalaradial), and fatty acid analogues that exploit the preferential binding of cPLA2 to arachidonic acid (AACOCF3 and MAFP). Consequently, the potency of each of the inhibitors toward the PLA2 enzyme for which it is selective is .1000-fold higher than its potency against the other type of PLA2 (43, 44, 52, 62, 63). The compounds have previously been found by others to inhibit PLA2 in intact cells at concentrations similar to those applied in the present study. Thus, AACOCF3 blocks arachidonic acid release from thrombin-stimulated platelets (64), from calcium ionophore-stimulated U937 monocytic cells (65), and from IL-1a-stimulated mesangial cells (66). In the macrophage-like cell line P388D1, where PAF induces arachidonic acid release both via cPLA2 and via sPLA2, MAFP was found to inhibit cPLA2 without directly affecting the sPLA2 response, while LY311727 selectively inhibited sPLA2-mediated release of arachidonic acid (36, 67). In the present study of HaCaT cells, we show that AACOCF3 and MAFP inhibit TNF-induced arachidonic acid release as well as TNF-mediated NF-kB activation, suggesting that cPLA2 may be involved in the TNF-activated signal transduction pathway leading to activation of NF-kB. We also synthesized EPACOCF3 and showed that this compound suppresses TNF-induced arachidonic acid release and NF-kB activation and inhibits in vitro cPLA2 enzyme activity with similar potency as AACOCF3. Since cPLA2 shows similar affinity for eicosapentaenoic and arachidonic acid (53), we propose that the nonhydrolyzable eicosapentenoic acid analogue EPACOCF3 inhibits cPLA2 by binding tightly to the active site of the enzyme, analogous to the inhibitory mechanism described for AACOCF3 (43, 68). Thus, the inhibitory effect of EPACOCF3 further strengthens the suggestion that cPLA2 is involved in TNF-mediated NF-kB activation. Both 12-epi-scalaradial and LY311727 inhibit TNF-induced arachidonic acid release, indicating that they can inhibit sPLA2 enzyme activity in intact HaCaT cells. Thus, the ability of these compounds to reduce TNF-mediated activation of NF-kB suggests

that also sPLA2 may be involved in the TNF signaling pathway mediating this response. It cannot be excluded that the compounds we have used may affect cellular components other than PLA2s. However, others have found that MAFP does not inhibit enzymes involved in phospholipid metabolism (67), and that the sPLA2 inhibitor LY311727, although structurally similar to indomethacin, does not inhibit cyclo-oxygenase (44). Barnette et al. (69) found that the irreversible sPLA2 inhibitor 12-epi-scalaradial may also inhibit Ca21 channels and phosphatidylinositol-specific phospholipase C. It is possible that the strong inhibitory effect of high doses of 12-epi-scalaradial (5 mM) on both TNF- and IL-1-induced NF-kB activation may be due to additional pharmacologic actions of this compound. However, the differential effects of lower doses of 12-epi-scalaradial on TNF and IL-1 responses taken together with the fact that LY311727, which employs a completely different inhibitory mechanism, produced a similar inhibitory pattern strongly indicate that sPLA2 may be the main target of both compounds. The fact that excess arachidonic acid counteracted the inhibitory effect of 12epi-scalaradial and LY311727 further strengthens this hypothesis. Important controls included in our study are the fatty acid analogues AACOCH3 and EPACHOHCF3. The fact that these analogues, which do not inhibit cPLA2 (Fig. 7A) (43), did not affect TNF-induced NF-kB activation indicates that the presence of a fatty acid analogue per se, even at high concentrations (50 mM), cannot mimic the effect of the specific cPLA2 inhibitors. Furthermore, the inhibitory activity of the compounds is due to intracellular effects rather than to a reduction of TNF receptor levels, as flow cytometric analyses showed that the level of TNFR p55 was unchanged after treatment for 2 h with AACOCF3 (25 mM) or AACOCH3 (25 mM) or for 1 h with 12-epi-scalaradial (5 mM; data not shown). The PLA2-generated lipid mediators involved in TNF-mediated activation of NF-kB may be arachidonic acid, other unsaturated fatty acids, lysophospholipids, or their metabolites. In a paper that was published after the completion of this study, Camandola et al. reported that arachidonic acid mediated activation of NF-kB in promonocytic U937 cells by a mechanism suggested to involve metabolization of arachidonic acid to PGs and leukotrienes (70). However, in a separate study (see Footnote 3), we show that TNFinduced release of arachidonic acid in HaCaT cells is not accompanied by measurable generation of metabolites, while IL-1 treatment results in a marked rise in both PGs and leukotriene B4. Furthermore, NDGA (10 mM), a well-known lipoxygenase inhibitor that completely blocked IL-1-induced leukotriene B4 production in HaCaT cells (see Footnote 3), did not reduce TNF-mediated NF-kB activation (data not shown). Thus, our data indicate that leukotrienes may not be the main signaling substances in TNFmediated activation of NF-kB in HaCaT cells. Our observations also emphasize the importance of considering cell specificity in signal transduction mechanisms, since we have found TNFR p55mediated NF-kB activation in human SW480 adenocarcinoma cells to be strongly inhibited by NDGA (2–10 mM), whereas the TNFR p75 pathway in SW480 cells proceeds independently of NDGA-sensitive components (see Footnote 4), similar to the TNFR p55 pathway in HaCaT cells. Our results suggest that although PLA2 is necessary for TNFmediated NF-kB activation, PLA2 activity alone is not sufficient to induce this response. We deduce this from the fact that IL-1 activates both cPLA2 and sPLA2 in HaCaT (see Footnote 3; Fig. 7 and Table I), and yet IL-1-mediated activation of NF-kB is not affected by inhibitor concentrations that clearly reduce PLA2 enzyme activity in vitro, suppress IL-1-induced PLA2 activity in intact cells, and markedly inhibit TNF-mediated activation of NF-kB. This

The Journal of Immunology suggests that cPLA2 or sPLA2, to contribute to activation of NFkB, must act together with other signaling molecules that are triggered by TNF but not by IL-1. The IL-1 signaling mechanism mediating NF-kB activation is reported to differ from the TNF signaling mechanism in that the IL-1R couples to the NIK kinase pathway via IL-1R-associated kinase and TRAF-6 (71), rather than via TRAF-2. Thus, our results may indicate that PLA2s are involved in TNF signaling events upstream of NIK kinase. Alternatively, PLA2s may be part of a NIK kinase-independent pathway or play a modulatory role in the signaling pathway proceeding through NIK kinase. This alternative or modulatory pathway may be identical with the TNFR p55 pathway characterized by us in SW480 adenocarcinoma cells, where we have shown that TNFR p55 employs additional signaling mechanisms not involved in a pathway common to TNFR p55- and TNFR p75-mediated NF-kB activation (72) (see Footnote 4). Balsinde and Dennis (67) recently showed that cPLA2 and sPLA2 are both necessary for platelet-activating factor-mediated arachidonic acid release in the macrophage-like cell line P388D1. They propose a model in which the two enzymes exhibit specific roles and where a rapid, cPLA2-generated burst of intracellular arachidonic acid is necessary for activation of sPLA2, which generates the bulk amounts of extracellular arachidonic acid (67). Our data would be compatible with a similar interdependence of the two PLA2s in HaCaT, since TNF-induced release of arachidonic acid is completely blocked by inhibitors with high selectivity against either cPLA2 or sPLA2. In accordance with our results, showing that cPLA2 and sPLA2 inhibitors reduce TNF-mediated NF-kB activation with similar efficiency, this model would predict that the TNF signal transduction pathway involves a sequential action of cPLA2 and sPLA2, where sPLA2-generated phospholipid hydrolysis products may be the main effectors triggering the subsequent events of the pathway. Secretory PLA2 is a proinflammatory mediator found to be highly elevated both in the circulation and locally in the tissue, in association with a number of pathologic conditions such as sepsis, fever, infections, atherosclerosis, chronic lung inflammation, rheumatoid arthritis, Crohn’s disease, and psoriasis (73–78)5. The main proinflammatory effect of sPLA2 is thought to be the generation of arachidonic acid as a precursor for eicosanoid hormones. Our study indicates a potentially new role for sPLA2, namely in the activation of NF-kB and of NF-kB-regulated expression of genes involved in inflammation. The ability of PLA2 inhibitors to block the TNF-mediated increase in nuclear NF-kB and cellular responses such as TNF-induced up-regulation of ICAM-1 expression should be of interest in the consideration of such compounds for the treatment of inflammatory conditions.

Acknowledgments We thank Unni Nonstad for excellent technical assistance.

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