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J. Biol. Chem., Vol. 276, Issue 32, 30527-30536, August 10, 2001

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Functional Coupling between Secretory and Cytosolic Phospholipase A₂ Modulates Tumor Necrosis Factor-- and Interleukin-1-induced NF-B Activation^*

Marit W. Anthonsen, Anita Solhaug, and Berit Johansen

From the UNIGEN Center for Molecular Biology, Faculty of Chemistry and Biology, Norwegian University of Science and Technology, N-7489 Trondheim, Norway

Received for publication, September 15, 2000, and in revised form, April 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tumor necrosis factor (TNF)- and interleukin (IL)-1 are potent activators of the transcription factor NF-B, induced during inflammatory conditions. We have previously shown that both secretory and cytosolic phospholipase A₂ (PLA₂) are involved in TNF-- and IL-1-induced NF-B activation. In this study, we have addressed the mechanism of PLA₂ involvement with respect to downstream arachidonic acid (AA) metabolites and the functional coupling between PLA₂s mediating NF-B activation. We show that in addition to inhibitors of secretory and cytosolic PLA₂s, 5-lipoxygenase inhibitors attenuate TNF-- and IL-1-stimulated NF-B activation. Exogenous addition of leukotriene B₄ (LTB₄) restored NF-B activation reduced by 5-lipoxygenase inhibitors or an LTB₄ receptor antagonist, thus identifying LTB₄ as a mediator in signaling to NF-B. TNF-- and IL-1-induced AA release from cellular membranes was accompanied by phosphorylation of cytosolic PLA₂. Inhibitors of secretory PLA₂ and of 5-lipoxygenase/LTB₄ functionality markedly reduced AA release and nearly completely abolished cytosolic PLA₂ phosphorylation. This demonstrates that secretory PLA₂, through 5-lipoxygenase metabolites, is an essential upstream regulator of cytosolic PLA₂ and AA release. Our results therefore suggest the existence of a functional link between secretory and cytosolic PLA₂ in cytokine-activated keratinocytes, providing a molecular explanation for the participation of both secretory and cytosolic PLA₂ in arachidonic acid signaling and NF-B activation in response to proinflammatory cytokines.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proinflammatory mechanistic action of PLA₂¹-generated lipid mediators is not understood in detail. Diverse PLA₂ enzymes hydrolyze the sn-2 bond of phospholipids releasing lysophospholipids and fatty acids. Both products are metabolized into bioactive lipids, like platelet-activating factor and eicosanoids. Many of the arachidonic acid (AA)-derived eicosanoids, including leukotrienes, prostaglandins, thromboxanes, and hydroxy fatty acids, are potent proinflammatory mediators, whose action is in part mediated by interaction with cell surface G-protein coupled receptors (1, 2). The production of these mediators is initiated by PLA₂ (3), and PLA₂ enzymes are considered important targets for development of anti-inflammatory therapies.

The PLA₂ enzymes comprise a heterogeneous family of enzymes with distinct enzymatic properties, including substrate specificity and Ca²⁺ requirement. Ten different groups of PLA₂ enzymes have been defined; eight are detected in human tissues (4-6), but only three groups of PLA₂ have been shown to be specifically involved in arachidonic acid release. The PLA₂ enzymes relevant to AA release in human tissues may be grouped as secretory or cytosolic enzymes. The cytosolic group IV PLA₂ includes three paralogs, , , and  (7-10). The  paralog (85 kDa), herein denoted cPLA₂, is ubiquitously expressed and is regulated by micromolar concentrations of Ca²⁺ and reversible phosphorylation (11). Several distinct snpPLA₂s (groups IIa (12), IID (13), V (14), and X (4)) have been identified. They require millimolar concentrations of calcium for catalytic activity and act on membrane phospholipids containing a variety of fatty acids, including AA, in the sn-2-position. Proinflammatory cytokines such as TNF- and IL-1 regulate the expression of snpPLA₂s of group IIa (12) and group V (15), and equally of cPLA₂ group IV, in vitro (16, 17). Group IIa secretory PLA₂ is involved in AA release during inflammatory conditions (18-20). Increased levels of snpPLA₂ in circulation and affected tissue have been found in association with various pathological conditions like, for example, rheumatoid arthritis, sepsis, infections, lung inflammation, and psoriasis (21-23).

Nuclear factor B (NF-B) is a transcription factor, which plays a critical role in immune and inflammatory responses (24, 25). NF-B is activated by a wide range of inducers, including ultraviolet irradiation, cytokines, inhaled occupational particles, lipoproteins, and bacterial or viral products (26). In resting cells, NF-B, prototypically a heterodimer of p50 and p65 subunits, resides in the cytoplasm in an inactive form bound to the inhibitory protein IB. Upon cellular activation, including in response to the proinflammatory cytokines TNF- and IL-1, IB is phosphorylated by an IB kinase complex and proteolytically degraded by proteasomes, leading to the activation of NF-B (27). NF-B then translocates into the nucleus, where it activates gene transcription of a number of genes involved in inflammatory processes.

Two different TNF receptors, TNFR p55 and TNFR p75, can mediate NF-B activation, but in most cells the TNFR p55 is the major signaling receptor. After receptor activation, members of the TNF receptor-associated factor protein family (28, 29) or RIP (30) are recruited to the receptor, which leads to subsequent activation of serine-specific kinases of the mitogen-activated protein kinase kinase kinase (MAP3K) family including NF-B-inducing kinase (NIK; Refs. 31 and 32). Activation of the IL-1 receptor triggers recruitment of the receptor-associated factors IL-1 receptor-associated kinase (33) and TNF receptor-associated factor 6 (34). IL-1 receptor has been found to activate TAK1 and NIK (32). In parallel to NIK, other MAP3K family members are also able to activate the IB kinase complex, including MEKK1 (35), MEKK2, and MEKK3 (36). The TNF--induced transduction pathway leading to activation of NF-B has also been proposed to involve phosphatidylcholine-specific phospholipase C (37), sphingomyelinase (38, 39), and protein kinase C  activated by ceramide (40).

In a previous study, we showed that selective inhibitors against either snpPLA₂ or cPLA₂ (41) inhibited activation of NF-B in human-derived keratinocytes. The NF-B inhibition was accompanied by a reduced expression of the cell surface adhesion molecule ICAM-1, illustrating the biological significance. The aim of the present study was to identify PLA₂-generated signaling molecules, to investigate their mechanism of action, and to examine the possible sequential interaction between secretory and cytosolic PLA₂ in NF-B activation. We present evidence herein that snpPLA₂ and 5-lipoxygenase activity regulate phosphorylation of cPLA₂ and cellular AA release, contributing to NF-B activation. Hence, a functional coupling between snpPLA₂ and cPLA₂ is of importance for PLA₂-mediated modulation of NF-B in response to either TNF- or IL-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Human interleukin-1 (IL-1; 5 × 10⁷ units/mg), leupeptin, and pepstatin were from Roche Molecular Biochemicals). TNF- (specific activity 7.6 × 10⁷ units/mg protein; Genentech Inc.) was a generous gift from Prof. Terje Espevik (Norwegian University of Science and Technology, Norway). Aprotinin, fatty acid-free bovine serum albumin (BSA), and nordihydroguaiaretic acid were from Sigma. Methyl arachidonyl fluorophosphonate (MAFP), AA, and leukotriene B₄ (LTB₄) were obtained from Cayman Chemicals. 12-epi-scalaradial and -pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655,238) were obtained from BIOMOL. Purified human recombinant snpPLA₂ group IIa, the LTB₄ receptor antagonist LY255283, and the snpPLA₂ inhibitor BM16.2269 were generous gifts from Dr. Jeff Browning (Biogen Inc.), Dr. David K. Herron (Lilly), and Prof. Ulrich Tibes (Roche Molecular Biochemicals), respectively. The snpPLA₂ inhibitor SB203347 was a generous gift from Dr. Lisa Marshall (SmithKline Beecham Pharmaceuticals). The two cPLA₂ polyclonal antibodies were generous gifts from Dr. Christina Leslie (National Jewish Medical and Research Center) and Dr. Ruth Kramer (Lilly). [5,6,8,9,11,12,14,15-³H]Arachidonic acid (specific activity, 212 Ci/mmol), [³²P]orthophosphate (specific activity, 6000-7000 Ci/mmol), and the ECL detection kit were purchased from Amersham Pharmacia Biotech.

Moloney murine leukemia virus reverse transcriptase was from Life Technologies, Inc.; Dynazyme DNA Polymerase was obtained from Finnzymes Oy; and RNasin was from Promega.

Cell Culture and Transfection-- The spontaneously immortalized skin keratinocyte cell line HaCaT (kindly provided by Prof. N. Fusenig, Heidelberg, Germany) was cultured at 37 °C in Dulbecco's modified Eagle medium supplemented with 10% (v/v) FCS, 0.3 mg/ml glutamine, and 0.1 mg/ml gentamicin. The pfLUC plasmid contains the mouse fos promoter cloned upstream of Photinus pyralis luciferase coding sequence, whereas the reporter plasmid pBIIX contains in addition two copies of a HIV-NF-B sequence cloned upstream of the mouse fos promoter (42). The plasmids pBIIX and pfLUC were kindly provided by Dr. M. Jäättelä (Danish Cancer Society, Copenhagen, Denmark). HaCaT cells were transfected using the calcium phosphate precipitation procedure according to standard protocols using a 10-fold excess of the plasmid of interest (pBIIX or pfLUC) over the selection plasmid encoding a puromycin selectable marker (pPur; CLONTECH). Briefly, each 100-mm plate with HaCaT cells at 50% confluency was co-transfected with 8 µg of pBIIX and 0.8 µg of pPur (CLONTECH) encoding a puromycin selectable marker. Transfections were also carried out using the plasmid pfLUC in combination with pPur. After 18 h, the calcium phosphate/DNA precipitate was removed by washing three times with phosphate-buffered saline. Culture medium was added to the cells, and 48 h after transfection the medium was supplemented with 0.5 µg/ml puromycin (Life Technologies). Transfected cells were selected in 0.5 µg/ml puromycin for 7-8 weeks, after which resistant cell clones were picked and analyzed for TNF-- or IL-1-induced luciferase expression. One clone of HaCaT-pBIIX was chosen and used for the experiments as described below.

Luciferase Assay-- Cells were seeded in 24-round multiwell plates (2.8 × 10⁵ cells/well). Treatment of the cells was carried out 2 days after reaching confluency. Treated cells were washed two times with phosphate-buffered saline and lysed, and luciferase activities were determined using the Luciferase Reporter Assay System (Promega) and Turner Luminometer model TD-20/20 (Turner Designs) as described by the manufacturer.

Measurement of [³H]Arachidonic Acid Release-- Cells were seeded in 24-round multiwell plates (2.8 × 10⁵ cells/well). Two days postconfluency, cells were labeled for 16 h with [³H]AA (0.4 µCi/ml) in the culture medium containing 0.5% FCS. About 90% of the added [³H]AA were incorporated by this procedure. After labeling, the cells were washed two times with PBS containing fatty acid-free BSA (2 mg/ml) in order to remove unincorporated radioactivity. Cells were then allowed to equilibrate at 37 °C before the addition of reagents. Treatments were carried out in the presence of 0.5% FCS and fatty acid-free BSA (0.1 mg/ml) to avoid reesterification of released [³H]AA. After treatment, the culture medium was harvested and cleared of detached cells by centrifugation (300 × g, 10 min). Cellular release of [³H]AA was assessed by liquid scintillation counting. Adherent cells were dissolved in 1 M NaOH in order to determine incorporated [³H]AA in the cells by liquid scintillation counting. The results are given as released [³H]AA in the supernatant relative to [³H]AA incorporated into the cells.

Permeabilization with Streptolysin O (SLO)-- Permeabilization of cells with SLO was performed as described previously (43). In brief, cells were seeded in 24-round multiwell plates (2.8 × 10⁵ cells/well). Two days postconfluency, the cells were incubated with activated SLO (100 ng/400 µl/well) at 4 °C for 15 min in a buffer containing 150 mM K⁺-glutamate, 5 mM nitrilotriacetic acid, 0.5 mM EGTA, 0.2% BSA, and 10 mM PIPES, pH 7.2 (permeabilization buffer). The cells were washed three times with the permeabilization buffer and then warmed to 37 °C, incubated for an additional 15 min at 37 °C, washed three times with a buffer containing 150 mM KCl and 20 mM PIPES, pH 7.2, and washed two times with culture medium. The cells were then preincubated with GTPS or GDPS before the addition of cytokines as described. Similar results of GTPS and GDPS were obtained in intact cells.

Metabolic Labeling of HaCaT Cells with [³²P]Orthophosphate and Immunoprecipitation of Group IV cPLA₂-- Since we were not able to observe clear mobility shifts of cPLA₂ when phosphorylated (Figs. 6 and 7), we used an in vivo labeling procedure to monitor cPLA₂ phosphorylation. HaCaT cells were seeded in 100-mm plates in RPMI 1640 medium supplemented with 0.3 mg/ml glutamine, 0.1 mg/ml gentamicin, and 10% FCS (8 × 10⁶ cells/plate). Two days postconfluency, the cells were incubated in culture medium containing 0.5% FCS and 0.1 mg/ml BSA for 16 h. Cells were washed two times each with PBS and phosphate-free RPMI medium before incubation for 2 h in phosphate-free RPMI medium containing [³²P]orthophosphate (0.6 mCi/plate). Appropriate inhibitors was added to the medium containing carrier free [³²P]orthophosphate, and the incubation was continued for 45 min before the cells were treated with TNF- or IL-1 (10 ng/ml) for 1 h. The cells were then washed twice in ice-cold PBS, scraped in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 40 mM -glycerophosphate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 200 µM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride) and homogenized by six passes through a 26-gauge needle. After precipitating cell debris by centrifugation (300 × g for 10 min), two different anti-cPLA₂ antibodies were added to the supernatants. After overnight incubation, 30 µl of Protein G-Sepharose (Amersham Pharmacia Biotech) was added before further incubation for 90 min at 4 °C. The immunocomplexes were washed three times with a low salt buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Triton X-100, 2 mM EDTA, 2 mM EGTA, 0.1% SDS), three times with a high salt buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.2% Triton X-100, 2 mM EDTA, 2 mM EGTA, 0.1% SDS), and once in 10 mM Tris. Immunocomplexes were boiled in 35 µl of Laemmli dissociation buffer and subjected to 10% polyacrylamide-SDS gel electrophoresis, followed by electroblotting onto nitrocellulose membranes (85 V for 4 h in 20% ethanol, 190 mM glycine, 25 mM Tris). ³²P-Labeled phosphorylated cPLA₂ was visualized by digital imaging and quantification of ³²P (PhosphorImager; Molecular Dynamics, Inc., Sunnyvale, CA).

In order to relate amounts of [³²P]cPLA₂ to total cPLA₂ protein, Western blot analysis using the ECL detection system was performed. After visualization of [³²P]cPLA₂ by digital imaging, membranes were blocked for 2 h in TBST (TBS containing 0.25% Tween) supplemented with 2% milk powder. Membranes were incubated with anti-cPLA₂ antibodies (1:5000) for 12 h at 4 °C and washed three times with TBST before incubation at room temperature with a 1:2500 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Dako). After washing three times with TBST, membranes were developed with the ECL detection reagent (Amersham Pharmacia Biotech). Protein levels were quantified using NIH Image, version 1.61. After normalization to cPLA₂ protein levels, ³²P incorporation into cPLA₂ was expressed as a percentage relative to untreated cells.

Total RNA Extraction and RT-PCR Analysis-- Total RNA was isolated from 2 days postconfluent HaCaT cells in 60-mm culture dishes with Trizol (Life Technologies) according to the protocol of the manufacturer. RNA concentrations were spectrophotometrically determined at 260 nm. First strand cDNA synthesis was performed with 2 µg of total RNA using random hexamers as primers in a final volume of 20 µl (5 ng/µl random hexamers, 1 mM dNTPs, 2 units/µl RNasin, and 10 units/µl Moloney murine leukemia virus reverse transcriptase). The reaction was carried out at 37 °C for 60 min. cDNAs encoding human PLA₂ enzymes were amplified from 3-5 µl of the cDNA reaction mixture using specific gene primers. PCR reactions for group IIa, IV, V, -actin, and 5-LO were performed as previously described (44, 45). For group IId, VI, and X PLA₂, the primers synthesized according to known human cDNA sequences were the following: 5'-GTTCCTCAGCATGGAGCTC-3' (forward primer) and 5'-TCCGAGACTATATTGGAGG-3' (reverse primer) for group IId; 5'-TGACAATTCTCAGGTGCTGC-3' (forward primer) and 5'-TCTTTCCAGGGAGAAGGGAT-3' (reverse primer) for group VI; and 5'-GGTGCCTCCGGTGGCCCTGCAG-3' (forward primer) and 5'-GTTCTCTGCCGGTCCGCACAGGCTCTG-3' (reverse primer) for group X. The PCR consisted of 35 cycles of 50 s at 94 °C, 50 s at 60 °C, and 30 s at 72 °C, resulting in PCR amplification products of 339 base pairs (IId), 727 base pairs (VI), and 417 base pairs (X).

Cytotoxicity Assay-- Cytotoxicity induced by different agents was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye uptake assay (46). Cells (55 × 10³ cells/well) were seeded in 96-round multiwell plates. The cells were incubated in the presence or absence of inhibitors in 100 µl of complete medium for 1 h at 37 °C before the addition of TNF- or IL-1 to a 10 ng/ml final concentration and further incubation for 1 h at 37 °C. Thereafter, 25 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/ml) was added to each well, and the incubation was continued for 3 h at 37 °C before the addition of 100 µl of extraction buffer (20% SDS, 50% dimethylformamide). After overnight incubation at 37 °C, the OD at 570 nm was measured using a microplate reader (Titertek Multiskan PLUS).

Statistical Analysis-- All data are expressed as means ± S.D. of separate experiments. Differences between means were determined by Student's t test for unpaired samples, and those at p < 0.05 were considered significant. Asterisks (*) and  in the figures indicate that values are statistically significant from the reference values ( for *, and * for , respectively).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of PLA₂ Isoforms and 5-LO in Human HaCaT Keratinocytes-- Several isoforms of PLA₂ have been detected in mammalian tissues and cell lines, among which are the different mammalian secretory PLA₂s, group I, IIa, V, and X. Group IIa, IV, V, VI, and X have been shown to be important for AA release under various conditions and are believed to mediate inflammatory responses (47-49). The expression pattern of secretory PLA₂s vary between tissues and cell lines (4, 12). These PLA₂s may either be functionally redundant or exhibit yet undefined specific cellular functions. In order to determine which isoforms of PLA₂ are expressed in human HaCaT keratinocytes and hence may contribute to NF-B signaling, we performed RT-PCR analysis on total RNA using specific PLA₂ oligonucleotides. We found that HaCaT keratinocytes express snpPLA₂s group IIa, IId, V, and X in addition to the ubiquitously expressed groups IV and VI cPLA₂ (Fig. 1). In addition, HaCaT keratinocytes express 5-LO (Fig. 1), as has previously been shown by others (50, 51). We did not observe any change in mRNA expression levels of these enzymes after treatment with TNF- or LPS for 1 or 4 h (Fig. 1), which are the durations of treatment used in this study. Thus, the enzymes necessary for AA release and conversion through the 5-LO pathway are present in unstimulated HaCaT cells.

View larger version (55K): [in this window] [in a new window]   Fig. 1.   RT-PCR analysis of distinct PLA2 isoforms and 5-LO in the human HaCaT keratinocytes. HaCaT cells were cultured in 60-mm dishes for 2 days after reaching confluency. After stimulation of the cells for 1 or 4 h with TNF- (10 ng/ml) or LPS (1 µg/ml), total RNA was extracted with Trizol, transcribed, and amplified using primers specific for the given isoforms of PLA2 or 5-LO as described under "Experimental Procedures." Lane NC shows the negative control of RT-PCR as amplification in the absence of cellular RNA.

Inhibitors of Both Secretory and Cytosolic PLA₂ Reduce NF-B-driven Luciferase Expression-- We have previously shown, by electrophoretic mobility shift assays of HaCaT nuclear extracts, that inhibitors toward snpPLA₂ (LY311727 and 12- epi-scalaradial) and cPLA₂/iPLA₂ (AACOCF3, (all-Z)-1,1,1- trifluoro-6,9,12,15,18-heneicosapentaen-2-one, and MAFP) reduce cytokine-induced nuclear translocation of NF-B (41). For the purpose of further studying the role of PLA₂ enzymes in NF-B activation, we generated HaCaT stable transfectants containing the NF-B-driven luciferase reporter construct, pBIIX. Treatment of the stably transfected HaCaT-pBIIX cells with TNF- or IL-1 for 1-4 h enhanced NF-B-dependent luciferase expression 4-15-fold. None of the clones transfected with the control plasmid pfLUC displayed TNF-- or IL-1-sensitive luciferase expression (data not shown). To assess the role of PLA₂s in NF-B activation and to confirm previous results obtained with electrophoretic mobility shift assays, we pretreated HaCaT cells with the selective, active site-directed secretory group IIa PLA₂ inhibitor SB203347 (52, 53) or an inhibitor of cPLA₂/iPLA₂, MAFP (54) before the addition of TNF- or IL-1. This treatment reduced luciferase expression in a dose-dependent manner (Fig. 2), completely abolishing B-dependent transcription at higher concentrations of the snpPLA₂ inhibitor SB203347 (Fig. 2, A and C). SB203347 shows a 40-fold selectivity for sPLA₂ over cPLA₂ (52). Since the IC₅₀ value of SB203347 in acid extracts of PMN is 4.7 µM (52), we expect that cPLA₂ activity is unaffected by this snpPLA₂ inhibitor at the concentrations applied. Other structurally distinct snpPLA₂ inhibitors (LY311727, 12-epi-scalaradial, and BM16.2269) and other cPLA₂ inhibitors (compounds similar to AACOCF3 (55) developed in our laboratory)² also behaved similarly and resulted in dose-dependent reduction of cytokine-induced luciferase activity. A potential problem associated with the use of chemical inhibitors to study receptor-mediated signaling is that receptor protein expression may be affected in addition to the signaling downstream of the receptor. To examine this possibility, the expression level of p55 TNF- receptor in HaCaT cells treated for 3 h with 5 or 10 µM 12-epi-scalaradial was examined by flow cytometric analysis (using an antibody generated against p55). No changes in the expression level was observed (data not shown), ruling out the possibility that the observed reduction in TNF--induced NF-B activation is due to an effect on TNF- p55 receptor levels. None of the inhibitors exerted cytotoxic effects at the concentrations applied, as examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and inhibitors or vehicles alone had no effect on B-dependent transcription (data not shown). The inhibitor MAFP is active against both group IV cPLA₂ and VI iPLA₂. However, we find that bromoenol lactone, which has a 1000-fold selectivity for inhibition of iPLA₂ versus cPLA₂, fails to inhibit cytokine-induced AA release. In addition, oleic acid release is not stimulated in HaCaT cells treated with cytokines, as would be expected if iPLA₂ (showing no fatty acid selectivity) was involved. This indicates that the inhibitory effect of MAFP on NF-B activation reflects the inhibition and involvement of cPLA₂, rather than iPLA₂, in this process. High concentrations of MAFP have been reported to stimulate phosphorylation of Jun N-terminal kinase/stress-activated protein kinases, p38, and p42/44 MAP kinases (56). We investigated lysates from HaCaT cells treated with 25 µM MAFP for up to 6 h by Western blot analysis using phosphorylation state-specific antibodies but did not observe any activation of these kinases.³ Hence, this reported effect of MAFP is not troublesome in our studies. Thus, these results clearly demonstrate that inhibitors toward either snpPLA₂ or cPLA₂ are effective in attenuating TNF-- and IL-1-induced NF-B activation in HaCaT keratinocytes, thus supporting the involvement of both snpPLA₂ and cPLA₂ in signaling preceding NF-B activation.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effect of PLA2 inhibitors on TNF-- and IL-1-induced NF-B-dependent transcription. HaCaT stable transfectants containing the NF-B-driven luciferase reporter plasmid pBIIX (HaCaT-pBIIX) were preincubated with PLA2 inhibitors for 45 min before stimulation with 10 ng/ml TNF- or IL-1 for 1 (B and D) or 2.5 h (A and C). The results are normalized to show -fold induction and are representative of four independent experiments. Data represent mean ± S.D. of three or six determinations. A, IL-1/SB203347; B, IL-1/MAFP; C, TNF-/SB203347; D, TNF-/MAFP.

Human Group IIa snpPLA₂ Augments IL-1-induced NF-B Activation-- The finding that inhibitors of snpPLA₂ reduce NF-B activation in response to TNF- or IL-1 suggests a role for snpPLA₂ in this process. In order to examine whether extracellular addition of human group IIa snpPLA₂ could activate NF-B, we added purified group IIa snpPLA₂ to cells alone or in combination with IL-1. Human recombinant group IIa snpPLA₂ at 1 µg/ml (which is within the range of physiologic concentration), did (although moderately) amplify the effect of IL-1 on NF-B activation (Fig. 3A). The effect of snpPLA₂ was slightly lower than that of 10 µM AA. However, like AA, snpPLA₂ (1 µg/ml) was unable to activate NF-B when used as sole agonist (Fig. 3C), thus showing that snpPLA₂ participates in NF-B induction but requires additional signaling from cytokine receptors. This finding together with the pronounced inhibitory effect of SB203347 suggests the participation of snpPLA₂ in cytokine-induced signaling to NF-B.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of exogenously added group IIa snpPLA2 or AA added in the presence of an snpPLA2 inhibitor on NF-B activation. HaCaT cells were grown to 2 days postconfluency. A, purified recombinant human group IIa snpPLA2 (1 µg/ml) or AA (10 µM) was added alone or in combination with IL-1 (10 ng/ml; 1 h). B and C, cells were pretreated for 45 min with SB203347 and treated with TNF- (10 ng/ml) or IL-1 (10 ng/ml) either alone or in combination with AA (10 µM) for 1.5 h. The data are representative of three independent experiments and are expressed as mean ± S.D. of triplicate determinations.

Exogenously Added AA Reverses the Effect of SB203347 on NF-B Activation-- To elucidate if the NF-B inhibition observed with PLA₂ inhibitors was due to reduced AA levels and, hence, if AA is the effective NF-B signaling component produced by PLA₂ activity, we examined the effect of the exogenous addition of AA on NF-B activation in the presence of SB203347. Indeed, we found that AA (10 µM) could overcome the inhibitory effect of SB203347 (Fig. 3, B and C), thus implying that AA participates in cytokine-induced activation of NF-B. In contrast, the same concentration of AA did not overcome the effect of MAFP on NF-B activation (data not shown). This may indicate that PLA₂ metabolites other than or additional to AA-derived metabolites may be effective mediators released by the activity of cPLA₂ (e.g. platelet-activating factor or lysophosphatidylcholine) or that MAFPs like AACOCF3 inhibit AA-metabolizing enzymes (57). AA added alone did not stimulate NF-B-dependent transcription, but it produced a synergistic effect when added together with either TNF- or IL-1 (data not shown). These results identify AA or AA-generated metabolites as molecular components contributing to cytokine-induced NF-B activation in keratinocytes.

5-LO Metabolites Mediate TNF-- and IL-1-elicited NF-B Activation-- AA is metabolized to leukotrienes and certain mono-, di-, and trihydroxy acids by the LO enzymes. To examine if the 5-LO pathway is of importance for PLA₂-mediated NF-B activation, we analyzed the effects of two different inhibitors of this pathway on NF-B activation. L-655,238 (a selective inhibitor of the 5-LO-activating protein FLAP) and LY255283 (an LTB₄ receptor antagonist (58)) reduced cytokine-induced NF-B activation in a dose-dependent manner (Fig. 4, A and B). To further examine the involvement of LTB₄ in activation of NF-B, we added LTB₄ to cells treated with the LTB₄ receptor antagonist or the 5-LO inhibitor L-655,238 to see whether exogenously added LTB₄ could rescue NF-B activation. LTB₄ partially reversed the NF-B inhibitory effect of L-655,238 in response to either TNF- (55% reversal) or IL-1 (80% reversal; Fig. 4, C and D), indicating involvement of other 5-LO metabolites in addition to LTB₄. In contrast to this partial effect, the inhibitory effect of the LTB₄ receptor antagonist LY255283 was completely relieved by the exogenous addition of LTB₄ (Fig. 4E). The sole addition of LTB₄ did not affect B-dependent transcription (Fig. 4E). The nonhydrolyzable GTP and GDP analogs, GTPS and GDPS, resulted in increased or reduced TNF--stimulated NF-B activation, respectively (Fig. 4F), suggesting participation of G-proteins in this process. This G-protein-mediated effect on NF-B could in part be due to leukotrienes, e.g. LTB₄, acting through their G-protein coupled receptors, although the possible involvement of other G-proteins would also contribute to this effect, e.g. Rac or Rho (59). Nevertheless, these results show that G-proteins and metabolites of 5-LO (specifically LTB₄, as indicated by the ability of the LTB₄ receptor antagonist to interfere with NF-B activation) contribute to TNF-- and IL-1-stimulated NF-B activation in human keratinocytes.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Involvement of 5-LO metabolites and G-proteins in cytokine-stimulated NF-B activation. HaCaT-pBIIX was pretreated for 45 min with various concentrations of L-655,238 (A) or LY255283 (B) before the addition of TNF- or IL-1 (10 ng/ml; 1 h). C-E, cells were pretreated 45 min with L-655,238 (7.5 µM; C and D) or LY255283 (10 µM; E) before the addition of LTB4 (10 nM) in combination with TNF- or IL-1 (10 ng/ml; 1 h). F, SLO-permeabilized cells were treated for 10 min with GTPS (50 µM) or GDPS (50 µM) prior to the addition of TNF- (10 ng/ml; 1 h). The data are representative of three independent experiments and are expressed as mean ± S.D. of triplicate determinations.

AA and 5-LO Metabolites Modulate Cytokine-stimulated Cellular AA Release-- Several investigators have suggested a sequential interplay between snpPLA₂ and cPLA₂ in response to different stimuli (20, 60, 61). Our present results show that selective inhibitors toward either snpPLA₂ or cPLA₂ attenuate TNF-- and IL-1-induced NF-B activation. Furthermore, both snpPLA₂ and cPLA₂ inhibitors reduce cytokine-stimulated AA release from HaCaT cellular membranes in a dose-dependent manner, resulting in complete inhibition of AA liberation at 1 µM MAFP or 1 µM AKH217,² 80% inhibition at 15 µM SB203347, and 75% inhibition at 5 µM 12-epi-scalaradial (41, 62). In addition, generation of LTB₄ was reduced by 60% in the presence of 20 µM LY311727 or 5 µM 12-epi-scalaradial and by 80% in the presence of 5 µM AACOCF3 (62). The participation of both PLA₂s in cellular AA release and NF-B activation could imply that the two PLA₂s act in a sequential manner in response to cytokines, with subsequent NF-B activation. In order to explore this issue, we examined if exogenously added AA could rescue inhibition of cellular AA release in the presence of PLA₂ inhibitors. AA (10 µM) was able to completely overcome the effect of both snpPLA₂ and cPLA₂ inhibitors on IL-1-stimulated cellular AA release (Fig. 5A). The effect of AA on PLA₂-inhibited TNF--stimulated NF-B activation was identical to that observed for IL-1 (data not shown). One problem associated with the approach of adding "cold" AA is that "cold" AA may reduce reincorporation of [³H]AA, which would lead to increased levels of [³H]AA measured extracellularly. However, since "cold" AA does not significantly increase the release of [³H]AA as seen in Fig. 5A, the effect of cold AA in the presence of SB203347 on [³H]AA stimulation arise from "adding back" an inhibited component of the IL-1-induced signaling. Hence, this demonstrates that both snpPLA₂ and cPLA₂ contribute to AA signaling in HaCaT keratinocytes. In addition, since the inhibitory effect of both snpPLA₂ and cPLA₂ inhibitors on [³H]AA release is relieved by excess AA, we may suggest that snpPLA₂ and cPLA₂ upon cytokine stimulation act in a sequential manner, possibly in a positive feedback model. The AA metabolites LTB₄, 5-oxoeicosatetraenoic acid, and 5-HETE has been found to activate cPLA₂ in neutrophils (63). We observed that the 5-LO inhibitor L-655,238 reduced cellular AA release by 85% in response to cytokines and that this effect was partially reversed by LTB₄ (Fig. 5B). Likewise, the nonhydrolyzable GTP and GDP analogs, GTPS and GDPS, exerted stimulatory or inhibitory effects on cytokine-inducible cellular AA release (Fig. 5C). The latter illustrates the participation of G-proteins in cytokine-stimulated activation of PLA₂, which could possibly be mediated through G-protein-coupled leukotriene (e.g. LTB₄ receptors). Thus, we may suggest that a sequential interplay between snpPLA₂ and cPLA₂ occurs in response to cytokines and that LTB₄ in this setting modulates cellular AA release.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   AA, 5-LO metabolites, and G-proteins contribute to cytokine-induced cellular [3H]AA liberation. A, postconfluent HaCaT cells were preincubated for 45 min with SB203347 (10 µM) or MAFP (10 µM) prior to a 1-h treatment with IL-1 (10 ng/ml) either alone or in combination with AA (10 µM). B, cells were treated with L-655,238 (5 µM) before the addition of LTB4 (10 nM) and TNF- (10 ng/ml; 1 h). C, SLO-permeabilized cells were treated for 10 min with GTPS (50 µM) or GDPS (50 µM) prior to a 1-h treatment with TNF- (10 ng/ml). The results are representative of three independent experiments, and data represent mean ± S.D. of triplicate determinations.

Cytokine-induced Phosphorylation of cPLA₂ Is Mediated by 5-LO Activity-- Phosphorylation of cPLA₂ is important for its activation and AA-releasing activity in response to cytokines (64-66). The results presented above show that cellular release of AA is reduced by 5-LO inhibitors. To explore if this reduced AA-release correlated with reduced phosphorylation of cPLA₂, we analyzed by in vivo phosphorylation experiments if cPLA₂ phosphorylation was changed in the presence of 5-LO inhibitors. HaCaT cells were metabolically labeled with [³²P]orthophosphate and pretreated with inhibitors before the addition of TNF- or IL-1 and immunoprecipitation of cPLA₂. Phosphorylation of cPLA₂ was increased 2-4-fold in the presence of TNF- or IL-1 (Fig. 6). The observed cPLA₂ phosphorylation in response to cytokines could possibly be brought about in a p38 kinase-dependent manner causing phosphorylation on both Ser-505 and Ser-727, as recently reported to occur in response to interferon- or A23187 (67, 68). We found that incorporation of [³²P]phosphate into cPLA₂ was strongly reduced in the presence of two different 5-LO inhibitors, the LTB₄ receptor antagonist and the nonselective LO inhibitor nordihydroguaiaretic acid (Fig. 6). Cellular AA release was also reduced under the same conditions (Fig. 5B, data not shown), and we may therefore suggest that the 5-LO pathway is involved in cytokine-evoked cPLA₂ phosphorylation and activation.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   LTB4 and 5-LO activity are implicated in TNF-- and IL-1-induced phosphorylation of cPLA2. HaCaT cells were metabolically labeled with [32P]orthophosphate for 2 h; incubated with nordihydroguaiaretic acid (NDGA; 10 µM), L-655,238 (10 µM), MK-886 (10 µM), or LY255283 (10 µM) for 45 min; and treated for 45 min with 10 ng/ml TNF- (upper panels) or IL-1 (lower panels). Cytosolic PLA2 was immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis and electroblotting. Incorporated 32P radioactivity was quantified by 32P digitalization using a PhosphorImager (Molecular Dynamics), normalized to cPLA2 protein levels in separate incubations (indicated by cPLA2), and are expressed as percentage of control. The experiment was repeated two times with similar results.

Catalytic Activity of snpPLA₂ Is Necessary for TNF-- and IL-1-induced Phosphorylation of cPLA₂-- Following up on the findings by Wijkander et al. (63) that secretory pancreatic PLA₂ induces phosphorylation of cPLA₂, we wanted to investigate if snpPLA₂ precedes cPLA₂ phosphorylation in response to TNF- and IL-1, leading to NF-B activation. Therefore, we examined whether inhibitors toward snpPLA₂ affected cytokine-induced phosphorylation of cPLA₂ as measured by metabolic labeling of HaCaT keratinocytes. A strong, almost complete reduction in incorporation of [³²P]phosphate into cPLA₂ was observed in cells treated with two various snpPLA₂ inhibitors (Fig. 7). Cellular AA release was attenuated by 75-85% by these inhibitors (Fig. 5A; Ref. 41). Hence, these results show that the catalytic activity of snpPLA₂ is necessary for TNF-- and IL-1-induced phosphorylation and activation of cPLA₂, in this way contributing to maximal NF-B activation.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   snpPLA2 regulates TNF-- and IL-1-induced phosphorylation of cPLA2. HaCaT cells were metabolically labeled with [32P]orthophosphate for 2 h, incubated with SB203347 (15 µM) or 12-epi-scalaradial (10 µM) for 45 min and treated for 45 min with 10 ng/ml TNF- or IL-1. Cytosolic PLA2 was immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis and electroblotting. Incorporated 32P radioactivity was determined, normalized, and expressed as described in the legend to Fig. 6. The experiment was repeated three times with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study suggests the existence of a functional link between snpPLA₂ and cPLA₂, conveyed by 5-LO metabolites, modulating TNF-- and IL-1-stimulated NF-B activation. These results also provide a molecular explanation to the participation of both secretory and cytosolic PLA₂s in NF-B activation. Several lines of evidence support the involvement of both PLA₂s through a positive feedback loop in which snpPLA₂, through 5-LO metabolites, modulates phosphorylation of cPLA₂: 1) inhibitors of both snpPLA₂ and cPLA₂ separately block NF-B activation; 2) the addition of group IIa snpPLA₂ moderately amplifies cytokine-stimulated NF-B activation; 3) inhibitors of the 5-LO pathway and an LTB₄ receptor antagonist partially prevent cytokine-stimulated NF-B activation; 4) 5-LO inhibitors attenuate cytokine-evoked cellular AA release and cPLA₂ phosphorylation; and 5) snpPLA₂ inhibitors abolish AA release and cytokine-induced phosphorylation of cPLA₂. In our experiments, we have used several structurally and mechanistically different inhibitors against snpPLA₂ (SB203347 and 12-epi-scalaradial) and 5-LO enzymes (L-655,238 and MK-886) as well as the LTB₄ receptor antagonist LY255283. In addition, in a previous study, we confirmed that even another snpPLA₂ inhibitor (the active site-directed inhibitor LY311727; Ref. 69) and several other cPLA₂ inhibitors (e.g. the trifluoromethylketones AACOCF3 and (all-Z)-1,1,1-trifluoro-6,9,12,15,18-heneicosapentaen-2-one) reduced nuclear translocation of NF-B in response to TNF- and IL-1 (41). In the previous study, we also confirmed that the inactive cPLA₂ inhibitor analogues AACOCH3 and (all-Z)-1,1,1-trifluoro-6,9,12,15,18-heneicosapentaen-2-ol had no effect on cytokine-induced NF-B activation and ensuing inhibitor specificity.

In this study, we found that exogenously added AA was able to restore cytokine-induced NF-B activation in cells treated with an inhibitor toward snpPLA₂, thus identifying that snpPLA₂-generated AA yields active NF-B signaling. Also, exogenously added AA reversed the effect of snpPLA₂ and cPLA₂ inhibitors on cellular [³H]AA release. Thus, AA metabolites, rather than other PLA₂-generated products such as lysophosphatidylcholine and platelet activating factor, are prominent AA- and NF-B-modulatory components in HaCaT cells. The inhibitory effect of the LTB₄ receptor antagonist on NF-B activation was completely overcome by the addition of LTB₄ (Fig. 4E), while that of the 5-LO inhibitor L-655,238 was only partially relieved after the addition of LTB₄ (Fig. 4, C and D). This could indicate that other 5-LO metabolites than LTB₄ are also functional in cytokine-induced NF-B activation. LTB₄ and 12-HETE are potent chemoattractants, e.g. for lymphocytes in human skin (70, 71), possibly connected to the participation of LTB₄ in NF-B activation and expression of adhesion molecules.

Based on our results, we propose a model for the involvement of snpPLA₂, 5-LO/LTB₄ and cPLA₂ in TNF-/IL-1-induced NF-B activation (Fig. 8). Interaction of TNF-/IL-1 with their respective receptors leads to signal transduction, resulting in activation of the MAP3K family (including NIK) and the IB kinases (IKK and -), leading to degradation of the IB inhibitor and activation of NF-B (31, 72). In parallel, TNF-/IL-1 may induce snpPLA₂ activity, resulting in AA/LTB₄ mobilization and subsequently cPLA₂ phosphorylation. In HaCaT keratinocytes, snpPLA₂ activity is found extracellularly in the medium and associated with the cellular surface in resting cells (62); thus, synthesis (see Fig. 1) or secretion of snpPLA₂s may not be necessary at this step (discussed below). Subsequently, snpPLA₂s may release AA, which through conversion to 5-LO metabolites induces kinase cascades that mediate phosphorylation and activation of cPLA₂. Regarding kinases promoting cPLA₂ phosphorylation and activation in response to TNF-/IL-1, we find that atypical PKC/ and p38, but not p42/44, MAP kinases are involved in snpPLA₂/LTB₄-mediated cPLA₂ and NF-B activation.³ The participation of p38 or p42/44 MAP kinases in TNF-/IL-1/sPLA₂/LTB₄-mediated cPLA₂ activation has recently been suggested (65, 73-75). Moreover, p38 MAP kinase and PKC/ have been implicated in TNF-/IL-1-stimulated NF-B activation (76-78). Thus, snpPLA₂/leukotrienes may contribute to NF-B activation by enhancing activity of distinct kinase cascades acting to phosphorylate cPLA₂ and to increase phosphorylation-mediated events leading to increased NF-B-dependent transcription. The leukotriene-evoked kinase cascades could couple to the known NF-B signaling upstream of the recently identified NIK (31) and the IB-kinases (IKK and -), e.g. via interaction with the RIP-associated adapter protein p62 as recently reported for PKC- (79) or downstream RIP. Alternatively, the PLA₂-generated metabolites may participate in signaling pathways that are parallel to the classical NIK-IKK-IB pathway, since parallel activation cascades through MAP kinase kinase kinases (TPL-2, TAK1, MEKK2, and MEKK3) are also able to activate NF-B (32, 36, 80). We found that PLA₂ activity alone (as examined by the addition of AA, LTB₄, or human group IIa snpPLA₂ separately or in combinations) is not sufficient to induce NF-B activation. Hence, PLA₂ activity contributes to NF-B activation but needs additional signals from TNF-/IL-1-receptors, probably initiating activation of the IKKs.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Proposed model for involvement of snpPLA2, LTB4, and cPLA2 in NF-B activation from TNF- and IL-1 receptors. Stimulation of TNF- and IL-1 receptors leads to signal transduction, resulting in activation of NIK, IKK, and NF-B. In parallel, by yet undefined mechanisms, TNF- or IL-1 initiates AA release by snpPLA2, generation of 5-LO metabolites (LTB4), and activation of cPLA2 through membrane-bound leukotriene (LT) receptors. Activation of leukotriene receptors may induce kinase cascades, which could interact with the NIK cascade, or other parallel MAP3K cascades, leading to phosphorylation and degradation of IB.

We found that the snpPLA₂ inhibitor SB203347 only at elevated concentrations (40-60 µM) was able to completely block NF-B activation, whereas the cPLA₂ inhibitor MAFP blocked NF-B activation by 55% at 25 µM. Thus, although snpPLA₂, AA, or LTB₄ are unable by themselves to activate NF-B, we propose that the PLA₂-generated positive feedback loop mediated by LTB₄ and other 5-LO metabolites may modulate NF-B activation in response to cytokines. In contrast, cytokine-induced cellular AA release was completely blocked by snpPLA₂ and cPLA₂ inhibitors, showing that PLA₂ enzymes are the main contributors to cytokine-elicited AA-release in these cells. Together these findings indicate a partial contribution of AA, 5-LO, and snp/cPLA₂s to NF-B activation. The involvement of this functional link between PLA₂s and the PLA₂/LTB₄-mediated positive feedback loop in NF-B activation may be dependent on the cellular presence of various PLA₂s and 5-LO.

An obvious requirement for our model for PLA₂-mediated NF-B activation is that the necessary PLA₂s and 5-LO are expressed. RT-PCR analysis (Fig. 1) showed that 5-LO in addition to PLA₂ groups IIa, IId, IV, V, VI, and X are expressed in unstimulated postconfluent keratinocytes. Other investigators have also found expression of both 5-LO and the 5-LO-activating protein FLAP, detected as mRNAs and proteins, in post-confluent HaCaT cells. 5-LO undergoes Ca²⁺-dependent translocation to the nuclear membrane upon inflammatory activation (81), and may also be regulated in a p38 MAP kinase-dependent manner (82). TNF- and IL-1 increase intracellular calcium levels (83), which could thus contribute to 5-LO activation. However, 5-LO has been found to be active per se in unstimulated post-confluent HaCaT cells (84). Thus, the enzymatic machinery necessary for immediate production of AA-generated 5-LO metabolites in response to cytokines is present in human HaCaT keratinocytes. Whether all snpPLA₂ enzymes expressed in HaCaT cells participate in TNF--/IL-1-induced AA release, cPLA₂ phosphorylation, and NF-B activation is uncertain. Group X snpPLA₂ may act directly on undisturbed cell membranes (49, 85, 86). Although group V snpPLA₂ can act directly on undisturbed neutrophils (75), group IIa, IId, and V snpPLA₂s are dependent on cellular activation to hydrolyze membranes of adherent cells (49, 85). We have previously shown that snpPLA2 activity is associated with HaCaT cells as well as in the culture medium (62). Heparin treatment of unstimulated HaCaT cells in order to detach extracellular proteoglycan-bound snpPLA₂ isoforms (groups IIa, IId, and V) led to increased snpPLA₂ enzyme activity detected in the culture medium, indicating that enzymatically active proteoglycan-bound snpPLA₂ isoforms are already associated with the cellular surface in unstimulated cells. Furthermore, we were not able to detect enhanced sPLA₂ activity in the medium after TNF-/IL-1 treatment, even in the presence of heparin. Hence, according to existing models and our previous results (62), group X snpPLA₂ may continuously degrade membrane phospholipids in unstimulated as well as stimulated HaCaT cells, whereas proteoglycan-bound snpPLA₂ isoforms (groups IIa, IId, and V) may participate in AA release upon TNF-/IL-1 treatment. The inhibitors against snpPLA₂ used in this study are designed against the group IIa enzyme. The group IId, V, and X enzymes are more closely related to group IIa than group I PLA₂, and we cannot exclude the possibility that the inhibitors also affect these enzymes. Thus, which snpPLA₂ isoforms participate in cytokine-induced AA and NF-B activation in HaCaT cells is uncertain and should be examined in future studies.

Our results illustrate that snpPLA₂ plays a regulatory role in cytokine-induced cPLA₂ phosphorylation and NF-B activation in human keratinocytes. In contrast, only cPLA₂ was found to contribute to activation of NF-B by LPS in human leukocytes (87). This difference may indicate cell type-specific PLA₂ signaling, which could be due to specific expression of PLA₂s and AA-metabolizing enzymes, as illustrated for IL-1-mediated 5-LO and NF-B activation (88, 89). Recent work by several laboratories has highlighted the role of snpPLA₂ in inflammatory mechanisms. Murakami et al. (90) found that heparin-binding group IIa and V sPLA2s amplify IL-1-induced PGE₂ synthesis, whereas Bidgood et al. (91) has shown that group IIa snpPLA₂ amplifies TNF--stimulated prostaglandin production in rheumatoid synoviocytes. Furthermore, group IIa snpPLA₂ enhances LPS-induced iNOS expression in an NF-B-dependent manner (92) and synergizes with PAF-induced Mac-I adhesion molecule expression, utilizing a 5-LO pathway (93). In agreement with our findings, snpPLA₂ alone had no significant effects in these studies but acted synergistically. In this context, our results describe a novel mechanism through which snpPLA₂ may contribute to cytokine-induced inflammation, thus as a regulator of cPLA₂ and NF-B activation. We have previously shown that snpPLA₂ as well as cPLA₂ inhibitors block TNF--stimulated expression of the adhesion molecule ICAM-1 in keratinocytes (41), illustrating the physiological significance of our findings. The snpPLA₂ is a proinflammatory enzyme that is highly elevated in circulation and locally in tissues in association with pathological conditions, and increased levels of snpPLA₂ may modulate the extent of immune mediated responses, e.g. as an upstream regulator of cPLA₂ and NF-B activation. Our results suggest an autocrine function for snpPLA₂ and AA-generated 5-LO metabolites in NF-B activation in keratinocytes. Therefore, our results may have relevance to the understanding of the shift between acute and chronic inflammation, in that a leukotriene autocrine effect may lead to sustained NF-B activation. Hence, therapeutic strategies aimed at inhibiting PLA₂ enzymes should be effective in treating skin inflammatory disorders.

	ACKNOWLEDGEMENTS

We thank Dr. Astrid Lægreid (Department of Physiology and Biomedical Engineering, Norwegian University of Science and Technology) for critical reading of the manuscript and professor Terje Espevik (Institute of Cancer Research, Norwegian University of Science and Technology) for generously providing the TNF- used in this study. We also highly appreciate receiving the LTB₄ receptor antagonist LY255283 (from Dr. David K. Herron), the snpPLA₂ inhibitor SB203347 (from Dr. Lisa Marshall), and cPLA₂ polyclonal antibodies (from Dr. Christina Leslie and Dr. Ruth Kramer).

	FOOTNOTES

^* This work was supported by a grant from the University of Trondheim (to M. W. A.), Norwegian Research Council Grant 123641/310, and Norwegian Cancer Society Grant A00038/003.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. Tel.: 47-73-551278; Fax: 47-73-596100; E-mail: Marit.W.Anthonsen@chembio.ntnu.no.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M008481200

² W. Sjursen, L. Skattebøl, and B. Johansen, manuscript in preparation.

³ M. W. Anthonsen, C. Solberg, and B. Johansen, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PLA₂, phospholipase A₂; cPLA₂, cytosolic phospholipase A₂; snpPLA₂, secretory nonpancreatic phospholipase A₂; AACOCF3, arachidonyl trifluoromethylketone; MAFP, methyl arachidonyl fluorophosphonate; HETE, hydroxyeicosatetraenoic acid; LO, lipoxygenase; LTB₄, leukotriene B₄; IL, interleukin; TNF, tumor necrosis factor; TNFR, TNF receptor; NIK, NF-B-inducing kinase; IKK, IB kinase; MAP, mitogen-activated protein; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1; RIP, receptor-interacting protein; BSA, bovine serum albumin; AA, arachidonic acid; FCS, fetal calf serum; PIPES, 1,4-piperazinediethanesulfonic acid; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; iPLA₂, calcium-independent PLA₂; sPLA₂, secretory PLA₂; GTPS, guanosine 5'-3-O-(thio)triphosphate; GDPS, guanyl-5'-yl thiophosphate; LPS, lipopolysaccharide; SLO, streptolysin O.

	REFERENCES

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