THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 32, Issue of November 15, pp. 19616-19823,199O Printed in U.S.A.

Correlation between Expression of &Lipoxygenase-activating Protein, 5=Lipoxygenase, and Cellular Leukotriene Synthesis*

(Received for publication, June 29, 1990)

Gregory K. Reid& Stacia KargmanS, Philip J. VickersS, Joseph A. Manein& Claire LL?veillb**, Diane EthierS, Douglass K. Miller& John W. Gillardn, Richard A. F. Dixon11 , and Jilly F. Evans+ From the $Departments of Pharmacology and !lMedicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, H9R 4P8, Canada, SDepartment of Biochemistry and Molecular Biology, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065, and J/Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486

Previous studies involving transfection of cDNAs for 5-lipoxygenase-activating protein (FLAP) and S-li- poxygenase into osteosarcoma cells have shown that both these proteins are essential for leukotriene syn- thesis (Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W., and Miller, D. K. (1990) Nature 343, 282-284). In the present study we show that FLAP is present in a va- riety of cells known to produce leukotrienes, but is absent from a number of cells which do not synthesize leukotrienes. Furthermore, differentiation of the hu- man promyelocytic HL-60 cell line towards granulo- cytic cells following exposure to dimethylsulfoxide is associated with the concurrent induction of both FLAP and 5-lipoxygenase and an increased capacity to syn- thesize leukotrienes. Cellular leukotriene synthesis in this system is functionally dependent on FLAP as shown by its inhibition by the leukotriene biosynthesis inhibitor MK-886, a compound which specifically binds to FLAP.

Leukotrienes are arachidonic acid metabolites formed by leukocytes and other myeloid cells following immunologic and inflammatory stimuli (l-4). Due to the biological properties of leukotrienes, which include enhancement of leukocyte chemotaxis and chemokinesis, production of vascular perme- ability changes, pulmonary constriction, mucous hypersecre- tion, vasoconstriction, and modulation of a number of immune responses, considerable effort over the past decade has been directed toward the development of inhibitors of leukotriene formation (5-8). Most inhibitors have been targeted against 5-lipoxygenase, the first enzyme in the leukotriene biosyn- thetic pathway, which catalyses both the oxygenation of ara- chidonic acid to 5-HPETE’ and the subsequent dehydration

* 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 USC. Section 1734 solely to indicate this fact.

** Present address: Immunology Laboratory, Ecole de Medecine Dentaire, Universite Laval, Quebec GlK 7P4, Canada.

1 The abbreviations used are: &HPETE, 5(S)-hydroperoxy- 6,8,11,14-eicosatetraenoic acid; LTAa, 5,6-oxido-7,9,11,-14-eicosate- traenoic acid; HPLC, high performance liquid chromatography; 5- HETE, 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid; LTB,, 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid; LTCd, 5(S)-hy- droxy-6(R)-S-glutathionyl-7,9(E)-11,14(Z)-eicosatetraenoic acid; LTD,, 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9(~)-11,14(Z)-eicosa- tetraenoic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MK-886, formerly designated as L-663,536 (3-[1- ~-chlorobenzyl)-5-isopropyl-3-tert-butylthioindol-2-yl]-2,2-dime-

of 5-HPETE to the unstable epoxide LTA,. Most of the known 5-lipoxygenase inhibitors appear to function through a redox mechanism potentially involving the generation of radical species which may be associated with drug toxicity (8- 10). Recently, a novel class of indole leukotriene biosynthesis inhibitors has been described which do not act directly on 5- lipoxygenase but rather prevent its membrane association and cellular activation (11, 12). This class is exemplified by MK- 886, a compound which inhibits leukotriene biosynthesis in all inflammatory cells so far examined (11-14). The specific protein target of MK-886 has been identified in neutrophils as a membrane-bound 18-kilodalton protein termed 5-lipox- ygenase-activating protein (FLAP) (15). Osteosarcoma cells cotransfected with FLAP and 5-lipoxygenase synthesized leu- kotrienes in response to ionophore challenge in contrast to cells transfected with FLAP or 5-lipoxygenase alone (16). Therefore, both FLAP and 5-lipoxygenase are necessary for cellular leukotriene synthesis.

In the present study we have investigated the occurrence of FLAP by immunoblot analysis in a variety of cells and have shown a positive relationship between the expression of FLAP and the ability of cells to produce leukotrienes. The relation- ship between FLAP, 5-lipoxygenase, and leukotriene synthe- sis has also been investigated during cellular differentiation of the human promyelocytic cell line HL-60 (17). The HL-60 cell line has been extensively characterized for its ability to differentiate towards different myeloid cell types in response to specific stimuli (l&19). In response to Me&O, HL-60 cells acquire morphological and functional characteristics of ma- ture neutrophils, including an increase in 5-lipoxygenase mRNA (20), an increased capacity to synthesize leukotrienes (21, 22) and an appearance of specific LTBl binding sites (23). We demonstrate here that the increased leukotriene synthetic ability of MezSO-differentiated HL-60 cells is as- sociated with the induction of both FLAP and 5-lipoxygenase. We show that leukotriene synthesis in the Me$O differen- tiated HL-60 cells is abolished by preincubation of the cells with MK-886, a compound that specifically binds to FLAP.

MATERIALS AND METHODS

Cell Culture-HL-60 cells, originally obtained from the American Type Culture Collection, were grown at 37 “C in Iscove’s modified Dulbecco’s medium, supplemented with 20% heat-inactivated fetal bovine serum (Sigma), 50 units/ml penicillin, 50 rg/ml streptomycin,

thylpropanoic acid); FLAP, 5-lipoxygenase-activating protein; Me&O. dimethvl sulfoxide: 100% 100.000 X P supernatant; lOOP, 100~006 X g pellet; ‘25VI-L:669,083, 3-i1-(4-hidrody-3-iodophenyl): methyl]-3-(4-azidophenylsulfonyl)-5-isopropylindol-2-yl)-2,2-dime- thylpropionic acid, where iodo is the radionuclide form of ‘?odine.

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Correlation between Expression of FLAP, 5-Lipoxygenase, and Cellular Leukotriene Synthesis 19819

and 2 mM L-glutamine (Flow Laboratories, Inc., McLean, VA) under an atmosphere of 6% CO1. To induce myeloid differentiation, cells were subcultured to 0.2 x lo6 cells/ml and exposed to 1.3% Me2S0 (Sigma) for up to 5 days. Morphological differentiation of cells was confirmed by staining with May-Grunwald-Giemsa stain. After ex- posure to Me$O for 5 days approximately 85% of cells morphologi- cally appeared to be mature granulocytes. Control cultures showed approximately 10% spontaneously differentiated cells, based on this morphological staining.

Cell Lysis and Subcellular Fractionation-Frozen cell pellets were thawed on ice in homogenization buffer (50 mM potassium phosphate buffer, pH 7.1, 0.1 M NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 60 pg/ml soybean trypsin inhibitor) at a density of 6.6 x lo7 cells/ml and sonicated at 4 “C by three 20-s bursts using a Cole-Parmer Instrument Co. (Chicago, IL) 4710 series ultrasonic homogenizer set at 75% duty cycle and power level 3. Sonicated cells were centrifuged at 10,000 x g for 15 min at 4 “C, and the resulting supernatant was recentrifuged at 100,000 X g for 60 min at 4 “C. The 100,000 x g supernatants (100s) were collected and the pellets (1OOP) rinsed with homogenization buffer and then resuspended in fresh homogenization buffer (i/3 volume of the original supernatant) using a Potter-Elvehjem homogenizer. Samples were immediately analyzed for 5lipoxygenase activity (100s) or lz51-L- 669,083 labeling (1OOP) or processed for immunoblot analysis (100s and 1OOP) as described below.

SDS-PAGE and Immunoblot Analysis of 5-Lipoxygenase and FLAP-loos and 1OOP samples were mixed with i/z-volume of SDS- PAGE samnle buffer (20 mM Tris-HCl. DH 6.8. 0.4% SDS. 4% glycerol, 0.24 M @-mercaptoethanol, and bromphendl blue) and boiled immediately for 5 min to prevent proteolysis (24). Proteins were then separated by SDS-PAGE on 10, 13.5, or lo-20% polyacrylamide gels by the method of Laemmli (25). Immunoblot analyses for B-lipoxy- genase (100s) and FLAP (1OOP) were performed after transferring proteins from the gels to nitrocellulose (26) or to PVDF membranes (15). Nonsnecific sites on the nitrocellulose were blocked with 3% gelatin, 20-mM Tris-HCl, pH 7.5, 0.5 M NaCl for 30 min at room temperature. Blots were washed twice with 0.05% Tween 20 or Triton X-100,20 mM Tris-HCl, pH 7.5,0.5 M NaCl (TTBS) and incubated for 120 min at room temperature with either a 1:200 dilution of 5- lipoxygenase antibody 432 (15) or a 1:150 dilution of FLAP antisera raised against either the human FLAP sequence l-39 or 42-51 (H5) in 1% gelatin/TTBS. Blots were washed twice with TTBS and incubated with 5 x lo5 cpm/ml ‘*‘I-protein A (low specific activity, Du Pont-New England Nuclear) in 1% eelatin/TTBS for 90 min at room temperature. Blots were washed f;e times with 50 mM phos- phate buffer, pH 7.1, 0.1 M NaCl, 0.5% Nonidet P-40, dried, and exnosed to Kodak XAR-5 film at -70 “C for 18-48 h. Autoradioeranhs were scanned using an LKB2202 Ultrascan laser densitomet& and the relative areas under the peaks for FLAP or 5-lipoxygenase used to compare amounts of protein.

RNA Isolation and Northern Blot Analyses-Total cellular RNA was isolated from HL-60 cells grown as described above using guan- idinium isothiocyanate extraction and cesium chloride gradient cen- trifugation, as previously described (27). Northern blot analysis was performed after electrophoresis of RNA on a 20 mM 3-(N-morpho- 1ino)propanesulfonic acid 1% (w/v)-agarose gel (28). The 5-lipoxy- genase probe used was the 813-base pair ClaI-BamHI fragment of human 5-lipoxygenase cDNA (20). The FLAP probe was the 326- base pair EcoRI-ScuI fragment of rat FLAP cDNA (16). Nitrocellulose blots were hybridized to radiolabeled probes (specific activity > 1 x lO* dpm/rg) prepared using a multiprime labeling kit (Amersham Corp.) and [32P]dCTP (3000 Ci/mmol). Blots were prehybridized, hybridized, and washed as previously described (29).

FLAP Photoaffinity Labeling and Zmmurwprecipitation-Samples of HL-60 1OOP (70 pg of protein) were preincubated in the absence or nresence of 1 IIM MK-886 for 2 min at 37 “C. then mixed with iz51- L-669,083 (2 x i05 cpm; 500-1000 Ci/mmol) and exposed to UV light from a 450-watt Hanovia light source at a distance of 15 cm from the samples for 2 min at room temperature. Samples were mixed with % volume SDS-PAGE sample buffer, boiled for 2 min, and proteins were separated by SDS-PAGE on 13.5% gels (25). For immunopre- cinitation of nhotoaffinitv labeled nroteins. samnles of the 1OOP (300 $) were prehrcubated in the absence or presence of 1 UM MK-886 for 2 min at 37 “C in 0.5 ml of 50 mM Tris HCl, pH 7.4, 25 mM EDTA. 20% zlvcerol. and incubated for a further 2 min at 37 “C with ““I-L-669,083 “(2 X ‘lo6 cpm; 500-1000 Ci/mmol). Photolysis was performed as above and samples mixed with 150 ~1 of 1% SDS, 100 mM Tris-HCl, pH 8.0, incubated for 15 min at room temperature,

and then boiled for 5 min. A 0.65-ml volume of 2% Triton X-100,600 mM NaCl, 20 mM Tris-HCl, pH 7.2, was added and samples incubated for 30 min at 4 “C. Nonspecific precipitates were removed by centrif- ugation at 15,000 x g for 15 min at 4 “C. To the resulting supernatants FLAP antiserum H5 (50 ~1) and protein A-Sepharose (4 mg) were added and the mix incubated for a further 60 min at 4 “C. The immune complex linked to protein A-Sepharose was pelleted by centrifugation at 15,000 x g for 15 s at 4 “C and washed twice with 1% Triton X-100,300 mM NaCl, 10 mM Tris-HCl, pH 7.2, and twice with 10 mM Tris-HCl, pH 7.0. The washed pelleted proteins were

Normal Cells Continuous Cell Lines

0m

rL

FIG. 1. Immunoblot analysis of FLAP in a selection of leu- kocytes and myeloid and lymphocytic cell lines. Membranes of cells were prepared and mixed with 2 x SDS sample buffer to a final concentration of 1 mg protein/ml as measured by the Pierce Chemical Co. BCA protein assay reagent. Amounts for each cell type loaded onto the lo-20% gradient gels were: rat peritoneal neutrophils (PMN, 5 pg), human platelets (20 fig), human monocytes (20 pg), human B- cells (20 rg), human T-cells (20 rg), human THP.l mono&c cells (20 rg), rat RAW 264.7 macrophage cells (40 rg), murine CXBGABMct-1 mastocytoma cells (20 rg), murine P815 mastocy- toma cells (20 fig), murine T-cell lines (20 pg of OKT3, E14, and BW5 cells), and murine YAC B-cells (20 rg). Following electropho- resis, the gels were transferred to PVDF membranes and immuno- blotted as described under “Materials and Methods.” Rat peritoneal neutrophils were prepared from casein-elicited rats as previously described (15). Human platelet membranes were a gift of Dr. S.-B. Hwang. Normal human monocytes and B-cells were purified from leukophoresed blood (University of Pennsylvania Hospital) by elu- triation (39). Normal human T-cells were obtained by rosetting mononuclear cells with sheep erythrocytes followed by centrifugation on a ficoll-hypaque gradient and hypotonic lysis to remove the eryth- rocytes (39). All cell lines were passaged in vitro except CXBG cells which were passaged weekly as described (40).

0 18 24 48 A. f 4.4

0 ia 24 48 B. - 1.4

-FLAP

+ 0.24

FIG. 2. RNA blot analysis of 5-lipoxygenase and FLAP in undifferentiated and Me&O-differentiated HL-60 cells. RNA was isolated and Northern blot analysis performed as described under “Materials and Methods.” 0, 18, 24, and 48 refer to hours of exposure of HL-60 cells to 1.3% Me*SO. Migration positions of marker RNA species are indicated on the right. Single RNA species corresponding to 2.7 and 1.0 kilobase for 5-lipoxygenase and FLAP, respectively, were detected. Equal RNA loading per lane was confirmed by ethid- ium bromide staining of ribosomal RN.4 (data not shown).

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19820 Correlation between Expression of FLAP, 5-Lipoxygenase, and Cellular Leukotriene Synthesis

OY 1. I

0 20 40 60 60 100

TIME OF DIFFERENTIATION &IRS)

FIG. 3. Immunoblot analysis of 5-lipoxygenase and FLAP protein in HL-60 cells following exposure to Me2S0. HL-60 cells were grown in the absence or presence of 1.3% Me2S0 and processed for SDS-PAGE immunoblot analyses as described under “Materials and Methods.” The areas of the peaks for 5-lipoxygenase (0) or FLAP (0) were compared relative to the maximum amount of immunoreactive protein observed after 48-h exposure to Me2S0. The insert shows autoradiograms from 100s samples for 5-lipoxygenase (5-LO) and 1OOP samples for FLAP where: S, reference standards of human leukocyte 5-lipoxygenase or FLAP; 1, control cells at 0.2 X lo6 cells/ml prior to exposure to Me&O; 2-6, HL-60 cells exposed to 1.3% Me*SO for 18, 24, 48, 72, and 96 h; and 7, 96-h control- undifferentiated cells at 1.6 x lo6 cells/ml.

0.7 I

04 1 0 20 40 60 60 100

TIME OF DIFFERENTIATION (HRS)

FIG. 4. 5-Lipoxygenase activity in 100s samples from HL- 60 cells following exposure to MezSO. HL-60 cells were grown in the absence or presence of 1.3% Me*SO and assayed for 5-lipoxy- genase activity as described under “Materials and Methods.” 5- Lipoxygenase activity is expressed as nanomoles of 5-HETE pro- duced/mg of protein/lo-min incubation as described under “Materials and Methods.”

resuspended in SDS-PAGE sample buffer, boiled for 5 min, and separated by SDS-PAGE on 13.5% gels (25). The gels were dried and exposed to XAR-5 film at -70 “C for 48-120 h.

5-Lipoxygenase Assay-5-Lipoxygenase activity was determined in 0.5-ml reaction volumes containing 100 pM arachidonic acid, 2 FM 15-hydroperoxy-11,13-eicosadienoic acid, 0.1 M Tris-HCl, 3 mM CaCl*, 1.6 mM EDTA, 2 mM ATP, 1 mM dithiothreitol, 2.5% glycerol, 30 mM potassium phosphate, and 100s protein sample (700-1700 pg of protein) at a final pH of 7.5. After incubation for 10 min at 37 “C, 0.5 ml of ethanol containing 13-hydroxylinoleic acid (3 nmol/ml) was added, samples mixed, and precipitated protein removed by centrif- ugation at 12,000 X g for 5 min at 4 “C. Aliquots of the resulting supernatants were analyzed by reversed-phase HPLC as previously described (24). Under these conditions 5-HETE and 5-HPETE are the major 5-lipoxygenase products and 5-lipoxygenase activity is measured as nmol5-HETE and 5-HPETE produced per mg of protein per lo-min incubation.

Leukotriene Synthesis in HL-60 Cells and Its Inhibition by MK- 886-HL-60 cells were harvested by centrifugation, washed once in Dulbecco’s phosphate buffered saline, and resuspended at 2 x lo7 cells/ml in Dulbecco’s phosphate buffered saline prewarmed to 37 “C. MK-886 in MezSO or Me2S0 alone was added, and the cells incubated for 5 min at 37 “C. Calcium ionophore A23187 was added from a 1 mM stock in Me&O to a final concentration of 1 NM and incubation

A) B) 100 P

LLbOS6\

66-

30-

Photoaffinity-Lab lmmunoprecipitates

02345

20- FLAP

FIG. 5. ‘2BI-L-669,083 photoaffinity labeling. A, T-L- 669,083 labeling in subcellular fractions from undifferentiated and 5- day Me&O-differentiated HL-60 cells. Photoaffinity labeling was carried out as described under “Materials and Methods” in the absence (-) or presence (+) of 1 pM MK-886. L = human leukocyte 100,000 X g membrane protein (10 rg); 1OOP = HL-60 100,000 X g membrane protein (70 pg). 0, no exposure to MeZSO; 5, 5 days exposure to 1.3% Me*SO. The migration positions of molecular weight markers are indicated. B, immunoprecipitation of ‘*“I-L-669,083 photoaffinity labeled proteins from HL-60 cells. FLAP H5 antiserum was used to immunoprecipitate photoaffinity labeled proteins from HL-60 1OOP samples after 0,2,3,4, and 5 days of exposure to Me*SO as described under “Materials and Methods.”

continued for a further 10 min at 37 “C. The concentration of Me*SO was constant in all samples and never exceeded 0.4%. A l-ml cell sample was added to 1 ml of ethanol containing prostaglandin BZ (1 nmol/ml). Samples were extracted as previously described (24) and analyzed by reversed-phase HPLC using the conditions indicated in the legend to Fig. 6.

RESULTS

A selection of myeloid-derived cell lines were investigated for the presence of FLAP by immunoblot analysis (Fig. 1). Using a peptide antibody to the rat FLAP l-39 amino acid sequence it had been shown earlier that FLAP was detected in similar amounts in both rat and human leukocyte mem- branes (15). When membrane fractions of normal human, rat, and murine leukocytes and a number of leukocytic cell lines were investigated for the presence of FLAP, only those cells able to produce leukotrienes contained significant concentra- tions of FLAP (Fig. 1). Human monocytes known to produce both LTB, and LTC, (30-33) contained FLAP but less than 25% of that found in neutrophils. FLAP was not detected in platelets or T-cells, and only a trace amount of FLAP was found in B-cells. In contrast to normal monocytes, FLAP was not detected in the human THP.l monocytic cell line. The rat macrophage cell line RW2647 had a small amount of FLAP, in contrast to murine CXBGABMct-1 mastocytoma cells which contained substantial amounts of FLAP. FLAP was absent in murine P815 mastocytoma cells, EL4, BW5, and YAC T-lymphocytic cell lines as well as murine B-cell hybridoma OKT3 cells (Fig. 1). The relative concentration of FLAP in these cells correlated well with previous reports of 5-lipoxygenase concentrations and to their known capacity to synthesize leukotrienes (4).

The human promyelocytic HL-60 cell line (17-23) was used to further investigate the expression of FLAP, 5-lipoxygenase, and leukotriene synthesis. In undifferentiated HL-60 cells FLAP mRNA was undetectable while a low concentration of 5-lipoxygenase mRNA was observed (Fig. 2). An increase was shown in both FLAP and 5-lipoxygenase mRNA in cells after exposure to Me2S0 (Fig. 2). These increased concentrations

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Correlation between Expression of FLAP, 5-Lipoxygenase, and Cellular Leukotriene Synthesis 19821

FIG. 6. Leukotriene production in HL-60 cells. A, undifferentiated HL-60 cells; B, 5-day Me?SO-differentiated HL- 60 cells: C. 5-dav MeTSO-differentiated HL-60 ‘ceils incubated with MK-886. Cell suspensions (2 X lo7 cells/ml) were incubated for 10 min at 37 “C with 1 pM A23187 in the absence (A and B) or presence (C) of 1 pM MK-886. Leuko- g trienes were extracted and analyzed by E reversed-phase HPLC on a Waters Cl8 N Novapak column eluting with acetoni- 4 trile/methanol/water/acetic acid (2&l&- 54:1) at a flow rate of 1.2 ml/min. Prod- ucts were monitored at 270 nm and LT&, LTD,, and LTB, concentrations calculated by comparison with an inter- nal standard prostaglandin Bz (PC&) correcting for relative absorption max- ima for LTCI, LTD., LTB4, and PGB,.

t

I , I I I I 0 5 10 15 20 25

TIME (mid

0 5 5001000 MK-888 Con5coentration hM1

FIG. 7. Inhibition of MK-886 of leukotriene synthesis in Me&O-differentiated HL-60 cells. 5-day MezSO-differentiated HL-60 cells (2 x lo7 cells/ml) were incubated for 3 min at 37 “C in the presence of various concentrations of MK-886. Calcium ionophore A23187 (1 f.tM) was added and cells incubated for a further 10 min at 37 “C. Leukotriene production was measured as described in the legend for Fig. 6. LTB,. LTC,. and LTD, concentrations were nooled fo; total leukitriene concentr&ion. Results are the means of dublicate determinations with the range of duplicates indicated.

of mRNA are associated with a similar increase in the levels of immunoreactive FLAP and 5-lipoxygenase after exposure to Me*SO (Fig. 3). Concentrations of immunoreactive 5-lipox- ygenase in the HL-60 cells plateau after 2-4&y exposure to Me2S0 while concentrations of FLAP decrease slightly after 2 days. Immunoblots of protein from undifferentiated cells were performed at both the initial subculture density of 0.2 x lo6 cells/ml and at the density reached after culture for 4 days (1.6 X lo6 cells/ml) to control for any possible changes in expression due to cell density. No differences in FLAP or 5- lipoxygenase immunoreactive concentrations were observed at these different cell concentrations.

B

jl L

I , I I I

0 5 10 15 20 25 0 5 10 15 20 25

TIME (mid TIME (mid

Enzyme activity and photoaffinity labeling studies were carried out to confirm the identities of 5-lipoxygenase and FLAP, respectively. 5-Lipoxygenase activity in cytosolic frac- tions from HL-60 cells increased for 4 days after exposure of HL-60 cells to Me*SO (Fig. 4). Undifferentiated HL-60 cells had lo-15% of the 5-lipoxygenase activity of 4-day MezSO- differentiated HL-60 cells (Fig. 4). The photoaffinity ligand ‘25I-L-669,O83 which was used in the initial characterization of FLAP was used to confirm levels of FLAP during HL-60 differentiation. Photoaffinity labeling of HL-60 proteins by ‘25I-L-669,O83 revealed a large number of nonspecifically la- beled proteins in both 100s (data not shown) and 1OOP (Fig. 5) fractions but only the labeling of an 18-kilodalton 1OOP protein consistently increased 3-4-fold after 5-day exposure to Me,SO (Fig. 5A). As shown in Fig. 5A the photoaffinity labeling of this 18-kilodalton protein was selectively inhibited by preincubation with MK-886. When ‘25I-L-669,O83 photo- affinity labeling was followed by immunoprecipitation with antisera raised against a peptide sequence of FLAP, only one radioactive band was immunoprecipitated (Fig. 5B). Detect- able amounts of this immunoprecipitated protein increased from 2 to 4 days following exposure of HL-60 cells to Me$O (Fig. 5). The photoaffinity labeling of the immunoprecipitated protein was inhibited by preincubation with MK-886 (data not shown).

Cellular production of leukotrienes following calcium ion- ophore activation of HL-60 cells was demonstrated in both undifferentiated and 5-day MezSO-differentiated HL-60 cells (Fig. 6). Leukotriene products identified were LTBI, LT&, and LTDl (Fig. 6B). After exposure to MezSO for 5 days approximately a 4-fold increase in each leukotriene product was observed with concentrations of 0.18,0.17, and 0.10 nmol/ 10’ cells increasing to 0.63, 0.82, and 0.35 nmol/lO’ cells for LTB4, LT&, and LTOI, respectively (Fig. 6, A and B). The identities of the leukotrienes were confirmed by coelution with the appropriate reference standards and also by radio- immunoassay for LTC, and LTD, (data not shown). Two unidentified peaks were observed in the HPLC profile after

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19822 Correlation between Expression of FLAP, 5-Lipoxygenase, and Cellular Leukotriene Synthesis

ionophore challenge of the Me,SO-differentiated HI,-60 cells (Fig. 6B). These migrate in a position similar to that of the all-trans-nonenzymatic LTB, isomers but were not defini- tively identified. Preincubation of MeSO-differentiated HL- 60 cells with 1 pM MK-886 prior to calcium ionophore chal- lenge abolished leukotriene synthesis (Fig. 6C). This inhibi- tion of leukotriene synthesis in MeSO-differentiated HL-60 cells was concentration-dependent with an ICsO of approxi- mately 100 nM (Fig. 7).

DISCUSSION

In the present investigation we have shown that only cells known to express 5lipoxygenase and to produce leukotrienes contain significant quantities of FLAP. Leukocytes have been shown to be the richest source of FLAP, which correlates well with their capacity to produce LTB, (4). While the B-cell line OKT3 did not express FLAP, normal human B-cells may contain a small amount of this protein (Fig. 1). Little leuko- triene production has been found in B-cells (4). In the present experiments the elutriated B-cells may contain up to 2% monocyte contamination and the latter cells may be the source of the FLAP measured.

Differentiation of HL-60 cells after exposure to MeSO provides a useful model system for studying the regulation of leukotriene synthesis. Previous studies had shown induction of 5-lipoxygenase at both the mRNA and protein level in MeSO-differentiated HL-60 cells (20, 34). The results of the present study show concurrent induction of both FLAP and 5lipoxygenase during MeSO-induced HL-60 differentiation that correlates well with their increased leukotriene synthetic capacity. Leukotriene synthesis in this system is inhibited by MK-886 in a concentration-dependent manner, with an I& similar to that observed for MK-886 inhibition of leukotriene synthesis in human leukocytes (11) and in FLAP/5-lipoxy- genase transfected osteosarcoma cells (16). In human leuko- cytes MK-886 has been shown to inhibit and reverse 5 lipoxygenase translocation. In Me&SO-differentiated HL-60 cells approximately 50% of the cytosolic 5-lipoxygenase has previously been shown to translocate to a membrane fraction after calcium ionophore challenge (34). This 5-lipoxygenase translocation is inhibited 70-90% by preincubation of the cells with 1 pM MK-886.’ How MK-886 binding to FLAP prevents 5-lipoxygenase membrane association and activation is presently unclear. The cellular products of 5-lipoxygenase activation are predominately leukotrienes derived from LTA, rather than 5-HETE and 5-HPETE, which are the major products observed for the isolated enzyme. Therefore, it ap- pears that the activation of 5-lipoxygenase in response to a cellular stimulation permits the concerted oxygenation and dehydration reactions, which are known to be intrinsic to the enzyme, leading to the formation of LTA,. Such a process may be facilitated by direct or indirect membrane association of 5-lipoxygenase with FLAP.

The concurrent induction of FLAP and 5-lipoxygenase mRNAs in HL-60 cells suggests that their genes may respond to similar transcriptional regulation. In this regard it is some- what surprising that the putative promoter region of the human 5-lipoxygenase gene (35) is similar to that of house- keeping genes that are constitutively expressed with little regulation (36). In another model myeloid system, namely ts- mutant-transformed macrophages, transcriptional control of the 5-lipoxygenase pathway has been shown to be a regulated and early event in macrophage differentiation (37). A com- parison of the FLAP and 5-lipoxygenase promoter regions

2 S. Kargman and G. Reid, unpublished data.

may yield interesting information on common transcriptional regulatory elements for expression of these proteins. At least one specific enzyme involved in the synthesis of leukotrienes, LTA, hydrolase, does not seem to be coexpressed with 5- lipoxygenase (38). In view of the diverse range of biological activities of leukotrienes, the differential regulation of pro- teins involved in their synthesis is of great interest for future research.

Acknowledgments-We thank P. Charleson for iodination of ‘251- L-669,083, M. Abramovitz for providing radiolabeled cDNA probes, G. Koo for the lymphocytic cell lines, J. Chan for the elutriated leukocytes, D. Gollenbach for THP.l cells, S. Hwang for platelet membranes, and B. Sholzberg for excellent secretarial services.

REFERENCES

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Gillard, R A Dixon and J F EvansG K Reid, S Kargman, P J Vickers, J A Mancini, C Léveillé, D Ethier, D K Miller, J W

5-lipoxygenase, and cellular leukotriene synthesis.Correlation between expression of 5-lipoxygenase-activating protein,

1990, 265:19818-19823.J. Biol. Chem. 

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