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Originally published online as doi:10.1189/jlb.0807531 on January 24, 2008

Published online before print January 24, 2008
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(Journal of Leukocyte Biology. 2008;83:1019-1027.)
© 2008 by Society for Leukocyte Biology

The role of diacylglyceride generation by phospholipase D and phosphatidic acid phosphatase in the activation of 5-lipoxygenase in polymorphonuclear leukocytes

Dana Albert*,1, Carlo Pergola{dagger},1, Andreas Koeberle{dagger}, Gabriele Dodt{ddagger}, Dieter Steinhilber* and Oliver Werz{dagger},2

* Institute of Pharmaceutical Chemistry, University of Frankfurt, Frankfurt, Germany; and
{dagger} Department of Pharmaceutical Analytics, Institute of Pharmacy, and
{ddagger} Interfakultäres Institut für Biochemie, Eberhard-Karls-University Tuebingen, Tuebingen, Germany

2 Correspondence: Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. E-mail: oliver.werz{at}uni-tuebingen.de


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ABSTRACT
 
Diacylglycerides (DAGs) such as 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulate 5-lipoxygenase (5-LO) enzyme activity and function as agonists for human polymorphonuclear leukocytes (PMNL) to induce 5-LO product synthesis. Here, we addressed the role of endogenous DAG generation in agonist-induced 5-LO activation in human PMNL. Preincubation of PMNL with the phospholipase D (PLD) inhibitor 1-butanol potently suppressed 5-LO product synthesis induced by the Ca2+ ionophore A23187 or thapsigargin (TG) and blocked A23187-evoked translocation of 5-LO from the cytosol to the nuclear membrane, analyzed by subcellular fractionation as well as by indirect immunofluorescence microscopy. Tertiary-butanol, a rather poor inhibitor of PLD, caused only moderate suppression of 5-LO and hardly inhibited 5-LO translocation. Interestingly, 1-butanol failed to inhibit 5-LO product formation when PMNL were stimulated with OAG (30 µM). Moreover, coincubation of A23187- or TG-stimulated PMNL with OAG reversed inhibition of 5-LO product formation by 1-butanol in a concentration-dependent manner (EC50, ~1 µM) and also restored 5-LO translocation. In addition, inhibition of phosphatidic acid phosphatase (PA-P) by propranolol or bromoenol lactone caused suppression of 5-LO product formation and of translocation, which could be reversed by addition of exogenous OAG. Together, our data suggest that in agonist-stimulated PMNL, the endogenous formation of DAGs via the PLD/PA-P pathway determines 5-LO activation.

Key Words: leukotriene • arachidonic acid • inflammation • 1-oleoyl-2-acetyl-glycerol • ionophore


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INTRODUCTION
 
Challenge of polymorphonuclear leukocytes (PMNL) with appropriate stimuli may cause the release of leukotrienes (LTs) and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid [5-H(P)ETE], which are mediators of inflammatory and allergic reactions, but also, may play a role in cardiovascular disorders and cancer [1 ]. 5-Lipoxygenase (5-LO) is the key enzyme in LT biosynthesis, converting liberated arachidonic acid (AA) to 5-H(P)ETE, which is metabolized further to the unstable epoxide LTA4 (for review, see ref. [2 ]). LTA4 is then converted to LTB4 or LTC4, depending on the cell type and the enzymes present.

A number of cofactors stimulate 5-LO catalysis in cell-free assays, including Ca2+ and Mg2+, ATP, phospholipids, and various glycerides (for review, see ref. [3 ]). Moreover, unidentified leukocyte proteins [2 ] as well as the coactosin-like protein [4 ], which directly interacts with 5-LO [5 ], enhance 5-LO catalysis. A well-balanced hydroperoxide level determines 5-LO activity by regulating the redox state of the active-site iron [6 , 7 ], and it is assumed that Ca2+ and monoglycerides or diacylglycerides (DAGs) mediate their stimulating effect by increasing the affinity of 5-LO toward hydroperoxides via the C2-like domain [8 , 9 ].

For 5-LO catalysis in the cell, the 5-LO-activating protein (FLAP), phosphorylations by the MAPK-activating protein kinase (MAPKAPK)-2 and ERKs, as well as association with the nuclear membrane (where FLAP resides) govern 5-LO product synthesis. Accordingly, stimuli [e.g., ionophores, thapsigargin (TG), chemokines], which elevate intracellular Ca2+ ([Ca2+]i) levels and activate MAPKAPK-2 and ERKs, resulting in 5-LO redistribution to the nuclear membrane, evoke cellular 5-LO product synthesis [2 ]. However, there are substantial differences of diverse stimuli in regard to the capacity for 5-LO product formation, despite similar efficacies for elevation of [Ca2+]i and activation of MAPKAPK-2/ERKs. Hence, additional mechanisms and factors may exist, eventually resulting in quite differential capacities to generate 5-LO products in response to select stimuli.

We recently showed that addition of the cell-permeable 1-oleoyl-2-acetyl-sn-glycerol (OAG) to isolated human PMNL induces substantial 5-LO product synthesis from exogenously added AA without concomitant elevation of [Ca2+]i, activation of MAPK, or 5-LO nuclear translocation [10 ]. Instead, OAG and other monoglycerides or DAGs were found to directly stimulate 5-LO catalysis [9 ]. It has been well-established that stimulation of PMNL with agonists inducing 5-LO product synthesis, which are Ca2+-ionophore A23187, TG, or chemokines, leads to a rapid generation of DAGs [11 12 13 14 15 16 ]. Hence, we attempted to investigate the role of in situ-generated DAGs in agonist-induced 5-LO activation. We found that selective inhibition of DAG formation via phospholipase D (PLD) and phosphatidic acid phosphatase (PA-P) in PMNL potently suppresses 5-LO product synthesis and 5-LO nuclear translocation in ionophore- or TG-activated PMNL, which is reversed by supplementation of exogenous OAG. We assume that in addition to Ca2+ and phosphorylations of 5-LO, the formation of DAGs via the PLD/PA-P pathway in response to ionophores is a determinant for cellular 5-LO activation.


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MATERIALS AND METHODS
 
BWA4C, ZM230487, and MK-886 were generous gifts by Dr. Lawrie G. Garland (Wellcome Research Laboratories, London, UK), by Dr. Robert M. McMillan (Zeneca Pharmaceuticals, Macclesfield, UK), and by Dr. Anthony W. Ford-Hutchinson (Merck-Frosst, Canada), respectively. 1-O-Oleyl-rac-glycerol (OG), 1-O-hexadecyl-2-acetyl-sn-glycerol (EAG), and D609 were from Alexis (Grünberg, Germany). 1,2-Dioctanoyl-sn-glycerol (DOG), OAG, Ca2+-ionophore A23187, AA, and TG were from Sigma (Deisenhofen, Germany). HPLC solvents were from Merck (Darmstadt, Germany). U-73122, bromoenol lactone (BEL), D609, and propranolol were from Calbiochem (Bad Soden, Germany).

Cells
Human PMNL were freshly isolated from leukocyte concentrates obtained at St. Markus Hospital (Frankfurt, Germany) or the Blood Center, University Hospital (Tuebingen, Germany). In brief, venous blood was taken from healthy adult donors, and leukocyte concentrates were prepared by centrifugation at 4000 g/20 min/20°C. PMNL were immediately isolated by dextran sedimentation, centrifugation on Nycoprep cushions, and hypotonic lysis of erythrocytes as described previously [17 ]. Cells (5x106 cells/ml; purity, >96–97%) were finally resuspended in PBS plus 1 mg/ml glucose (PG buffer).

Determination of 5-LO product formation
For assays of intact cells, freshly isolated PMNL (5x106) were finally resuspended in 1 ml PG buffer plus 1 mM CaC12 (PGC) buffer. After preincubation with diverse inhibitors at 37°C for 12 (for OAG-stimulated samples) or 15 min, the reaction was started by addition of exogenous AA at the indicated concentrations. A23187 or TG was added simultaneously with exogenous AA; OAG was added 3 min prior to AA. After 10 min at 37°C, the reaction was stopped with 1 ml methanol and 30 µl 1 N HCl, and 200 ng prostaglandin B1 and 500 µl PBS were added. Formed 5-LO metabolites were extracted and analyzed by HPLC as described [18 ]. 5-LO product formation is expressed as ng 5-LO products per 106 cells, which includes LTB4 and its all-trans isomers, 5(S),12(S)-di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid and 5-H(P)ETE. 5-HETE and 5-H(P)ETE coelute as one major peak; integration of this peak represents both eicosanoids. Cysteinyl LTs (LTC4, D4, and E4) were not detected, and oxidation products of LTB4 were not determined.

For assays in broken cell preparations, freshly isolated PMNL (5x106) were finally resuspended in 1 ml PBS plus 1 mM EDTA, cooled on ice for 5 min, and sonicated (5x10 s). Samples were preincubated with the test compounds, and 1 mM ATP was added. After 5–10 min at 4°C, samples were prewarmed for 30 s at 37°C, and 2 mM CaCl2 and 20 µM AA were added to start 5-LO product formation. The reaction was stopped after 10 min at 37°C by addition of 1 ml ice-cold methanol, and the formed metabolites were analyzed by HPLC as described for intact cells.

Subcellular fractionation by mild detergent lysis
Subcellular localization of 5-LO by cell fractionation was investigated as described previously [19 ]. In brief, freshly isolated PMNL (3x107) in 1 ml PGC buffer were preincubated with the indicated test compounds for 15 min at 37°C; OAG and A23187 were added as indicated, and the samples were further incubated for 10 min and then chilled on ice to stop the reaction. Nuclear and non-nuclear fractions were obtained after cell lysis by 0.1% Nonidet P-40 (NP-40). Aliquots of these fractions were immediately mixed with the same volume of 2x SDS-PAGE sample-loading buffer, heated for 6 min at 95°C, and analyzed for 5-LO protein by SDS-PAGE and Western blotting (WB).

SDS-PAGE and WB
Total cell lysates (20 µl) and aliquots of nuclear and non-nuclear fractions (25 µl) were mixed with 4 µl glycerol/0.1% bromphenol blue (1:1, vol/vol) and analyzed by SDS-PAGE using a Mini Protean system (Bio-Rad, Hercules, CA, USA) on a 10% gel. After electroblot to nitrocellulose membrane (Amersham Pharmacia, Piscataway, NJ, USA), membranes were blocked with 5% nonfat dry milk in 50 mM Tris/HCl, pH 7.4, and 100 mM NaCl (TBS) for 1 h at room temperature. Membranes were washed and then incubated with primary antibody overnight at 4°C. Anti-5-LO antiserum (1551, AK7, kindly provided by Dr. Olof Rådmark, Karolinska Institute, Stockholm, Sweden) was affinity-purified on a 5-LO column. The membranes were washed with TBS and incubated with a 1:1000 dilution of alkaline phosphatase-conjugated IgG (Sigma) for 2 h at room temperature. After washing with TBS and TBS plus 0.1% NP-40, proteins were visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma) in detection buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2).

Indirect immunofluorescence microscopy (IFM)
Human PMNL (1.5x106 in 500 µl PGC buffer) were incubated at 37°C for 15 min with the indicated compounds. Cells were then centrifuged at 30 g for 1 min onto poly-L-lysine (MW 150,000–300,000, Sigma-Aldrich, St. Louis, MO, USA)-coated glass coverslips in the wells of a 12-well plate and activated by addition of 2.5 µM A23187 for 3 min at 37°C. Cells were fixed in methanol (–20°C, 30 min) and permeabilized in acetone (–20°C, 3 min), followed by two wash steps with PBS. The staining was performed by incubating the coverslips with the anti-5-LO serum (1551, AK-7) for 30 min at room temperature. The coverslips were then washed five times with PBS, incubated with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA; diluted 1:300 in PBS) for 10 min at room temperature in the dark, and washed five times with PBS. The DNA was stained with 0.1 µg/ml diamidino-2-phenylindole (DAPI) in PBS for 3 min at room temperature in the dark. The coverslips were then washed twice and mounted on glass slides with Mowiol (Calbiochem) containing 2.5% n-propyl gallate (Sigma). The fluorescence was visualized with a Zeiss Axiovert 200M microscope.

Statistics
Statistical evaluation of the data was performed by one-way ANOVA, followed by a Tukey-honestly significant difference (HSD) post-hoc test. Where appropriate, Student’s t-test for paired observations was applied. A P value <0.05 (*), <0.01 (**), or <0.001 (***) was considered significant.


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RESULTS
 
Inhibition of PLD by 1-butanol suppresses cellular 5-LO product formation in stimulated PMNL
Using an inhibitor approach, we attempted to investigate if reduced generation of DAGs from endogenous sources by inhibition of relevant PL-mediated pathways may affect agonist-induced 5-LO product synthesis in PMNL. The routes for cellular generation of DAG and the corresponding pharmacological inhibitors of the PLs and committed enzymes involved are illustrated in Figure 1. The hydrolysis of PIP2 to DAGs and inositol-1,4,5-trisphosphate is catalyzed by PIP2-PLC, which is blocked by U-73122. PLD generates PA from PC and is blocked by 1-butanol. PA is further converted by PA-P into DAGs; the latter step is inhibited by propranolol or by BEL. Finally, the PC-specific PLC catalyzes the formation of DAGs from PC and is blocked by D609.


Figure 1
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  		Figure 1. PL pathways leading to DAG formation and their pharmacological inhibition. PC, Phosphatidylcholine; PIP2, phosphatidylinositol-4,5-bisphosphate.

Previous studies showed that upon stimulation of PMNL with A23187, DAGs are rapidly generated via the PLD/PA-P pathway [11 12 13 ]. PLD catalyzes a transphosphatidylation reaction, where a phosphatidyl-PLD intermediate uses water as a nucleophilic acceptor to produce PA [20 ]. Short-chain primary aliphatic alcohols, such as 1-butanol, can substitute for water and generate phosphatidylalcohols, which are only poor substrates for PA-P and thus, prevent DAG synthesis [21 ]. 5-LO product synthesis in PMNL was induced by stimulation with A23187 or TG, which are assumed to activate PMNL functions mainly by an elevation of the [Ca2+]i levels [17 , 22 , 23 ], as well as by exogenously added OAG, which apparently activates 5-LO by direct interaction [9 , 10 ]. Exogenous AA (20 µM) was supplied together with the stimuli to exclude the necessity of cytosolic PLA2 (cPLA2) activation and thus, endogenous AA release.

5-LO product synthesis in PMNL induced by the A23187 or TG was strongly suppressed by 1-butanol at reasonable concentrations (Fig. 2 ) that also block PLD [21 ]. It should be noted that 1-butanol, up to 100 mM, caused no significant inhibition of 5-LO in cell-free assays, excluding direct 5-LO inhibitory effects of 1-butanol (not shown). Also, exposure of PMNL to 100 mM 1-butanol for 30 min caused no significant cytotoxic effects (visualized by trypan blue staining and light microscopy; not shown). Of interest, 1-butanol completely failed to suppress 5-LO product synthesis when PMNL were activated by OAG (Fig. 2) . Thus, the PLD inhibitor 1-butanol selectively suppresses cellular 5-LO product synthesis in PMNL stimulated with A23187 or TG.


Figure 2
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  		Figure 2. Effects of 1-butanol on agonist-induced 5-LO product synthesis in human PMNL. Freshly isolated PMNL (5x106 in 1 ml PGC buffer) were preincubated with 1-butanol at the indicated concentrations at 37°C for 15 min. A23187 (2.5 µM) and TG (1 µM) were added together with 20 µM AA; OAG (30 µM) was added 3 min prior to AA. After 10 min at 37°C, the reaction was terminated, and 5-LO products formed were determined by HPLC. Results are given as mean + SE; n = 4. Data were analyzed by ANOVA, followed by Tukey-HSD post-hoc test: **, P < 0.01; ***, P < 0.001, versus without (w/o) 1-butanol.

Exogenously supplied OAG reverses inhibition of 5-LO product synthesis by 1-butanol
Inhibition of cellular 5-LO product formation by 1-butanol in A23187- and TG-activated PMNL could be a result of a lack of DAGs generated by the PLD/PA-P pathway. Therefore, PMNL were supplemented with exogenous OAG to overcome reduced supply of endogenous DAGs. In fact, coaddition of 100 µM OAG to PMNL completely reversed the inhibitory effect of 1-butanol (Fig. 3A ). Concentration-response experiments (Fig. 3B) revealed that 1 µM OAG is sufficient to overcome 5-LO suppression by 1-butanol in A23187-activated PMNL. Tertiary-butanol that hardly inhibits PLD [21 ] reduced 5-LO product synthesis in A23187-activated PMNL in a less-efficient manner (Fig. 3C) , and other alcohols such as isopropanol or ethanol (up to 1%, vol/vol) only moderately inhibited 5-LO (not shown). As observed for 1-butanol, OAG also restored the moderate suppression of 5-LO product synthesis by tertiary-butanol (Fig. 3C) . Note that suppression of 5-LO by other well-defined pharmacological inhibitors of LT synthesis, including the 5-LO inhibitor ZM230487, quercetin, BWA4C, or the FLAP inhibitor MK-886, was not significantly affected by OAG (Fig. 3C) .


Figure 3
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  		Figure 3. Exogenous OAG reverses inhibition of 5-LO product formation by 1-butanol in PMNL. (A) Freshly isolated PMNL (5x106 in 1 ml PGC buffer) were preincubated with 1-butanol at the indicated concentrations at 37°C for 12 min. Then, OAG (100 µM) or solvent (DMSO) was added, and after 3 min, A23187 (2.5 µM) or TG (1 µM) was added together with 20 µM AA each. After 10 min at 37°C, the reaction was terminated, and 5-LO products formed were determined by HPLC. Results are given as mean + SE; n = 3. Data were analyzed by Student’s t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus corresponding samples that did not receive OAG (solid bars). (B) Concentration response of OAG. PMNL were preincubated with 100 mM 1-butanol for 12 min at 37°C. OAG was added at the indicated concentrations, and after another 3 min, 2.5 µM A23187 plus 20 µM AA were added. After 10 min at 37°C, 5-LO products formed were determined by HPLC. Results are given as mean + SE; n = 5. Data were analyzed by ANOVA, followed by Tukey-HSD post-hoc test: **, P < 0.01; ***, P < 0.001, versus 1-butanol in the absence of OAG (second bar from left). (C) Effects of tertiary-butanol and pharmacological 5-LO inhibitors. PMNL were preincubated with 30 mM 1-butanol (1-BuOH) or tertiary-butanol (t-BuOH), 100 nM BWA4C, 10 nM MK-886, 1 µM quercetin, or 100 nM ZM230487 for 12 min at 37°C as indicated. OAG (30 µM) or solvent (DMSO) was supplemented as indicated, and after another 3 min, 2.5 µM A23187 plus 20 µM AA were added. After another 10 min at 37°C, 5-LO products formed were determined. Results are given as mean + SE; n = 3. Data were analyzed by Student’s t-test; *, P < 0.05, versus the corresponding sample that did not receive OAG. (D) Comparison of various glycerides. PMNL were preincubated with 100 mM 1-butanol for 12 min at 37°C. Then, DOG, OG, EAG, and OAG were added at 10 and 100 µM as indicated, and after another 3 min, 5-LO product formation was induced by addition of 2.5 µM A23187 plus 20 µM AA. After 10 min at 37°C, 5-LO products formed were determined by HPLC. Results are given as mean + SE; n = 3. Data were analyzed by ANOVA, followed by Tukey-HSD post-hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus 1-butanol in the absence of glyceride (second bar from left).

Next, OAG-related glycerides, such as DOG, a DAG, the ether-linked EAG, and the monoglyceride OG, were tested for possible reversal of 5-LO inhibition by 1-butanol. We previously found that besides OAG, EAG but not OG or DOG stimulated PMNL for 5-LO product synthesis [10 ]. Along these lines, DOG and OG failed to reverse 5-LO inhibition in A23187-stimulated PMNL, whereas EAG could impair the inhibitory effect of 1-butanol (Fig. 3D) .

Inhibition of PA-P by propranolol or BEL suppresses cellular 5-LO product formation in stimulated PMNL
Propranolol and BEL inhibit PA-P, the subsequent enzyme after PLD in DAG formation [24 , 25 ]. As shown in Figure 4A , propranolol suppressed 5-LO product synthesis in PMNL in response to A23187 but not so when PMNL were stimulated with OAG. In broken cell preparations, propranolol caused no significant 5-LO inhibition (not shown), implying that the compound does not directly interfere with 5-LO enzyme activity. Again, supplementation of A23187-activated PMNL with exogenous OAG (100 µM) reversed the effects of propranolol. Of interest, a combination of propranolol and 1-butanol gave no additive or synergistic inhibitory effects. Thus, inhibition of A23187-induced 5-LO product synthesis was quantitatively the same when both inhibitors were applied to PMNL as compared with cells that received only 1-butanol (not shown). Similarly, inhibition of PA-P by 10 or 30 µM BEL [25 ] reduced 5-LO product formation in A23187-stimulated PMNL, and coaddition of OAG (30 µM) reversed the suppressive effects of BEL (Fig. 4B) .


Figure 4
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  		Figure 4. Effects of propranolol and BEL on agonist-induced 5-LO product synthesis in human PMNL. Freshly isolated PMNL (5x106 in 1 ml PGC buffer) were preincubated with propranolol (A) or BEL (B; or DMSO as solvent) at the indicated concentrations at 37°C for 15 min. A23187 (2.5 µM) and OAG (100 µM) were added together with 20 µM AA, each as indicated. After 10 min at 37°C, 5-LO products formed were determined by HPLC. 5-LO product formation in the absence of inhibitors (100%, control) was 105.6 ± 18.1, 51.8 ± 5.4, and 142.7 ± 44.23 ng/106 cells in PMNL stimulated with A23187, OAG, or A23187 + OAG, respectively. Results are given as mean + SE; n = 3. Data were analyzed by ANOVA, followed by Tukey-HSD post-hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus solvent sample (w/o).

Effect of PLD or PA-P inhibition on agonist-induced 5-LO translocation
Redistribution of 5-LO from the cytosol to the nuclear membrane in agonist-stimulated PMNL is a determinant for induction of 5-LO product formation [19 , 26 ]. The effects of 1-butanol or propranolol on 5-LO translocation upon stimulation with A23187 were analyzed by crude subcellular fractionation using mild detergent lysis and WB analysis [19 ], as well as by indirect IFM. As shown in Figure 5A , in subcellular fractionation studies, 5-LO was in the non-nuclear fraction of resting cells. Stimulation with A23187 caused 5-LO redistribution from the non-nuclear to the nuclear fraction, and this was blocked by pretreatment of PMNL with 100 mM 1-butanol (but not by 100 mM tertiary-butanol used as a negative control) and by 300 µM propranolol or 30 µM BEL. Supplementation of A23187-activated PMNL with 30 µM OAG reversed the inhibitory effects of 1-butanol, propranolol, or BEL and 5-LO enriched in the nuclear fraction. OAG alone failed to induce 5-LO translocation and did not affect 5-LO translocation induced by AA or A23187 (not shown and ref. [10 ]). Essentially, the same pattern of subcellular localization of 5-LO was obtained by microscopic analysis, shown in Figure 5B . Thus, in resting PMNL (Fig. 5B , a), a diffuse redistribution of 5-LO was detected in the cytosolic regions not associated with nuclear DNA (DAPI staining in blue). A23187 treatment caused enrichment of 5-LO at nuclear membrane structures (Fig. 5B , b). The nuclear localization is supported by the overlay of the staining for 5-LO (green) and costaining for DNA using DAPI (blue). Whereas OAG alone had no significant effect (Fig. 5B , c), coincubation of PMNL with A23187 plus OAG gave a more granular staining around the nuclei (Fig. 5B , d). Pretreatment with 1-butanol (Fig. 5B , e) or propranolol (Fig. 5B , f) prevented 5-LO translocation, and coaddition of OAG was able to reverse the inhibitory effects, allowing 5-LO to associate with nuclear membrane structures (Fig. 5B , g and h), again 5-LO seemingly agglomerated around the nucleus. Together, the generation of DAG via PLD/PA-P appears to play an important role for A23187-induced 5-LO translocation in PMNL.


Figure 5
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  		Figure 5. 1-Butanol, propranolol, or BEL suppresses agonist-induced 5-LO translocation in PMNL; reversal by OAG. (A) Crude subcellular fractionation. Freshly isolated PMNL (3x107 in 1 ml PGC buffer) were preincubated with 100 mM 1-butanol or tertiary-butanol, 300 µM propranolol, or 30 µM BEL, as indicated at 37°C for 12 min. Then, 30 µM OAG was added as indicated, and after another 3 min, cells were stimulated with 2.5 µM A23187. After 10 min, the samples were chilled on ice, lysed using 0.1% NP-40, and separated into nuclear (nuc) and non-nuclear fractions. 5-LO protein was determined in these fractions by WB. Correct loading of the gel and transfer of proteins to the nitrocellulose membrane were confirmed by Ponceau staining (not shown). The results shown are representative of at least three independent experiments. (B) Indirect IFM. PMNL were preincubated with 1-butanol (100 mM), propranolol (300 µM), OAG (30 µM), or solvent (DMSO) control to OAG as indicated, centrifuged onto poly-L-lysine-coated glass coverslips, and activated by A23187 (2.5 µM) as indicated. After 3 min, cell were fixed, permeabilized, and incubated with anti-5-LO serum (1551, AK-7). After addition of Alexa Fluor 488 goat anti-rabbit IgG, the fluorescence was analyzed. The DNA was stained with 0.1 µg/ml DAPI for 3 min at room temperature. Upper panels (a–h) show staining for 5-LO; the respective lower panels (a'–h') show staining for 5-LO (green) and DNA (blue) in an overlay (light blue). (a) Resting, (b) A23187, (c) OAG, (d) OAG + A23187, (e) 1-butanol + solvent + A23187, (f) 1-butanol + OAG + A23187, (g) propranolol + solvent + A21387, (h) propranolol + OAG + A23187. The slides shown are representative of four similar samples.

Effects of inhibitors of PIP2-PLC (U-73122) and PC-PLC (D609)
Next, we attempted to examine the role of PIP2-PLC in agonist-induced 5-LO activation using the PIP2-PLC inhibitor U-73122 [27 ]. As found previously [28 ], regardless of the stimulus, U-73122 (0.3 up to 30 µM) led to a concentration-dependent reduction of 5-LO product formation that was not reversed by OAG (Fig. 6A ), and also, 5-LO activity in cell-free assays was potently inhibited by U-73122, indicating that the compound directly interferes with 5-LO catalysis, and a role of PIP2-PLC in agonist-induced 5-LO activation cannot be investigated using this compound. Similarly, D609, an inhibitor of PC-PLC [29 ], potently suppressed cellular 5-LO product synthesis, regardless of the presence of OAG, and also blocked 5-LO activity in cell-free assays (at reasonable concentrations [29 , 30 ]; Fig. 6B ), implying that D609 is a direct inhibitor of 5-LO and is thus not suitable to examine a role of PC-PLC in A23187-activated PMNL. In addition, the effects of U-73122 and D609 on 5-LO subcellular redistribution were studied. In contrast to 1-butanol, propranolol, or BEL, neither U-73122 (10 µM) nor D609 (50 µM) blocked A23187-induced translocation of 5-LO to the nuclear fraction (Fig. 6C) .


Figure 6
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  		Figure 6. Effects of U-73122 and D609 on product formation and translocation of 5-LO. Freshly isolated PMNL (5x106 in 1 ml PGC buffer) were preincubated with U-73122 (A) or D609 (B) at the indicated concentrations at 37°C for 15 min. A23187 (2.5 µM) or OAG (30 µM) were added together with 20 µM AA, each as indicated, and after 10 min at 37°C, 5-LO products formed were determined by HPLC. For cell-free assays (homogenates), PMNL (5x106 in 1 ml PG buffer containing 1 mM EDTA) were sonicated on ice. U-73122 (A) or D609 (B) were added together with 1 mM ATP. After 10 min, samples were prewarmed for 30 s at 37°C, and 2 mM CaCl2 plus 20 µM AA were added to start the 5-LO reaction. After 10 min, 5-LO products were determined by HPLC. Results are given as mean + SE; n = 3–4. (C) Effects of U-73122 and D609 on 5-LO translocation. Freshly isolated PMNL (3x107 in 1 ml PGC buffer) were preincubated with 10 µM U-73122 or 50 µM D609 (or DMSO as vehicle, control) at 37°C for 15 min. Cells were then stimulated with 2.5 µM A23187 for 10 min at 37°C as indicated. 5-LO subcellular localization was analyzed after cell lysis using 0.1% NP-40, separation into nuclear and non-nuclear fractions, and WB. Correct loading of the gel and transfer of proteins to the nitrocellulose membrane were confirmed by Ponceau staining (not shown). The results shown are representative of at least four independent experiments.


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DISCUSSION
 
Previous studies with intact PMNL showed that OAG functions as a potent agonist evoking 5-LO product formation from exogenous AA in a protein kinase C (PKC)-independent manner [10 ]. It was found that OAG and certain other DAGs directly stimulate 5-LO by interaction with a putative phopholipid binding site within the C2-like domain, enabling Ca2+-independent 5-LO catalysis at low peroxide tone [9 ]. As a result of these favorable effects of DAGs on 5-LO activity, we attempted to assess the role of endogenous DAG generation in agonist-evoked 5-LO product synthesis in intact PMNL. Our data suggest that DAGs generated via the PLD/PA-P route are major contributors for cellular 5-LO product formation in PMNL and are seemingly determinants for agonist-evoked nuclear membrane translocation of 5-LO.

The rapid generation of DAGs by the PLD/PA-P pathway in PMNL challenged by A23187 has been well-established before [11 12 13 ], and PLD signaling is known to play a pivotal role in PMNL activation [12 , 14 , 15 , 31 32 33 34 ]. Agonists that cause activation of PLD in PMNL, including A23187 [11 , 12 , 14 ], TG [35 ], fMLP [12 , 36 ], C5a [33 ], or LTB4 [34 ], elicit 5-LO product synthesis [2 ], implying a possible interrelation. 1-Butanol and propranolol efficiently suppressed PLD and PA-P activity, respectively, in PMNL [37 38 39 ], and inhibition of PLD- and PA-P-mediated DAG production reduced LT formation in IgE-challenged RBL-2H3 cells [40 , 41 ]. It was suggested that PLD activation may be a prerequisite for IgE-dependent and ionomycin-stimulated LT release from RBL-2H3 cells [41 ].

Our present results imply a pivotal role of the PLD/PA-P pathway and cellular DAGs in 5-LO activation. Thus, the PLD inhibitor 1-butanol concentration-dependently and effectively blocked 5-LO product synthesis in PMNL activated by A23187 or TG at concentrations (10–100 mM) that act on PLD [21 , 38 , 39 ]. Tertiary-butanol, a poor substrate for PLD, was much less efficient to inhibit cellular 5-LO product synthesis, and other alcohols such as ethanol or isopropyl alcohol failed in this respect. Interference of 1-butanol on the basis of AA liberation (by affecting cPLA2 activity) can be excluded, as exogenous AA was supplemented as substrate for 5-LO, thus circumventing the need for endogenous AA supply. We also conclude that 1-butanol does not directly interfere with 5-LO catalysis itself, as it caused no significant inhibition of 5-LO activity in cell-free assays. Importantly, supplementation of DAGs that are able to stimulate 5-LO in PMNL (i.e., OAG and EAG but not OG or DOG [10 ]) reversed the inhibitory effect of 1-butanol. Moreover, when cellular 5-LO activity was induced by OAG, 1-butanol failed to block 5-LO product synthesis. These results indicate that the inhibitory effect of the PLD inhibitor 1-butanol on cellular 5-LO activation is a result of inhibition of DAG formation.

In the synthesis of DAGs from PC, PLD first generates PA that is then dephosphorylated by PA-P, eventually yielding DAGs. Accordingly, inhibitors of PA-P should exert essentially the same effect on 5-LO activation as the PLD inhibitor 1-butanol. In fact, the PA-P inhibitors propranolol or BEL suppressed A23187-induced 5-LO product synthesis and 5-LO translocation at concentrations that typically suppress PA-P-mediated responses in intact cells [15 , 24 , 25 , 33 34 35 , 41 ]. BEL inhibits PLA2 isoenzymes [42 , 43 ], which may limit AA supply. However, BEL blocked 5-LO product formation when exogenous AA (20 µM) was provided, excluding PLA2 inhibition as an underlying mechanism. Again, exogenous supplementation of OAG reversed inhibition of 5-LO product synthesis as well as 5-LO translocation in A23187-activated PMNL by propranolol or BEL. This reversal of 5-LO inhibition by OAG was specific for the action of 1-butanol or propranolol and BEL but was not observed for pharmacological inhibitors of 5-LO product synthesis (i.e., BWA4C, quercetin, ZM230487, or MK-886), implying that OAG selectively restores 5-LO activity as a result of a lack of DAGs caused by inhibition of PLD (1-butanol) or PA-P (propranolol, BEL). Apparently, depletion of endogenous DAGs as a result of inhibition of the PLD/PA-P pathway prevents activation of 5-LO in PMNL, but exogenous OAG substitutes for the lack of the endogenous DAGs. Attempts to immediately assess the role of DAGs generated by PC-PLC and PIP2-PLC using D609 and U-73122 as selective inhibitors, respectively, failed as a result of direct interference with 5-LO. Nevertheless, the failure of U-73122 and D609 to inhibit A23187-induced 5-LO translocation rather excludes a contribution of these pathways in 5-LO activation.

Upon PMNL stimulation, 5-LO moves from the cytoplasm to the nucleus to associate with the nuclear membrane, where FLAP resides, which determines the capacity for 5-LO product synthesis (for review, see ref. [44 ]). In both assays investigating 5-LO subcellular redistribution, 1-butanol (but not tertiary-butanol) and propranolol or BEL blocked A23187-induced 5-LO translocation to nuclear structures, and these suppressive effects were reversed by external supply of OAG. However, addition of OAG alone is not sufficient to induce 5-LO translocation in PMNL and did not increase 5-LO translocation when combined with A23187 or AA [10 ]. 5-LO possesses an N-terminal regulatory C2-like domain that binds PC vesicles and Ca2+ [45 ]. The selective association of 5-LO with the nuclear membrane is accounted by the specificity of 5-LO to bind PC (enriched in the nuclear membrane) in a Ca2+-dependent manner [46 47 48 ]. DAGs stimulate 5-LO via the C2-like domain mediated by three Trp residues (W13, W75, W102) [9 ]. It is conceivable that in agonist-stimulated cells, DAGs are required for association of 5-LO with the nuclear membrane via the C2-like domain. Along these lines, a firm anchoring of PKC{epsilon} at the plasma membrane via the C2 domain that depends on the PLD/PA-P-mediated generation of DAGs has been demonstrated in RBL-2H3 cells [49 ]. For PKC{alpha}, it was shown that the C2 domain mediates the initial Ca2+- and phosphatidylserine-dependent membrane binding of the enzyme, whereas the C1 domain is involved in subsequent membrane penetration and DAG binding [50 ].

For activation of 5-LO in intact cells, an agonist has to meet certain criteria: provoke a pronounced and rapid elevation of the [Ca2+]i and/or cause activation of MAPKAPK-2 and ERK1/2, and these criteria are fulfilled by most stimuli (e.g., A23187, TG, fMLP, C5a, zymosan, LTB4), eliciting 5-LO product synthesis in leukocytes [2 ]. For A23187- or TG-induced 5-LO product synthesis from exogenous AA, the pronounced elevation of [Ca2+]i has been regarded to be sufficient, as MAPK activity was dispensable [17 , 51 ]. Based on the present findings, DAGs, in addition to Ca2+, play a major role in A23187- or TG-induced 5-LO activation. As mentioned before, both stimuli cause rapid activation of PLD and PA-P in PMNL [12 13 14 , 35 ], and inhibition of DAG formation blocked 5-LO translocation and product synthesis under the same conditions. Apparently, in the absence of DAGs, elevation of [Ca2+]i is not sufficient to enable translocation and activity of 5-LO in PMNL. Nevertheless, OAG alone caused no 5-LO translocation in PMNL [10 ], and after depletion of [Ca2+]i, OAG even failed to evoke 5-LO product formation [10 ], implying that Ca2+ and DAGs are determinants. Possibly, OAG reduces the Ca2+ requirement for 5-LO activation, supported by the lower concentrations of Ca2+ (~200 nM) sufficient for substantial activation of 5-LO in intact cells as compared with purified 5-LO (requiring 1–10 µM Ca2+). On the other hand, OAG also activated p38 MAPK and ERK1/2 (not shown and ref. [10 ]), and 1-butanol or propranolol blocked the activation of these MAPKs induced by A23187 (not shown). However, the selective kinase inhibitors SB203580 (p38 MAPK) and U0126 (ERK pathway) did not abolish 5-LO product synthesis induced by OAG [10 ] or by A23187 [17 ], implying that PLD/PA-P inhibition does not act via MAPK to reduce 5-LO activity.

Taken together, generation of DAGs via the PLD/PA-P pathway seems to be a critical process in LT biosynthesis. It appears that in agonist-stimulated PMNL, elevated levels of Ca2+ and DAG generation are required for 5-LO nuclear membrane translocation and induction of substantial 5-LO product synthesis. These findings may add to the understanding of the complex machinery of 5-LO activation in intact cells by translating molecular mechanisms from biochemical studies of 5-LO enzyme modulation in vitro into a cellular context.


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ACKNOWLEDGEMENTS
 
This study was supported by grants from the European Union (QLG1-CT-2001-01521 and LSHM-CT-2004-005033). We thank Sven George and Bianca Jazzar for expert technical assistance.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received August 10, 2007; revised November 28, 2007; accepted December 17, 2007.


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