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(Journal of Leukocyte Biology. 2003;73:191-200.)
© 2003 by Society for Leukocyte Biology

Cell type-dependent activation of 5-lipoxygenase by arachidonic acid

Eva Bürkert*, Dagmar Szellas*, Olof Rådmark{dagger}, Dieter Steinhilber* and Oliver Werz*

* Institute of Pharmaceutical Chemistry, University of Frankfurt, Germany; and
{dagger} Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, Stockholm, Sweden

Correspondence: Dr. Oliver Werz, Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany. E-mail: o.werz{at}pharmchem.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. We show that stimulation of polymorphonuclear leukocytes (PMNL), rat basophilic leukemia (RBL)-1, or transfected HeLa cells with arachidonic acid (AA) caused prominent 5-LO product formation that coincided with the activity of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase. 5-LO product formation in AA-stimulated PMNL and RBL-1 cells was independent of Ca2+. However, in HeLa cells expressing a 5-LO mutant lacking potential 5-LO phosphorylation sites, removal of Ca2+ caused a prominent loss of 5-LO activity. For Mono Mac 6 (MM6) cells, AA failed to activate ERKs, and AA-induced 5-LO product formation was only minute. Also, activation of ERKs by phorbol esters did not lead to prominent 5-LO product synthesis. Instead, 5-LO activation in MM6 cells required Ca2+ or alternative signaling pathways induced by hyperosmotic stress. In summary, mechanisms for activation of 5-LO differ considerably between cell types.

Key Words: leukotriene • Ca2+ • MAP kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biosynthesis of proinflammatory leukotrienes (LTs) from arachidonic acid (AA) mainly occurs in myeloid cell lineages upon stimulation [1 ]. As a result of their potent physiological and pathophysiological actions, in vivo generation of LTs is tightly regulated and depends on an orchestrated interplay among several enzymes. 5-Lipoxygenase (5-LO) is the key enzyme in LT biosynthesis, catalyzing the first two steps in the conversion of AA to LTA4, which is further converted to LTB4 or LTC4 depending on the enzymes present [2 ].

On stimulation, 5-LO redistributes from a soluble cell compartment to the nuclear membrane, where liberated AA is transferred via membrane-bound 5-LO-activating protein to 5-LO for metabolism; for review, see refs. [1 , 3 ]. Elevated, intracellular Ca2+ is considered to be important for activation of 5-LO in the cell, and it was shown that 5-LO binds Ca2+ at a C2 domain that stimulates 5-LO catalysis in vitro and in vivo and mediates membrane association [4 5 6 7 ]. In fact, several stimuli for LT synthesis, such as ionophores, thapsigargin, N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor, C5a, zymosan, crystals, and microbes, are capable to raise intracellular Ca2+ levels (see ref. [8 ] and references therein). Also, stimulation of human polymorphonuclear leukocytes (PMNL) with exogenous AA causes elevation of intracellular Ca2+ and leads to the generation of LTs [9 10 11 12 13 ].

In addition to this Ca2+-mediated enzyme activation, alternative signal transduction pathways such as protein phosphorylation have been proposed to stimulate 5-LO activity. Thus, it was recently shown that 5-LO can be phosphorylated by p38 mitogen-activated protein kinase (MAPK)-regulated MAPK-activated protein kinases (MKs) and by extracellular signal-regulated kinase (ERK)1/2 [13 , 14 ], activating the enzyme for LT synthesis in various cell types [8 , 12 13 14 15 16 ]. Of interest, a synergism between the ERK1/2 and p38 MAPK pathways in 5-LO activation was apparent in AA-stimulated PMNL or HeLa cells, and prominent 5-LO phosphorylation rates by these kinases in vitro required the presence of AA or other unsaturated fatty acids [12 , 13 ].

Aside from being a substrate for eicosanoid biosynthesis, free AA mediates a number of biological actions in leukocytes, including neutrophil degranulation, chemotaxis, adherence, and superoxide formation (see ref. [17 ] and references therein). At the molecular level, free AA can modify the activity of phospholipases, protein kinases (PKs; e.g., PKC, PKA, Ca2+-calmodulin-dependent kinase), G-proteins, adenylate and guanylate cyclases, as well as ion channels (for review, see ref. [18 ]). Notably, AA leads to activation of phosphatidylinositol 3-kinase (PI-3K) [19 ] and MAPK pathways in various cell types [17 , 20 21 22 23 24 25 26 ], and activation of MAPK pathways by AA appears to be cell type-specific [17 , 26 ]. In this study, we investigated 5-LO product generation and activation of MAPKs in response to exogenous AA in several cell types as well as the signaling mechanisms involved.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI-1640 medium was from Gibco (Grand Island, NY), and fetal calf serum (FCS) was obtained from Boehringer Mannheim (Mannheim, Germany). Insulin was a gift from Aventis (Frankfurt, Germany). Human transforming growth factor-ß1 (TGF-ß1) was purified from outdated platelets as described [27 ]. Calcitriol was kindly provided by Dr. Herbert Wiesinger (Schering AG, Berlin). SB203580, Fura-2/AM, and 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) were purchased from Calbiochem (Frankfurt, Germany), and U0126 was from Promega (Madison, WI). Calcium ionophore A23187, sucrose, and AA were from Sigma Chemical Co. (Deisenhofen, Germany), and high-pressure liquid chromatography (HPLC) solvents were from Merck (Darmstadt, Germany).

Cell culture and transient transfections
Rat basophilic leukemia (RBL)-1 cells were maintained in RPMI-1640 medium supplemented with 10 mM HEPES, pH 7.4, 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, 1 mM sodium pyruvate, 1x nonessential amino acids, and 10 µg/ml bovine insulin at a density of 2 x 105 cells/ml. Cells were harvested for experiments 3 days after splitting. Mono Mac (MM) 6 cells were cultured and differentiated with TGF-ß and calcitriol as described [28 ].

Human PMNL were freshly isolated from leukocyte concentrates obtained at St. Markus Hospital (Frankfurt, Germany). In brief, venous blood was taken from healthy adult donors and subjected to centrifugation at 4000 g for 20 min at 20°C for preparation of leukocyte concentrates. Thereafter, PMNL were immediately isolated by dextran sedimentation, centrifugation on Nycoprep cushions (PAA Laboratories, Linz, Austria), and hypotonic lysis of erythrocytes as described previously [29 ] and were finally resuspended (5x106 cells/ml; purity >96–97%) in phosphate-buffered saline (PBS) plus 1 mg/ml glucose (PG buffer) or alternatively, in PG and 1 mM CaCl2 (PGC buffer) as indicated.

HeLa cells were maintained and transiently transfected using the Ca2+ phosphate method as described [12 ]. The mutated plasmids pcDNA3.1–5LO-S271A, pcDNA3.1–5LO-S663A, and pcDNA3.1–5LO-S271A–S663A were prepared from pcDNA3.1–5LO by replacement of the EcoRV to NotI fragment with the corresponding DNA fragments from pT3–5LO-S663A or pT3–5LO-S271A–S663A as described [13 ]. For incubations, cells were finally resuspended in PG or PGC buffer.

Determination of 5-LO product formation
PMNL (5x106), RBL-1 cells (3x106), MM6 cells (3x106), or transformed HeLa cells (2x106) were finally resuspended in 1 ml PG or PGC buffer and were preincubated at 37°C with the indicated additives as described. The reaction was started by addition of AA or AA plus ionophore A23187 at the indicated concentrations. When cell integrity was examined by light microscopy and trypan blue exclusion, cells were viable (>90%) in the presence of 80 µM AA for at least 1 h. Final volume of the incubations was 1 ml. After 10 min at 37ºC, the reaction was stopped with 1 ml methanol and 30 µl 1 N HCl, and 200 ng prostaglandin B1 (internal standard) and 500 µl PBS were added. After centrifugation (10 min, 800 g), the samples were applied to C-18 solid-phase extraction columns (100 mg), which were conditioned with 1 ml methanol and 1 ml water. The columns were washed with 1 ml water and 1 ml 25% methanol; 5-LO metabolites were extracted with 300 µl methanol and were analyzed by HPLC as described [30 ] using UV light detection at 235 nm {5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid [5-H(p)ETE]} and 280 nm {all-trans isomers of LTB4, LTB4, and 5(S),12(S)-di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid [5(S),12(S)-DiHETE]}, respectively. The respective molar absorptions were used for calculation. 5-LO product formation is expressed as ng of 5-LO products per 106 cells, which includes 5-H(p)ETE as well as LTB4 and its all-trans isomers. 5-HETE and 5-H(p)ETE coelute as one major peak, and integration of this peak represents both eicosanoids. Cysteinyl LTs (LTC4, D4, and E4), and oxidation products of LTB4 were not determined.

MAPK activation
Freshly isolated PMNL (5x106), RBL-1 cells (3x106), and MM6 cells (3x106) were resuspended in PGC buffer; final volume was 100 µl. After addition of the indicated stimuli, samples were incubated at 37°C, and the reaction was stopped after the indicated times by addition of 100 µl ice-cold 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer [SDS-b; 20 mM Tris/HCl, pH 8, 2 mM EDTA, 5% SDS (w/v), 10% ß-mercaptoethanol], vortexed, and heated for 6 min at 95°C. Total cell lysates (20 µl) were analyzed for phosphorylated MAPK by SDS-PAGE and Western blotting (WB).

SDS-PAGE and WB
Total cell lysates (20 µl) were mixed with 4 µl glycerol/0.1% bromphenol blue (1:1, vol/vol) and were analyzed by SDS-PAGE on a 10% gel. After electroblot to nitrocellulose-membrane (Amersham Pharmacia, Little Chalfont, UK) blocking with 5% nonfat dry milk for 1 h at room temperature (RT), membranes were washed and incubated with primary antibodies (Ab) overnight at 4°C. Phospho-specific Ab recognizing ERK1/2 (Thr202/Tyr204) or p38 MAPK (Thr180/Tyr182) and anti-ERK1/2 Ab were obtained from New England Biolabs, Inc. (Beverly, MA); anti-p38 MAPK Ab was from Santa Cruz Biotechnology (Santa Cruz, CA). All Ab were used at 1:2000 dilution. The membranes were washed and incubated with 1:1000 dilution of alkaline phosphatase-conjugated immunoglobulin G (Sigma Chemical Co.) for 2 h at RT. After washing, proteins were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma Chemical Co.) in detection buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2).

Measurement of intracellular Ca2+ levels
Freshly isolated PMNL (1x107), RBL-1, or HeLa cells (3x106 each) were resuspended in 1 ml PGC buffer and incubated with 2 µM Fura-2/AM for 30 min at 37°C. Cells were washed, resuspended in 1 ml PGC buffer, and transferred into a thermally controlled (37°C) fluorimeter cuvette in a spectrofluorometer (Aminco-Bowman series 2, Polytec, Waldbronn, Germany) with continuous stirring. The fluorescence emission at 510 nm was measured after excitation at 340 and 380 nm, respectively. Intracellular Ca2+ levels were calculated according to the method of Grynkiewicz et al. [31 ]. Maximal fluorescence was obtained by lysing the cells with 1% Triton-X 100 and minimal fluorescence, by chelating Ca2+ with 10 mM EDTA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell type-dependent induction of 5-LO product formation by AA
Freshly isolated human PMNL, RBL-1, and differentiated MM6 cells were stimulated with AA alone or for comparison, with AA plus ionophore in the presence of 1 mM extracellular Ca2+, respectively, and the formation of LTB4 and its all-trans isomers as well as 5-H(p)ETE (thereafter referred as "5-LO products") was determined. As can be seen from Figure 1 , left panel, in PMNL stimulated with ionophore, formation of these 5-LO products was already high in the absence (41.5±10 ng/106 cells) or at quite low concentrations of exogenous AA, peaking at 20 µM AA (111.6±19 ng/106 cells) with a subsequent decline. In agreement with previous reports [10 , 12 , 32 ], exposure of PMNL to AA alone (>=20 µM) resulted in a moderate but dose-dependent activation of 5-LO, and product formation was maximal (17.3±8.4 ng/106 cells) at the highest AA concentration (80 µM) used. It should be noted that PMNL were immediately isolated from leukocyte concentrates after venipuncture to minimize possible influences on 5-LO activation pathways by cytokines (or adenosine) that can be released during storage from surrounding cells.



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  		Figure 1. 5-LO product formation in PMNL, RBL-1, and MM6 cells in the presence of Ca2+. Freshly isolated PMNL (5x106; left), RBL-1 cells (3x106; middle), or MM6 cells (3x106; right) were finally resuspended in PGC buffer and incubated with the indicated concentrations of AA in the absence or presence of ionophore (optimal concentrations of ionophore were 2.5 µM for PMNL, 1 µM for RBL-1 cells, and 5 µM for MM6 cells). After 10 min at 37°C, the reaction was terminated, and the formation of 5-LO products was determined by HPLC as described. Results are given as mean + SE; n = 4–6.

 
Treatment of RBL-1 cells with ionophore plus AA caused considerable 5-LO product formation at AA concentrations >=2.5 µM, peaking at 40 µM (462±41 ng/106 cells), whereas no significant amounts of 5-LO products were formed in the absence of exogenous AA. When RBL-1 cells were stimulated with AA alone, at least 20 µM AA was necessary for significant induction of 5-LO product formation (Fig. 1 , middle panel). Notably, for RBL-1 cells, most prominent amounts of 5-LO products (866±57 ng/106 cells) were formed when cells were exposed to 60 µM AA in the absence of ionophore.

A different pattern was observed for MM6 cells. Although these cells generated substantial amounts of 5-LO products when stimulated with AA plus ionophore (e.g., 257±71 ng/106 cells at 10 µM AA), no significant 5-LO product formation (<10 ng/106 cells) was detectable after exposure to AA alone (2.5 up to 80 µM; Fig. 1 , right panel).

Ca2+ influx and effects of Ca2+ depletion on 5-LO activation
AA causes moderate mobilization of intracellular Ca2+ in various cell types, and it was found that elevated, intracellular Ca2+ (>{approx}200 nM) stimulates 5-LO for product formation [1 ]. To examine if AA-induced 5-LO product formation is related to an elevation of intracellular Ca2+, we measured the cellular Ca2+ influx. As shown in Figure 2 , AA (40 µM) failed to induce a significant Ca2+ influx in RBL-1 cells or MM6 cells, and only a moderate increase of the Ca2+ levels (to 120 nM) was observed for AA-stimulated PMNL. In control experiments, ionomycin strongly increased the intracellular Ca2+ levels in all three cell types.



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  		Figure 2. Mobilization of intracellular Ca2+ to Fura-2-loaded cells in PGC buffer. AA (40 µM) or ionomycin (1 µM) was added as indicated in the figure, and the fluorescence was measured. Intracellular-free Ca2+ was calculated as described. The monitored curves show one typical experiment out of three to four.

 
To investigate if at least basal Ca2+ levels are required for AA-induced 5-LO product formation, cells from above were incubated with AA in a Ca2+-free buffer. To ensure the absence of extracellular Ca2+, 1 mM EDTA was added; to remove extracellular and intracellular Ca2+, 1 mM EDTA plus 30 µM BAPTA-AM were added. As shown in Figure 3 , left panel, for PMNL, removal of Ca2+ did not abrogate AA-induced 5-LO product formation; in contrast, at AA concentrations, >=40 µM depletion of Ca2+ strongly increased the amounts of 5-LO products formed. For RBL-1 cells, no appreciable change in the formation of 5-LO products was found after removal of Ca2+ (Fig. 3 , middle panel). Finally, for MM6 cells, 5-LO product formation by AA was low also after depletion of Ca2+ (Fig. 3 , right panel). Together, AA-induced formation of 5-LO products in PMNL and RBL-1 cells is Ca2+-independent, obviously involving alternative mechanisms for enzyme activation other than Ca2+ mobilization.



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  		Figure 3. 5-LO product formation in PMNL, RBL-1, and MM6 cells in the absence of Ca2+. Freshly isolated PMNL (5x106; left), RBL-1 cells (3x106; middle), or MM6 cells (3x106; right) were finally resuspended in PG buffer. Then, 1 mM Ca2+, 1 mM EDTA, and 30 µM BAPTA-AM were added as indicated. After 10 min at 37°C, AA at the indicated concentrations was added, and cells were incubated for another 10 min at 37°C. The reaction was terminated, and the formation of 5-LO products was determined by HPLC as described. Results are given as mean + SE; n = 4–5.

 
Kinetics of 5-LO product formation in intact cells
The kinetics of 5-LO product formation in PMNL are shown in Figure 4A . Stimulation with 10 µM AA plus 2.5 µM ionophore in the presence of Ca2+ rapidly activates 5-LO. After 45 s, product formation was ~90% of maximum. A different pattern was obtained for cells stimulated with AA alone, particularly after depletion of Ca2+. Thus, after a lag phase of ~45 s, 5-LO product formation proceeded continuously (up to 10 min). Similar results were obtained for RBL-1 cells (Fig. 4B) . Therefore, elevation of intracellular Ca2+ by ionophore rapidly activates 5-LO with a subsequent, sudden termination of enzyme activity, whereas 5-LO activation by AA alone is rather delayed, and enzymatic activity is prolonged.



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  		Figure 4. Kinetics of 5-LO product formation in PMNL and RBL-1 cells; effects of Ca2+. (A) Freshly isolated PMNL (5x107) were stimulated with 2.5 µM ionophore plus 10 µM AA in 10 ml PGC buffer or stimulated with 60 µM AA in 10 ml PG buffer containing 1 mM EDTA or 1 mM EDTA plus 30 µM BAPTA-AM. After the indicated times at 37°C, aliquots of these incubations (1 ml) were added to the same volume of ice-cold methanol to stop the 5-LO reaction, and 5-LO products were determined. (B) RBL-1 cells (3x107) were stimulated with 1 µM ionophore plus 10 µM AA in 10 ml PGC buffer or were stimulated with 60 µM AA in 10 ml PG buffer containing 1 mM EDTA plus 30 µM BAPTA-AM. After the indicated times at 37°C, aliquots of these incubations (1 ml) were added to the same volume of ice-cold methanol, and 5-LO products were determined. Results are representative of at least three separate experiments.

 
AA-induced activation of p38 MAPK and ERKs
It was shown that depending on the stimulus, activation of 5-LO in intact cells (for example, by cell stress stimuli) may occur by Ca2+-independent pathways involving MAPK pathways [12 , 14 15 16 ]. Thus, we investigated if MAPK pathways are involved in 5-LO activation by AA.

Isolated PMNL, RBL-1, and MM6 cells were incubated in PGC buffer with increasing amounts of AA for 1.5 min, and ionophore (RBL-1, PMNL) or ionophore plus phorbol 12-myristate 13-acetate (PMA; MM6 cells) were used as positive controls [13 ]. MAPK activation was determined by WB using phosphospecific Ab against the activated, dually phosphorylated forms of p38 MAPK and ERKs. Whereas AA led to a dose-dependent activation of p38 MAPK and ERKs in PMNL (ERK2) and RBL-1 cells (ERK1/2); (which correlated to the induction of 5-LO product formation), only p38 MAPK but not ERKs was activated by AA in MM6 cells (Fig. 5A ). As aforementioned, no 5-LO products were formed in MM6 cells under these conditions (see Fig. 1A ). In control incubations, PMA plus ionophore led to prominent ERK1/2 activation in MM6 cells, conditions that also caused substantial 5-LO product formation [16 ].



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  		Figure 5. Activation of ERK1/2 and p38 MAPK by AA. Freshly isolated PMNL (5x106), MM6 cells (3x106), and RBL-1 cells (3x106) were resuspended in 100 µl PGC buffer, respectively. (A) To determine the dose-response of AA for MAPK activation, cells were stimulated with AA for 1.5 min at 37°C. (B) To determine the time course of MAPK activation induced by AA, cells were incubated with 40 µM AA for the indicated times at 37°C. Incubations were terminated by addition of the same volume of SDS-b. Samples were electrophoresed and analyzed for dually phosphorylated ERK1/2 and p38 MAPK by immunoblotting. Equal amounts of protein were evaluated with anti-ERK1/2 and anti-p38 MAPK Ab (not shown). Results are representative of at least three separate experiments.

 
In Figure 5B , the time course of MAPK activation is shown. In RBL-1 cells and PMNL, ERK as well as p38 MAPK were rapidly activated in response to 40 µM AA within 30 s, and maximal activation was detected after 1–1.5 min, compatible with the respective kinetics for 5-LO product synthesis (Fig. 4) . In MM6 cells, ERK1/2 activation was not at all detectable over a range of 10 min. Collectively, AA activates p38 MAPK in all three cell types and ERKs in PMNL and RBL-1 cells but not in MM6 cells.

Effects of ERK and p38 MAPK inhibitors on AA-induced 5-LO product formation
The involvement of MAPK in 5-LO product formation in RBL-1 cells was further assessed using the specific MAPK kinase (MEK)1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580. AA-induced 5-LO product formation in RBL-1 cells was partially inhibited by 10 µM SB203580 as well as by 3 µM U0126 (to 42±7% and 43±15%, respectively), concentrations that completely suppressed the activities of the kinases (not shown). A combination of both inhibitors led to additive effects and strongly (82±10%) prevented 5-LO activation (Fig. 6 ). Removal of Ca2+ by EDTA and BAPTA caused slightly further 5-LO inhibition by U0126 (69±16%), but the magnitude of 5-LO inhibition by SB203580 or by the combination of U0126 and SB203580 was hardly affected by Ca2+ depletion. Of interest, when cells were stimulated with AA plus ionophore (leading to a substantial increase in intracellular Ca2+), only marginal 5-LO inhibition (<=14%) was observed. Therefore, the MAPK inhibitors specifically interfere with 5-LO product formation induced by AA but only moderately suppress product formation induced by a stimulus (ionophore) that leads to prominent elevation of intracellular Ca2+.



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  		Figure 6. Effects of SB203580 and U0126 on AA-induced 5-LO product formation in RBL-1 cells. RBL-1 (5x106) cells were resuspended in 1 ml PGC buffer or in 1 ml PG buffer containing 1 mM EDTA plus 30 µM BAPTA-AM and were preincubated with 10 µM SB203580 and/or 3 µM U0126 for 30 min at 37°C as indicated. Then, cells were treated with 60 µM AA alone or with 10 µM AA plus 1 µM ionophore (iono) as indicated in the figure and were incubated for another 10 min at 37°C, and 5-LO products were determined. The control values (100%) in the absence of inhibitors were 787 ± 271, 903 ± 241, and 327 ± 56 ng/106 cells, incubated with 60 µM AA plus 1 mM Ca2+, 60 µM AA plus EDTA/BAPTA-AM, and 10 µM AA, 1 mM Ca2+ plus 1 µM ionophore, respectively. Results are given as mean + SE;n = 3–4. Student’s t-test; *P < 0.05; **P < 0.01.

 
Requirement of Ca2+ for AA-induced 5-LO activation in transfected HeLa cells
To further dissect the pathways by which AA activates 5-LO, we investigated 5-LO product formation in HeLa cells, transfected with wild type (wt) 5-LO or with the mutated proteins S271A–5-LO, S663A–5-LO, or S271A–S663A–5-LO [which lack putative phosphorylation sites for MKs (Ser-271) and ERKs (Ser-663), respectively]. Ionophore-induced 5-LO product formation and crude enzyme activity in cell homogenates were about the same for all three 5-LO enzymes (not shown; compare refs. [12 , 13 ]). In agreement with previous studies [12 , 13 ], HeLa cells transfected with wt 5-LO and stimulated with AA produced considerable amounts of 5-LO products, but mutation of Ser-271 or Ser-663 to alanine caused reduced 5-LO product synthesis (Fig. 7A ). Depletion of intracellular Ca2+ (by BAPTA-AM) or extracellular Ca2+ (by EDTA) caused only moderate suppression of AA-induced product formation of the wt 5-LO (<19%), whereas a combination of both chelators decreased the formation of 5-LO products to ~55% (Fig. 7A) . Also, stimulation with AA leads to a significant elevation of intracellular Ca2+ in HeLa cells (up to ~350 nM; Fig. 7B ). Thus, Ca2+ appears to be partially required for AA-induced 5-LO activation in HeLa cells. Of interest, depletion of Ca2+ markedly reduced 5-LO product formation of S271A–5-LO and S663A–5-LO, indicating that the AA-induced activation of mutated 5-LO enzymes strongly depends on Ca2+. Extracellular Ca2+ seemed to play a minor role for activation of the S663A mutant, whereas removal of intracellular Ca2+ by BAPTA-AM gave more reduced 5-LO product synthesis (72±4%) as compared with S271A–5-LO (49±9%; Fig. 7A ). Notably, the mutant S271A–S663A–5-LO (lacking both phosphorylation sites) was not active at all in the absence of Ca2+ (not shown). Taken together, Ca2+ and 5-LO phosphorylation seems to be required for AA-induced 5-LO product formation in HeLa cells.



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  		Figure 7. Role of Ca2+ on 5-LO product formation in transfected HeLa cells. HeLa cells were transiently transformed with plasmid DNA (10 µg) encoding wt 5-LO, S271A–5-LO, or S663A–5-LO as described in Materials and Methods. (A) 5-LO product formation. Cells (2x106) were resuspended in 1 ml PG buffer containing the indicated additives (1 mM CaCl2, 1 mM EDTA, 30 µM BAPTA-AM). After 10 min at 37°C, 40 µM AA was added, and cells were incubated for another 10 min at 37°C. 5-LO product formation was determined by HPLC. The control values (100%) in the presence of Ca2+ were 420 ± 69, 137 ± 15, and 202 ± 48 ng/106 cells for wt 5-LO, S271A–5-LO, and S663A–5-LO, respectively. Results are given as mean + SE; n = 3. Student’s t-test; **P < 0.01. (B) Influx of Ca2+. Cells transfected with wt 5-LO were loaded with Fura-2, the indicated stimuli were added, and the fluorescence was measured. Intracellular-free Ca2+ was calculated as described. The monitored curves show one typical experiment out of three to four.

 
Hyperosmotic stress induces 5-LO product formation in MM6 cells
5-LO product formation with respect to 5-LO phosphorylation was further investigated in MM6 cells. Priming with PMA for 10 min led to considerable activation of ERKs (and p38 MAPK; Fig. 4A ). However, PMA priming increased 5-LO product synthesis after subsequent stimulation with AA (40 µM) only 1.5- to twofold (not shown). Thus, at low, intracellular Ca2+ concentrations (present in MM6 after AA challenge; see Fig. 2 ), activation of ERKs (and p38 MAPK) appears not sufficient for prominent 5-LO product formation from exogenous AA in MM6 cells; compare ref. [16 ].

We determined if hyperosmotic stress, which activated 5-LO and MAPK pathways in PMNL and a B cell line (BL41-E95-A) [8 , 15 ], could induce 5-LO product synthesis also in MM6 cells. Compared with isotonic conditions, preincubation of MM6 cells with sucrose for 5 min led to a dose-dependent up-regulation (up to eightfold) of 5-LO product synthesis induced by 40 µM AA (Fig. 8A ). Most prominent effects were observed when cells were preincubated with sucrose for 3–5 min prior addition of AA (Fig. 8B) . In parallel, ERKs became strongly activated by sucrose in a dose- and time-dependent manner (Fig. 8A and 8B , lower panels), although some discrepancies at higher concentrations (1 and 1.2 M) of sucrose were apparent between 5-LO product formation and ERK activation (Fig. 8A) . As can be seen from Figure 8C , removal of Ca2+ by EDTA or by EDTA plus BAPTA-AM only slightly abrogated 5-LO product formation induced by AA plus sucrose, and sucrose treatment caused no enhancement of AA-induced mobilization of Ca2+ (not shown), implying Ca2+-independent activation pathways for 5-LO.



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  		Figure 8. Characterization of sucrose-induced 5-LO product formation and activation of ERK1/2 in MM6 cells. (A) Dose-response curve. To determine 5-LO product formation (upper panel), sucrose was added to MM6 cells (3x106) in PGC buffer to a final volume of 1 ml, 5 min before stimulation with 40 µM AA. After another 10 min at 37°C, 5-LO product formation was determined. Results are given as mean + SE; n = 3. Student’s t-test; **P < 0.01. To determine ERK1/2 activation (lower panel), sucrose was added to MM6 cells (3x106) in PGC buffer to a final volume of 100 µl. After 5 min at 37°C, 40 µM AA was added and incubated for 1.5 min. Then, samples were analyzed for dually phosphorylated ERKs by WB. (B) Time course of 5-LO product formation. MM6 cells (3x106 in PGC buffer) were stimulated with 0.8 M sucrose at 37°C; final volume was 1 ml. After the indicated times, 40 µM AA was added, the samples were incubated for another 10 min, and 5-LO products were determined by HPLC. Results are given as mean + SE; n = 3. Student’s t-test; **P < 0.01. To determine ERK1/2 activation, MM6 cells were stimulated with 0.8 M sucrose at 37°C. After the indicated times, samples were analyzed for dually phosphorylated ERK1/2 by WB. Results are representative of at least three separate experiments. (C) MM6 cells (3x106) were incubated first in PG buffer containing 1 mM Ca2+, 1 mM EDTA, and 30 µM BAPTA-AM as indicated for 10 min and were then stimulated with 0.8 M sucrose at 37°C; final volume was 1 ml. After 5 min, 40 µM AA was added, and after another 10 min, 5-LO products were determined by HPLC. Results are given as mean + SE; n = 3. (D) MM6 cells (3x106 in PGC buffer) were preincubated with 10 µM SB203580 and/or 3 µM U0126 for 30 min at 37°C as indicated and then stimulated with 0.8 M sucrose; final volume was 1 ml. After 5 min, 40 µM AA was added, and after another 10 min, 5-LO products were determined by HPLC. Results are given as mean + SE; n = 4.

 
The effects of kinase inhibitors on 5-LO product formation in MM6 cells were determined. Of considerable interest, neither U0126 (3 µM) nor SB203580 (10 µM) alone nor the combination of both inhibitors markedly suppressed 5-LO product formation induced by sucrose (Fig. 8D) , suggesting that additional mechanisms besides phosphorylation events involving ERK1/2 or p38 MAPK are involved in sucrose-induced 5-LO product formation in MM6 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we show that AA differentially induces 5-LO product formation depending on the cell type. In this study, LTB4 and its all-trans isomer as well as 5-H(p)ETE are the representative 5-LO products that were determined, whereas their metabolites and the formation of cys-LTs were not assessed. Thus, one should keep in mind that the signaling components studied (Ca2+, p38 MAPK, ERKs) are germane only with respect to these representative 5-LO metabolites. Although all cell types examined in this study produced considerable amounts of 5-LO products in response to AA plus ionophore, striking differences were observed when cells had been stimulated with AA alone. Thus, AA-induced 5-LO product formation was prominent in RBL-1 cells, freshly isolated human PMNL, and transfected HeLa cells, but not in MM6 cells. In parallel, in RBL-1, PMNL, and MM6 cells, p38 MAPK was activated (phosphorylated) in response to AA. ERK1/2 became rapidly activated in response to AA only in PMNL and RBL-1 cells, but AA failed to activate ERK1/2 in MM6 cells. However, activation of ERKs in MM6 cells by PMA caused no prominent 5-LO product synthesis from exogenous AA. The AA-induced 5-LO product synthesis in PMNL or RBL-1 cells appeared to be independent of Ca2+ but instead required the activity of p38 MAPK and ERK1/2 (see ref. [13 ] for effects of MAPK inhibitors on PMNL). For AA-stimulated HeLa cells, phosphorylation and Ca2+ seem to be involved in 5-LO product synthesis. Collectively, our data suggest that depending on the cell type, Ca2+ and phosphorylation events differentially determine AA-induced 5-LO product formation. In this respect, also other ligand receptor interactions may use different and cell type-dependent signaling pathways for 5-LO activation that have yet to be described.

Today, it is generally accepted that upon cell stimulation, elevation of intracellular Ca2+ can activate 5-LO for LT biosynthesis [1 , 3 ]. Ca2+ binds to the C2 domain of 5-LO and thereby stimulates 5-LO reactions [6 , 33 ] and promotes 5-LO association with the nuclear membrane [5 , 7 ]. Nevertheless, 5-LO product formation in vitro and in vivo can also be substantial in the absence of Ca2+ [8 , 10 , 34 35 36 ]. Recently, we demonstrated Ca2+-independent activation of 5-LO in PMNL stimulated by cell stress [8 ].

It was shown that ERKs and p38 MAPK-regulated MKs can phosphorylate 5-LO in vitro, and activation of these kinases correlated to increased 5-LO product synthesis without concomitant mobilization of intracellular Ca2+ [8 , 13 , 14 ]. Phosphorylation events in conjunction with Ca2+ are also important for the activity of cytosolic phospholipase A2 (cPLA2) and for PKC, and it was suggested that phosphorylation increases the affinity of these enzymes toward Ca2+ [4 , 37 , 38 ]. In analogy with cPLA2, it appears that activation of 5-LO occurs at high intracellular Ca2+ levels (created by ionophore) without the need of phosphorylation [8 , 13 ], whereas enzyme activation by phosphorylation might circumvent the necessity for elevated Ca2+ levels. Notably, Mg2+, which is abundant in cells (0.1–1 mM), can bind and activate 5-LO and might thus substitute for Ca2+ [35 ].

Formation of LTs in PMNL in response to exogenous AA was reported by several groups, including ours [9 10 11 12 13 ]. In vivo, peripheral blood leukocytes can use exogenous AA derived from neighboring platelets or endothelial cells to generate LTs via transcellular routes [39 ], thus implying possible relevance of our present findings to such in vivo scenarios. It was proposed that besides being a 5-LO substrate, AA may contribute to LT formation at multiple sites. AA causes moderate mobilization of intracellular Ca2+ (Fig. 2 and ref. [9 ]), which depends on the initial conversion to LTB4. It was suggested that this initial burst of LTB4 biosynthesis occurs in the absence of measurable Ca2+ mobilization [11 ], and it is conceivable that phosphorylation may facilitate such an initial enzyme activation. In this regard, it is of interest that AA leads to rapid and dose-dependent activation of the 5-LO kinases MK2 and ERK1/2 [17 , 22 , 26 ], and AA is required for efficient phosphorylation of 5-LO by MK2 and ERK2 [12 , 13 ]. Of interest, depletion of the potential MK2 or ERK1/2 phosphorylation sites in 5-LO specifically impaired AA-induced 5-LO product synthesis in HeLa cells [12 , 13 ]. Intriguingly, it was shown for human and rat neutrophils that conversion of AA by 5-LO to 5-HETE is necessary for substantial activation of the ERK cascade [26 , 40 ], implying autoregulatory loops for 5-LO activation by ERKs. Taken together, it is reasonable to speculate that one possible (Ca2+-independent) mechanism by which AA stimulates 5-LO product formation might be the induction and promotion of 5-LO phosphorylation.

In this study, AA failed to substantially elevate the intracellular Ca2+ levels in PMNL and RBL-1 cells (Fig. 2) but caused rapid and marked activation of ERK1/2 and p38 MAPK that correlated to 5-LO product synthesis (Fig. 5) . As found previously for PMNL [13 ] and also for RBL-1 cells, pharmacological inhibition of these 5-LO kinase pathways blocked AA-induced 5-LO product formation. In contrast, the kinase inhibitors failed to suppress 5-LO product synthesis in PMNL [13 ] and RBL-1 cells (Fig. 6) treated with ionophore plus AA. Under these conditions, Ca2+ may be the predominant activator of 5-LO, and phosphorylation should be of minor importance. Such interpretations are favored by the experiments using transfected HeLa cells (Fig. 7) . It was shown that AA activates p38 MAPK and ERK pathways in HeLa cells [17 ], and incubation of HeLa cells transfected with 5-LO gave prominent 5-LO product formation in response to AA [12 , 13 ]. Whereas the activity of wt 5-LO was reduced to ~40% by Ca2+ depletion in AA-stimulated HeLa cells, removal of Ca2+ had a more striking effect on 5-LO product formation of mutated 5-LO enzymes (~20% remaining activity) lacking phosphorylation sites (Fig. 7) . These data imply that in HeLa cells, Ca2+ (which was significantly elevated after stimulation with AA) and phosphorylation seem to contribute to AA-induced 5-LO product formation. This is in accordance with previous findings [12 , 13 ] showing that for 5-LO phosphorylation site mutants expressed in HeLa cells, AA-induced 5-LO activity was strongly, but not completely, reduced.

It is puzzling that in PMNL, depletion of Ca2+ strongly increased AA-induced 5-LO product formation and that in RBL-1 cells, elevated, intracellular Ca2+ levels (created by ionophore) led to lower 5-LO activity compared with stimulation with AA alone or even in the absence of Ca2+ (Figs. 1 and 2) . In the presence of Ca2+, enzymatically active 5-LO in vitro becomes rapidly inactivated (t1/2=1 min) by lipid hydroperoxides formed during catalysis (turnover-dependent inactivation), whereas in the absence or at low concentrations of Ca2+, inactivation is considerably prolonged [41 , 42 ]. Accordingly, 5-LO kinetic data obtained from intact cells demonstrate that ionophore-induced 5-LO product formation was terminated after 1–1.5 min, and AA-induced 5-LO product synthesis (particularly after omission of Ca2+) proceeded continuously up to 10 min (Fig. 4) . Thus, in cells depleted of Ca2+, 5-LO inactivation may be delayed, leading to higher amounts of products formed after 10-min incubations.

In contrast to PMNL and RBL-1 cells, only p38 MAPK but not the ERKs is activated by AA in MM6 cells (Fig. 5) ; the reason for this is unclear. It was found that AA activates the p38 MAPK in a cell type-specific manner. Thus, AA stimulates p38 MAPK in endothelial cells, neutrophils, HL60, and HeLa cells but not in Jurkat cells [17 ]. Examination of the signal transduction pathways by which AA stimulates ERKs in human and rat neutrophils revealed the involvement of pertussis toxin-sensitive G proteins, nonreceptor tyrosine kinases, PI-3K, phospholipase C, Raf-1, and MEK [26 , 40 ]. The responsivness of these components to AA in MM6 cells remains to be determined. Also, the reason for the inability of MM6 cells to form 5-LO products in response to AA alone is not clear and cannot be explained by low ERK activity, as PMA, which substantially activated ERK1/2, hardly increased (at most about twofold) 5-LO product formation. Thus, in contrast to PMNL and RBL-1 cells, phosphorylation events involving ERKs and MKs are not sufficient to activate 5-LO in MM6 cells. In this respect, we showed in our previous reports that PMA enhanced 5-LO product formation in MM6 only after subsequent stimulation with ionophore, and it appeared that kinase activity plus Ca2+ are necessary for full activation of 5-LO [13 , 16 ].

Preincubation with sucrose induced ERK1/2 activation as well as Ca2+-independent up-regulation (up to eightfold) of 5-LO product synthesis. However, sucrose-induced 5-LO activation was only marginally (<15–20%) attenuated by SB203580 or/and U0126, suggesting that the MAPK pathways are of minor importance. This is in sharp contrast to our recent report, where hyperosmotic, stress-induced 5-LO product formation in PMNL or B-lymphocytes was blocked by the p38 MAPK inhibitor SB203580 [8 , 15 ]. Therefore, alternative, hyperosmotic, stress-induced signal transduction pathways contribute to 5-LO activation in MM6 cells, and 5-LO phosphorylation (or phosphorylation of other proteins) by ERKs and/or p38 MAPK-regulated MKs seems less important. Conceivable, alternative routes could be the stress-inducible c-jun NH2-terminal kinase (JNK) or PI-3K pathways. However, JNKs were not activated in sucrose-treated MM6 cells (not shown), and the specific PI-3K inhibitor wortmannin (up to 1 µM) barely reduced 5-LO product formation alone or in combination with U0126 and SB203580 (not shown). The identification of the respective pathways by which sucrose induces 5-LO activation in MM6 cells remains an interesting task for the future.

Together, our findings show that exogenous AA is a potent activator of 5-LO depending on the cell type and emphasize that Ca2+ and MAPK pathways contribute to activation of 5-LO. It also becomes clear that the relative importances of Ca2+/phosphorylation vary between different cell types and that a given stimulus (as AA and possibly other receptor-coupled stimuli) uses different mechanisms in different cells. Thus, a simple scheme for activation of 5-LO in intact cells is not generally applicable, particularly as it appears that unknown players still remain to be identified.


   ACKNOWLEDGEMENTS
 
We thank Astrid Neuss for expert technical assistance. This study was supported by grants from the Deutsche Pharmazeutische Gesellschaft, the Swedish Medical Research Council (03X-217), the EU (QLG1-CT-2001–01521), and the Verum Foundation.

Received July 10, 2002; revised September 13, 2002; accepted September 17, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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