* Institute of Pharmaceutical Chemistry, University of Frankfurt, Germany; and
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, 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
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Key Words: leukotriene • p38 MAP kinase • arachidonic acid • cytosolic phospholipase A2 • cell stress • Mono Mac 6
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Formation of LTs in leukocytes depends on the release of sufficient amounts of AA from endogenous pools by activated phospholipase A2 (PLA2) or via transcellular migration of AA, released from surrounding cells such as platelets or endothelial cells. Depending on the cell type, 5-LO can be present in the cytosol but also in a soluble pool of the nucleus of resting cells (for review on 5-LO, see ref. [3 ]). On cell stimulation by Ca2+-mobilizing agents (e.g., ionophore A23187, thapsigargin), soluble 5-LO translocates to the nuclear membrane where it colocalizes with 5-lipoxygenase-activating protein (FLAP) and cytosolic PLA2 (cPLA2). This migration to the nuclear membrane is believed to be necessary for substantial formation of LTs, and it is assumed that phosphorylation of 5-LO could influence its translocation to the nucleus and/or the association with the nuclear membrane and could stimulate its catalytic activity [4 5 6 ]. Moreover, the capacity of 5-LO to metabolize AA depends on several cofactors, such as Ca2+, adenosine 5'-triphosphate (ATP), phosphatidylcholines (membranes), hydroperoxides, and cellular proteins. In fact, it was demonstrated that 5-LO may interact with Grb-2, a coactosin-like protein, a transforming growth factor-ß (TGF-ß) receptor-associated protein, and a putative RNase III [7 , 8 ].
Hypertonicity (HT) leading to cell shrinkage has been shown to inhibit several neutrophil functions, such as superoxide production, phagocytosis, elastase release, adhesion, and cytokine production. However, the cellular mechanisms underlying these effects remain largely undefined [9 10 11 12 13 14 ]. Osmotic cell shrinkage triggers rapid tyrosine phosphorylation in a variety of cell types and was shown to activate mitogen-activated protein kinase (MAPK) pathways [13 , 15 16 17 18 19 20 ]. It is interesting that HT, which activated MAPK by itself, suppressed the activation of these enzymes by other stimuli [11 , 13 ].
Recently, cellular stress was shown to activate p38 MAPK and downstream MAPK-activated protein kinases (MAPKAPKs), which can phosphorylate 5-LO in vitro, and it was suggested that such phosphorylation increases 5-LO product formation in various leukocytes [6 , 21 , 22 ].
In this study, we present that HT, leading to cell shrinkage, reversibly abrogates ionophore-induced release of AA and 5-LO product formation, as well as translocation of 5-LO to the nuclear membrane in polymorphonuclear leukocytes (PMNL) and Mono Mac 6 (MM6) cells. We show that HT does not prevent rapid Ca2+ influx but prevents the ionophore-induced activation of p38 MAPK-regulated 5-LO kinases.
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-32P]ATP (110
TBq/mmol) was purchased from Amersham (Buckinghamshire, England).
Nycoprep was from PAA Laboratories (Linz, Austria); phorbol
12-myristate 13-acetate (PMA), Ca2+ ionophore
A23187, and thapsigargin were from Sigma Chemical Co. (Deisenhofen,
Germany); and high-pressure liquid chromatography (HPLC) solvents were
from Merck (Darmstadt, Germany). Fura-2 AM was from Calbiochem (Bad
Soden, Germany).
Cells and cell incubations
MM6 cells were cultured and differentiated with TGF-ß and
calcitriol as described [25
]. Cells were harvested by
centrifugation [200 g, 10 min at room temperature (RT)]
and washed once in phosphate-buffered saline (PBS), pH 7.4. Human PMNL
were isolated immediately from fresh leukocyte concentrates obtained at
St. Markus Hospital (Frankfurt, Germany) as described
[26
]. Cells were resuspended in isotonic PBS containing
1 mg/ml glucose and 1 mM CaCl2 (PGC buffer). Hyperosmolar
solutions were added prior to stimulation with the indicated agonists
at 37°C at a final volume of 1 ml (see below). In some experiments,
cells were washed by centrifugation (200 g, 5 min at RT)
immediately after treatment with hyperosmolar solutions and were
resuspended in 1 ml isotonic PGC buffer.
Determination of 5-lipoxygenase product formation
For assays of intact cells, freshly isolated PMNL
(5x106) or differentiated MM6 cells (3x106)
were finally resuspended in PGC buffer and preincubated as indicated.
The reaction was started by addition of ionophore A23187, with or
without exogenous AA at the indicated concentrations. Final volume of
the incubations was 1 ml. 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
[27
]. 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 [5(S),12(S)-DiHETE] and
5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid
[5-H(p)ETE].
Permeabilization of PMNL
Freshly isolated PMNL were permeabilized using nystatin,
according to the method of Krump et al. [28
], with some
minor changes. In brief, PMNL were resuspended in KS solution (115 mM
KCl, 50 mM sucrose), collected by centrifugation (200 g, 5
min at RT), and finally resuspended in this solution at 5 x
107 cells/ml. After incubation in the absence or presence
of 50 µg/ml nystatin for 10 min at 4°C, 0.3 M KCl was added to the
cell suspension for 3 min at 4°C. Then, cells were washed by
centrifugation and resuspended in KS solution containing 0.3 M KCl to
remove nystatin, leading to resealing the plasma membrane. After 5 min
at 37°C, cells were centrifuged, transferred into PGC buffer, and
challenged by 2.5 µM ionophore in the absence or presence of 0.3 M
KCl, and 5-LO translocation was determined as described below.
Determination of release of [3H]-labeled AA from PMNL
and MM6 cells
Freshly isolated PMNL or differentiated MM6 cells were
resuspended at 2 x 106/ml in RPMI-1640 medium
containing 4.8 nM [3H]AA (corresponding to 0.25 µCi/ml,
specific activity 200 Ci/mmol) and were incubated for 120 min at 37°C
in 5% CO2 atmosphere. Thereafter, cells were collected by
centrifugation and washed once with PBS and twice with PBS containing 2
mg/ml fatty acid-free albumin to remove unincorporated
[3H]AA. Labeled PMNL (5x106) or MM6 cells
(3x106) were resuspended in 1 ml PGC buffer containing 2
mg/ml fatty acid-free albumin and were preincubated as indicated. The
reaction was started by addition of ionophore A23187. Final volume of
the incubations was 1 ml. After 5 min at 37°C, the samples were put
on ice for 2 min, and cells were centrifuged at 200 g for 5
min at RT. Aliquots (200 µl) of the supernatants were measured in a
beta-counter (Micro Beta Trilux, Perkin Elmer, Foster City, CA) to
detect the amounts of [3H]-labeled AA released into the
cell medium.
Subcellular redistribution of 5-LO
Subcellular localization of 5-LO was performed as described
previously [29
]. In brief, MM6 cells
(1x107) or human PMNL (3x107) were
resuspended in PGC buffer and preincubated as indicated at 37°C.
After addition of ionophore at the indicated concentrations, samples
were incubated for another 10 min at 37°C, chilled on ice for 3–5
min, and nuclear and nonnuclear fractions were obtained after cell
lysis by 0.1% Nonidet P-40 (NP-40). Aliquots of nuclear and nonnuclear
fractions were mixed immediately with the same volume of 2 x
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
sample loading buffer (SDS-b), heated for 6 min at 95°C, and analyzed
for 5-LO protein by SDS-PAGE and immunoblotting.
Measurement of intracellular calcium levels
Freshly isolated PMNL (1x107 in 1 ml PGC buffer)
were incubated with 2 µM Fura-2 AM for 30 min at 37°C. Cells were
washed, resuspended in PGC buffer (final vol, 1 ml), and transferred
into a thermally controlled (37°C) fluorimeter cuvette in a
spectrofluorometer (Aminco-Bowman Series 2, Thermo Spectronic,
Rochester, NY) 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. [30
], and
Fmax (maximal fluorescence) was obtained by
lysing the cells with 1% Triton-X 100 and Fmin
(minimal fluorescence), by chelating Ca2+ with
10 mM ethylenediaminetetraacetate.
Immunoblot analysis of total cell lysates and subcellular
fractions; antibodies
Total cell lysates (20 µl) and aliquots (25 µl) of pair-wise
subcellular fractions (cytosol and nucleus, corresponding to equal
amounts of cells) 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) on a 10% gel. After electroblot to
nitrocellulose membrane (Amersham Pharmacia, Little Chalfont, UK),
membranes were blocked with 5% nonfat dry milk in 50 mM Tris/HCl, pH
7.4, and 100 mM NaCl Tris-buffered saline (TBS) for 1 h at RT.
Membranes were washed and then incubated with primary antibody
overnight at 4°C. Anti-5-LO antiserum (AK7, 1551) was
affinity-purified on a 5-LO column, and phospho-specific antibodies
recognizing p38 MAPK (Thr180/Tyr182) were obtained from New England
Biolabs (Beverly, MA) and were used as 1:2000 dilution. Antibody
against p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA).
The membranes were washed with TBS and incubated with 1:1000 dilution
of alkaline phosphatase (AP)-conjugated immunoglobulin G (Sigma
Chemical Co.) for 2 h at RT. After washing with TBS and TBS plus
0.1% NP-40, proteins were visualized with the AP substrates, 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).
In-gel kinase assay
Incubations were stopped by addition of the same volume of SDS-b
and heated for 6 min at 95°C. Total cell lysates of PMNL and MM6
cells corresponding to 0.5 x 106 cells were analyzed
for 5-LO kinase activity by in-gel kinase assay using purified 5-LO
(0.2 mg/ml) as substrate as described [6
].
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Figure 1. Hypertonicity suppresses ionophore-induced 5-LO product formation
in PMNL and MM6 cells. (A) Isolated PMNL. To 5 x 106
cells in PGC buffer, solutions of the indicated compounds
were added directly prior to stimulation with 2.5 µM A23187. (B) MM6
cells. Cells (1.5x106) in PGC buffer were
preincubated with 100 nM PMA for 10 min at 37°C. Then, solutions of
the indicated compounds were added directly prior to stimulation with 5
µM A23187. Final incubation volume was 1 ml, and the samples were
incubated at 37°C for 10 min. 5-LO product formation was determined
by HPLC. The control values (100%) were 51.8 ± 6.7
ng/106 PMNL and 83.2 ± 6.6 ng/106 MM6
cells. Results are given as mean + SE,
n = 3–5.
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Figure 2. Effects of exogenous AA on hypertonicity-suppressed 5-LO product
formation. (A) Isolated PMNL. NaCl at the indicated concentrations was
added to 5 x 106 cells and resuspended in PGC
buffer to a final volume of 1 ml, directly prior to
stimulation with 2.5 µM A23187 or with 2.5 µM A23187 plus 40 µM
AA as indicated. After 10 min at 37°C, 5-LO product formation was
determined. The control values (100%) were 51.8 ± 6.7 and
75.4 ± 6.4 ng/106 cells in the absence or presence of
exogenous AA, respectively. (B) MM6 cells. When 5-LO product formation
was determined in the absence of exogenous AA, cells were preincubated
with 100 nM PMA for 10 min at 37°C. Sucrose at the indicated
concentrations was added to 1.5 x 106 cells and
resuspended in PGC buffer to a final volume of 1 ml. Then,
cells were stimulated with 5 µM A23187 or with 5 µM A23187 plus 40
µM AA as indicated at 37°C for 10 min, and 5-LO product formation
was determined. The control values (100%) were 83.4 ± 6.6 and
460 ± 25.7 ng/106 cells in the absence or presence of
exogenous AA, respectively. Results are given as mean +
SE, n = 3–4.
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Figure 3. Rapid termination of 5-LO product formation by hypertonicity. Freshly
isolated PMNL (2x108), resuspended in 14 ml PGC buffer,
were stimulated with 2.5 µM A23187. After the time periods indicated
in the figure, aliquots (700 µl) corresponding to 5 x
106 cells were mixed with the same volume of ice-cold
methanol or alternatively transferred to tubes containing 300 µl 1 M
NaCl solution (0.3 M NaCl, final concentration, 1 ml final volume) and
incubated for a further 10 min at 37°C. For all samples, 5-LO product
formation was determined by HPLC. Similar results were obtained in two
additional independent experiments.
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Figure 5. Hyperosmolarity blocks translocation of 5-LO and reduces 5-LO product
formation. (A) Isolated PMNL. NaCl at the indicated concentrations was
added to 5 x 106 (5-LO product formation) or 3 x 107 (5-LO translocation) cells, resuspended in PGC
buffer to a final volume of 1 ml. Then, cells were
stimulated with 2.5 µM A23187 or with 2.5 µM A23187 plus 40 µM AA
as indicated at 37°C for 10 min. (B) MM6 cells. Cells
(1.5x106 for 5-LO product formation or 1x107
for 5-LO translocation) were primed for 10 min at 37°C with 100 nM
PMA. Then, NaCl at the indicated concentrations was added, and cells
were stimulated with 5 µM A23187 or with A23187 (5 µM) plus AA (40
µM) for 10 min. 5-LO product formation was determined by HPLC.
Results are given as mean + SE, n =
3–4. 5-LO translocation was determined by Western blot analysis of
5-LO in subcellular fractions (non-nuclear, nuclear) following cell
fractionation by detergent lysis (0.1% NP-40). Pair-wise samples
(non-nuclear, nuclear) correspond to the identical cell numbers.
Similar results were obtained in two additional, independent
experiments.
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Figure 4. Hypertonicity blocks the release of [3H]AA from
phospholipids. Freshly isolated human PMNL (2x106/ml in
RPMI-1640 medium) were prelabeled with 0.25 µCi/ml
[3H]AA for 120 min at 37°C and 5% CO2.
After removal of unincorporated [3H]AA, cells
(5x106 in 1 ml PGC buffer, containing 2 mg/ml fatty
acid-free albumin) were treated with the indicated additives, and 2.5
µM A23187 was added and incubated for 5 min at 37°C. Free
(nonesterified) [3H]AA was determined as described in
Materials and Methods. Results are given as mean + SE,
n = 5.
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Cell shrinkage reversibly blocks translocation and 5-LO product
synthesis
We attempted to determine whether cell destruction, intracellular
hyperosmolarity, or cell shrinkage reduced translocation and product
formation of 5-LO. To determine if HT-mediated effects were reversible,
PMNL and MM6 cells were exposed to hypertonic solutions for 5 min and
were centrifuged, and hyperosmolar medium was then replaced by isotonic
buffer. PMNL were stimulated with ionophore subsequently, and MM6 cells
were primed with PMA for 10 min and then stimulated with ionophore. As
shown in Figure 6
, reconstitution of isotonic conditions (fresh buffer) restored
activity and translocation of 5-LO in both cell types completely. When
NaCl-treated PMNL were transferred to fresh buffer and re-exposed to
hypertonic NaCl, 5-LO product formation was suppressed again
(unpublished results).
 View larger version (25K): [in a new window] |
Figure 6. Inhibition of 5-LO translocation and 5-LO product formation by
hypertonicity is reversible. (A) Isolated PMNL. Sucrose or NaCl (at the
indicated final concentrations) was added to 5 x 106
(5-LO product formation) or 3 x 107 (5-LO
translocation) cells, resuspended in PGC buffer as
indicated in the figure. Cells were stimulated with 2.5 µM A23187 for
10 min at 37°C or alternatively centrifuged, resuspended in fresh PGC
buffer, and stimulated with 2.5 µM A23187 for 10 min at 37°C as
indicated. (B) MM6 cells. Cells (1.5x106, 5-LO product
formation, or 1x107, 5-LO translocation) were primed for
10 min at 37°C with 100 nM PMA. Then, NaCl (0.3 M) was added as
indicated, and cells were stimulated with 5 µM A23187 for 10 min at
37°C. Alternatively, non-PMA-primed cells were treated with NaCl (0.3
M, final concentration), centrifuged, resuspended in fresh PGC buffer,
and primed with 100 nM PMA for 10 min at 37°C prior to stimulation
with 5 µM A23187 for another 10 min at 37°C. 5-LO product formation
and 5-LO translocation were determined as described in the legend to
Figure 4
. The control values (100%) for 5-LO product formation were
51.8 ± 6.7 and 83.2 ± 6.6 ng/106 PMNL and MM6
cells, respectively.
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Figure 7. Cell shrinkage suppresses 5-LO translocation and 5-LO product
formation. (A) Isolated PMNL. Sucrose, NaCl, or urea (at the indicated
final concentrations) was added to 5 x 106 (5-LO
product formation) or 3 x 107 (5-LO translocation)
cells, resuspended in PGC buffer as indicated in the
figure. Then, cells were stimulated with 2.5 µM A23187 for 10 min at
37°C. (B) MM6 cells. Cells (1.5x106, 5-LO product
formation, or 1x107, 5-LO translocation) were primed for
10 min at 37°C with 100 nM PMA. Then, NaCl or urea was added as
indicated, and cells were stimulated with 5 µM A23187 for 10 min at
37°C. 5-LO product formation and 5-LO translocation were determined
as described in the legend to Figure 4
. The control values (100%) for
5-LO product formation were 51.8 ± 6.7 and 83.2 ± 6.6
ng/106 PMNL and MM6 cells, respectively.
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Figure 8. 5-LO translocation in nystatin-permeabilized PMNL. Freshly isolated
PMNL were permeabilized by nystatin as described in Materials and
Methods and incubated with 2.5 µM ionophore in the absence or
presence of additional 0.3 M KCl for 10 min as indicated in the figure.
5-LO translocation was determined by Western blot analysis of 5-LO in
subcellular fractions (non-nuclear, nuclear), following cell
fractionation by detergent lysis (0.1% NP-40). In contrast to
untreated cells (lanes 1 and 2), hypertonic KCl could not prevent 5-LO
translocation in PMNL treated with nystatin (lanes 3 and 4), where cell
shrinkage was prevented. Similar results were obtained in two
additional, independent experiments.
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Figure 9. Effects of hypertonicity on ionophore-induced
Ca2+ influx. Fura-2-loaded PMNL
(1x107 in PGC buffer) were treated as indicated at 37°C,
and the fluorescence was measured; final volume was 1 ml. Intracellular
free Ca2+ was calculated as described. The
monitored curves show one typical experiment out of four to five.
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Figure 10. Hypertonicity prevents activation of p38 MAPK and downstream 5-LO
kinases. Freshly isolated PMNL (5x106), resuspended in PGC
buffer, were stimulated with the indicated additives at 37°C. Final
volume was 100 µl. After 2.5 min, the reaction was stopped by
addition of SDS-b, and the samples were boiled at 95°C for 6 min. (A)
Determination of p38 MAPK activation. Samples were electrophoresed and
analyzed for dually phosphorylated p38 MAPK by immunoblotting (upper
panel), and equal amounts of protein were evaluated with anti-p38 MAPK
antibodies (lower panel). (B) Activation of p38 MAPK-regulated 5-LO
kinases. Samples were electrophoresed on a polyacrylamide gel that
contained 0.2 mg/ml 5-LO as substrate. In-gel kinase assay was
performed as described in Materials and Methods. Arrows indicate the
expected positions for MK2 and MK3.
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Particularly in the absence of exogenous substrate, suppression of 5-LO product formation by HT was prominent; thus, limited substrate supply seems to contribute to low 5-LO product synthesis. One reason for limited substrate supply should be impaired activity of cPLA2, which is assumed to release AA at the nuclear membrane for LT formation in PMNL and monocytes [31 ]. In fact, HT strongly suppressed ionophore-induced release of AA (Fig. 4) . Cytosolic PLA2 is regulated by Ca2+ binding to its C2 domain and/or phosphorylation by members of the MAPK family, depending on the cell type and the stimulus used [32 , 33 ]. In this study, HT prevented ionophore-induced activation of p38 MAPK, implying that reduced kinase activities could possibly contribute to low AA release. However, opposite effects were observed for human platelets, where HT increased ionophore-induced liberation of AA and the activity of cPLA2, accompanied by elevated MAPK activities [34 ]. Further studies are necessary to elucidate the mechanisms by which AA release is suppressed in leukocytes by HT.
Another reason for impaired 5-LO product synthesis could be that HT impaired 5-LO translocation. For efficient LTA4 formation in intact cells, 5-LO has to translocate from a soluble compartment to the nuclear membrane, where it colocalizes with cPLA2 and FLAP [35 , 36 ], and an orchestrated interplay between these enzymes is considered important for substantial LT synthesis. HT blocked translocation of 5-LO from the cytosol to the nucleus, with a clear correlation with the dose response for inhibition of 5-LO product formation (Fig. 5) . We found before that in MM6 cells, stimulation with ionophore alone failed to induce 5-LO translocation and product formation, but after priming with PMA, subsequent ionophore stimulation led to 5-LO redistribution to the nucleus, and 5-LO product synthesis from endogenous substrate occurred [29 ]. Nonprimed cells (5-LO remains in the cytosol) gave prominent 5-LO product formation after stimulation with ionophore plus exogenous AA. We concluded that in presence of exogenous AA, 5-LO in the cytosol has ample supply of substrate, and 5-LO product formation can occur without translocation. In this study, also in the presence of exogenous AA, HT still reduced 5-LO product formation to about 60–70% of control, indicating that translocation also may be beneficial for conversion of exogenous substrate. Indeed, it was found that FLAP also stimulated the conversion of exogenous AA [37 ]. Taken together, the strong suppression of 5-LO product formation by HT in cells stimulated with ionophore alone seems to be because of the inhibition of cPLA2, causing low substrate release, but also as a result of the inability of 5-LO to translocate to the nuclear membrane, where AA is presented by FLAP for efficient metabolism.
When Ca2+ ionophore activates 5-LO in PMNL and other types of leukocytes, one important event is Ca2+ binding to the N-terminal C2-like domain of 5-LO [38 ], which in turn promotes membrane association similar to cPLA2 activation by Ca2+ [31 ]. Thus, elevation of intracellular Ca2+ is a determinant for activation of cPLA2 and 5-LO, and it seemed possible that HT could prevent the activation of these enzymes as a result of interference with ionophore-induced Ca2+ influx. However, in agreement with others [13 , 39 , 40 ], HT caused no significant suppression of agonist-induced Ca2+ mobilization compared with isotonic conditions (Fig. 9) . Importantly, elevated Ca2+ levels in ionophore-stimulated cells were not decreased when hypertonic NaCl was added 10–15 s after ionophore stimulation. This is in contrast to 5-LO product formation, which was immediately blocked after NaCl addition (Fig. 3) .
p38 MAPK-regulated MKs can phosphorylate 5-LO in vitro [6 ], and there is increasing evidence that p38 MAPK and MKs lead to activation of 5-LO in PMNL and MM6 cells [6 , 22 ]. Different types of cell stress (sodium arsenite, osmotic stress) could activate p38 MAPK pathways and 5-LO in PMNL, also after chelation of Ca2+. The p38 MAPK inhibitor SB203580 was quite efficient to inhibit cell stress-induced 5-LO product formation (IC50, 5 µM), whereas ionophore-induced 5-LO product synthesis was less sensitive (IC50, 30 µM) [22 ]. Also, in rat fibroblasts transfected with 5-LO-GFP, a high concentration of SB203580 (15 µM, which may not be specific for p38 MAPK) was required to inhibit ionophore-induced 5-LO translocation to the nucleus [41 ]. In comparison, for inhibition of MK activity in PMNL stimulated with ionophore, 3 µM SB203580 was sufficient [6 ]. Taken togeher, Ca2+ mobilizing agents or HT by themselves are sufficient for p38 MAPK activation (Fig. 10) , and each stimulus itself can activate 5-LO for product formation [22 ]. However, it appears that ionophores leading to Ca2+ mobilization activate 5-LO primarily by the Ca2+ effect (compare above), which does not depend on p38 MAPK activity.
In view of the 5-LO stimulatory effects of Ca2+ and HT-induced p38 MAPK activity, it is puzzling that when PMNL were subjected to HT in combination with ionophore, 5-LO product synthesis was downregulated. A change in phosphorylation processes may be one factor involved, because a combination of ionophore and HT gave reduced activity of p38 MAPK and MKs in PMNL (Fig. 10) . However, downregulation of p38 MAPK and MKs cannot be the entire explanation, because SB203580 did not counteract ionophore-induced 5-LO activity in PMNL efficiently [6 , 22 ]. However, the protein-kinase inhibitor calphostin C suppressed ionophore-induced 5-LO product formation from endogenous AA in MM6 cells and PMNL [29 ]. Calphostin C prevented translocation of 5-LO to the nuclear membrane, and when exogenous substrate was added, 5-LO product synthesis was prominent, probably because of cytosolic 5-LO still acting on the added AA. Thus, calphostin C, as HT in combination with ionophore, may also impair other phosphorylation events of importance for cellular 5-LO activity.
In analogy with our observations, others showed that in PMNL, HT alone activates p38 MAPK but at the same time, prevents p38 MAPK activation in response to agonists such as lipopolysaccharide [11 ]. Junger et al. [13 ] showed that in PMNL, HT by itself stimulated p38 MAPK and degranulation, whereas HT blocked activation of p38 MAPK and reduced degranulation in response to fMLP. To date, it is unclear how HT can uncouple ionophore-stimulated p38 MAPK activation in PMNL. It was suggested that HT could render p38 MAPK refractory to other stimuli or could uncouple signaling pathways upstream of p38 MAPK [11 , 13 ]. A further complication is that these effects seem to be cell-type specific. In a human B-cell line (BL41-E95-A), ionophore and HT (or other types of cell stress) were required for substantial p38 MAPK activation, which in turn, was necessary for 5-LO product synthesis. Thus, in B cells, ionophore and HT act in conjunction to stimulate p38 MAPK and 5-LO [21 ]. Remarkably, in the B cells, translocation of 5-LO to the nucleus did not occur under any conditions, and exogenous AA was always required for the cytosolic 5-LO activity.
HT was shown to result in dramatic changes in the cytoskeleton [42 , 43 ], which might cause a sustained interaction of 5-LO with cytoskeletal proteins such as actin, possibly retaining the enzyme within this locus. In fact, 5-LO binds actin in vitro, and it was postulated that in resting leukocytes, 5-LO might be bound to the cytoskeleton [5 , 7 ]. 5-LO interacts with coactosin-like protein (CLP, an actin-binding protein), and in vitro, 5-LO can inhibit actin polymerization and compete with F-actin for CLP binding [8 , 44 ]. It was shown that in neutrophils, HT induced sustained polymerization of actin [43 ], and inhibition of actin polymerization by cytochalasin B increased fMLP-induced LT synthesis [45 , 46 ]. It was speculated before that the translocation process may involve phosphorylation of 5-LO and other proteins and that the cytoskeleton may be involved [5 ]. Possibly, the effects of HT on Ca2+-induced 5-LO activation may involve alterations in the actin cytoskeleton, which prevent nuclear translocation of 5-LO. Moreover, osmotic changes of cytoskeletal structures may also affect other protein interactions and intracellular signal-transduction mechanisms relevant for cellular 5-LO activity. Rearrangement of the actin cytoskeleton was also suggested to be the reason for impaired 5-LO activity in PMNL subjected to sulphatide-induced cell spreading [47 ].
Collectively, we show here that HT, via cell shrinkage, prevents ionophore-induced 5-LO translocation and product formation. Downregulation of p38 MAPK pathways may be one factor involved, but other mechanisms putatively involving the cytoskeleton could also be operative. Inhibition of ionophore-activated LT formation is another example of suppression of Ca2+-induced phagocyte functions by HT.
Received October 2, 2001; revised November 22, 2001; accepted November 26, 2001.
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dmark, O. P. (2000) The molecular biology and regulation of 5-lipoxygenase Am. J. Respir. Crit. Care Med. 161,S11-S15dmark, O. (2000) 5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases Proc. Natl. Acad. Sci. USA 97,5261-5266dmark, O. (1999) Interaction of 5-lipoxygenase with cellular proteins Proc. Natl. Acad. Sci. USA 96,1881-1885dmark, O. (1999) 5-Lipoxygenase binds calcium Biochemistry 38,4441-4447[Medline]dmark, O. (2001) Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes Blood 97,2487-2495This article has been cited by other articles:
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  M. Rakonjac, L. Fischer, P. Provost, O. Werz, D. Steinhilber, B. Samuelsson, and O. Radmark Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production PNAS, August 29, 2006; 103(35): 13150 - 13155. [Abstract] [Full Text] [PDF]
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