Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, Stockholm, Sweden
Correspondence: Dr. Olof Rådmark, Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: olof.radmark{at}mbb.ki.se
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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Key Words: 5-lipoxygenase • mitogen-activated protein kinase • heat shock protein • Mono Mac 6 • hypertonicity • sodium arsenite
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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The three major mitogen-activated protein (MAP) kinase (MAPK) families,
the extracellular signal-regulated kinases (ERKs), the c-jun
NH2-terminal kinases/stress activated protein kinases
(JNKs/SAPKs), and the p38 MAPKs are protein serine/threonine kinases
that require dual phosphorylation on tyrosine and threonine residues
for activity [6
]. In contrast to the ERKs, which are
mainly activated by growth factors and other mitogenic stimuli, JNKs
and p38 MAPKs are activated in response to a number of stress stimuli.
In mammalian cells, p38 MAPK is activated by oxidative stress,
hyperosmolarity, UV light, heat shock, sodium arsenite (SA), endotoxin,
and inflammatory cytokines such as interleukin (IL)-1 and tumor
necrosis factor (TNF)-
[7
]. The activated kinase
phosphorylates downstream MAPK-activated protein (MAPKAP) kinases and
certain transcription factors [8
9
10
11
]. SB203580, at
concentrations below 10 µM, has been demonstrated to be an inhibitor
of p38 MAPK with no or minor effects on JNK, ERK, and several other
protein kinases [12
] and is thus considered a useful
tool to evaluate p38 MAPK-dependent events in vivo. Recently we showed
that p38 MAPK-regulated MKs can phosphorylate 5-LO in vitro and that
up-regulation of MK activity in PMNLs is connected with increased 5-LO
activity [13
]. Tyrosine kinase signaling also is
important for LT synthesis and translocation of 5-LO to the nuclear
membrane in granulocytic cells, and it has been demonstrated that 5-LO
can occur in a phosphorylated form after stimulation of HL60 cells with
ionophore [14
]. p38 MAPK is also involved in the
phosphorylation and activation of cPLA2, which releases
arachidonic acid (AA) as substrate for LTs and prostaglandins
[15
], and stimuli of cell stress
(H2O2, SA, sorbitol) enhanced ionophore-induced
AA release in platelets [16
].
In addition to well-established functions of LTs as inflammatory mediators, several findings imply that LTs also are involved in the primary adaptive immune response. Thus, LTB4 enhanced both lymphokine-driven proliferation of B lymphocytes [17 ] and IL-4-induced immunoglobulin (Ig) E production in normal peripheral blood mononuclear cells [18 ]. In mice subjected to targeted 5-LO gene disruption, ovalbumin-induced IgG and IgE production is reduced [19 ]. Also, 5-LO is expressed in dendritic cells [20 , 21 ], and mobilization of dendritic cells to lymph nodes depends on the LTC4 transporter multidrug resistance protein 1 [22 ]. B-lymphocytes express 5-LO protein and produce 5-LO metabolites in cell homogenates, but in contrast to PMNLs or monocytes, cellular 5-LO activity in BL41-E95-A cells is suppressed by selenium-dependent peroxidases [23 , 24 ]. However, LT synthesis in B cells is strongly enhanced after increase in the cellular redox status, via depletion of glutathione or by addition of hydrogen peroxide or hydroperoxides [23 , 25 , 26 ]. Oxidative stress (exposure to H2O2, diamide, or ionizing irradiation) is reported to trigger tyrosine phosphorylation and MAPK activation in B-lymphocytes, resulting in cell activation [27 28 29 30 31 ]. In B-lymphocytes, p38 MAPK is required for CD40-induced gene expression and proliferation [32 ] and IgM-induced apoptosis [33 ]. Here we demonstrate that stimuli of cellular stress up-regulate LT synthesis in BL41-E95-A cells and that stress-induced 5-LO activity is blocked by the p38 MAPK inhibitor SB203580. Cellular stress also led to increased activation of p38 MAPK and phosphorylation of MAPKAP kinase (MK) substrates in vitro (5-LO and Hsp27), which coincided with LT synthesis.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (110 TBq/mmol) was purchased from
Amersham-Pharmacia, Uppsala, Sweden. Human transforming growth factor
(TGF)-ß1 was purified from outdated platelets as previously described
[35
], and calcitriol was obtained from Biomol, Plymouth
Meeting, PA.
Cells and cell culture
BL41-E95-A cells were kindly provided by Dr. H.-E. Claesson
(Karolinska Institute, Stockholm) and maintained in RPMI 1640 medium
with glutamine supplemented with 10% fetal calf serum, 100 µg/mL of
streptomycin, and 100 U/mL of penicillin. Cultures were seeded at a
density of 2 x 105 cells/mL. Monocyte-macrophage
6 (MM6) cells were cultured and differentiated with TGF-ß and
calcitriol as previously described [36
]. Cells were
harvested by centrifugation [200 g, 10 min, room
temperature (RT)], washed once in phosphate-buffered saline (PBS), pH
7.4, and finally resuspended in PBS containing 1 mM Ca2+
and 1 mg/mL of glucose.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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Western blot
For preparation of total cell lysates, cells were resuspended in
PBS with 1 mM Ca2+ and 1 mg/mL of glucose, lysed by
addition of the same volume of 2x sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (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 at 95°C for 6 min.
Aliquots of cell lysates or subcellular fractions were analyzed by
SDS-PAGE on a 4–15% linear gradient gel. After electroblotting to
nitrocellulose membrane (Hybond C; Amersham-Pharmacia), membranes were
blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 50 mM
Tris/HCl, pH 7.4, and 100 mM NaCl) for 1 h at RT. Membranes were
washed and then incubated with antisera overnight at 4°C. The
membranes were washed with TBS and incubated with a 1:1,000 dilution of
alkaline phosphatase (AP)-conjugated IgGs (Sigma) for 2 h at RT.
After washing with TBS and TBS plus 0.1% Nonidet P-40 (NP-40),
proteins were visualized with the AP substrates nitro blue 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).
In vitro kinase assay
For preparation of samples, incubations were stopped by addition
of 2 v of ice-cold lysis buffer (20 mM Tris/HCl, pH 7.4, 150 mM
NaCl, 2 mM EDTA, 1% Triton X-100, 0.5 % NP-40, 50 mM NaF, 2 mM
Na3VO4, 25 mM ß-glycerophosphate, 10 mM
sodium pyrophosphate, 10 mM 4-nitrophenyl phosphate, 1 mM
phenylmethylsulfonyl fluoride, 5 µM ZnCl2, 10 µg/mL of
leupeptin, and 60 µg/mL of soybean trypsin inhibitor). During 10 min
in this buffer, the suspension was vortexed repeatedly (5-s bursts) to
assure complete lysis. Supernatants were obtained by centrifugation of
the lysates (16,000 g, 10 min, 4°C) and kept on ice.
Aliquots corresponding to 0.2 x 106 BL41-E95-A cells
were immediately mixed with the same volume of kinase buffer (25 mM
HEPES, pH 7.5, 25 mM MgCl2, 25 mM ß-glycerophosphate, 2
mM dithiothreitol, 0.1 mM Na3VO4) containing
ATP (50 µM) and [-32P]ATP (2 µCi/mL), and 1 µg
of recombinant Hsp27 was added as substrate. The final volume was 20
µL, and incubation time was 30 min at 30°C. The reaction was
terminated by addition of SDS-b and heating at 95°C for 6 min.
Samples were separated by SDS-PAGE (see Western blot), and
phosphorylated proteins were visualized by autoradiography of the dried
gels.
In-gel kinase assay
Incubations were stopped by addition of the same volume of
SDS-b and heating for 6 min at 95°C. Total cell lysates of
BL41-E95-A cells 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
[13
].
Subcellular fractionation by detergent lysis
Subcellular localization of 5-LO was investigated as described
previously [39
]. In brief, BL41-E95-A cells (3 x
107) or MM6 cells (1 x 107) were
resuspended in 1 mL of PBS with 1 mM Ca2+ and 1 mg/mL
glucose. After addition of the indicated stimuli, samples were
incubated for 5 min at 37°C and chilled on ice. Nuclear and
non-nuclear fractions were obtained after cell lysis by 0.1% NP-40.
Aliquots of nuclear and non-nuclear fractions were immediately mixed
with the same volume SDS-b, heated for 6 min at 95°C, and analyzed
for 5-LO protein by Western blot using affinity purified 5-LO antiserum
1551.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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and IL-1 (added together with A23187 plus AA), also enhanced
cellular 5-LO activity. As observed for PMNL and MM6 cells stimulated
with ionophore plus exogenous AA, 5-HETE was always the major 5-LO
metabolite (
75–80 %) released from BL41-E95-A cells, and no
agonist dependent shift in the ratio of 5-HETE, LTB4 and
its all-trans isomers was observed. As found by others
[23
, 26
] 5-LO activity in BL41-E95-A cells
depended on the presence of exogenous substrate, and maximal 5-LO
product formation was obtained at 40 µM AA. Cell stress (0.8 M
sorbitol, 1 mM SA) induced upregulation of 5-LO activity was similar
(about fivefold) also at 2.5, 10 or 20 µM AA (data not shown). 5-LO
product formation also correlated to the concentration of ionophore
used, at 1 µM A23187 5-LO product formation was about 30–50%,
compared with 10 µM ionophore. Osmotic stress (0.8 M sorbitol)
upregulated 5-LO activity to the same extent (four- to fivefold)
also at 1 µM ionophore. Treatment of BL41-E95-A cells with
stress stimuli (1 mM SA, 0.3 M NaCl or 0.8 M sorbitol) did not affect
the integrity of the cells as determined by light microscopy with
trypan blue exclusion (not shown), indicating that 5-LO product
formation occurred in intact cells and was not due to crude catalytic
activity of 5-LO in broken cell preparations.
 View larger version (19K): [in a new window] |
Figure 1. Cellular stress stimulates 5-LO activity in BL41-E95-A cells.
BL41-E95-A cells (1.5 x 107 in 1 mL of PBS containing
1 mg/mL of glucose and 1 mM CaCl2) were incubated for 10
min at 37°C with the indicated stimuli, and 5-LO activity was
determined by HPLC as described in Material and Methods. (A) All
incubations received 10 µM ionophore A23187 plus 40 µM AA (control
conditions). In addition, sorbitol, SA (at the indicated
concentrations), and 1 ng/mL of TNF- and IL-1, respectively, were
added as indicated. (B) Cells were stimulated with 40 µM AA or with
40 µM AA plus 1 µM thapsigargin (thaps), in the presence or absence
of 0.8 M sorbitol. Results are given as mean ± SE;
n = 3–4.
|
The effects of cellular stress were prominent, when cells were stimulated simultaneously with stress-inducers and ionophore plus AA and were less prominent with longer preincubation periods. The time-course of the effect of osmotic stress on 5-LO product formation is shown in Figure 2 . BL41-E95-A cells were treated with 0.3 M NaCl for 0–10 min before the addition of ionophore plus AA, and 5-LO activity was determined. Addition of NaCl together with ionophore gave the best effect, about fivefold activation. Repeated addition of NaCl (10 min after the first addition) gave no activation, however transfer of cells which had been treated with NaCl for 10 min to fresh buffer, and renewed stimulation with 0.3 M NaCl restored the up-regulative effect (Fig. 2) , again indicating that hyperosmolarity did not compromise cell integrity.
 View larger version (22K): [in a new window] |
Figure 2. Time-course of osmotic stress-induced 5-LO activity. BL41-E95-A cells
(1.5 x 107 in PBS containing 1 mg/mL of glucose and 1
mM CaCl2) were preincubated with 0.3 M NaCl at 37°C for
the indicated times and subsequently stimulated with ionophore A23187
and AA (10 and 40 µM, respectively) for another 10 min.
Alternatively, cells were washed after NaCl treatment by centrifugation
(500 g, 3 min, RT), resuspended in fresh buffer, and
stimulated with 0.3 M NaCl together with A23187 plus AA (10 and 40
µM, respectively). 5-LO activity was determined by HPLC. Results are
expressed as means + SE of three independent
experiments.
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View this table: [in a new window] |
Table 1. 5-LO Activity in BL41-E95-A Cells
|
 View larger version (47K): [in a new window] |
Figure 3. Expression and activity of p38 MAPK and MKs. (A) Expression of p38 MAPK
and MK2 in MM6 cells (lanes 1 and 3) and BL41-E95-A cells (lanes 2 and
4). Total cell lysates corresponding to equal amounts of cells
(0.5 x 106) were analyzed by Western blot using
antibodies against p38 MAPK and MK2. (B) In vitro kinase assay with
Hsp27 as substrate. BL41-E95-A cells (5 x 106 in 1 mL
of PBS containing 1 mg/mL of glucose and 1 mM CaCl2) were
preincubated pairwise with or without 10 µM SB203580 for 30 min at
37°C. Two and one-half minutes after addition of the indicated
stimuli at 37°C, cells were lysed and supernatants were assayed for
MK activity. Concentrations: Ionophore, 10 µM; sorbitol, 0.8 M; SA, 1
mM; H2O2, 10 µM; NaCl, 0.3 M; and diamide,
100 µM. Phosphorylated Hsp27 was separated by SDS-PAGE and visualized
by autoradiography. (C) Determination of 5-LO kinase activity in total
cell extracts of MM6 and BL41-E95-A cells. Cells (2.5 x
107 in 1 mL of PBS containing 1 mg/mL of glucose and 1 mM
CaCl2) were incubated at 37°C with the indicated
additives (ionophore, 10 µM; sorbitol, 0.8 M; SB203580, 3 µM).
After 3 min, cells were lysed by addition of SDS-b, vortexed, and
heated at 95°C for 6 min. Aliquots corresponding to 0.5 x
106 cells were analyzed for 5-LO kinase activity in a
SDS-10% polyacrylamide gel, which was polymerized in the presence of
0.2 mg/mL of purified recombinant 5-LO as described in Materials and
Methods. Positions of standard proteins are indicated. Similar results
were obtained in two additional independent experiments.
|
Activation of p38 MAPK was demonstrated by Western blot analysis of BL41-E95-A cell lysates with an antibody recognizing the dually phosphorylated kinase. As shown in Figure 4A , relatively small amounts of phosphorylated (activated) p38 MAPK were present in unstimulated BL41-E95-A cells, and treatment with 10 µM ionophore and/or 40 µM AA caused no significant p38 MAPK activation, whereas addition of sorbitol led to a strongly increased signal. Activation of p38 MAPK was not dependent on the presence of ionophore and AA; treatment of BL41-E95-A cells with sorbitol or SA alone was sufficient for kinase activation (data not shown). The time courses for activation of p38 MAPK and 5-LO were compared. For cells stimulated with sorbitol, ionophore, and AA, both p38 MAPK phosphorylation (Fig. 4B) and 5-LO products were maximal after 2 min (Fig. 4C) .
 View larger version (26K): [in a new window] |
Figure 4. Activation of p38 MAPK correlates to 5-LO activity. (A) Activation of
p38 MAPK. BL41-E95-A cells (1.5 x 107) resuspended in
100 µL of PBS containing 1 mg/mL of glucose and 1 mM
CaCl2 were stimulated at 37°C without or with ionophore
(10 µM), AA (40 µM), and sorbitol (0.8 M) as indicated. After 2.5
min, incubations were terminated by addition of the same volume of
SDS-b, and total cell lysates were analyzed by SDS-PAGE and
immunoblotting using a specific antibody that detects the dually
phosphorylated form of p38 MAPK (upper panel). Equal sample loading was
demonstrated with anti-p38 MAPK antibody (lower panel). (B) Time-course
of p38 MAPK activation. BL41-E95-A cells (1.5 x 107)
resuspended in 100 µL of PBS containing 1 mg/mL of glucose and 1 mM
CaCl2 were stimulated at 37°C with 0.8 M sorbitol as
indicated. All incubations received 10 µM A23187 plus 40 µM AA
simultaneously with sorbitol. After the indicated times, incubations
were terminated by addition of the same volume of SDS-b, and samples
were analyzed for phosphorylated p38 MAPK and p38 MAPK. (C) Time-course
of 5-LO product formation. BL41-E95-A cells (1.5 x
108 in 10 mL of PBS containing 1 mg/mL of glucose and 1 mM
CaCl2) were stimulated with 0.8 M sorbitol plus ionophore
A23187 and AA (10 and 40 µM, respectively). After the indicated times
at 37°C, aliquots (1 mL, corresponding to 1.5 x 107
cells) were mixed with 1 mL of methanol. 5-LO activity was determined
as described in the Materials and Methods section.
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 View larger version (23K): [in a new window] |
Figure 5. Effects of SB203580 on 5-LO activity in BL41-E95-A cells. Intact
BL41-E95-A cells (1.5 x 107 in PBS containing 1 mg/mL
of glucose and 1 mM CaCl2), or corresponding homogenates
were preincubated with the indicated concentrations of SB203580 for 30
min at 37°C (intact cells) or 10 min at 4°C (homogenates). To
intact cells, H2O2 (10 µM) or NaCl (0.3 M)
was added together with ionophore A23187 and AA (10 and 40 µM,
respectively). To homogenates, 1 mM ATP was added, and the 5-LO
reaction was started by addition of CaCl2 and AA (1 and 40
µM, respectively). After 10-min incubations at 37°C, 5-LO activity
was determined as described in Materials and Methods. Results are
expressed as means ± SE of three independent
experiments.
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 View larger version (51K): [in a new window] |
Figure 6. Subcellular distribution of 5-LO. For determination of 5-LO
distribution in resting and activated cells, 1 x 107
MM6 cells and 3 x 107 BL41-E95A cells were
resuspended in 1 mL of PBS containing 1 mg/mL of glucose and 1 mM
CaCl2 and incubated with or without activators
for 5 min at 37°C. For activation, MM6 cells were incubated with 100
nM PMA at 37°C for 10 min before the addition of 5 µM ionophore (5
min). BL41-E95-A cells were treated with 10 µM ionophore, 40 µM AA,
and 0.8 M sorbitol for 5 min. Cell fractionation and 5-LO Western
blotting were performed as described in experimental procedures.
Pairwise samples (nonnuclear and nuclear) correspond to the identical
cell numbers.
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Expression and functionality of p38 MAPK and MK2 in B-lymphocytes have been reported [44 ]. For example, p38 MAPK is involved in the mouse IgM-induced apoptosis of human B cells [33 ] and in CD40-induced gene expression and proliferation [32 ]. Oxidative stress has been shown to trigger protein tyrosine phosphorylation and MAPK activation in B-lymphocytes [27 ]. Oxidants such as H2O2, diamide, and phenylarsine oxide increase tyrosine phosphorylation via inhibition of protein tyrosine phosphatases [28 29 30 31 , 40 ]. Activation of p38 MAPK involves dual phosphorylation on both Thr and Tyr in the TGY motif [7 ], and many of the kinases in the cascade upstream of p38 MAPK are tyrosine kinases; thus it appears reasonable that p38 MAPK in BL41-E95-A cells could be sensitive to the redox state of the cells, via the regulation of tyrosine kinase activity. Recently, we established that 5-LO is phosphorylated by p38 MAPK-dependent MKs, and we suggested that this phosphorylation might regulate cellular 5-LO activity [13 ]. Because low cellular 5-LO activity in B cells can be enhanced by oxidative stress, we investigated the involvement of p38 MAPK in this process.
We found that cellular stress (hyperosmolarity, SA, and oxidative
stress), protein tyrosine phosphatase inhibitors, or cytokines TNF-
plus IL-1 up-regulate ionophore-induced LT synthesis in the human B
cell line BL41-E95-A. Cell stress as well as the proinflammatory
cytokines TNF and IL-1 are potent activators of p38 MAPK
[7
] and have been shown to activate MKs in various
leukocytes capable of phosphorylating 5-LO in vitro
[13
]. Activation of 5-LO by stress stimuli was
transient, and maximal 5-LO activity was obtained when the stimuli were
added simultaneously with ionophore and AA (Fig. 2)
. It is well
documented that, dependent on the nature of the stimulus, activation of
p38 MAPK is rapid and declines within minutes [45
,
46
]. There was no absolute requirement of ionophore
A23187 for 5-LO activation, because sorbitol treatment of BL41-E95-A
cells in the presence of AA alone caused a four- to fivefold increase
in 5-LO activity versus control incubations (Fig. 1B)
. In agreement
with others [23
, 26
], 5-LO product
formation in BL41-E95-A cells depended on the presence of exogenous
substrate and correlated to the AA concentrations. In the absence of
exogenous AA, LT synthesis was not detectable under any circumstances,
probably because these B cells failed to release AA from endogenous
pools [47
]. However, the concentration of AA had no
substantial effect on the degree of up-regulation of 5-LO activity by
cell stress.
The effects of calcium ionophore A23187, the "standard stimulus"
for cellular LT formation, on kinase activation in BL41-E95-A cells and
MM6 cells were actually quite different (Fig. 3C)
. In MM6 cells,
ionophore activated MK2 (probably also MK3), and ionophore is a potent
stimulus for LT biosynthesis [13
, 39
]. In
contrast, ionophore neither activated p38 MAPK (Fig. 4A)
nor stimulated
p38 MAPK-dependent kinase activities of BL41-E95-A cells (Fig. 3B
3C) ,
and ionophore together with exogenous AA gave only marginal 5-LO
activity. However, when different p38 MAPK activators were given to
BL41-E95-A cells together with ionophore and AA, both the cellular 5-LO
activity (Table 1
and Fig. 1
) and kinase activity increased
considerably (Fig. 3B
3C
; Fig. 4A
4B
), and the appearance of
phosphorylated (active) p38 MAPK correlated with the up-regulation of
5-LO activity (Fig. 4)
. Both cellular 5-LO activity (Fig. 5)
and MK
activation (Fig. 3C)
were suppressed by the p38 MAPK inhibitor SB203580
at comparable concentrations. Although this compound at low doses (<3
µM) is assumed to be a highly specific inhibitor for p38 MAPK, it was
recently shown that higher concentrations of SB203580 (3–5 µM) can
also reduce phosphatidylinositol 3-kinase (PI 3-kinase)-regulated
protein kinase B (PKB) activity [48
], a kinase pathway
that is also activated by oxidative stress and heat shock. In fact,
similar concentrations of SB203580 were necessary for inhibition of
5-LO activity (Fig. 5)
. However, PKB was inactivated by osmotic shock
[49
], and the PI 3-kinase inhibitor wortmannin (
1
µM) failed to reduce 5-LO activity in BL41-E95-A cells (data not
shown); thus the PI 3-kinase/PKB pathway is probably not involved in
5-LO activation. It thus appears that activation of p38 MAPK-regulated
MKs and subsequent 5-LO phosphorylation correlate with cellular 5-LO
activity in BL41-E95-A cells.
BL41-E95-A is the third cell type for which increased activity of MKs seems to correlate with increased 5-LO activity, which has also been found previously for human PMNLs and the monocytic cell line MM6. Thus, MK2 in PMNL and MM6 cells could phosphorylate 5-LO in vitro, and it seems that MK3 is also a 5-LO kinase candidate [13 ]. In-gel kinase analysis of total lysates from BL41-E95-A cells indicated that MK2 (47 and 55 kDa) is also a 5-LO kinase in these cells, but MK3 activity (40 kDa) was not detected. The 55-kDa band seemed to contain other kinases as well, as observed previously for samples from MM6 cells [13 ]. For PMNL and MM6 cells, activation of kinases which can phosphorylate 5-LO is always accompanied by increased translocation of 5-LO to the nucleus [39 ]. It has been reported that 5-LO in the Burkitt lymphoma B cell line BL41-E95-A, as well as in B-CLL cells, is associated with the nucleus [26 ]. However, by Western blot analysis of subcellular fractions (after cell lysis with NP-40), we were unable to detect 5-LO in nuclear fractions from BL41-E95-A cells, after treatment with kinase stimuli together with ionophore and exogenous arachidonate (Fig. 6) . Although we can not strictly exclude that NP-40 lysis might detach 5-LO from the nuclear membrane of BL41-E95-A cells, this might indicate that LT biosynthesis in these cells (in the presence of exogenous arachidonate) could occur in a nonnuclear compartment. For eosinophils it was published that 5-LO is present also in cytoplasmic lipid bodies [50 ], and lipid bodies in U937 cells contain MAPKs [51 ]. FLAP is expressed in BL41-E95-A cells [23 ], and membrane-bound FLAP (abundant in the nuclear membrane) is thought to donate AA to 5-LO during LT synthesis at the nuclear membrane [5 ]. MK886, which binds to FLAP, suppresses ionophore-induced 5-LO product formation in B cells treated with oxidative stress (diamide) at low concentration of AA (1–2.5 µM), whereas higher AA concentrations abolish the inhibitory action of this drug [26 ]. Also in our hands, MK886 (0.1–1 µM) only moderately suppressed 5-LO product formation induced by ionophore and sorbitol at 10 or 40 µmM AA (data not shown). As previously discussed [39 ], it appears that also in BL41-E95-A cells, non-nuclear 5-LO can be active in the presence of exogenous AA.
Similarities between cytosolic phospholipase A (cPLA) 2 and 5-LO suggest how phosphorylation could regulate 5-LO activity. A C2 domain in cPLA;I2 mediates Ca2+ stimulation of activity (see ref. 52 and references therein), and similar findings have been made for 5-LO [2 ]. Both enzymes also can be phosphorylated by MAPKs. For cPLA2, it has been suggested that the C2 domain, together with another phosphorylated part of the protein, could determine membrane association and thus activity [52 ]. Similar mechanisms might apply to 5-LO. In summary, we show that in BL41-E95-A cells, activation of the p38 MAPK pathway is required for efficient LT synthesis, which implies a new role for this MAPK in B cells. As discussed above, selenium-dependent peroxidases are potent determinants of 5-LO activity in lymphocytes, and the redox status is coupled to the activity of tyrosine kinases. Thus, treatment of BL41-E95-A cells with pro-oxidative agents could stimulate 5-LO in two ways, by promoting formation of the ferric form of the lipoxygenase active site and by promotion of phosphorylation of 5-LO.
Received January 22, 2001; revised May 9, 2001; accepted May 11, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDetermination of 5-LO activityRESULTSDISCUSSIONREFERENCES |
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 E. Burkert, D. Szellas, O. Radmark, D. Steinhilber, and O. Werz Cell type-dependent activation of 5-lipoxygenase by arachidonic acid J. Leukoc. Biol., January 1, 2003; 73(1): 191 - 200. [Abstract] [Full Text] [PDF]
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 O. Werz, D. Szellas, D. Steinhilber, and O. Radmark Arachidonic Acid Promotes Phosphorylation of 5-Lipoxygenase at Ser-271 by MAPK-activated Protein Kinase 2 (MK2) J. Biol. Chem., April 19, 2002; 277(17): 14793 - 14800. [Abstract] [Full Text] [PDF]
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E. Burkert, O. Radmark, B. Samuelsson, D. Steinhilber, and O. Werz Hypertonicity suppresses ionophore-induced product formation and translocation of 5-lipoxygenase in human leukocytes J. Leukoc. Biol., March 1, 2002; 71(3): 477 - 486. [Abstract] [Full Text] [PDF]
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O. Werz, E. Burkert, B. Samuelsson, O. Radmark, and D. Steinhilber Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes Blood, February 1, 2002; 99(3): 1044 - 1052. [Abstract] [Full Text] [PDF]
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