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INTRODUCTION |
During the last few years, it has become clear that sphingolipids,
in addition to being structural constituents of cell membranes, are
sources of important signaling molecules. Particularly, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP),1 have emerged as a new
class of potent bioactive molecules, implicated in a variety of
cellular processes such as cell differentiation, apoptosis, and
proliferation (1-4). Interest in SPP focused recently on two distinct
cellular actions of this lipid, namely its function as extracellular
ligand activating specific G protein-coupled membrane receptors and its
role as intracellular second messenger (5). Important clues to a
specific intracellular action of SPP were the following findings.
First, activation of various plasma membrane receptors, such as the
platelet-derived growth factor receptor (6, 7), the Fc
RI (8), and
the Fc
RI antigen receptors (9), was found to rapidly increase
intracellular SPP production through stimulation of sphingosine kinase.
Second, inhibition of sphingosine kinase stimulation strongly reduced or even prevented cellular events triggered by these tyrosine kinase-linked receptors, such as receptor-stimulated DNA synthesis, Ca2+ mobilization, and vesicular trafficking (6, 8, 9).
Finally, intracellular SPP was found to mimic the receptor responses,
i.e. it stimulated DNA synthesis and mobilized
Ca2+ from internal stores (10-14). We recently reported
that the G protein-coupled muscarinic acetylcholine receptor subtypes
m2 and m3 expressed in HEK-293 cells also induce a rapid and transient SPP production by sphingosine kinase. Furthermore, intracellular injection of SPP rapidly mobilized Ca2+ in intact HEK-293
cells and inhibition of sphingosine kinase markedly inhibited
Ca2+ signaling by these and other G protein-coupled
receptors (14).
To characterize the mechanisms leading to sphingosine kinase activation
by G protein-coupled receptors and the cellular role of this pathway,
we investigated whether the formyl peptide receptor stimulates
sphingosine kinase in myeloid differentiated human leukemia (HL-60)
cells, whether G proteins participate in this process, and, finally,
whether sphingosine kinase activation is involved in specific cellular
responses to receptor stimulation in HL-60 cells. It is well
established that most, if not all, cellular responses to formyl peptide
receptors in HL-60 cells and neutrophils, including phospholipase C
(PLC) stimulation, Ca2+ mobilization, superoxide
production, and enzyme release, are mediated by pertussis toxin
(PTX)-sensitive Gi-type G proteins (15). Here, we report
that formyl peptide receptor activation rapidly stimulates sphingosine
kinase in differentiated HL-60 cells and that G proteins are involved
in this process. Moreover, it is shown that inhibition of sphingosine
kinase by DL-threo-dihydrosphingosine (DHS) or
N,N-dimethylsphingosine (DMS) does not affect PLC
stimulation and superoxide production but largely inhibits
receptor-stimulated Ca2+ mobilization and enzyme release.
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EXPERIMENTAL PROCEDURES |
Materials--
N-Formyl-methionyl-leucyl-phenylalanine
(fMLP), lucigenin, dibutyryl cAMP, cytochalasin B, staurosporine,
p-nitrophenyl-
-D-glucuronide, and
p-nitrophenyl-
-D-glucosaminide were obtained
from Sigma. GTP
S and GDP
S were from Boehringer Mannheim.
Myo-[2-3H]inositol (24.4 Ci/mmol) was from NEN
Life Science Products, D-erythro-[3H]sphingosine (15 Ci/mmol) was from ARC, RPMI 1640 medium was from Life Technologies,
Inc., bisindolylmaleimide I (GF 109203X) was from Calbiochem, and PTX
was from List Biological Laboratories. All other materials were from
previously described sources (14). Immediately before use, stock
solutions of the lipids made in methanol or isopropanol were dried down
and dissolved in Hanks' balanced salt solution (118 mM
NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM
D-glucose, and 15 mM HEPES, pH 7.4), containing
in addition 1 mg/ml fatty acid-free bovine serum albumin.
HL-60 Cell Culture--
HL-60 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 150 units/ml
penicillin, and 150 µg/ml streptomycin in 5% CO2.
Differentiation into neutrophil-like cells was induced by culturing
HL-60 cells for 48 h in the presence of 0.5 mM
dibutyryl cAMP. For PTX treatment, cells were incubated for 20 h
with 50 ng/ml PTX (16, 17).
Assay of Sphingosine Kinase in Intact HL-60
Cells--
Sphingosine kinase-catalyzed formation of SPP was measured
as described before (14). In brief, differentiated HL-60 cells (1.8 × 106 cells) equilibrated in Hanks' balanced
salt solution/bovine serum albumin for 5 min at 37 °C were incubated
with [3H]sphingosine (~105 cpm; final
concentration, 30 nM) for 30 s to 10 min at 37 °C in a total volume of 200 µl. The reactions were stopped by addition of 2 ml of ice-cold methanol, followed by 1 ml of chloroform. After
vigorous vortexing and incubation for 1 h at 37 °C, particulate matter was pelleted by centrifugation, and the supernatant was evaporated to dryness in a SpeedVac centrifuge. After redissolving in
20 µl of methanol, the samples were spotted onto Silica gel 60 TLC
plates (Merck), together with authentic unlabeled sphingosine and SPP.
Separation of the products was achieved by TLC in 1-butanol:acetic acid:water (3:1:1) as a solvent system. Sphingosine and SPP spots were
visualized by staining with ninhydrin spray. After scraping, radioactivity in the spots was measured by liquid scintillation counting. Formation of [3H]SPP is expressed as
cpm/1.8 × 106 cells and corrected for time 0 count
rates, amounting to 80-100 cpm.
Assay of Sphingosine Kinase Activity in HL-60
Cytosol--
Differentiated HL-60 cells were suspended in ice-cold
lysis buffer (1.2 × 106 cells/ml) containing 100 mM potassium phosphate buffer (pH 7.4), 1 mM
EDTA, 10 mM MgCl2, and 1 mM
2-mercaptoethanol and disrupted on ice by 50-100 strokes in a
pre-cooled Dounce homogenizer. After centrifugation for 20 min at
50,000 × g and 4 °C, the supernatant (cytosolic
fraction) was collected and used directly in sphingosine kinase assays.
Enzyme activity was determined for 20 min at 37 °C in a total volume
of 200 µl, containing 10 µM
[3H]sphingosine (~105 cpm), 1 mM ATP, 10 mM MgCl2, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mg/ml fatty
acid-free bovine serum albumin, 100 mM potassium phosphate
buffer (pH 7.4), and HL-60 cytosol (120-200 µg of protein). Reactions were terminated by addition of 2 ml of ice-cold methanol. Extraction, TLC separation, and quantification of [3H]SPP
were carried out as described above. Sphingosine kinase activity is
expressed as pmol SPP formed per mg protein and 20 min.
[Ca2+]i Measurements--
Intracellular
free Ca2+ concentration ([Ca2+]i) was
determined with the fluorescent Ca2+ indicator dye Fura-2
in a Hitachi spectrofluorimeter as described before (18). In some
experiments, Ca2+ was omitted from the medium, and in
addition 5 mM EGTA was added 2 min before stimulus exposure.
Assay of PLC--
Measurement of PLC-catalyzed inositol
phosphate formation was performed as described in detail previously
(19). In brief, after labeling for 48 h with
myo-[3H]inositol (2.5 µCi/ml) in
inositol-free medium, differentiated HL-60 cells were equilibrated for
10 min at 37 °C in Hanks' balanced salt solution/bovine serum
albumin with or without the indicated agents. Cells were then incubated
with 10 µM fMLP at 37 °C in the presence of 10 mM LiCl for 20 s and 10 min to measure formation of
[3H]inositol 1,4,5-trisphosphate (IP3) and
total [3H]inositol phosphates, respectively, as reported
before (19).
Measurement of Superoxide Anion Generation--
Generation of
superoxide anions in differentiated HL-60 cells was measured with the
lucigenin assay (20) as described previously (18).
Measurement of Enzyme Release--
Release of
-glucuronidase
and N-acetyl-
-glucosaminidase enzymes from HL-60 cells
was assessed as described (21). Briefly, differentiated HL-60 cells
(3.5-5 × 106 cells) were suspended in 500 µl of
Hanks' balanced salt solution and incubated for 5 min at 37 °C in
the presence of cytochalasin B (5 µg/ml). Reactions initiated by the
addition of fMLP were conducted for 5 min at 37 °C and terminated by
placement of the tubes on ice. After cell pelleting, activities of
-glucuronidase and N-acetyl-
-glucosaminidase enzymes
in supernatant fluids and cell lysates were determined as described
(22). Release of the enzymes is given as a percentage of the total
cellular content.
Data Presentation and Analysis--
Unless otherwise stated,
results are presented as the means ± S.E. of three independent
experiments, each done in duplicates or triplicates. Statistical
analysis was performed by Student's two-tailed t test for
unpaired data.
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RESULTS |
Formyl Peptide Receptor and G Protein Stimulation of Sphingosine
Kinase in HL-60 Cells--
First, we investigated whether the formyl
peptide receptor activates the sphingosine kinase in dibutyryl
cAMP-differentiated HL-60 cells. For this, formation of
[3H]SPP from [3H]sphingosine was determined
for various periods of time at 37 °C in the absence and presence of
the receptor agonist fMLP, which was applied simultaneously with the
radiolabel. Cellular uptake of [3H]sphingosine was fast,
reaching 76% of total added after 30 s and 84% after 5 min of
incubation, and was not influenced by fMLP (data not shown). Basal
conversion of [3H]sphingosine by sphingosine kinase to
[3H]SPP was also rapid, and within 2-5 min a plateau of
[3H]SPP was reached (Fig.
1A). Activation of the formyl
peptide receptor by 10 µM fMLP induced a rapid and
transient increase in [3H]SPP production (Fig.
1B). After 2 min, the increase in [3H]SPP
reached a maximum of 63 ± 11% (n = 6) above
basal [3H]SPP formation in unstimulated cells. Comparable
increases in [3H]SPP production were observed in cells
stimulated for 2 min with 0.1 µM and 1 µM
fMLP. When the measurements were performed in the presence of the
sphingosine kinase inhibitors, DHS (30 µM) or DMS (15 µM) (6, 8, 23-25), basal and fMLP-stimulated formation of [3H]SPP were inhibited by 80-90% (data not shown).
Together, these results indicate that the formyl peptide receptor is
capable of stimulating SPP production by sphingosine kinase in
differentiated HL-60 cells.

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Fig. 1.
Formyl peptide receptor-stimulated SPP
formation in HL-60 cells. Formation of [3H]SPP from
[3H]sphingosine was determined without (A) and
with 10 µM fMLP (B) for the indicated periods
of time in differentiated HL-60 cells (3-6 experiments) as described
under "Experimental Procedures." In B, values are
expressed as percentages of [3H]SPP production relative
to unstimulated cells. *, significantly different from unstimulated
cells (p < 0.05).
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To investigate the signaling pathway leading to sphingosine kinase
activation, we studied first whether Gi-type G proteins are
involved in the formyl peptide receptor action. Thus, formation of
[3H]SPP was measured in control and PTX-treated HL-60
cells. Stimulation of control cells for 2 min with 10 µM
fMLP increased [3H]SPP levels by 67 ± 17% over
unstimulated cells (Fig. 2A).
PTX treatment (50 ng/ml, 20 h) had no effect on basal
[3H]SPP production but completely blocked fMLP-induced
stimulation of sphingosine kinase, indicating that activation of
sphingosine kinase by the formyl peptide receptor is mediated by
Gi-type G proteins. To directly assess the role of G
proteins in sphingosine kinase stimulation, we studied the effects of
direct G protein activation on [3H]SPP formation in
intact cells and in a cell-free system. Direct activation of
heterotrimeric G proteins by AlF4
in
intact HL-60 cells led to a rapid and transient production of
[3H]SPP, in a manner similar to the receptor agonist fMLP
(Fig. 2B). [3H]SPP levels in cells stimulated
for 1, 2, and 5 min with AlF4
amounted
to 117, 142, and 46%, respectively, above unstimulated control
cells.

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Fig. 2.
G protein stimulation of sphingosine kinase
in intact HL-60 cells. Formation of [3H]SPP from
[3H]sphingosine was determined for 2 min in control and
PTX-pretreated HL-60 cells in the absence (Basal) and
presence of 10 µM fMLP (A) or in control HL-60
cells for the indicated periods of time in the absence
(Basal) and presence of
AlF4 (10 mM NaF plus 10 µM AlCl3) (B). In A,
values are expressed as percentages of [3H]SPP production
relative to unstimulated control cells. *, significantly different from
basal SPP formation (p < 0.05).
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Sphingosine kinase activity has been found to be present in the cytosol
of various cell types (4), and purification of a cytosolic sphingosine
kinase from rat kidney has recently been reported (26). Therefore, to
study G protein regulation of sphingosine kinase in a cell-free system,
the effects of various guanine nucleotides on sphingosine kinase
activity were investigated in cytosol prepared from differentiated
HL-60 cells. Addition of the stable GTP analog GTP
S stimulated SPP
production by sphingosine kinase in HL-60 cell cytosol by about 50% in
a concentration-dependent manner, with an EC50
value of less than 1 µM and a maximal effect at 10-100 µM (Fig. 3A). On
the other hand, addition of GTP (100 µM) and the stable
GDP analog GDP
S (500 µM) did not alter basal
sphingosine kinase activity (Fig. 3B). However, GDP
S (500 µM) almost fully blocked sphingosine kinase stimulation
by GTP
S (100 µM). Together, these results indicate
that G proteins are signal transduction components in the pathway
leading to sphingosine kinase activation in myeloid differentiated
HL-60 cells.

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Fig. 3.
G protein stimulation of sphingosine kinase
activity in cytosol of HL-60 cells. Sphingosine kinase activity
was determined in HL-60 cytosolic fractions in the presence of the
indicated concentrations of GTP S (A) or in the absence
(Basal) and presence of 100 µM GTP S, 500 µM GDP S, GTP S plus GDP S, or 100 µM
GTP as described under "Experimental Procedures" (B).
Values are expressed as percentages of basal sphingosine kinase
activity, amounting to 45 ± 6 pmol SPP/mg protein and 20 min
(n = 14). *, significantly different from basal
sphingosine kinase activity (p < 0.05).
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Role of Sphingosine Kinase in Formyl Peptide Receptor Signaling in
HL-60 Cells--
Previous studies in other cell types suggested that
sphingosine kinase activation represents a Ca2+ signaling
pathway for some plasma membrane receptors (8, 9, 14). Because fMLP
stimulated sphingosine kinase, we investigated the role of this pathway
in Ca2+ mobilization in HL-60 cells. Stimulation of HL-60
cells with fMLP (0.1 nM to 0.1 µM) in the
presence of 1 mM extracellular Ca2+ markedly
increased [Ca2+]i (Fig.
4A). Maximal increase by about
500 nM was observed at 0.1 µM fMLP. In cells
pretreated for 1 min with the sphingosine kinase inhibitor DHS (30 µM), [Ca2+]i increases were reduced
by 80-90% at all fMLP concentrations examined, even at a
supramaximally effective fMLP concentration of 10 µM
(data not shown). Half-maximal inhibition of fMLP (0.1 µM)-stimulated [Ca2+]i increase by
DHS was observed at 5.9 ± 0.4 µM (Fig. 4B). A similar inhibition was observed with DMS, another
sphingosine kinase inhibitor (IC50 = 5.2 ± 0.9 µM). As exemplified in Fig. 4C, treatment of
HL-60 cells with DHS (30 µM) also markedly inhibited the
fMLP (1 µM)-stimulated [Ca2+]i
increase measured in the absence of extracellular Ca2+.

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Fig. 4.
Effects of sphingosine kinase inhibitors on
fMLP-stimulated [Ca2+]i increases. In the
presence of 1 mM extracellular Ca2+, increases
in [Ca2+]i induced by fMLP at the indicated
concentrations were determined in HL-60 cells pretreated for 1 min
without (Control) and with 30 µM DHS
(A), or [Ca2+]i increases induced by
0.1 µM fMLP were determined in cells pretreated for 1 min
with DHS or DMS at the indicated concentrations (B). In
B, values are given as percentages of control, the 100%
value reflecting the [Ca2+]i increase induced by
0.1 µM fMLP in the absence of inhibitors (519 ± 71 nM, n = 6). C, in the absence of
extracellular Ca2+, fMLP (1 µM)-induced
[Ca2+]i increase was determined in HL-60 cells
pretreated for 1 min without (Control) and with 30 µM DHS. Superimposed tracings are shown. Addition of fMLP
is indicated by the arrow.
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DHS and DMS by themselves did not cause an increase in
[Ca2+]i in HL-60 cells. To dismiss the
possibility that the sphingosine kinase inhibitors might deplete
intracellular Ca2+ stores, we studied the effect of DHS (30 µM) on [Ca2+]i
elevations induced by the endoplasmic reticulum Ca2+-ATPase
inhibitor, thapsigargin, in the absence of external Ca2+.
In contrast to fMLP stimulation, maximal [Ca2+]i
increases induced by thapsigargin (1 µM) were not affected by DHS (control cells, 73 ± 4 nM;
DHS-treated cells, 87 ± 4 nM). The time to peak
[Ca2+]i after thapsigargin addition, however, was
significantly (p < 0.05) increased from 60 ± 8 s in control cells to 103 ± 9 s in DHS-treated cells
(data not shown). The inhibitory action of DHS and DMS on fMLP-induced
[Ca2+]i increase was not caused by inhibition of
PLC stimulation. Preincubation of HL-60 cells for 10 min with 30 µM DHS or 15 µM DMS, strongly reducing
Ca2+ mobilization, neither altered basal nor inhibited fMLP
(10 µM)-stimulated production of either IP3
or total inositol phosphates (Table I). Furthermore, release of 45Ca2+ induced by
IP3 (0.3-20 µM) from
45Ca2+-preloaded saponin-permeabilized cells,
measured as described before (27), was not affected by pretreatment of
HL-60 cells for 10 min with 30 µM DHS. Finally, because
DHS and DMS can act as protein kinase C (PKC) inhibitors (28), we
examined whether the inhibition of Ca2+ signaling might be
due to PKC inhibition. However, in contrast to DHS and DMS,
pretreatment of HL-60 cells with the PKC inhibitors staurosporine (100 nM, 3 min), or bisindolylmaleimide I (100 nM, 15 min) had no effect on the fMLP (0.1 µM)-induced
[Ca2+]i elevation, amounting to 605 ± 36 nM in control cells and 637 ± 26 and 591 ± 99 nM in staurosporine- and bisindolylmaleimide I-treated
cells, respectively (data not shown).
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Table I
Lack of effect of DHS and DMS on basal and fMLP-stimulated PLC
activities in HL-60 cells
Formation of [3H]IP3 and total [3H]inositol
phosphates was determined in differentiated HL-60 cells pretreated for
10 min without (None) and with 30 µM DHS or 15 µM DMS in the absence (Basal) and presence of 10 µM fMLP as described under "Experimental Procedures."
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Finally, the role of sphingosine kinase in two well characterized
functional responses of myeloid differentiated HL-60 cells to formyl
peptide receptor activation was examined, namely production of
superoxide anions by NADPH oxidase and enzyme exocytosis. Pretreatment of differentiated HL-60 cells for 1 min with 30 µM DHS
did not affect the superoxide production induced either by a maximally effective concentration of fMLP (1 µM) or by a
half-maximally effective one (10 nM) (Fig.
5). On the other hand, staurosporine (100 nM) strongly inhibited (by 70 ± 0.5%) the fMLP (1 µM)-stimulated production of superoxide anions (data not
shown).

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Fig. 5.
Lack of effect of sphingosine kinase
inhibition on fMLP-stimulated superoxide production.
Following pretreatment of HL-60 cells for 1 min with vehicle
(Control) or 30 µM DHS, superoxide production
induced by 1 µM and 10 nM fMLP was determined
as described under "Experimental Procedures." Values are given as
percentages of maximum superoxide production measured with 1 µM fMLP. There was no significant difference
between control and DHS-treated cells.
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In contrast to superoxide production, formyl peptide
receptor-stimulated enzyme release was strongly affected by the
sphingosine kinase inhibitors. As illustrated in Fig.
6A, pretreatment of HL-60
cells for 5 min with 30 µM DHS or 15 µM DMS
inhibited, by 45-50%, the fMLP (1 µM)-stimulated
release of
-glucuronidase. A similar inhibition was observed when
the effect of DMS on fMLP (1 µM)-stimulated release of
N-acetyl-
-glucosaminidase was examined (Fig.
6B). Pretreatment with DMS inhibited receptor-stimulated release of this enzyme half-maximally and maximally (about 85% inhibition) at 12 and 20 µM DMS, respectively. Basal
enzyme release was not affected by the sphingosine kinase inhibitors.
In contrast to DHS and DMS, pretreatment of HL-60 cells with
staurosporine (100 nM, 3 min) did not affect
fMLP-stimulated
-glucuronidase release (data not shown).

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Fig. 6.
Effects of sphingosine kinase inhibitors on
fMLP-stimulated enzyme release. Following pretreatment of HL-60
cells for 5 min with vehicle (Control), 30 µM
DHS or 15 µM DMS (A), or DMS at the indicated
concentrations (B), release of -glucuronidase
(A) and N-acetyl- -glucosaminidase
(B) was determined in the absence (Basal) and
presence of 1 µM fMLP as described under "Experimental
Procedures." *, significant inhibition of fMLP-stimulated enzyme
release (p < 0.001, 4-11 experiments).
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DISCUSSION |
In the present study, we demonstrate that sphingosine kinase
catalyzing formation of SPP from sphingosine is rapidly activated by
formyl peptide receptors in myeloid differentiated HL-60 cells, a
process apparently mediated by Gi-type G proteins, and that sphingosine kinase activity is also stimulated by direct G protein activation, both in intact cells and in a cell-free system.
Furthermore, by using the two sphingosine kinase inhibitors DHS and
DMS, evidence is provided suggesting that the sphingosine kinase
pathway plays an important role in functional responses of HL-60 cells
triggered by activation of the formyl peptide receptor. The time course and the magnitude of fMLP-stimulated SPP production in HL-60 cells were
comparable with those reported for the G protein-coupled muscarinic
receptors expressed in HEK-293 cells and for various tyrosine
kinase-linked receptors in other cellular systems (6-9, 14). The
sphingosine kinase inhibitors DHS and DMS effectively suppressed basal
and fMLP-stimulated SPP formation, indicating that sphingosine kinase
is in fact responsible for SPP production in HL-60 cells.
The present study demonstrates for the first time that G proteins are
involved in sphingosine kinase activation. First, similar to most other
cellular responses of formyl peptide receptors in HL-60 cells and
neutrophils (15), the fMLP-induced sphingosine kinase stimulation was
completely abolished in PTX-treated cells, indicating participation of
Gi-type G proteins. Second, direct activation of
heterotrimeric G proteins by AlF4
also
markedly increased SPP production in intact HL-60 cells, with a time
course similar to that observed for the formyl peptide receptor
agonist. Third, the stable GTP analog GTP
S stimulated sphingosine
kinase activity in HL-60 cytosolic fractions with a
concentration-response curve in the µM range, and this
stimulation was prevented by the stable GDP analog, GDP
S. The
identity of the GTP-binding protein mediating the stimulatory effect of
GTP
S, which can activate both heterotrimeric and low molecular
weight GTP-binding proteins, on HL-60 cytosolic sphingosine kinase
activity is presently under investigation.
In agreement with our previous findings on muscarinic receptors in
HEK-293 cells (14), we provide evidence that the sphingosine kinase
pathway is involved in the formyl peptide receptor-mediated Ca2+ mobilization in HL-60 cells. Short-term pretreatment
of the cells with the sphingosine kinase inhibitors DHS and DMS
potently reduced the fMLP-induced increases in
[Ca2+]i, with IC50 values (5-6
µM) in the ranges of those described for inhibition of
sphingosine kinase activity (6, 8, 23-25). Control experiments
excluded that suppression of Ca2+ signaling by DHS and DMS
was caused by PKC inhibition, depletion of internal Ca2+
stores, and perturbation of the PLC/IP3 pathway. It is
highly unlikely that the intracellularly formed SPP is released from the cells and stimulates the cells in an autocrine manner,
i.e. by activation of plasma membrane sphingolipid
receptors. First, there was no evidence of fMLP-stimulated release of
[3H]SPP into the medium. Second, similar to results
reported before in promyelocytic HL-60 cells (18), extracellularly
added SPP only marginally increased [Ca2+]i in
differentiated HL-60 cells (by
30 nM at 1 µM SPP). Finally, desensitization of this small
SPP-induced [Ca2+]i increase by 70% following
overnight treatment of the cells with 1 µM SPP did not
affect the formyl peptide receptor-induced [Ca2+]i increase (data not shown). The lag time
between peak [3H]SPP formation and
[Ca2+]i increase most likely results from the
time span required for extracellularly applied
[3H]sphingosine to cross the plasma membrane and reach
intracellular sphingosine kinase to be converted to
[3H]SPP. Because SPP and IP3 are generated in
a similar time-frame, there may be a concerted action of SPP and
IP3 in releasing Ca2+ from internal stores. In
this sense, we found that DHS caused a delay in Ca2+
release induced by thapsigargin, without reducing the maximal [Ca2+]i increase by this agent. In lacrimal
acinar cells, the thapsigargin-induced [Ca2+]i
rise due to leakage of Ca2+ from IP3-sensitive
stores has been shown to be dependent on basal levels of
IP3 (29). Thus, the absence of SPP after DHS treatment may
explain the delay in Ca2+ release induced by thapsigargin.
Our data thus suggest that although the formyl peptide receptor
strongly stimulates PLC-catalyzed IP3 formation, an intact
sphingosine kinase/SPP pathway is required for the receptor-elicited
Ca2+ mobilization, similar to the results reported before
for other receptors in other cell types (8, 9, 14). Recently,
Ca2+ mobilization in Chinese hamster ovary cells by the G
protein-coupled receptors for endothelin-1 and thrombin, both strongly
stimulating the PLC/IP3 pathway, has been reported to be
independent of the IP3 receptor (30). Thus, an alternative
or additional mechanism, which may be the sphingosine kinase/SPP
pathway, appears to be involved in Ca2+ signaling by these
receptors, similar to the results shown here for the formyl peptide
receptor in HL-60 cells. At present, the connection between the two
pathways in Ca2+ signaling is not clear.
To investigate the physiological role of the sphingosine kinase in
chemoattractant signaling, we studied the effects of the sphingosine
kinase inhibitors on superoxide production and enzyme release.
Production of superoxide anions by the NADPH oxidase in phagocytic
cells involves several membrane and cytosolic proteins and can be
turned on by various distinct mechanisms, including increase in
[Ca2+]i and PKC activation but also by
Ca2+- and PKC-independent pathways (31-33). Treatment of
HL-60 cells with the sphingosine kinase inhibitor, DHS (30 µM), causing marked inhibition of fMLP-induced
[Ca2+]i increase, had no effect on superoxide
production induced by fMLP, both at maximally and half-maximally
effective concentrations of this stimulus, whereas the PKC inhibitor
staurosporine strongly inhibited the fMLP response. These results
indicate that sphingosine kinase is apparently not involved in the
signaling pathway to NADPH oxidase. In contrast to superoxide
production, there is a tight correlation between rises in
[Ca2+]i and enzyme exocytosis in HL-60
granulocytes (34). Pretreatment of differentiated HL-60 cells with DHS
and DMS strongly inhibited the fMLP-stimulated release of
-glucuronidase and N-acetyl-
-glucosaminidase. Moreover, the inhibition of fMLP-induced
N-acetyl-
-glucosaminidase release and
[Ca2+]i increase by DMS exhibited a similar
concentration dependence, strongly suggesting that inhibition of enzyme
release is, at least to a major extent, due to blockade of
Ca2+ mobilization. Because the fMLP-stimulated enzyme
release was not completely inhibited by DHS or DMS at concentrations
effectively blocking fMLP-induced [Ca2+]i rises,
it appears that the activated receptor generates additional signals
required for exocytosis, e.g. stimulation of phosphatidylinositol 3-kinase (34). Finally, the differential sensitivity of the cellular events triggered by the formyl peptide receptor to DHS and DMS (i.e. no effect on PLC stimulation
and superoxide production, but marked inhibition of Ca2+
mobilization and enzyme release) underlines the specificity of the
sphingosine kinase inhibitors.
In conclusion, this study demonstrates for the first time that formyl
peptide receptors stimulate through PTX-sensitive Gi-type G
proteins SPP production by sphingosine kinase in myeloid differentiated HL-60 cells and that this enzyme is also stimulated by direct G protein
activation. Furthermore, evidence is provided that the sphingosine
kinase/SPP pathway is involved in Ca2+ and exocytosis
signaling pathways but apparently not in superoxide production in
phagocytic cells.