Sphingosine 1-Phosphate, a Diffusible Calcium Influx Factor Mediating Store-operated Calcium Entry*
Kiyoshi Itagaki and
Carl J. Hauser
From the
Department of Surgery, Division of Trauma, New Jersey Medical School,
Newark, New Jersey 07103
Received for publication, February 19, 2003
, and in revised form, May 6, 2003.
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ABSTRACT
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Store-operated calcium entry (SOCE) is a fundamental mechanism of calcium
signaling. The mechanisms linking store depletion to SOCE remain
controversial, hypothetically involving both diffusible messengers and
conformational coupling of stores to channels. Sphingosine 1-phosphate (S1P)
is a bioactive sphingolipid that can signal via cell surface G-protein-coupled
receptors, but S1P can also act as a second messenger, mobilizing calcium
directly via unknown mechanisms. We show here that S1P opens calcium entry
channels in human neutrophils (PMNs) and HL60 cells without prior store
depletion, independent of G-proteins and of phospholipase C. S1P-mediated
entry has the typical divalent cation permeability profile and inhibitor
profile of SOCE in PMNs, is fully inhibited by 1 µM
Gd3+, and is independent of
[Ca2+]i. Depletion of PMN calcium
stores by thapsigargin induces S1P synthesis. Inhibition of S1P synthesis by
dimethylsphingosine blocks thapsigargin-, ionomycin-, and platelet-activating
factor-mediated SOCE despite normal store depletion. We propose that S1P is a
"calcium influx factor," linking calcium store depletion to
downstream SOCE.
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INTRODUCTION
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Store-operated calcium entry
(SOCE)1 is the primary
mechanism of regulated calcium entry into "non-excitable" cells
like immunocytes. The mechanisms governing SOCE are controversial but have
centered on two main hypotheses
(1). In one model, emptying of
endoplasmic reticulum (ER) calcium stores leads to the release of small
diffusible messenger molecules that act on channels to increase
Ca2+ entry
(2). Randriamampita and Tsien
(3) found strong suggestions of
such a messenger molecule in extracts from Jurkat T-cells. They termed this
molecule "calcium influx factor" (CIF).
Other hypotheses suggest that store depletion leads to calcium channel
activation via direct physical interactions between cell membranes and
intracellular structures. The "exocytosis model"
(4,
5) of physical interactions
suggests that vesicles bearing calcium channels fuse with the cell membrane
after store depletion. The "conformational coupling" model
suggests that ER calcium store depletion can lead to direct physical
interactions between ER proteins such as the IP3 receptor and cell
membrane calcium channels such as TRPC3
(6,
7). No specific molecular
mechanisms have been proposed however, that might initiate such physical
interactions. Moreover, data has continued to accumulate supporting the
existence of an unidentified, low molecular weight CIF
(8,
9).
Sphingosine 1-phosphate (S1P) is a universally bioactive, small
(Mr 380) sphingolipid metabolite. S1P is derived from the
metabolism of sphingolipids and, most notably, from the actions of sphingosine
kinase (10,
11) on sphingosine. In 1991,
Zhang et al. (12)
showed that S1P induces proliferation in Swiss 3T3 fibroblasts. Since that
time, the actions of S1P have been studied in detail, and it has been found to
be an important extracellular messenger molecule affecting cell growth,
differentiation, adhesion, motility, and survival in many organisms and cell
types (13,
14). The calcium-mobilizing
effects of S1P are often mediated through G-protein coupled receptors (GPCR),
and S1P is the natural ligand for at least five cell-surface GPCRs now known
as S1PR15 (previously Edg1, -5, -3, -6, and -8)
(1518).
S1P is also known to enter cells directly, however
(19,
20), and in some systems S1P
can regulate calcium homeostasis by acting as a second messenger
(17,
21). The mechanisms by which
S1P acts in this intracellular role have not yet been identified, although
direct effects on ER calcium stores have been suggested
(22). In yeast, however,
intracellular S1P accumulation clearly mediates its biologic effects via
calcium entry into the cell
(23).
Polymorphonuclear leukocytes (PMN) are central effectors of the innate
immune response to inflammation in humans. Calcium entry into the PMN
typically occurs through store-operated mechanisms, and we have noted
dysfunctional PMNs store-operated calcium entry (SOCE) after injury
(24). Human PMN have been
found to mobilize Ca2+ in response to extracellular
stimuli via the activation of SphK
(14), but yet it has also been
shown that neutrophil-like HL-60 cells show a complete absence of specific S1P
binding (21), suggesting an
absence of functional S1PR. We therefore hypothesized that S1P might mobilize
PMN calcium by acting as a second messenger to initiate SOCE.
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EXPERIMENTAL PROCEDURES
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Sphingosine 1-PhosphateS1P was purchased from Sigma (St.
Louis, MO). This preparation of S1P is formulated to be soluble in methanol,
which is the optimal vehicle for S1P
(25). A 5 mM stock
solution was prepared according to the manufacturer's recommendations. The
concentration (14 µl/ml) of methanol vehicle used has no effect
calcium release or entry. Binding of S1P to plasma proteins has been reported
to attenuate its biologic effects
(26). We confirmed this in
preliminary studies of S1P calcium mobilization in PMN. Therefore, we felt
that BSA-free environments were optimal for evaluating the biologic effects of
S1P, and avoided the use of BSA in S1P preparation.
Other MaterialsSphingosine, pertussis toxin, and other
general chemicals were also purchased from Sigma.
D-erythro-N,N-Dimethylsphingosine (DMS) was
purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA).
D-erythro-[3H]Sphingosine (1.85 MBq) was
purchased from PerkinElmer Life Sciences (Boston, MA). Thapsigargin and
Fura-2AM were purchased from Molecular Probes (Eugene, OR). PAF, fMLP,
ionomycin, and U73122
[GenBank]
were purchased from Calbiochem (La Jolla, CA).
Neutrophil PreparationsStudies were performed in compliance
with the Institutional Review Board of the University of Medicine and
Dentistry of New Jersey (UMDNJ)-New Jersey Medical School. Informed consent
was obtained for blood withdrawn from healthy human volunteers. A detailed
description of our techniques for PMN isolation is available elsewhere
(25). Briefly, neutrophils
were isolated from heparinized whole blood using a one-step centrifugation
procedure. The neutrophil layer was collected, osmolarity was restored, and
cells were washed and suspended in HEPES buffer. The number and purity of PMN
were checked by flow cytometry. Cells were loaded with 1 µM
Fura-2AM and kept on ice until for study. Aliquots of 2 x 106
cells were re-warmed just prior to each experiment.
HL-60 Cell PreparationsHL60-G cells, a subclone of the
human promyeloblastic leukemia HL60 cell line, were the kind gift of Dr.
George Studzinski (UMDNJ, Newark, NJ). Cells were cultured at 37 °C in
RPMI 1640 with L-glutamine (Invitrogen) supplemented with 20%
heat-inactivated, fetal bovine serum (Invitrogen). 1.3% Me2SO was
added for 25 days to differentiate them into PMN-like cells.
Differentiation was assured by assessment of CD11b using flow cytometry
(27,
28).
Divalent Cation Measurements by
SpectrofluorometryIntracellular calcium
([Ca2+]i) was monitored by Fura
fluorescence at 505 nm, using 340/380-nm dual wavelength excitation at 37
°C with constant stirring. Calibration was performed after each experiment
using digitonin (Rmax) and EGTA
(Rmin). [Ca2+]i
was calculated from the 340/380-nm fluorescence ratio as per the methods of
Grynkiewicz et al.
(29). More detailed
descriptions of our techniques are available elsewhere
(30).
Measurement of Total PMN S1P Production by Thin Layer Chromatography
(TLC)PMN isolated as above (2 x 106 in 100 µl
for each reaction) were incubated in Hanks' balanced salt solution buffer with
1 mM calcium. Aliquots were incubated with DMS or vehicle as
indicated for 10 min prior to reactions. At T = 0, 100 µl of a
mixture containing [3H]sphingosine (PerkinElmer Life Sciences,
70,000 cpm/reaction) with or without 1 µM TG (final
concentration, 500 nM) was added to warmed PMN to begin the
reaction. Aliquots were allowed to incubate at 37 °C for 0, 2, 5, and 10
min. The reactions were stopped by the addition of 1 ml of ice-cold
methanol:chloroform (2:1). The samples were then vortexed for 10 s and spun
for 10 min at 2000 x g. Supernatants were collected, and a
second 100-µl mixture of methanol:chloroform (2:1) was applied, vortexed,
and spun as before. The combined supernatants were dried, resuspended in 60
µl of methanol, and applied to Silica Gel 60 TLC plates. Unlabeled
sphingosine and sphingosine 1-phosphate were applied to control lanes to
identify [3H]sphingosine and [3H]sphingosine
1-phosphate, respectively. Lipids were separated by TLC using a
1-butanol/acetic acid/water (3:1:1) mixture. Standard control lipids were
visualized using ninhydrin (for S1P and SP) and molybdenum blue (for S1P). The
radioactivity of the corresponding areas of the experimental lanes was
measured by liquid scintillation counting. The mean ± S.E. of three
observations at each time point is shown. More detailed methods can be found
elsewhere (31).
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RESULTS
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S1P Induces Calcium Influx DirectlyS1P was applied to human
PMN in the absence of external calcium (0.3 mM EGTA added) in order
to study calcium store release and influx
(Fig. 1). After equilibration,
cells were treated with S1P or vehicle (methanol). PMN show no store release
transient at all in response to S1P. In contrast, dose-dependent calcium
influx is seen on re-addition of external 1 mM
Ca2+. Calcium influx is immediate under these
conditions, rises to a typical plateau, and subsequently decays. In other
experiments no store release transient was seen in
Ca2+-free media out to 900 s, although brisk store
release responses to the GPCR agonists formyl-Met-Leu-Phe (fMLP) and
platelet-activating factor (PAF) continued to be present during this time
(data not shown).

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FIG. 1. Sphingosine 1-phosphate induces Ca2+ influx in
human PMN. PMN were Fura-loaded in HEPES buffer with 1 mM
Ca2+ and 0.1% BSA. Immediately prior to study, PMNs were
spun for 5 s at 4500 rpm, washed once with BSA-free/Ca2+
free buffer, spun again, and resuspended in BSA-free, nominally
Ca2+-free (0.3 mM EGTA) buffer for study. S1P
was added at t = 30 s in the concentrations shown. For the zero S1P
control, 3 µl of vehicle (methanol) was added. 1 mM
Ca2+ was added to the medium at t = 50 s.
Dose-dependent calcium entry is immediately noted. The morphology of calcium
entry is typical of SOCE in the PMN. Traces represent the mean ± S.E.
of 34 experiments at each S1P dose.
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S1P Induction of Calcium Influx Is Independent of ER Store
DepletionAlthough no store release transient was seen in response
to S1P in the experiments presented in Fig.
1, we evaluated the possibility that undetected store release
might have occurred. Fura-loaded PMN samples in calcium-free media were
treated with 10 µM S1P or methanol vehicle. The cells were
stimulated 30 s later with fMLP to assess the status of
IP3-releasable ER calcium stores
(Fig. 2). fMLP-induced store
release was morphologically identical with and without prior S1P, and no
statistically significant evidence of S1P-initiated store depletion could be
found at this time point. Moreover, the degree of SOCE seen (on
Ca2+ re-addition at t = 300,
Fig. 2) was clearly a
reflection of the presence of S1P rather than prior store depletion by fMLP.
In longer experiments (data not shown), a slow but significant depletion of
Ca2+ stores was noted after S1P exposure, but this was
limited to a 50% fall in fMLP-releasable stores in comparison to
vehicle-treated cells after 15 min. These findings therefore confirm that
calcium entry in response to S1P occurs in the absence of significant ER
calcium store depletion.

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FIG. 2. S1P does not induce emptying of PMN calcium stores. PMN were studied
in BSA-free, nominally calcium-free conditions. Cells were treated with 10
µM S1P or methanol at 30 s. The cells were then stimulated with
10 nM fMLP (EC90) at 60 s. 1 mM
Ca2+ was added to the medium at t = 300 s. The
store release transients seen on fMLP treatment were indistinguishable. After
S1P treatment, however, SOCE was magnified severalfold. The traces
shown are the mean [Ca2+]i ±
S.E. from four separate experiments.
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S1P Induction of Calcium Influx Is Independent of
G-proteinsPrior studies have shown that PMN-like HL60 cells show
no specific binding of S1P
(21) and that endogenous S1P
production in PMN activates the cells at S1P concentrations far lower than
those required for signaling through any of the known S1PR
(22). These findings, plus the
lack of GPC store depletion by S1P noted in
Fig. 2, make it very unlikely
that S1P acts via GPC, cell-surface S1PR in human PMN. Nonetheless, we studied
the possibility that S1P-mediated Ca2+ influx might
depend on prior store release by cell surface G-protein-coupled S1PR.
S1PR15 can all signal through the pertussis toxin
(PTX)-sensitive G
i
(15,
18). Activation of
G
i in the PMN activates phospholipase C
(PLC) and
causes IP3-dependent release of cell calcium stores within seconds.
To further exclude the possibility that PMN responses to S1P were initiated
via G
i-coupled S1PR, we treated PMN with pertussis toxin
(PTX, 1 µg/ml, for 3 h at 37 °C) prior to stimulation with S1P.
G
i-coupled calcium store release in the neutrophil is fully
inhibited at this PTX dose
(3234).
We found that, in the neutrophil, PTX had no effect upon S1P-induced calcium
entry (Fig. 3A),
whereas [Ca2+]i responses to fMLP
were uniformly abolished (Fig.
3B). Thus PMN calcium influx in response to S1P was
independent of PTX-sensitive G
i-linked S1PR.

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FIG. 3. Calcium mobilization by S1P is pertussis toxin (PTX)-insensitive.
PMN were incubated with PTX (1 µg/ml) or vehicle for 3 h at 37 °C. In
the upper panel, PMN were suspended in nominal
Ca2+, BSA-free buffer. At 30 s cells were treated with 5
µM S1P. 1 mM Ca2+ was added to
the medium at t = 50 s. Calcium entry in response to S1P was
indistinguishable in the presence and absence of PTX. Note, again, that S1P
elicits no measurable store emptying in this time frame
(Fig. 2). In the lower
panel, cells incubated identically with PTX were exposed to 10
nM fMLP at 30 s. PTX completely blocked the
Ca2+ store release response to fMLP. The subsequent
calcium entry response was limited to the level of the "leak."
(i.e. that Ca2+ entry always seen on
re-calcification of the medium irrespective of prior stimulation or store
depletion. PMN [Ca2+]i changes due
to leak can vary from 20 to 40 nM in PMN using our methods.) PMN
incubated 3 h without PTX responded normally to fMLP (lower panel, upper
trace).
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In addition, however, the S1P2 and SIP3 receptors are
also known to bind to Gq/11
(18). The Gq/11
pathway is not PTX-inhibited, but like G
i, Gq/11
acts through phospholipase C
(PLC) and IP3 to release cell
calcium stores (15,
18). Thus S1P acting on
S1P2 or SIP3 could potentially deplete PMN calcium
stores in a PTX-insensitive manner but in a PLC-dependent fashion. We
therefore incubated PMN with or without the PLC inhibitor U73122
[GenBank]
(20
µM, 5 min at 37 °C) prior to stimulation with S1P. This
agent inhibits calcium store depletion by both G
i and
Gq. U73122
[GenBank]
had no effect whatsoever on calcium entry in response to
S1P (Fig. 4), whereas, again,
[Ca2+]i mobilization in response to
fMLP was fully blocked. These experiments demonstrate that PMN calcium entry
after S1P is independent of G
i, Gq, the
PLC/IP3 pathway, and the depletion of cell
Ca2+ stores.

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FIG. 4. Calcium influx caused by S1P is insensitive to PLC inhibition. PMN
were treated with 20 µM U73122
[GenBank]
(upper trace) for 15 min
in the cuvette in the presence of 1 mM calcium and 0.1% fatty-acid
free BSA. Recordings were begun at t = 0. At 30 s 10 nM
fMLP was applied and no [Ca2+]i flux
was detected. At 100 s 20 µM S1P was applied. S1P caused calcium
influx. In the lower trace (displaced for clarity) PMN were exposed
to S1P at the time marked without prior exposure to U73122
[GenBank]
.
[Ca2+]i flux is indistinguishable in
the presence and absence of U73122
[GenBank]
. Representative traces are shown, three to
four experiments were done per condition. No S1P-mediated
[Ca2+]i flux is seen in the absence
of external Ca2+. S1P is bound strongly by BSA, and this
slow pattern of calcium influx is typical of PMN responses to S1P in
BSA-containing medium. The BSA was required to prevent cell clumping after
adding the U73122
[GenBank]
.
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S1P-stimulated Ca2+ Entry Is Independent of
G-protein Expression in HL60 CellsTo further exclude the
possibility that G-protein-coupled, cell-surface S1PR might play a role in PMN
Ca2+ entry response to S1P, we evaluated the PMN-like,
HL60-G cell line. We found that in their basal (undifferentiated) state, these
HL60 cells showed no Ca2+ store release at all in
response to fMLP (Fig.
5A, lower trace) or any other typical PMN GPCR
agonist (complement fragment 5a, interleukin-8, GRO-
, and leukotriene
B4, data not shown). After treatment with 1.3% Me2SO,
however, the cells differentiate into a PMN-like (CD11b+, data not
shown) phenotype. We found that such "HL60-PMN" cells showed brisk
Ca2+ store release responses to fMLP
(Fig. 5A, upper
trace) as well as all the other PMN agonists noted above. S1P-induced
Ca2+ store release remained totally absent despite this
generalized increase in the expression of functional GPCR
(Fig. 5B). Conversely,
S1P induced very similar amounts of calcium entry in HL60-PMN and in HL60
cells (Fig. 5B). These
findings support the lack of involvement of GPCR in S1P-mediated PMN calcium
mobilization.

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FIG. 5. Responses of HL-60 cells to S1P. Undifferentiated HL60 or
differentiated HL60-PMN cells (see "Experimental Procedures") were
exposed to 10 nM fMLP (A) or to S1P (B).
Undifferentiated cells showed no response to fMLP and a typical small
Ca2+ "leak" upon re-addition of external
Ca2+. HL60-PMNs show a morphologically normal
Ca2+ store release transient after fMLP stimulation,
followed by typical SOCE. Similar induction of Ca2+
store release was seen in response to multiple GPCR agonists (see text). In
B, neither HL60 nor HL60-PMN showed any Ca2+
store release response to S1P, but brisk Ca2+ entry was
seen upon re-addition of external Ca2+. Only the
Ca2+ "leak" was seen in the absence of S1P,
which was very similar (note the change in scale) in HL60
(A) and HL60-PMN (B). These findings demonstrate that
S1P-mediated Ca2+ entry is independent of the overall
degree of GPCR expression in PMN-like cells.
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Ionic Selectivity of SIP-induced Ca2+
EntryHaving determined that PMN calcium entry responses to S1P
were independent of store depletion and of GPCR, we compared the known
attributes of PMN SOCE with S1P-mediated Ca2+ entry. We
have previously reported that total SOCE in human PMN reflects the combined
contributions of Ca2+-specific and
Sr2+-permeable pathways
(30). We therefore exposed PMN
to 10 µM S1P and serially added 1 mM
Sr2+ and 1 mM Ca2+ to
the medium (Fig. 6). We
detected both Sr2+ influx and Ca2+
influx on serial cation addition. As with TG, ionomycin, fMLP, and PAF
stimulation in our prior studies, we found that, after S1P stimulation,
additional calcium entry through Ca2+-specific pathways
always persisted after Sr2+ entry had reached
equilibrium. Thus S1P activates influx through the specific and nonspecific
PMN Ca2+ entry pathways and acts upon them in a fashion
consistent with that seen previously with traditional agonists eliciting SOCE
in the PMN.

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FIG. 6. S1P elicits both Sr2+ and
Ca2+ entry in PMN. After Fura-2AM loading, PMN were
spun and washed in buffer without BSA or Ca2+. Cells
were spun and resuspended in cuvettes in BSA/Ca2+-free
media. S1P was applied at 30 s. Sr2+ and
Ca2+ were added at 300 and 450 s, respectively. S1P
causes dose-dependent entry of each cation in relative proportions similar to
those seen after TG, ionomycin, and PAF stimulation in our prior studies
(24). Representative traces
are shown, n = 34 per condition.
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Sensitivity of SIP-induced Ca2+ Entry to
Inorganic Channel BlockersTo ensure that the influx of
Ca2+ seen after S1P stimulation was due to entry through
channel mechanisms, we studied the effects of the inorganic calcium channel
blockers lanthanum (La3+), nickel
(Ni2+), zinc (Zn2+), and
gadolinium (Gd3+) on S1P-induced influx. PMN were
treated with 1 mM La3+,
Ni2+, or Zn2+ prior to S1P
stimulation (Fig. 7).
La3+ and Zn2+ abolished all PMN
calcium influx in response to S1P, but Ni2+ was
ineffective. This pattern of sensitivity is identical to that of PMN responses
to GPC agonists and thapsigargin we described in prior studies
(30).

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FIG. 7. Inhibition of S1P-induced calcium entry by inorganic channel
blockers. PMN were stimulated with S1P in the presence of the heavy metal
and lanthanide channel-blockers La3+,
Ni2+, and Zn2+ (all 1
mM), and the media were re-calcified at the times shown.
La3+ abolishes Ca2+ entry.
Experiments performed in the presence of Zn2+ show
traces overlapping those done in the presence of La3+
(data not shown). In the presence of Ni2+,
[Ca2+]i traces were
indistinguishable from those seen in the absence of any blockers (data not
shown). Representative traces are shown, n = 34 per
condition.
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Gadolinium (Gd3+) is another lanthanide inhibitor of
calcium entry channels, but at sub-micromolar concentrations
Gd3+ is thought to be specific for the store-operated
and mechanically sensitive calcium channels that are likely to be composed of
TRP proteins (1,
30,
3538).
We therefore performed more detailed studies of PMN calcium entry responses to
S1P in the presence of Gd3+
(Fig. 8). S1P-initiated calcium
influx was inhibited by nanomolar concentrations of Gd3+
with an EC50 of
250 nM and complete inhibition at 1
µM. Again, this pattern was very similar to that we have
previously reported for store-operated calcium entry channels in the PMN
(30).

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FIG. 8. Gadolinium (Gd3+) inhibits S1P-induced calcium
entry. PMN were treated with the concentrations of
Gd3+ shown for 1 min in the presence of calcium prior to
study. 10 µM S1P was added at t = 30 s.
Gd3+ caused dose-dependent suppression of S1P-induced
Ca2+ entry. Complete blockade of
Ca2+ entry was seen at 1 µM.
Representative traces are shown, n = 3 for each concentration.
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Direct Depletion of ER Calcium Stores Activates S1P
SynthesisThe stimulation of S1P production from sphingosine by GPC
agonists is well known (39,
40), but if S1P links store
depletion to SOCE, then direct store depletion should elicit S1P synthesis.
This has not been examined. We therefore studied PMN synthesis of
[3H]S1P from [3H]sphingosine after thapsigargin (TG)
stimulation using thin-layer chromatography
(31). TG depletes ER stores
directly by inhibition of the ER calcium ATPase. We found that TG initiated
rapid incorporated of [3H]sphingosine into [3H]S1P
(Fig. 9). The kinetics of
[3H]S1P formation seen after TG store depletion in PMN were very
similar to those seen in HL-60 cells stimulated by formyl peptide
(39), with rapid synthesis
noted over the first 2 min and subsequent formation of a plateau lasting more
than 10 min. Moreover, the conversion of [3H]sphingosine to
[3H]S1P in response to TG was completely inhibited by the SphK
inhibitor DMS (13,
14).

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FIG. 9. Direct store depletion causes PMN S1P synthesis. PMN (2 x
106) were incubated with DMS or vehicle as indicated for 10 min.
Reactions were started (t = 0) by adding [3H]sphingosine
with or without TG (see text). The reactions were stopped at 0, 2, 5, and 10
min. Total [3H]S1P was collected and assayed by TLC. The mean
± S.E. of three observations at each time point is shown. TG initiated
rapid synthesis of S1P. No significant S1P synthesis was seen in the absence
of TG. DMS abolished S1P production in TG-treated cells to the level of
unstimulated cells.
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Inhibition of S1P Synthesis Uncouples Store Depletion from
SOCEA wide variety of external stimuli can cause cells to
metabolize sphingosine to S1P via the actions of SphK (reviewed in Olivera and
Spiegel (41)). This conversion
is inhibited by DMS (13,
14). If direct store depletion
initiates S1P synthesis and thus SOCE, then inhibition of S1P synthesis by DMS
should inhibit SOCE resulting from direct store depletion. We therefore
assessed whether SOCE initiated by TG (500 nM) or ionomycin (100
nM) could be inhibited by DMS. Store depletion by TG
(Fig. 10A) as well as
ionomycin (Fig. 10B)
proceeded normally in calcium-free media in the presence of DMS. Upon
re-addition of calcium to the medium, however, we saw dose-dependent
inhibition of SOCE by DMS with 100% inhibition at about 15 µM.
These data clearly demonstrate that blockade of SphK and the resultant loss of
cellular S1P synthesis abolishes the link between ER calcium store depletion
and calcium entry into the PMN.

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FIG. 10. DMS inhibits PMN SOCE initiated by direct store depletion. PMN were
incubated for 1 min with increasing doses of DMS in calcium-free media. In
A, 500 nM TG was added at 30 s. 1 mM
Ca2+ was added after the completion of store release. In
B, 100 nM ionomycin (Iono) was added at 30 s.
Again, 1 mM calcium was added after the completion of store
release. In each case, store release was unaffected by DMS, but SOCE was
suppressed in a dose-dependent manner. In the presence of 15 µM
DMS, calcium entry was suppressed to the level of the "leak"
current. Representative traces are shown, n = 34 for each
concentration of DMS.
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We also examined this issue in HL60 cells
(Fig. 11) and found that
TG-initiated Ca2+ store release was unaffected by DMS
and completely independent of the state of cell differentiation. Conversely,
DMS inhibited TG-initiated SOCE completely. The SOCE response to TG, which,
unlike responses to cell-surface agonists, is GPCR-independent, was noted to
be markedly increased by differentiation of the cells.

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FIG. 11. DMS inhibits HL60 SOCE initiated by direct store depletion. HL60
(upper panel) and HL60-PMN (lower panel) cells were studied
in a like manner to the PMN studied in
Fig. 10. Quantitative
Ca2+ store release was unaffected by the differentiation
of the cells (note the difference in scale) or by the presence of
DMS. Again though, 10 µM DMS was found to inhibit TG-initiated
SOCE to the level of "leak." SOCE response to TG (which is
GPCR-independent) was markedly increased by differentiation of the cells.
|
|
DMS-inhibitable SOCE Is
[Ca2+]i-independentTG induces
both S1P synthesis and DMS-inhibitable calcium entry. To determine whether
such DMS-inhibitable calcium entry truly represents SOCE, it is important to
know whether it occurs in a manner that is independent of elevations in
[Ca2+]i. We therefore studied the
effects of DMS on store depletion-dependent entry of manganese
(Mn2+) into
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-loaded PMN (Fig.
12). Under these conditions, ER stores are depleted without
elevations of [Ca2+]i, and
store-operated divalent cation entry can be assessed as the quenching of Fura
by Mn2+ entry
(24). As expected, in the
absence of prior store depletion by TG (A), PMN showed no sign of
Mn2+ entry. Cells that have undergone prior store
depletion by TG, however (B), allow rapid entry of
Mn2+ from the media. Such Mn2+
entry was blocked by prior PMN incubation with DMS (C). Thus DMS
inhibits store-operated influx of divalent cations in a
[Ca2+]i-independent fashion.

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FIG. 12. DMS inhibits SOCE in a calcium-independent manner. Fura-loaded PMN
were incubated in 20 µM BAPTA for 9 min at room temperature.
A, cells were exposed to vehicle only (Me2SO) at 30 s.
Samples were then observed in the cuvette for 10 min. At that time, a mixture
of 500 µM Mn2+ and 1.5 mM
Ca2+ was added where indicated to quench Fura. In
B, the cells were exposed to TG (500 nM) at 30 s. Again,
500 µM Mn2+ and 1.5 mM
Ca2+ were added at the time indicated. In C,
cells were pretreated for 1 min with 10 µM DMS and then exposed
to TG (500 nM) at 30 s. Again, after allowing 10 min for store
depletion, Mn2+ and Ca2+ were
added. Fluorescent intensity (FI) was monitored at both 340/380 nm (not shown)
and at 360 nm, as shown (FI360). No 340/380 nm
[Ca2+]i release transient was seen
at any time (data not shown). On Mn2+ addition, a small
initial drop in total fluorescence at 360 nm is noted due to quenching of Fura
in the media, but no Mn2+ entry into the cells was
detected in the absence of prior store depletion (A). In contrast,
prior TG treatment (B) caused rapid Mn2+ entry.
TG-dependent Mn2+ entry was blocked by prior treatment
with DMS (C). Linear curve-fitting (SigmaPlot) was then performed on
the FI360 after Mn2+ addition (n =
34 independent experiments per condition). This revealed a mean slope
of 2.3 ± 0.6 units/s after treatment with vehicle only. The
FI360 slope decreased to 14.9 ± 0.9 units/s in
TG-treated cells but was returned to unstimulated levels (3.1 ±
0.4 units/s) by DMS pretreatment (p < 0.01, analysis of
variance/Tukey's test). The data show that DMS inhibits the calcium entry
response to TG store depletion in a
[Ca2+]i-independent fashion.
|
|
Inhibition of S1P Synthesis Uncouples GPC Receptors from
SOCEStimulation of PMN by GPC agonists is known to cause S1P
synthesis (21) as well as
store depletion. As seen in Figs.
10 and
11, blockade of S1P synthesis
by DMS uncouples pharmacologic store depletion from SOCE. We therefore
evaluated the dependence of SOCE after physiologic GPC store depletion upon
S1P synthesis. To accomplish this, we stimulated PMN with 100 nM
PAF in the presence and absence of DMS. DMS had no discernable effect on store
depletion by PAF (Fig. 13,
AC). When calcium was added to the media, brisk
normal SOCE was seen in the absence of DMS
(Fig. 13A). After PMN
pretreatment with 10 µM DMS, however, the SOCE response to PAF
was abolished (Fig.
13B). Finally (Fig.
13C), when DMS was added after PAF-induced store
depletion only minimal inhibition of SOCE was seen. Thus inhibition of
PAF-induced SOCE by DMS requires the presence of the inhibitor at the time of
store depletion and SphK activation. These findings confirm that the
activation of SOCE by physiologic GPC store depletion depend on S1P synthesis
by SphK. These experiments also address the issue of whether S1P receptors
linked to Gq (i.e. S1PR2 and S1PR3)
might activate cation channels directly in the PMN, as has been suggested in
some cell types (42,
43). PAF receptors (PAFrs) do
indeed activate Gq/11
(44), but the inhibition of
PAF-mediated calcium entry by DMS (Fig.
13B) suggests that PAFr/Gq-dependent calcium
entry requires S1P synthesis. Moreover, the data in
Fig. 9C show that
calcium entry after PAFr/Gq activation requires SphK activity
subsequent to ER store depletion.

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FIG. 13. DMS inhibits G-protein-coupled receptor-initiated SOCE. PMN were
exposed to 100 nM PAF at t = 30 s in calcium-free medium.
In A (control) no DMS was used. The DMS vehicle (Me2SO)
was applied at 150 s. In B, DMS was applied 100 s prior to PAF. In
C, DMS was added at 150 s, allowing >1 min for full DMS effect.
All media were re-calcified at t = 220 s to measure SOCE. The
traces represent the mean
[Ca2+]i ± S.E. of three
experiments. Each experimental trace is shown compared with an identical
"blank" control trace where only PAF vehicle (3 µl of ethanol)
was used at t = 30. This "blank" trace demonstrates the
nonspecific calcium "leak." DMS given prior to store depletion
(B) completely eliminated PAF-initiated SOCE. DMS given after store
depletion (C) caused only minimal inhibition.
|
|
DMS Does Not Inhibit Calcium EntryPrior studies observing
inhibition of calcium flux by DMS have proposed that it might inhibit calcium
entry channels directly (45).
This is unlikely in the light of the findings presented in
Fig. 13. If DMS were to act by
direct inhibition of calcium channels, its inhibition of
Ca2+ entry should be independent of whether it was given
before or after store depletion, as long as it was present at the time of
calcium entry. Such was clearly not the case. Conversely, if DMS acts by
inhibiting S1P synthesis at the time of store depletion, its effect should be
diminished if given after store depletion and SphK activation have begun, and
such was indeed the case (Fig. 13, B
versus C). Moreover, as seen in
Fig. 9, DMS clearly inhibits
the synthesis of S1P stimulated by TG.
To further discount any possibility that inhibition of calcium entry by DMS
could reflect channel blockade, we evaluated the effects of DMS on calcium
entry initiated directly by exogenous S1P
(Fig. 14). If DMS were to
inhibit Ca2+ entry through SOCE channels, it should act
like La3+ or Gd3+
(Fig. 5) and block the calcium
entry responses elicited by S1P. In fact, we found that DMS caused no
inhibition of S1P-initiated Ca2+ or
Sr2+ entry at any concentration. Rather, DMS enhanced
cation entry responses to S1P in a dose-dependent fashion. Because DMS
increases rather than decreases S1P-induced increases in
[Ca2+]i, it clearly cannot act by
blocking S1P-induced cation entry. Nonetheless, the synergistic ability of
exogenous DMS and S1P to increase
[Ca2+]i is unexplained, and several
possible mechanisms might be advanced. DMS might interfere with S1P
degradation as well as its synthesis. Or, exogenous DMS and S1P might interact
to produce cell-membrane changes allowing cationic influx. This latter
explanation seems less likely, because the influx seen was dose-responsive and
formed typical "plateaus" at all concentrations, this suggesting
regulated rather than pathologic cation entry. Last, entry of
Sr2+ and Ca2+ always occurred in
the same relative proportions as those seen when normal PMN are stimulated
with thapsigargin (30).
Importantly, none of these events can underlie the actions of DMS other
conditions. Where used to inhibit endogenous S1P formation, DMS uniformly acts
to decrease calcium entry, even at high concentrations
(Fig. 10A).

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FIG. 14. DMS does not block calcium influx. In this experiment PMN were
incubated with DMS at the concentrations shown or with vehicle (3 µl of
Me2SO) for 1 min prior to recordings in
Ca2+-free, BSA-free media. At t = 30 s, 10
µM S1P was applied. Sr2+ (1 mM)
was added at 300 s, and 1 mM Ca2+ was added
at 450 s. DMS caused dose-dependent enhancement of the entry of each cation.
Control experiments using DMS alone demonstrated no calcium entry above the
normal "leak" seen on cation re-addition (data not shown). The
data show that DMS does not inhibit Ca2+ entry into PMN.
Rather, it suggests that a previously undescribed synergism may exist between
the cellular effects of exogenous S1P and DMS. These effects remain to be
studied. Representative traces are shown, n = 3 independent
experiments.
|
|
 |
DISCUSSION
|
---|
In 1993, Randriamampita and Tsien
(3) described a soluble
messenger molecule in cellular extracts that could elicit SOCE, which they
termed calcium influx factor (CIF). In partially characterizing this molecule,
they suggested it was released from organelles both into the cytoplasm and
into the extracellular medium. They also suggested CIF would be a
phosphorylated molecule with a molecular mass less than 500 Da, bearing
hydroxyl and amino groups on adjacent carbons. Although other mechanisms of
inducing SOCE have been proposed, the possibility that SOCE, at least in some
forms, might require the action of a CIF has never been discounted. The
identity of that CIF, however, has remained unknown.
S1P is an important extracellular signaling molecule and growth factor that
conforms to all the characteristics initially suggested for CIF
(3). S1P is found in yeast,
plant, insect, and mammalian cells. It is synthesized rapidly from sphingosine
by SphK in response to a wide variety of extracellular signals. Once
synthesized, S1P can either pass outward through plasma membranes to act upon
the many cell surface GPC receptors of the Edg/S1PR class, or it can
act as an intracellular second messenger to mobilize cell calcium
(46,
47). S1P may also be an
intermediary in the activation of
[Ca2+]i by other second messengers,
such as lysophosphatidic acid
(48).
The multiple routes by which S1P production is linked to calcium
mobilization have complicated analyses of its mechanisms of action in other
cell types. Because PMN had been shown to mobilize Ca2+
through an SphK-mediated pathway
(14) we hypothesized S1P might
be an important PMN activator. Our initial experiments showed that S1P did
indeed mobilize Ca2+ in PMN, but we saw no trace of the
transient store release spike typically expected when stimulating neutrophil
GPCR. We therefore hypothesized that S1P might act primarily as a second
messenger in PMN by eliciting SOCE.
We have shown here that direct exposure of normal human PMN to S1P
activates calcium influx pathways with a wide variety of characteristics
typical of PMN SOCE. Exogenous S1P-mediated Ca2+ entry
occurs rapidly without measurable depletion of calcium stores and is
independent of Edg/S1PR signaling through G
i,
Gq, or phospholipase C. Rapid activation of
Ca2+ entry by exogenous S1P appears to occur over a dose
range of approximately one order magnitude of extracellular S1P concentrations
that may be attainable in normal plasma and are likely to be present in
disease states (17,
26). S1P was fully sufficient
to initiate SOCE de novo in PMN, and its synthesis was necessary for
SOCE under all conditions tested. In vivo, S1P acts downstream from
the afferent, store depletion arm of physiologic SOCE. The amplitude and
morphology Ca2+ entry in PMN treated with S1P is similar
to that seen in PMN treated with TG or ionomycin
(30) and indistinguishable
from physiologic PMN SOCE responses to PAF
(24).
We also studied the linkage between endogenous S1P production and
activation of PMN SOCE. Isolated store depletion by TG was shown to induce the
rapid and DMS-inhibitable formation of S1P from sphingosine; S1P synthesis
occurred over 12 min with the subsequent formation of a plateau of S1P
concentration, and TG-initiated, DMS-inhibitable SOCE was shown to be
[Ca2+]i-independent. We found that
DMS had no effect at all upon the degree of direct Ca2+
store depletion by TG or ionomycin or upon G-protein-coupled store depletion
by PAF. Yet DMS blocked Ca2+ entry responses to TG,
ionomycin, and PAF quantitatively and in a dose-dependent fashion. This
inhibition seen was not due to channel blockade, because it was dependent upon
the inhibitor being present at the time of store depletion rather than at the
time of calcium entry. Moreover, DMS enhances Ca2+ entry
responses to exogenous S1P. Rather, the inhibitory effects of DMS were all
compatible with its observed inhibition of store depletion-initiated S1P
synthesis. These findings all demonstrate a clear, direct linkage between ER
store depletion and the activation of SphK and SOCE.
Thus, in summary, the synthesis of S1P from sphingosine by SphK is a
necessary step, downstream from store depletion either by direct or
G-protein-coupled mechanisms, and upstream from channel activation and calcium
entry. In effect, S1P synthesis occurs in direct response to store depletion
and is both necessary and sufficient for the initiation of store-operated
calcium entry in the PMN.
The characteristics of PMN calcium entry seen after stimulation by S1P are
also clearly those of SOCE. The pronounced sensitivity to
Gd3+ inhibition and the ionic selectivity of the
conductance was typical of cation entry through SOCE channels in the PMN
(30). These findings are also
compatible with those of Su et al.
(49), who noted that lymphoid
cells have two SOCE channels, one of which is also nonselective and may
require diacylglycerol production as well as store depletion for activation.
Thus multiple linkage mechanisms must clearly exist to connect store depletion
to total "stimulated calcium entry"
(7).
The mechanisms by which S1P activates SOCE channels remain unknown. Despite
being a diffusible messenger molecule with the characteristics of a
"CIF," S1P may only activate some store depletion-stimulated
calcium entry mechanisms. Other similar messengers may exist. Moreover, S1P
may act directly on channels or via downstream mechanisms that require the
interaction of membrane channels with the cytoskeleton. In this regard, S1P
has been shown to regulate myosin light chain phosphatase
(50). Also, it has been
suggested that membrane sphingolipids may play a role in the assembly of
oligomeric proteins (such as calcium channel proteins) that are located in
lipid rafts (51). This gives
rise to the speculation that conformational events based upon membrane lipid
composition could also play a role in S1P-mediated calcium entry. Thus the
finding that S1P acts as a soluble CIF in this instance in no way discounts
the potential involvement and importance of direct physical interactions
between membrane channels and intracellular structures. Rather, it remains to
be determined whether SOCE requires elements of both hypotheses, and whether
the soluble calcium influx factor S1P may link store depletion to downstream
conformational events.
 |
FOOTNOTES
|
---|
* This work was supported by a grant from the Foundation for UMDNJ (to K. I.)
and by Grant GM-59179 from the National Institutes of Health (to C. J. H.).
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Surgery, University of
Medicine and Dentistry/New Jersey Medical School, MSB G-524, 185 South Orange
Ave., Newark, NJ 07103. Tel.: 973-972-2894; Fax: 973-972-6803; E-mail:
hausercj{at}UMDNJ.edu.
1 The abbreviations used are: SOCE, store-operated calcium entry; S1P,
sphingosine 1-phosphate; CIF, calcium influx factor; PMN, polymorphonuclear
leukocytes; DMS,
D-erythro-N,N-dimethylsphingosine; SphK,
sphingosine kinase; IP3, inositol 1,4,5-trisphosphate; ER,
endoplasmic reticulum; GPCR, G-protein-coupled receptor; BSA, bovine serum
albumin; PAF, platelet-activating factor; fMLP, formyl-Met-Leu-Phe; PTX,
pertussis toxin; PLC, phospholipase C; GPC, G-protein-coupled; GPCR, GPC
receptor agonist; PAFr, PAF receptor. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. K. B. Kannan, Zoltan Fekete, Eleonora Feketeova, and Qi Lu
for their assistance with cell preparations and flow cytometry and Linda
Vetrecin for her assistance in the preparation of the manuscript.
 |
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