1 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY
14853, USA
2 Department of Molecular Medicine, Veterinary Medical Center, Cornell
University, Ithaca, NY 14853, USA
Author for correspondence (e-mail:
bab13{at}cornell.edu)
Accepted 15 April 2003
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Summary |
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Key words: Ceramides, Lipid rafts, IgE receptors, Phospholipase D, Mast cells
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Introduction |
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Membrane structural heterogeneity is likely to be particularly important
for enzymes that have lipids as substrates, including phospholipases. For
example, it is well established that defects in membrane bilayers modulate
phospholipase A2 (PLA2) activity
(Burack and Biltonen, 1994;
Burack et al., 1997
), and a
recent study showed that binding of ADP-ribosylation factor 6 (ARF6) to
bilayers containing phosphatidylinositol (4,5)-bisphosphate (PIP2)
creates defects that could be involved in the activation of phospholipase D
(PLD) (Ge et al., 2001
). These
lipases generate important lipid second messengers, as well as amphiphilic
products that can modulate membrane structure and influence processes such as
vesicle budding and exocytosis (Liscovitch
et al., 2000
; Cohen and Brown,
2001
; Ivanova et al.,
2001
). Ceramides are the products of sphingomyelin hydrolysis by
sphingomyelinases, and these endogenous, long-chain neutral lipids have been
implicated as signal transduction mediators in certain cell types
(Kolesnick et al., 2000
).
Short-chain, cell-permeable ceramides are commonly used to mimic the
mechanisms of action of naturally occurring ceramides in signal transduction,
and effects of these ceramides on specific pathways in cell signaling have
been described (Mathias et al.,
1991
; Huwiler et al.,
1996
; Hannun and Obeid,
2002
). Among such studies, Nakamura et al. showed that
C2-ceramide (C2-cer) inhibits antigen-stimulated PLD
activation in RBL mast cells, and they concluded that this results from
inhibition of Ca2+ influx and consequent inhibition of
Ca2+-dependent protein kinase C (PKC) isozymes upstream of PLD
(Nakamura et al., 1996
). Other
studies have implicated more-direct effects of short-chain ceramides on PLD
activity (Singh et al., 2001
;
Venable et al., 1996
).
Specific binding of these short-chain ceramides to ion channels or enzymes is
commonly suggested to be responsible for the biological effects observed.
However, it has been shown that membrane-destabilizing properties of
C2-cer correlate with inhibition of platelet signaling, and it has
been suggested that this may be a more general mechanism by which short-chain
ceramides affect cell signaling (Simon and
Gear, 1998
).
In the present study, we evaluated the effects of various ceramides on
lipid order in RBL mast cell plasma membranes, and compared these effects with
inhibition of signaling mediated by FcRI in these cells. We find that
disruption of lipid order by certain short-chain ceramides is highly
correlated with their inhibition of antigen-stimulated Ca2+
mobilization and activated PLD1. Our results point to activation of PLD as a
critical step for antigen-stimulated release of Ca2+ from internal
stores, and they indicate that perturbation of lipid order by short-chain
ceramides may be a generally useful strategy for investigating the role of
membrane structure in cellular signaling.
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Materials and Methods |
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Cells
RBL-2H3 cells (Barsumian et al.,
1981) were maintained in monolayer culture in Minimum Essential
Medium supplemented with 20% fetal bovine serum (Atlanta Biologicals,
Norcross, GA) and 10 µg/ml gentamicin sulfate. All tissue culture reagents
were obtained from Gibco (Grand Island, NY) unless otherwise noted. Cells were
harvested 3-5 days after passage.
Fluorescence anisotropy measurements of lipid order in plasma
membrane vesicles
Plasma membrane vesicles were isolated from RBL-2H3 cells by chemically
induced cell blebbing and labeled with DPH-PC as described previously
(Gidwani et al., 2001). After
washing the vesicles to remove unincorporated probe, 2 ml of the membrane
suspension (
150 µM in phospholipid) in buffered saline solution (BSS:
135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5.6 mM
glucose, 20 mM HEPES, pH 7.4) was placed in a 10x10x40 mm acrylic
cuvette and stirred continuously in a thermostatic sample chamber at 37°C.
Fluorescence anisotropy measurements were made as described previously
(Gidwani et al., 2001
) before
and after C6-cer, C2-cer or C2-dhcer were
added in successive increments from stock solutions in ethanol or dimethyl
sulfoxide (DMSO). Control experiments showed that the solvents added (<1%
v/v total) had negligible effects on anisotropy values. Mol% ceramide was
based on the known amount of ceramide added and the amount of plasma membrane
phospholipid determined by an organic phosphate assay as described previously
(Gidwani et al., 2001
).
Fluorescence resonance energy transfer (FRET) measurements of
proximity between cell-surface molecules
For donor labeling, RBL-2H3 cells were harvested in 1.5 mM EDTA, 135 mM
NaCl, 20 mM HEPES, pH 7.4, pelleted by centrifugation (200 g,
8 minutes), and washed once in BSS. Cell aliquots at
5x106/ml in BSS were incubated with various FRET donors:
Alexa488-IgE (1 hour at 37°C), Alexa488-anti-GD1b (1 hour at
37°C), Alexa488-cholera toxin B (30 minutes at room temperature),
FITC-anti-transferrin receptor (3 hours at 4°C) or Alexa488-anti-CD43 (3
hours at 4°C). The cells were pelleted by centrifugation, washed twice in
BSS to remove unbound antibodies, and resuspended at 1x106/ml
in BSS in thermostated cuvettes. Prior to each experiment, donor-labeled
samples were examined in a fluorescence microscope to confirm uniform plasma
membrane staining. Steady-state donor fluorescence (Alexa488 or FITC;
excitation, 490 nm; emission 520 nm) was monitored as a function of time in an
SLM 8000C spectrofluorometer; FRET acceptor (Cy3-anti-Thy-1, 1.5 µg/ml
final concentration) was added to the cuvette, and time-dependent donor
quenching was quantified. Various ceramides were then added to the cuvette
from concentrated stock solutions in ethanol or DMSO and changes in donor
fluorescence were monitored. Percent disruption of FRET between the
cell-surface molecules by the ceramides was calculated according to Eqn 1:
![]() | (1) |
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Ca2+ measurements
Cells were harvested as described above and loaded with indo-1, a
fluorescent indicator of intracellular free Ca2+
([Ca2+]i), as previously described
(Paar et al., 2002), during
which time they were also sensitized with excess IgE. Suspensions of
indo-1-loaded cells (1x106 cells/ml) in BSS with 0.3 mM
sulfinpyrazone to minimize leakage of indo-1 were stirred at 37°C in
acrylic cuvettes, and [Ca2+]i was monitored with an SLM
8000C spectrofluorometer (excitation, 330 nm; emission, 400 nm). For
measurements in the absence of extracellular Ca2+, indo-1-loaded
cells were washed twice and resuspended at 1x106 cells/ml in
Ca2+-free BSS with sulfinpyrazone. [Ca2+]i is
represented as indo-1 fluorescence intensity, which is nearly proportional to
[Ca2+]i under the conditions of these experiments
(Pierini et al., 1997
). The
Ca2+ response was quantified by integrating the area under the
antigen response curve from 0 to 10 minutes post-stimulation in the presence
of extracellular Ca2+ and until the response returned back to
baseline level in the absence of extracellular Ca2+. Percent
inhibition of Ca2+ mobilization by the ceramides or butanols was
calculated using Eqn 2:
![]() | (2) |
In vitro assays for PLD activity
Recombinant PLD1, ARF1 and PKCßII were purified as described
previously (Walker et al.,
2000). For recombinant PLD2, monolayers of Spodoptera
friguperda 21 (Sf21) cells were infected with baculovirus encoding human
PLD2. After 72 hours infection, the cells were harvested, washed twice in 2 ml
Solution F (8.1 mM Na2HPO4, 1.5 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl, 1 mM DTT, 0.1 mM PMSF,
2.5 mM EDTA), and lysed by nitrogen cavitation at 4°C for 60 minutes at
1100 psi. Membranes and cytosol were separated by spinning at 174000
g for 1 hour, and the membrane-bound fraction of PLD2 were
isolated, resuspended in solution F and aliquoted for assays. Lipid vesicles
with [3H]phosphatidylcholine (PC), phosphatidylethanolamine,
PIP2 and 11 mol% cholesterol were prepared as previously described
(Brown et al., 1993
). To
prepare vesicles with ceramide, an appropriate volume of the ceramide stock in
ethanol was added to the lipid mixture in chloroform to a
phospholipid:ceramide ratio of 2:1; they were then dried, hydrated and
sonicated similar to lipids for control vesicles. All assays were conducted at
37°C for 30 minutes as described (Brown
et al., 1993
). PLD activity was quantified as previously described
(Brown et al., 1995
).
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Results |
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Short-chain ceramides reduce the extent of FRET between lipid raft
markers on intact cells
DPH-PC is not suitable for measurements of lipid order in intact cells, in
part because it is difficult to maintain its plasma membrane localization. As
an alternative, we used FRET between lipid-raft-associated proteins at the
cell surface (Kenworthy and Edidin,
1998; Varma and Mayor,
1998
). For our experiments, Alexa488-labeled IgE or
anti-ganglioside mAb AA4 serve as the energy transfer donors, and Cy3-labeled
anti-Thy-1 bound to Thy-1, an abundant GPI-anchored protein, serves as the
energy transfer acceptor. As shown previously, these antibodies label RBL
cells uniformly at the plasma membrane and co-redistribute when IgE is
crosslinked at 4°C (Holowka et al.,
2000
). As shown in Fig.
3A for Alexa488-IgE bound to Fc
RI on RBL cells, addition of
excess Cy3-anti-Thy-1 causes time-dependent donor quenching, which attains a
new steady state level within a few minutes at 37°C. This quenching of
Alexa488 fluorescence is the result of FRET to Cy3 because it is not observed
when Thy-1 is pre-occupied with excess unlabeled anti-Thy-1, and
Cy3-anti-Thy-1 is added subsequently (A.G., unpublished). Similar FRET are
observed when either Alexa488-cholera toxin B, bound to ganglioside
GM1, or Alexa488-anti-GD1b (AA4), bound to a
GD1b ganglioside (Guo et al.,
1989
), are used as the donor to Cy3-anti-Thy-1. Quenching of donor
fluorescence is not observed when either Alexa488-anti-CD43 mAb, bound to CD43
(leukosialin) (Shelley et al.,
1989
), or FITC-anti-transferrin receptor mAb, bound to transferrin
receptor (Testa et al., 1993
),
are used as the donor, and the same amount of Cy3-anti-Thy-1 is added as
acceptor (A.G., unpublished). Both of these donor-labeled cell-surface
proteins have been shown to be excluded from lipid rafts
(Cinek and Horejsi, 1992
;
Harder et al., 1998
;
Melkonian et al., 1999
),
indicating that FRET detected between acceptor-labeled anti-Thy-1 and
donor-labeled IgE, anti-ganglioside, or cholera toxin B is due to enhanced
proximity resulting from their mutual preferences for a lipid raft
environment.
To evaluate whether short-chain ceramides disrupt lipid raft structure on intact cells using FRET as an indicator, these amphiphiles were added once donor quenching reached a steady state, and the relief of this quenching was monitored. As shown in Fig. 3A, addition of 8 µM C6-cer causes a rapid, time-dependent increase in Alexa488-IgE fluorescence. This increase in donor fluorescence by C6-cer addition was not observed in the absence of the bound acceptor, indicting that it is not due to a direct effect on donor fluorescence (A.G., unpublished). A similar reduction in donor quenching by Cy3-anti-Thy-1 is observed if 8 µM C6-cer is added to cells in which Alexa488-anti-GD1b is employed as the FRET donor, consistent with disruption of lipid raft structure.
The effects of different short-chain ceramides on FRET between Alexa488-IgE
and Cy3-anti-Thy-1 are summarized in Fig.
3B. 8 µM C6-cer inhibits FRET by about 25%, and a
similar amount of inhibition by C2-cer required four times more of
this derivative, consistent with a lower membrane partition coefficient for
this less hydrophobic amphiphile. Similarly,
Fig. 2 shows that 4 times
more C2-cer than C6-cer is required to achieve the same
reduction in membrane order. Higher concentrations of these ceramides (e.g. 16
µM C6-cer) caused significant changes in donor fluorescence in
the absence of acceptors, possibly due to membrane disruption (A.G.,
unpublished). 32 µM C2-dhcer inhibits FRET by less than 5%
(Fig. 3B), consistent with the
lack of effect of this derivative on lipid order measured by DPH-PC anisotropy
(Fig. 2). FRET measurements
were also carried out by pretreating donor-labeled cells with ceramide just
prior to acceptor addition; the resulting inhibition of FRET for each of these
ceramides was found to be similar to that shown in
Fig. 3B (A.G., unpublished).
These results show that short-chain ceramides reduce proximity between
lipid-raft-associated components on intact cells in parallel with their
capacity to disrupt lipid order in plasma membrane vesicles as measured by
DPH-PC fluorescence anisotropy.
Short-chain ceramides inhibit antigen-stimulated Ca2+
mobilization in parallel to disruption of lipid order
A study by Nakamura et al. found that Ca2+ mobilization via
FcRI is effectively inhibited by 30 µM C2-cer in parallel
to inhibition of antigen-stimulated degranulation
(Nakamura et al., 1996
). To
investigate whether these effects are mediated by disruption of lipid order,
we compared the effects of different ceramides on Ca2+ mobilization
in RBL cells to their effects on lipid order. As shown in first half of
Fig. 4A, crosslinking
IgE-Fc
RI with multivalent antigen leads to a typical biphasic
Ca2+ response. In the second trace in
Fig. 4A, addition of 32 µM
C2-cer causes little perturbation of the basal Ca2+
level, but substantially inhibits both the initial and the sustained phases of
the response to antigen. For the integrated Ca2+ response over 10
minutes of stimulation, this represents an average of 80% inhibition for three
separate experiments (Fig. 4E).
As for the energy transfer results, 8 µM C6-cer inhibits
Ca2+ mobilization to a similar extent as 32 µM
C2-cer, but the addition of C6-cer causes a significant
increase in [Ca2+] that complicates quantitation of its inhibitory
effects on antigen-stimulated responses (A.G., unpublished). In contrast to
these ceramides, 32 µM C2-dhcer causes much less inhibition of
this response (Fig. 4B).
Long-chain C16-cer at 8 µM final concentration has a small
potentiating effect on antigen-stimulated Ca2+ mobilization, and it
causes a small transient response itself when added to the cells
(Fig. 4C). These results are
summarized for multiple experiments in Fig.
4E. The relative potencies of these ceramides parallel their
effects on lipid order detected by anisotropy measurements on plasma membrane
vesicles (Fig. 2) or on model
membranes for C16-cer (Massey,
2001
), and they also parallel effects on FRET on the RBL cell
surface (Fig. 3B).
|
Because the initial phase of the Ca2+ response was inhibited by
C2-cer almost as effectively as the sustained phase
(Fig. 4A), we tested the effect
of this ceramide on the Ca2+ response observed in the absence of
extracellular Ca2+. Under these conditions, Ca2+ influx
does not occur, and only a transient response due to Ca2+ release
from intracellular stores is triggered by stimulated inositol
(1,4,5)-trisphosphate (IP3) production
(Meyer et al., 1988;
Smith et al., 2001
). As shown
in Fig. 4D, substantial
inhibition of this transient antigen-stimulated response by C2-cer
is observed, indicating an effect of C2-cer at, or upstream of,
Ca2+ release from stores. As summarized for multiple experiments in
Fig. 4E, 32 µM
C2-cer inhibits this transient Ca2+ response by
60%, whereas 32 µM C2-dhcer does not inhibit this response.
These results show that a principal step in Ca2+ mobilization
inhibited by short-chain ceramides is at, or upstream of, Ca2+
release from stores.
PLD is upstream of antigen-stimulated Ca2+ release from
stores in RBL-2H3 cells
Previously, our laboratory demonstrated that cholesterol-dependent lipid
order is important for initiation of signaling via FcRI
(Field et al., 1997
;
Sheets et al., 1999
).
Therefore, we investigated whether C2-cer inhibits
antigen-stimulated tyrosine phosphorylation under conditions in which these
short-chain ceramides inhibit Ca2+ mobilization. Consistent with a
previous report (Nakamura et al.,
1996
), we could not detect significant inhibition of tyrosine
phosphorylation with 32 µM C2-cer in whole cell lysates (A.G.,
unpublished), possibly because of the relatively small reductions in membrane
order that these amphiphiles cause compared with that caused by cholesterol
depletion (Fig. 2)
(Gidwani et al., 2001
).
In previous studies, C2-cer was reported to inhibit in vitro PLD
activity (Singh et al., 2001)
as well as antigen-stimulated PLD activity in RBL cells in vivo
(Nakamura et al., 1996
). To
evaluate whether PLD might be involved in antigen-stimulated Ca2+
mobilization in RBL cells, we investigated the effects of n-butanol, a
substrate for transphosphatidylation by PLD that prevents phosphatidic acid
(PA) production and inhibits stimulated degranulation in RBL cells
(Lin and Gilfillan, 1992
;
Cissel et al., 1998
). As shown
in Fig. 5A,B, n-butanol
inhibits Ca2+ mobilization in RBL cells by
60% at a final
concentration of 0.5% (v/v). By contrast, t-butanol, which does not undergo
this transphosphatidylation reaction, inhibits the Ca2+ response by
<10% at the same concentration (Fig.
5A,B). In the absence of extracellular Ca2+, transient
Ca2+ mobilization is also inhibited by n-butanol, but not by
t-butanol (Fig. 5B), similar to
the effects of C2-cer and C2-dhcer
(Fig. 4E). These results
suggest that stimulated production of PA by PLD is an important step in the
antigen-stimulated release of Ca2+ from intracellular stores.
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Differential inhibition of in vitro PLD activities by ceramides
Parallel inhibition of antigen-stimulated Ca2+ mobilization by
n-butanol and C2-cer suggested that the effects of
C2-cer on this response might be mediated by direct inhibition of
stimulated PLD activity. Therefore, we systematically investigated the effects
of C2-cer, C2-dhcer and C16-cer on the enzyme
activity of both the known isoforms PLD1 and PLD2
(Colley et al., 1997;
Hammond et al., 1995
) in a
previously established in vitro assay
(Brown et al., 1993
). Sonicated
phospholipid/cholesterol vesicles containing 3H-PC, with or without various
ceramides at a mole ratio of 2:1 phospholipid:ceramide, were used as
substrates for preparations of PLD1 and PLD2 with or without their activators.
PLD2 does not usually respond to small GTPases or PKC in the exogenous
substrate assay (Colley et al.,
1997
), although relatively minor stimulation by ARF has been
reported (Lopez et al., 1998
).
Fig. 6A shows relative enzyme
activity for PLD2 in the presence of ARF1; C2-cer inhibits enzyme
activity by
50% compared with control vesicles. By contrast,
C2-dhcer and C16-cer enhance PLD2 activity by
1.6-fold and
2.7-fold, respectively, relative to the control.
Similar trends were observed in the absence of ARF1 (A.G., unpublished).
|
Fig. 6B shows the effects of
ceramides on PLD1 activity in the presence of its activators ARF1 and
PKCßII. C2-cer very effectively inhibits PLD1 activity, to the
extent of 85%, whereas C2-dhcer and C16-cer have very
small effects. Under these conditions, ARF1 and PKCßII activate PLD1 by
6-8 fold. PLD1 in the absence of these activators is also inhibited by
C2-cer to a similar extent (90% inhibition; A.G.,
unpublished). As represented in Fig.
6C, the pattern of inhibition of activated PLD1 by the various
ceramides is highly correlated with the inhibition profile for Ca2+
mobilization in RBL cells, consistent with the butanol results that implicate
activated PLD as being upstream of antigen-stimulated Ca2+ release
from stores in RBL cells. Together, these results provide evidence that
short-chain ceramides exert primary effects on signaling by inhibition of PLD1
via perturbation of membrane structure.
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Discussion |
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Our findings are consistent with these latter results and support the
hypothesis that short-chain ceramides inhibit signaling by disruption of
cholesterol-dependent lipid order in biological membranes. In addition, our
results indicate that PLD is a particularly sensitive molecular target for
these order-disrupting effects, and that this may be relevant to the effects
of short-chain ceramides in many biological processes. PLD1 and PLD2 have been
implicated in a wide variety of signaling and vesicle transport events
(Exton, 1999), including
antigen-stimulated degranulation in RBL mast cells
(Brown et al., 1998
;
Way et al., 2000
;
Choi et al., 2002
), but the
lack of potent pharmacological agents and useful dominant-negative constructs
have made it difficult to define the roles for PLDs in many of these studies.
ARF-dependent PLD activation has been implicated in COPI-mediated coated
vesicle budding from Golgi cisternae
(Ktistakis et al., 1995
;
Chen et al., 1997
), and the
inhibition of membrane protein maturation in the Golgi by C6-cer
(Rosenwald and Pagano, 1993
)
may also reflect a role for ordered lipids in this process. Additionally, PA
has been shown to be an important co-factor for phosphatidylinositol
4-phosphate 5-kinase [PI(4)P5K]
in the synthesis of PIP2
(Honda et al., 1999
), and this
provides one example of a PLD-dependent signaling pathway that is potentially
relevant to many biological processes.
Cockcroft and colleagues have described evidence for a role for
ARF-dependent activation of PLD in the regulation of PIP2 synthesis
in stimulated RBL cell exocytosis (Way et
al., 2000) and ruffling
(O'Luanaigh et al., 2002
).
Consistent with these studies, our recent characterization of a mutant RBL
cell line that is defective in antigen-stimulated Cdc42 and Rac activation
(Field et al., 2000
) led us to
postulate that Rho-family-dependent synthesis of PIP2 is necessary
for sustained IP3 production and Ca2+ mobilization that
leads to exocytosis in these cells
(Hong-Geller et al., 2001
).
The model in Fig. 7 summarizes
this hypothesis, and indicates how activation of PLD could play an important
role in Ca2+ mobilization via stimulated PIP2 synthesis.
In this model, Syk tyrosine kinase-dependent phosphorylation of the adaptor
protein LAT leads to the activation of a guanine nucleotide exchange factor
(GEF) for Cdc42 that promotes GTP binding and leads to the production of PA
and PIP2. Consistent with this model, the mutant RBL cells
defective in Cdc42 activation are also defective in antigen-stimulated PA
production (Field et al.,
2000
).
|
Our fluorescence anisotropy measurements of lipid order in plasma membrane
vesicles from the RBL cells show that biologically active short-chain
ceramides decrease cholesterol-dependent lipid order in these membranes in
proportion to the amount of these ceramides added up to 25 mole percent, the
highest value tested (Fig. 2).
In these measurements, C6-cer is 4-5 times more effective than
C2-cer on a per mole basis, and this is consistent with the
fourfold greater hydrophobic partition coefficient expected for the
longer acyl chain of this ceramide
(Tanford, 1980
). We used FRET
between lipid raft components as a measure of the amount of lipid order in
intact cells, and we found that C2-cer and C6-cer
reduced FRET between Fc
RI and Thy-1 in proportion to their expected
membrane partition coefficient. Although Fc
RI does not fractionate with
lipid raft components in sucrose gradients in the absence of crosslinking
(Field et al., 1995
;
Field et al., 1997
), it has a
detectable association with lipid rafts in intact cells, indicated by its
co-redistribution with crosslinked Thy-1 (D.H., unpublished)
(Holowka and Baird, 2001
).
Similar FRET results for Alexa488-anti-GD1b as the donor probe and
Cy3-anti-Thy-1 as the acceptor, together with the lack of detectable FRET with
non-raft donors anti-transferrin receptor mAb or anti-CD43 mAb, support these
conclusions. For both the anisotropy and FRET measurements,
C2-dhcer has little or no effect on lipid order, and this
correlates with the lack of biological activity of this ceramide, despite its
very similar chemical structure to C2-cer
(Fig. 1).
The physical basis for this dramatic difference in effects on lipid order
and biological activity for C2-cer and C2-dhcer is not
fully understood, but it has been suggested
(Simon and Gear, 1998) that
more-favorable hydrogen bonding for C2-cer due to the allylic
double bond (Fig. 1) cause
formation of two rigid planes of H-bonded atoms that results in a bulkier,
more-rigid headgroup structure. This configuration would yield a cone-shaped
lipid that could disrupt the packing of saturated phospholipids and
cholesterol in a liquid-ordered bilayer. The propensity for these uncharged,
short-chain ceramides to flip rapidly from the outer to inner leaflet of the
plasma membrane is high (Bai and Pagano,
1997
), and this is probably important for their effects on enzymes
such as PLD1, which act at the inner leaflet. Thus, the unique physical
properties of C2-cer and C6-cer make them particularly
effective as membrane perturbants with substantial functional consequences. In
contrast to these, long-chain ceramides such as C16-cer are more
cylindrical in shape and should pack well into a liquid-ordered membrane, much
like sphingomyelin. Thus, long-chain ceramides naturally produced by the
action of sphingomyelinases would not be expected to cause the same functional
effects as short-chain ceramides in situations where perturbation of lipid
order is the basis for these effects.
As summarized in Fig. 6C,
the inhibitory effects of C2-cer on antigen-stimulated
Ca2+ mobilization and on in vitro PLD1 activity are not observed
with C2-dhcer or C16-cer, and thus conform to the
criteria for functional inhibition by membrane perturbation. For measurement
of Ca2+ responses, it is clear that the concentration of
C2-cer used (32 µM) to obtain 60-80% inhibition in the
presence or absence of extracellular Ca2+ does not cause
significant leakiness in the cells, as judged by the minimal increase in
indo-1 fluorescence observed upon addition of this ceramide
(Fig. 4A,D). Furthermore, the
rapid onset of changes in FRET (Fig.
3A) and the short incubation time with C2-cer prior to
antigen stimulation (Fig. 4A,D)
make it unlikely that the effects observed are due to a metabolic derivative
of this ceramide. Although we do not have a direct measure of the
incorporation of C2-cer, C2-dhcer and C16-cer
into the cells, all three exhibit significant effects on the Ca2+
response, and the previous study by Simon and Gear showed that radiolabeled
C2-cer and C2-dhcer both undergo efficient incorporation
into platelets at similar concentrations of ceramides as those used in our
experiments (Simon and Gear,
1998
). Furthermore, the transient increase in intracellular
Ca2+ that we observe upon addition of C16-cer
(Fig. 4C) and the small
potentiation of antigen-stimulated Ca2+ mobilization observed due
to this ceramide (Fig. 4C,E)
indicates that it is also incorporated significantly into the cells.
Our evidence for the involvement of PLD1 activation in the initiation of
antigen-stimulated Ca2+ mobilization is based on the differential
sensitivity of Ca2+ mobilization to inhibition by n-butanol but not
by t-butanol (Fig. 5), and on
its sensitivity to inhibition by ceramides with the same structural
discrimination as PLD1 activity in in vitro assays
(Fig. 6C). By contrast, PLD2
activity under similar assay conditions is less sensitive to inhibition by
C2-cer, and shows marked enhancement by C2-dhcer and
C16-cer (Fig. 6A).
This pattern is less consistent with that predicted for inhibition by
disruption of lipid order. However, it is possible that these ceramides have
multiple effects on the activity of PLD2 under these in vitro assay
conditions, such that the net effects include a component of inhibition that
is due to disruption of lipid order. Using a mixed-micelle assay system, it
has been found that PLD2 activity is effectively inhibited by
C2-cer (Singh et al.,
2001). However, they also found that C16-cer inhibits
PLD2 activity in this assay; it is unclear to what extent these effects depend
on the presence of detergents in their assay. Both PLD1 and PLD2 have been
implicated in antigen-stimulated degranulation in RBL cells
(Choi et al., 2002
), and PLD1
has been shown to be recruited to the plasma membrane due to antigen
stimulation (Brown et al.,
1998
), whereas PLD2 is constitutively localized there
(Choi et al., 2002
). Our
results are most consistent with a role for PLD1 in the initiation of
antigen-stimulated Ca2+ mobilization, but we cannot exclude a role
for PLD2 in this process as well. As depicted in
Fig. 7, we suggest that
generation of PA by activated PLD is involved in stimulated PIP2
synthesis, which provides a pool of this substrate for sustained production of
IP3 by PLC
, a requirement for stimulated exocytosis
(Beaven and Kassessinoff, 1997
;
Scharenburg and Kinet, 1998). Other roles for PA, such as a direct effect on
PLC
activity (Jones and Carpenter,
1993
), cannot be excluded by our results.
In the study by Nakamura et al., inhibition of antigen-stimulated
Ca2+ mobilization and degranulation by C2-cer was found
to correlate with inhibition of Ca2+ influx
(Nakamura et al., 1996). In a
separate study, Mathes et al. reported that C2-cer inhibits the
calcium release-activated calcium current (ICRAC) activated by
IP3 in patch-clamped RBL cells
(Mathes et al., 1998
). These
studies indicate that C2-cer can inhibit store-operated
Ca2+ influx, but it is unclear whether this effect of
C2-cer is due to disruption of lipid order and/or inhibition of
PLD. Comparing the effects of butanols, C2-dhcer and
C16-cer on IP3-activated ICRAC should help to
answer this question. Such comparisons may have general utility in providing
evidence for the involvement of PLD in diverse cellular processes.
Although PLD activity is frequently reported to be a target for inhibition
by short-chain ceramides, the mechanism for this effect remains incompletely
defined. Our results indicate that this inhibition correlates with disruption
of lipid order, and Powner et al. recently reported that PLD1 in stimulated
RBL cells is Triton X-100 insoluble, suggesting possible association with
lipid rafts (Powner et al.,
2002). In our in vitro assays, PLD1 activity is sensitive to
C2-cer in the absence of activators ARF1 and PKCßII,
indicating that activation per se is not the ceramide-sensitive step. By
analogy to the importance of membrane defects for the catalytic activity of
PLA2 (Burack and Biltonen,
1994
), we propose that PLD might also depend on membrane defects
for its catalytic activity. Boundary regions between more-fluid and less-fluid
membrane domains have been shown to provide defects for PLA2
(Burack et al., 1997
) and,
similarly, the interface between liquid-ordered and fluid regions of
biological membranes could provide such defects for PLD catalytic activity.
Furthermore, the study by Ge et al. indicates that ARF GTPases can contribute
to defect formation (Ge et al.,
2001
). In this context, short-chain ceramides would inhibit PLD
activity by reducing order and thereby reducing potential interfaces. Future
systematic studies with purified PLD isoforms and model membranes of
well-defined composition will be necessary to test this hypothesis. Regardless
of the molecular mechanism for these effects on PLD, our present results show
that long- and short-chain ceramides, by virtue of their differential effects
on membrane lipid order, can serve as useful probes for evaluating the role of
plasma membrane structure and heterogeneity in cellular signaling.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: Department of Pharmacology and The Institute for Chemical
Biology, Vanderbilt University Medical Center, 412 Preston Research Building,
Nashville, TN 37232-6600, USA
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References |
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