From the Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082
Received for publication, October 27, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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This study presents evidence that
phosphoinositide 3-kinase (PI3K) plays a concerted role with
phospholipase C Activation of mast cells by the cross-linking of the high affinity
receptor for IgE (Fc It is noteworthy that the proposed involvement of PI3K in the
regulation of a non-capacitative Ca2+ influx pathway is
reminiscent of our finding of a PI(3,4,5)P3-sensitive Ca2+ entry mechanism in platelets (19) and Jurkat T cells
(20). Consequently, we hypothesize that this
PI(3,4,5)P3-sensitive Ca2+ entry is a conserved
mechanism that plays a crucial role in the regulation of
receptor-mediated Ca2+ signaling among these hematopoietic
cells. This hypothesis is corroborated by the present data showing the
involvement of this PI(3,4,5)P3-mediated Ca2+
influx in Fc Materials--
D-myo-Inositol
1,3,4,5-tetrakisphosphate, potassium salt
(Ins(1,3,4,5)P4)
1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4,5-trisphosphate, protonated form (PI(3,4,5)P3),
1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4,5-trisphosphate, protonated form
(di-C8-PI(3,4,5)P3),
1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3,4-bisphosphate, protonated form (PI(3,4)P2),
1-O-(1,2-di-O-palmitoyl-sn-glycero- 3-O-phosphoryl)-D-myo-inositol
4,5-bisphosphate, protonated form (PI(4,5)P2), and
1-O-(1,2-di-O-palmitoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol 3-monophosphate, protonated form (PI(3)P) were synthesized as previously reported (21, 22). The identity and purity of all inositol
phosphates and inositol lipids were verified by 1H and
31P NMR and high resolution mass spectrometry. Published
data from this and other laboratories have shown that
PI(3,4,5)P3 and other inositol lipids are cell-permeant and
can readily fuse with cell membranes to exert cellular responses in
different types of cells, including platelets (19), NIH3T3 cells (23),
adipocytes (24), and Jurkat T cells (20). Fura-2 acetoxymethyl ester
(fura-2 AM), fluo-3 acetoxymethyl ester (fluo-3 AM), and 2-aminoethyoxy diphenylborate (2-APB) were purchased from Calbiochem. Leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and the antigen dinitrophenol-conjugated human serum albumin (DNP-HSA) were products from Sigma. [3H]Inositol was purchased from PerkinElmer
Life Sciences. DNP-specific monoclonal IgE was a kind gift from Dr.
Henry Metzger (Chemical Immunology Section, National Institute of
Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
Health). The construct expressing hemagglutinin (HA)-tagged Cell Culture--
RBL-2H3 cells (from ATCC) were maintained in
monolayer culture in 75-cm3 plastic tissue culture flasks
containing 15 ml of Eagle's minimum essential medium with 10% fetal
bovine serum and 0.1% gentamicin at 37 °C in the presence of 5%
CO2.
Fluorescence Spectrophotometric Measurement of
PI(3,4,5)P3-Induced [Ca2+]i
Response--
[Ca2+]i was monitored by change in
the fluorescence intensity of fura-2-loaded cells. RBL-2H3 cells
(1 × 107 cells/ml) were suspended in 1 ml of assay
buffer consisting of 4.3 mM
Na2HPO4, 24.3 mM
NaH2PO4, 4.3 mM
K2HPO4, 113 mM NaCl, 5 mM glucose, pH 7.4. fura-2 loading was achieved by exposing
the cells to 10 µM fura-2 AM in the presence of 0.5%
bovine serum albumin and 2 mM probenacid in the dark for
1 h at 37 °C. The cells were then pelleted by centrifugation at
1000 × g for 10 min, washed with assay buffer twice,
and resuspended at 8 × 105 cells/ml in the same
buffer containing 1 mM Ca2+. The effect of
various inositol lipids on [Ca2+]i was examined
by fura-2 fluorescence in a Hitachi F-2000 spectrofluorimeter at
37 °C with excitation and emission wavelengths at 340 and 510 nm,
respectively, as described in the literature (25, 26). The maximum
fura-2 fluorescence intensity (Fmax) in RBL-2H3
cells was determined by adding 4-bromo-A23187 (1 µM), and
the minimum fluorescence (Fmin) was determined
following depletion of external Ca2+ by 5 mM
EGTA. The [Ca2+]i was calculated according to the
equation [Ca2+]i = Kd(F Fluorescence Spectrophotometric Measurement of Antigen-induced
[Ca2+]i Response--
RBL-2H3 cells were
sensitized with 1 µg/ml mouse DNP-specific IgE overnight. Cells were
collected, suspended in assay buffer, loaded with fura-2, and activated
by the antigen DNP-HSA (1 µg/ml). Changes in
[Ca2+]i were monitored by fura-2 fluorimetry as
described above.
All the above fura-2 experiments were also repeated with fluo-3-loaded
cells in a similar manner. fluo-3 displays a lower Kd (864 nM at 37 °C) and a longer
excitation wavelength (505 nm) of the dye-Ca2+ complex
vis à vis fura-2 (28). Consistent results were
obtained with both fluorescence dyes.
Transient Transfection--
[3H]Inositol Phosphate Turnover Analysis--
The
examination of phosphoinositol turnover was carried out according to a
modification of the procedure described previously (20). In brief,
RBL-2H3 cells were incubated with
myo-[2-3H]inositol (10 µCi/106
cells/ml) in Eagle's minimum essential medium supplemented with 10%
fetal bovine serum. The cells were then washed with the medium, sensitized with DNP-specific IgE, and washed with 20 mM
Hepes, pH 7.4, containing 285 mM NaCl, 11 mM
KCl, 1.3 mM Na2HPO4, 1 mM KH2PO4, 8.3 mM
NaHCO3, 1.6 mM MgSO4, 2.2 mM MgCl2, 2.2 mM CaCl2, and 5.6 mM glucose. Aliquots containing 1 × 106 cells were each resuspended in 0.3 ml of the
aforementioned assay buffer and transferred to 1.5-ml microcentrifuge
tubes. Each sample was incubated with 0.3 µg of DNP-HSA for the
indicated times and quenched by adding 0.25 ml of 6% trichloroacetic
acid. The tubes were centrifuged for 2 min at 12,000 × g. The supernatant (200 µl) was analyzed by high pressure
liquid chromatography on a 5-µm Adsorbosphere Sax column (4.6 × 200 mm) equilibrated with H2O. The
[3H]inositol phosphates were eluted with a linear
gradient of 0-0.9 M
NH4H2PO4 in 60 min at a flow rate
of 1 ml/min. Fractions were collected every 1 ml, and their
radioactivity was measured by liquid scintillation. Synthetic
[3H]Ins(1,3,4,5)P4,
[3H]Ins(1,4,5)P3,
[3H]Ins(4,5)P2, and Ins(4)P were used as
standards. The respective retention times were 60, 48, 43, and 31 min.
Preparation of RBL-2H3 Cell Plasma Membrane Vesicles--
For
the Ca2+ release assay, the plasma membrane was purified as
previously described (20). In brief, RBL-2H3 cells (4 × 108 cells) were washed with phosphate-buffered saline and
suspended in 5 ml of PM buffer consisting of 20 mM Hepes,
pH 7.2, 110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 5 mM
KH2PO4, 1 mM dithiothreitol, 1 mM EGTA, 1 mM AEBSF, and 20 µg/ml leupeptin.
The cell suspension was homogenized in a Dounce homogenizer using a
loose pestle with five strokes up and down. The homogenate was
centrifuged at 1500 × g for 10 min. The pellet was
suspended in 3.125 ml of PM buffer and mixed with 5.5 ml of 69% (w/w)
sucrose to make a final 44% (w/w) sucrose-membrane mixture. The
mixture was overlaid with 42.3% (w/w) sucrose, and the two-phase
suspension was subjected to centrifugation at 90,000 × g for 2 h in a swinging bucket rotor. The membrane
material at the interface of the phases contained the greatest
enrichment in plasma membranes based on the activity of
(Na+-K+)-ATPase. This fraction was collected,
suspended in 5 ml of 10 mM Hepes, pH 7.5, containing 1 mM dithiothreitol, 1 mM AEBSF, and 20 µg/ml
leupeptin, and centrifuged at 25,000 × g for 10 min. The pellet was suspended in 1 ml of the same buffer.
PI(3,4,5)P3 Induces [Ca2+]i
Increase in RBL-2H3 Mast cells by Stimulating Ca2+
Influx--
To investigate the involvement of PI3K in
antigen-stimulated Ca2+ response in mast cells, we first
examined the effect of exogenous PI(3,4,5)P3 on
[Ca2+]i in RBL-2H3 cells. Exposure of
fura-2-loaded RBL-2H3 cells to PI(3,4,5)P3, ranging from 1 to 20 µM, elicited an instantaneous [Ca2+]i increase in a dose-dependent
manner (Fig. 1A, upper panel).
This PI(3,4,5)P3 effect became saturated at 20 µM, beyond which no significant enhancement in the
amplitude of Ca2+ response was noted (data not shown). The
PI(3,4,5)P3-induced [Ca2+]i rise was
largely attributable to Ca2+ influx, because the increase
could be abolished by Ca2+ depletion with 5 mM
EGTA (Fig. 1A, lower panel). With regard to other
phosphoinositides examined, PI(3,4)P2 at high doses (20 µM) could also elicit [Ca2+]i
increase but to a much lesser extent than PI(3,4,5)P3 (Fig.
1B, upper panel), however. The potency was
~10% of that of PI(3,4,5)P3. On the other hand,
PI(4,5)P2 and PI(3)P did not show any appreciable effect on
[Ca2+]i (lower panel). These data
suggest the presence of a PI(3,4,5)P3-sensitive
Ca2+ influx mechanism in RBL-2H3 mast cells.
PI(3,4,5)P3 Does Not Disturb Internal Ca2+
Stores in RBL-2H3 Cells--
Data from several groups suggest that
PI(3,4,5)P3 might stimulate Ins(1,4,5)P3
production by activating PLC
Furthermore, we obtained evidence that thapsigargin-sensitive
Ca2+ pools were not disturbed by PI(3,4,5)P3.
RBL-2H3 cells were exposed to 20 µM
PI(3,4,5)P3 in a Ca2+-depleted medium, followed
by 1 µM thapsigargin. As shown in Fig. 2, PI(3,4,5)P3 did not affect
the extent of thapsigargin-induced Ca2+ response vis
à vis that without PI(3,4,5)P3 pretreatment.
Together, these results bore out the premise that
PI(3,4,5)P3-induced Ca2+ response was
independent of PLC activity and was solely attributable to
Ca2+ influx from the medium.
Role of PI3K in Fc
First, the effect of the PI3K inhibitor LY294002 on antigen-stimulated
Ca2+ response in fura-2-loaded RBL-2H3 cells was assessed.
In this study, LY294002 was used in lieu of wortmannin to inhibit PI3K in cells in light of the nonspecific effect of wortmannin on myosin light-chain kinase, one of the key regulatory enzymes in mast cells
(8). RBL-2H3 cells were sensitized with DNP-specific IgE overnight,
loaded with the fluorescence indicator, and pretreated with varying
concentrations of LY294002. After stimulation with the antigen DNP-HSA
(1 µg/ml), changes in [Ca2+]i were monitored by
fluorimetry. In line with the earlier reports that the
antigen-stimulated Ca2+ response was largely attributable
to Ca2+ influx (11, 31), depletion of external
Ca2+ by EGTA substantially diminished the Ca2+
signal following antigen stimulation (Fig.
3A, inset).
Fig. 3 indicates that LY294002 pretreatment attenuated the amplitude of
antigen-induced Ca2+ response in a
dose-dependent manner. The effect of LY294002 on the
Ca2+ response attained maximum at 200 µM,
beyond which no further decrease was noticed (data not shown).
Presumably, the residual Ca2+ response following LY294002
treatment was attributed to Ins(1,4,5)P3-induced Ca2+ release and the consequent capacitative
Ca2+ entry. This premise was corroborated by the
concomitant treatment of IgE-sensitized RBL-2H3 cells with LY294002
(200 µM) and 2-APB (40 µM) (Fig.
3B, trace c). For 2-APB (40 µM)
alone, a 55% reduction in the Ca2+ response was noted
(trace b), which was due to the inhibition of
Ins(1,4,5)P3-induced Ca2+ release and the
accompanied capacitative Ca2+ entry.
Second, an independent biochemical approach was taken to confirm the
above results, in which RBL-2H3 cells were transiently transfected with
a vector expressing HA epitope-tagged
As shown, the level of
Moreover, the extent of inhibition on the Ca2+ response
displayed a direct correlation with that of the antigen-stimulated
release of Ins(1,3,4,5)P4 Is Not Physiologically
Relevant in Antigen-induced Ca2+ Response in RBL-2H3
Cells--
In cells, the 3-phosphorylation of Ins(1,4,5)P3
by Ins(1,4,5)P3-specific kinase accounts for a major
pathway for the formation of Ins(1,3,4,5)P4 (33).
Consequently, Ins(1,4,5)P3 accumulation in response to
antigen stimulation in RBL-2H3 cells was accompanied by an increase in
Ins(1,3,4,5)P4 (Fig.
5A, left panel).
Structurally, Ins(1,3,4,5)P4 represents the head group of
PI(3,4,5)P3, and displays in vitro
cross-reactivity with PI(3,4,5)P3 in many aspects of biochemical functions (19, 20). Moreover, Ins(1,3,4,5)P4
has been implicated as a second messenger in facilitating
Ca2+ entry into non-excitable cells (34, 35). Consequently,
a crucial issue that warranted investigation was whether
Ins(1,3,4,5)P4 played a role in the regulation of
Fc
In this study, RBL-2H3 cells were pretreated with the
Ins(1,4,5)P3 3-kinase inhibitor adriamycin (10 µM) (25), which completely blocked
Ins(1,3,4,5)P4 synthesis while not interfering with
Ins(1,4,5)P3 formation (Fig. 5B, left
panel). It is noteworthy that the complete inhibition of
Ins(1,3,4,5)P4 production had no appreciable effect on
antigen-induced Ca2+ response vis à vis
the control (Fig. 5, A and B, right
panels). Together, these data argue against any second-messenger
role of Ins(1,3,4,5)P4 in regulating receptor-mediated
Ca2+ entry in RBL-2H3 mast cells.
PI(3,4,5)P3 Directly Elicits Ca2+ Efflux
from Ca2+-loaded Plasma Membrane Vesicles--
The above
observations also prompted a question of how PI(3,4,5)P3
facilitated Ca2+ entry across plasma membranes. In
principle, PI(3,4,5)P3 could directly sensitize a
Ca2+-entry mechanism or it might mediate the action through
the stimulation of other signaling components. For example, data from
several laboratories suggest the involvement of Btk in
PI(3,4,5)P3-regulated Ca2+ entry in platelets
and B cells (36, 37). This premise is consistent with the notion that
Fc
To address whether PI(3,4,5)P3-mediated Ca2+
entry required any cytoplasmic component such as Btk, we examined the
Ca2+-transporting activity of PI(3,4,5)P3 in
plasma membrane vesicles prepared from RBL-2H3 cells. Western blot
analysis confirmed that this plasma membrane preparation was devoid of
Btk (Fig. 6A).
The inside-out plasma membrane of RBL-2H3 cells displayed
ATP-dependent Ca2+ sequestering activity. These
membrane vesicles were loaded with Ca2+ by exposing them to
ATP in the presence of thapsigargin (1 µM) and oligomycin
(5 µg/ml). The Ca2+-loaded membrane vesicles were washed
and analyzed for Ca2+ release induced by
PI(3,4,5)P3, which was equivalent to Ca2+
influx in intact cells. Fura-2 fluorescence indicates that addition of
di-C8-PI(3,4,5)P3 (20 µM) caused
immediate Ca2+ release followed by slow re-uptake, which
was presumably due to small quantities of residual ATP in the milieu
(Fig. 6B). This finding suggests that
PI(3,4,5)P3 might activate a Ca2+ entry
mechanism on plasma membranes independent of cytoplasmic components. It
is noteworthy that the Ca2+ release was not augmented by
the subsequent challenge with PI(3,4,5)P3, which is in line
with our observation found in the membrane vesicles prepared from
Jurkat T cells (19). The lack of Ca2+ response was likely
due to desensitization or saturation of the binding site instead of the
depletion of Ca2+, since the addition of 1 µM
4-bromo-A23187 following PI(3,4,5)P3 treatment triggered
the release of large amounts of Ca2+ (Fig. 6B,
inset).
This study presents pharmacological and biochemical evidence that
PI3K plays a concerted role with PLC Ca2+ signaling in response to Fc in initiating antigen-mediated
Ca2+ signaling in mast cells via a
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-sensitive Ca2+ entry pathway.
Exogenous PI(3,4,5)P3 at concentrations close to its
physiological level induces instantaneous Ca2+ influx into
RBL-2H3 cells. This PI(3,4,5)P3-induced intracellular Ca2+ increase is independent of phospholipase C activity or
the depletion of internal stores. Moreover, inhibition of PI3K by
LY294002 or by overexpression of the dominant negative inhibitor
p85
suppresses the Ca2+ response to the cross-linking of the
high affinity receptor for IgE (Fc
RI). Concomitant treatment of
RBL-2H3 cells with LY294002 or
p85 and 2-aminoethyl diphenylborate,
a cell-permeant antagonist of D-myo-inositol
1,4,5-trisphosphate receptors, abrogates antigen-induced Ca2+ signals, whereas either treatment alone gives rise to
partial inhibition. Conceivably, PI(3,4,5)P3-sensitive
Ca2+ entry and capacitative Ca2+ entry
represent major Ca2+ influx pathways that sustain elevated
[Ca2+]i to achieve optimal physiological
responses. This study also refutes the second messenger role of
D-myo-inositol 1,3,4,5-tetrakisphosphate in
regulating Fc
RI-mediated Ca2+ response. Considering the
underlying mechanism, our data suggest that PI(3,4,5)P3
directly stimulates a Ca2+ transport system in plasma
membranes. Together, these data provide a molecular basis to account
for the role of PI3K in the regulation of Fc
RI-mediated
degranulation in mast cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI)1
leads to the secretion and generation of an array of mediators that
induce immediate allergic inflammation (for review, see Ref. 1).
Although the Fc
RI-mediated signaling cascade has been characterized,
the regulatory mechanism governing mast cell degranulation is only
partially understood. Fc
RI is a heterotrimeric protein complex
(
2) that contains immunoreceptor tyrosine-based
activation motifs (ITAMs) in both the
and
subunit cytoplasmic
domains (2). The protein-tyrosine kinase (PTK) Lyn is associated
with the
subunit at the resting state (3), and its action is
promoted by Fc
RI cross-linking (4). Lyn phosphorylates ITAMs of the
and
subunits, resulting in the recruitment of Lyn and Syk, respectively, through Src homology-2 (SH2) domain-mediated interactions with phosphotyrosine residues (5, 6). Activation of these newly
recruited PTKs, in turn, facilitates the translocation and phosphorylation of multiple substrates, including phospholipase C
(PLC
) isozymes and phosphoinositide 3-kinase (PI3K) (ref review, see
Ref. 1). The activated PLC
hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to
D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol, which induce the release
of Ca2+ from intracellular stores and the activation of
protein kinase C, respectively. On the other hand, stimulation of
PI3K results in transient accumulation of micromolar levels of
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and
phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Although
the involvement of PI3K in antigen-induced degranulation is established
(7-11), a clear consensus regarding how lipid products derived from
the action of PI3K mediate the cellular responses has yet to emerge.
Putative downstream targets for PI(3,4,5)P3 and
PI(3,4)P2 include SH2- or pleckstrin homology (PH)
domain-containing signaling enzymes such as PLC-
, Akt, and Bruton's
tyrosine kinase (Btk) (12, 13), which transduce the signal to the
respective signaling pathways. In addition, data from several
laboratories suggest a role of PI3K in the regulation of intracellular
Ca2+ ([Ca2+]i) increase in mast cells
(9, 11, 14). Evidence suggests that PI3K may mediate
[Ca2+]i elevation in mast cells via two distinct
mechanisms. First, in vitro data indicate that
PI(3,4,5)P3 stimulates Ins(1,4,5)P3 production
by activating PLC
isozymes (15, 16). This PLC
activation may be
attained directly by facilitating membrane translocation (15, 16) or
indirectly via Btk (14, 17). Second, PI3K may increase
[Ca2+]i by facilitating Ca2+
mobilization across plasma membranes (11). This premise was prompted by
data showing the inhibitory effect of PI3K inhibitors on
antigen-induced Ca2+ response (9, 11, 14), and is in line
with the notion that two distinct Ca2+ influx pathways
(capacitative versus non-capacitative) are operative in
antigen-stimulated RBL-2H3 cells (18).
RI-mediated Ca2+ response in RBL-2H3 mast
cells. Considering the crucial role of Ca2+ influx in the
secretion of inflammatory mediators, this mechanism provides a
molecular basis whereby PI3K regulates mast cell function. Moreover,
given that PI3K and PLC
are the downstream effectors of the
Fc
RI-mediated tyrosine kinase cascade, we propose that these two
enzymes act in a concerted manner to initiate Ca2+ response
to antigen stimulation. In stimulated cells, PI3K and PLC
act on the
mutual substrate PI(4,5)P2 to generate
PI(3,4,5)P3 and Ins(1,4,5)P3, respectively,
which activate Ca2+ channels at different cellular
compartments to provide the elevated [Ca2+]i
required for optimal physiological responses.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85
(HA·
p85) was provided by Professor Alex Toker (Harvard Medical
School). Monoclonal anti-HA and rabbit anti-Btk antibodies were
purchased from Roche Molecular Biochemicals and BD Biosciences, respectively.
Fmin)/(Fmax
F), where Kd denotes the apparent
dissociation constant (Kd = 224 nM) of
the fluorescence dye-Ca2+ complex (27).
p85 is a deletion mutant that
lacks a region required for tight association with p110 but is still
able to bind to appropriate phosphotyrosine targets. Thus,
p85 can
compete with native p85 for binding to essential signaling proteins and
behaves as a dominant negative mutant. Transient transfection of
HA·
p85 was carried out using the LipofectAMINE Plus reagent
according to the protocol supplied by the manufacturer. In brief,
Opti-MEM medium (1.5 ml) containing the indicated amount of the
HA·
p85 expression vector (3-12 µg) was incubated with 120 µl
of the Plus reagent at 25 °C for 15 min, and 12 µl of the
LipofectAMINE reagent in 1.5 ml of Opti-MEM was added. In parallel, an
empty pCMV/blue plasmid (12 µg) was subjected to the same treatment
as a negative control. The mixture was incubated at 25 °C for 15 min
and added to 9 ml of serum-free Opti-MEM (Life Technologies). RBL-2H3
cells in a T-75 flask were washed with serum-free Opti-MEM and were
added to the aforementioned transfection medium. After 3 h at
37 °C, the transfection media were replaced with 15 ml of Eagle's
minimum essential medium containing 10% fetal bovine serum. The
transfected cells were allowed to grow for 2 days to express foreign
DNA. The collected cells were analyzed for antigen-induced
Ca2+ response by fluorescence spectrometry and for
HA·
p85 expression by Western blot analysis using anti-HA antibody.
-Hexosaminidase Secretion--
The release of mast cell
mediators by exocytosis was monitored by the
-hexosaminidase assay.
RBL-2H3 cells were grown in 12-well plates and passively sensitized
with DNP-specific IgE. The IgE-sensitized cells were washed twice with
Tyrode's buffer consisting of 10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM
CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% bovine serum albumin. Secretion was
initiated by exposing cells to the antigen DNP-HSA (1 µg/ml). After
1 h, the reaction was terminated by placing the plate on ice. The
enzyme activities of
-hexosaminidase in 50 µl of supernatants and
attached cells solubilized with 0.5% Triton X-100 were measured with
200 µl of 1 mM p-nitrophenyl
N-acetyl-
-D-glucosaminide in 0.1 M sodium citrate, pH 4.5, at 37 °C for 1 h. The
reaction was stopped by the addition of 500 µl of 0.1 M
NaHCO3. The release of the product p-nitrophenol
was measured by monitoring the absorbance at 400 nm. The percentage of
degranulation was calculated by dividing the absorbance of the
supernatant over the combined absorbance of the supernatant and cell lysate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, upper panel, time course
and dose dependence of the effect of PI(3,4,5)P3 on
[Ca2+]i in RBL-2H3 cells; lower panel,
PI(3,4,5)P3-induced [Ca2+]i increase
was abrogated in a Ca2+-depleted medium, indicating the
increase was due to Ca2+ influx. External Ca2+
was depleted by treating medium with 5 mM EGTA for 10 min.
B, phosphoinositide specificity in eliciting
[Ca2+]i increase in RBL-2H3 cells. Upper
panel, PI(3,4)P2; lower panel,
PI(4,5)P2 and PI(3)P. [Ca2+]i was
analyzed by fura-2 fluorimetry as described under "Experimental
Procedures." The arrows indicate the time of addition of
individual compounds. Traces are representative of three independent
experiments. Consistent results were obtained when another fluorescence
dye fluo-3 was used in the above experiments.
isozymes via discrete mechanisms (9,
11, 14). This raised a concern that PI(3,4,5)P3-induced
[Ca2+]i increase might, in part, be due to
Ca2+ release from endoplasmic reticulum stores. To refute
this possibility, we first examined the effect of two distinct
pharmacological inhibitors that blocked
Ins(1,4,5)P3-induced Ca2+ mobilization. These
included the PLC inhibitor U73122 (10 µM) and the
cell-permeant antagonist of Ins(1,4,5)P3 receptors 2-APB (40 µM) (29, 30). Although these inhibitors were
effective in attenuating Fc
RI-mediated Ca2+ response in
RBL-2H3 cells (Figs. 3 and 4), they did not display significant
effect on PI(3,4,5)P3-induced [Ca2+]i
response. The extents of the [Ca2+]i increase
elicited by 20 µM PI(3,4,5)P3 in RBL-2H3 cells pretreated with U73122 and 2-APB were 95 ± 2%
(n = 3) and 98 ± 2% (n = 3),
respectively, of that of the untreated control.
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Fig. 2.
PI(3,4,5)P3 does not affect
thapsigargin (TG)-sensitive internal Ca2+
pools. In the presence of 5 mM EGTA, fura-2-loaded
RBL-2H3 cells were treated with, in tandem, 20 µM
PI(3,4,5)P3 and 1 µM thapsigargin.
Inset, in the presence of 5 mM EGTA,
fura-2-loaded cells were treated with 1 µM thapsigargin
alone. The arrows indicate the time of addition of
individual compounds. Traces are representative of three independent
experiments.
RI-induced Ca2+ Response in
RBL-2H3 Cells--
The existence of a
PI(3,4,5)P3-sensitive Ca2+ entry mechanism
suggests a link between PI3K and receptor-activated Ca2+
signaling in RBL-2H3 cells. Because both PI3K and PLC
are downstream effectors in Fc
RI-mediated tyrosine kinase cascades, we hypothesized that PI3K acted in concert with PLC
in initiating the
Ca2+ response to Fc
RI cross-linking. To test this
hypothesis, a combination of pharmacological and molecular genetic
approaches was employed to characterize the role of PI3K in
Fc
RI-induced Ca2+ response.
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Fig. 3.
A, inhibitory effect of LY294002 on
Fc RI-mediated Ca2+ response in RBL-2H3 cells. Cells were
sensitized with DNP-specific IgE, loaded with fura-2, and activated
with the antigen DNP-HSA (Ag). Traces a-d
represent Ca2+ responses in the presence of 0, 40, 100, and
200 µM LY294002, respectively. The maximum inhibitory
effect was attained at 200 µM. The inset
depicts the effect of Ca2+ depletion on FceRI-mediated
Ca2+ response. Traces a and b denote
the Ca2+ responses of the control and in the presence of 5 mM EGTA, respectively. B, inhibitory effect of
2-APB (40 µM) alone (trace b) and of the
combination of 2-APB (40 µM) and LY294002 (200 µM) (trace c). Trace a depicts the
Ca2+ response of the control. [Ca2+]i
was analyzed as described under "Experimental Procedures."
Traces are representative of three independent experiments.
Consistent results were obtained when another fluorescence dye fluo-3
was used in the above experiments.
p85 (HA·
p85). It is well
documented that deletion of the binding motif for the catalytic p110
subunit of p85 confers PI3K dominant negative activity (32). We have
applied this dominant negative inhibitor in Jurkat T cells as part of
our effort to demonstrate the role of PI3K in T cell receptor-mediated
Ca2+ signaling (20). Western analysis using anti-HA
antibody verified the expression of HA·
p85 in transiently
transfected RBL-2H3 cells (Fig.
4A).
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Fig. 4.
Overexpression of
p85 inhibits Fc
RI-mediated
Ca2+ response and secretion in RBL-2H3 cells.
A, expression levels of HA·
p85 in RBL-2H3 cells that
had been transfected with 1 µg/ml of a control pCMV/blue plasmid
(a) or with increasing amounts of the HA·
p85-expressing
plasmid (b, 0.25 µg/ml; c, 0.5 µg/ml;
d, 0.75 µg/ml; e, 1 µg/ml) for 2 days. The
Western analysis was carried out using antibodies against the HA tag.
The loading amounts of individual samples were calibrated in reference
to actin as internal standard (data not shown). Data are representative
of three independent experiments. B,
p85 inhibited
Fc
RI-mediated Ca2+ response in a
dose-dependent manner. C,
p85 inhibited
Fc
RI-mediated secretion of
-hexosaminidase in a
dose-dependent manner. Cells expressing different levels of
p85 (a-e, as indicated above) were collected after
overnight sensitization with DNP-specific IgE. The collected cells were
loaded with fura-2 and stimulated with DNP-HSA (Ag) to test
for Ca2+ response and
-hexosaminidase secretion
(a-e in accordance with the above designations). In
addition, cells expressing the highest level of
p85 were treated
with 2-APB (40 µM) for 10 min before antigen stimulation.
As shown, the concerted action of
p85 and 2-APB abrogated the
Ca2+ signal (trace f in B) and
-hexosaminidase secretion (vertical bar f in
C). Traces are representative of three
independent experiments. Vertical bars shown are means ± S.D. (n = 3). Consistent results were obtained when
another fluorescence dye fluo-3 was used in the above
experiments.
p85 expression displayed a direct correlation
with the amount of cDNA used in the transient transfection. Accordingly, transfected RBL-2H3 cells expressing varying levels of
HA·
p85 were tested for Ca2+ response to Fc
RI
cross-linking. As shown, the expression of
p85 attenuated the
Ca2+ signal, ranging from 10 to 50%, in a
dose-dependent manner (Fig. 4B, traces
a-e). In line with the data shown in Fig. 3B, the
antigen-induced Ca2+ response was nearly abrogated when the
cells expressing the highest level of HA·
p85 were treated with
2-APB (40 µM) (trace f).
-hexosaminidase (Fig. 4C). This observation
reaffirms the close relationship among PI3K activity,
[Ca2+]i increase, and mast cell degranulation.
RI-mediated Ca2+ response in RBL-2H3 cells.
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Fig. 5.
Evidence that Ins(1,3,4,5)P4 is
not involved in Fc RI-mediated Ca2+
response in RBL-2H3 cells. A, left panel,
kinetics of [3H]Ins(1,4,5)P3 and
[3H]Ins(1,3,4,5)P4 production in
[3H]inositol-labeled RBL-2H3 cells in response to Fc
RI
cross-linking as described under "Experimental Procedures." Results
are given as means of three independent experiments. A,
right panel, Fc
RI-mediated Ca2+ response in
RBL-2H3 cells. Cells were sensitized with DNP-specific IgE, loaded with
fura-2, and activated with the antigen DNP-HSA as described under
"Experimental Procedures." B, left panel,
effect of adriamycin on kinetics of
[3H]Ins(1,4,5)P3 and
[3H]Ins(1,3,4,5)P4 production in
[3H]inositol-labeled RBL-2H3 cells in response to Fc
RI
cross-linking. [3H]Inositol-labeled RBL-2H3 cells were
pretreated with 10 µM adriamycin for 1 h before
Fc
RI cross-linking. Results are given as means of three independent
experiments. As shown, the Ins(1,4,5)P3 3-kinase inhibitor
completely blocked the formation of Ins(1,3,4,5)P4 without
affecting Ins(1,4,5)P3 production. B,
right panel, effect of adriamycin (10 µM) on
Fc
RI-mediated Ca2+ response in RBL-2H3 cells. No
appreciable difference was noted in the Ca2+ response
between the control and adriamycin-treated cells, even though the
inhibitor completely inhibited Ins(1,3,4,5)P4 synthesis.
Traces are representative of three independent
experiments.
RI-induced Ca2+ influx was impaired in Btk-deficient
mast cells (38). Btk, a member of the Tec non-receptor PTK family,
contains a pleckstrin (PH) domain that selectively binds
PI(3,4,5)P3 in vitro (39, 40). Consequently,
PI(3,4,5)P3 promotes the translocation and subsequent
phosphorylation of Btk by Src family kinases (41). Once activated, Btk
can phosphorylate and activate a number of protein substrates such as
PLC
(42, 43) and c-Jun N-terminal kinases (44). However, the
mechanism by which Btk regulates receptor-mediated
[Ca2+]i increase remains enigmatic, because it
has been reported to be independent of increased PLC
activity (36,
37).
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Fig. 6.
Evidence that PI(3,4,5)P3-induced
Ca2+ response does not require any cytoplasmic component
such as Btk. A, Western blot analysis of Btk in the plasma
membrane vesicle and cell lysate of RBL-2H3 cells. No appreciable
amounts of Btk were detectable in the membrane vesicles. The blots are
representative of three independent experiments. B,
Ca2+ release from plasma membrane vesicles by
di-C8-PI(3,4,5)P3 (20 µM).
RBL-2H3 cell plasma membranes were prepared as described under
"Material and Methods" and were treated with 30 µM
Mg2+-ATP, 1 mM CaCl2, 1 µM thapsigargin, and 2.5 µg/ml oligomycin on ice for 10 min. The membrane vesicles were washed with 10 mM Hepes, pH
7.0, four times, and suspended in the same buffer. The assay medium
consisted of 0.2-0.25 mg of membrane proteins and 1 µM
fura-2 in 2 ml of 10 mM Hepes, pH 7.0. After the external
Ca2+ concentration returned to a near-base level, the
membrane vesicles were stimulated with 20 µM
di-C8-PI(3,4,5)P3 as indicated. The
inset indicates the Ca2+ response following the
sequential additions of 25 µM PI(3,4,5)P3 and
1 µM 4-bromo-A23187. Traces are representative
of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in the regulation of
Ca2+ influx in RBL-2H3 mast cells via a
PI(3,4,5)P3-sensitive Ca2+ entry pathway.
First, exogenous PI(3,4,5)P3 at concentrations close to its
physiological levels induces instantaneous Ca2+ influx into
RBL-2H3 cells. Second, inhibition of PI3K by LY294002 or by
overexpression of the dominant negative inhibitor
p85 suppressed Fc
RI-mediated Ca2+ response. Third,
PI(3,4,5)P3 was capable of stimulating Ca2+
efflux from Ca2+-loaded plasma membrane vesicles,
suggesting that PI(3,4,5)P3 interacts with a
Ca2+-entry system on plasma membranes independent of
cytoplasmic components. This unique Ca2+ influx mechanism
has also been demonstrated in platelets (19) and Jurkat T cells (20) in
our laboratory. In addition, a recent report indicates that the
Ca2+ influx induced by the cross-linking of IgG-receptor
(Fc
R) in human neutrophils was inhibited by PI3K inhibitors (45).
Together, these data underscore a novel function of PI3K in the
regulation of receptor-mediated Ca2+ responses in these
hematopoietic cells. This premise is in line with the notion that
receptor-stimulated tyrosine kinase cascades that lead to the
translocation and activation of PI3K of PLC
bear a high degree of
similarity in the signaling mechanism among these cell types.
RI cross-linking is
essential to the antigen-induced secretion of inflammatory mediators in RBL-2H3 cells (31, 46), and therefore is one of the key early events in
mast-cell signal transduction. The present data support our hypothesis
that PI3K and PLC
play a concerted role in initiating antigen-induced Ca2+ signaling (Fig.
7).
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Fig. 7.
A working hypothesis to account for the
concerted action of PLC and PI3K in initiating
Ca2+ response following Fc
RI
cross-linking in mast cells.
PI3K and PLC act on the mutual substrate PI(4,5)P2 to
generate two important second messengers, PI(3,4,5)P3 and
Ins(1,4,5)P3, respectively, that initiate the
Ca2+ response to Fc
RI cross-linking by activating
Ca2+ transport systems in different cell compartments. It
is well understood that the major part of Fc
RI-mediated
Ca2+ signals in mast cells is attributable to
Ca2+ influx. According to our hypothesis, this
Ca2+ influx is regulated by two discrete Ca2+
entry mechanisms on plasma membranes: 1) the capacitative
Ca2+ entry that is secondary to the depletion of the
intracellular Ca2+ store by Ins(1,4,5)P3, and
2) the PI(3,4,5)P3-sensitive Ca2+ entry that is
independent of the filling state of internal Ca2+ stores.
Considering the temporal relationship between these two mechanisms,
PI(3,4,5)P3-senitive Ca2+ entry should precede
capacitative Ca2+ entry, because it is independent of
signals from store depletion. Consequently, the concerted action of
these two discrete pathways allows the cells to sustain elevated
[Ca2+]i to achieve optimal physiological
responses in mast cells as well as other types of hematopoietic cells.
In the literature, Ins(1,3,4,5)P4, an immediate metabolite of Ins(1,4,5)P3, has been implicated in mediating Ca2+ entry in many non-excitable cells, including sea urchin eggs (34) and Xenopus oocytes (35). It was found that microinjection of Ins(1,3,4,5)P4 into the whole cells caused [Ca2+]i increase through a mechanism dependent on external Ca2+. In addition, several research groups have isolated an Ins(1,3,4,5)P4-binding protein, GAP1IP4BP, from platelet plasma membranes (47-50). However, the present study refutes the second messenger role of Ins(1,3,4,5)P4 in regulating FceRI-mediated Ca2+ inflow in mast cells. This finding is consistent with earlier data in mouse lacrimal acinar cells (51) and Jurkat T cells (20, 25) that have ruled out the involvement of Ins(1,3,4,5)P4 in receptor-activated Ca2+ influx. Considering the largely shared structural motifs, it is plausible that the microinjected Ins(1,3,4,5)P4 in sea urchin eggs and Xenopus oocytes might mimic PI(3,4,5)P3 to activate the PI(3,4,5)P3-sensitive Ca2+ entry mechanism, resulting in increased [Ca2+]i.
The involvement of PI3K and Btk in receptor-dependent
Ca2+ signals in various hematopoietic cells has been the
subject of many recent investigations (36-38). However, the link
between Btk and PI(3,4,5)P3-sensitive Ca2+
entry remains inconclusive. Although Btk is an essential component for
the activating phosphorylation of PLC-2 (43), a recent study by
Watson and colleagues indicates that the Ca2+ entry pathway
mediated by PI(3,4,5)P3 and Btk in platelets and megakaryotes was independent of increased PLC activity (36). Moreover,
data from Kinet and co-workers (37) suggest that Btk exerted its
regulation on receptor-mediated Ca2+ signals by protecting
PI(3,4,5)P3 from degradation by endogenous SH-2 containing
inositol phosphatase. Therefore, Btk may play an auxiliary role by
binding PI(3,4,5)P3 via its PH domain, thereby enhancing
PI(3,4,5)P3 accumulation. This PI(3,4,5)P3
sequestration hypothesis is consistent with our observation that
PI(3,4,5)P3 could directly stimulate Ca2+
transport in plasma membrane vesicles in the absence of Btk or any
other cytoplasmic component. Nevertheless, the present data could not
rule out the possibility that Btk might play the role as a regulator of
the Ca2+ channel activity, which is under investigation.
In summary, this PI(3,4,5)P3-sensitive Ca2+
entry pathway not only provides molecular insights into the role of
PI3K in the regulation of mast cell degranulation but also represents a
potential target for the modulation of cell function in mast cells.
However, questions remain outstanding regarding the regulatory
mechanism underlying PI(3,4,5)P3-mediated Ca2+
entry and its relationship with Ca2+-release activated
Ca2+ channels located on plasma membranes. Studies are
currently under way in this laboratory to further understand these issues.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Henry Metzger
(NIAMS/National Institutes of Health) for providing DNP-specific IgE
and helpful comments on the manuscript, and to Dr. Alex Toker (Harvard
Medical School) for the construct expressing HA-tagged p85.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM53448.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: College of
Pharmacy, The Ohio State University, 500 West 12th Ave., Columbus, OH
43210-1291. Tel.: 614-688-4008; Fax: 614-688-8556; E-mail: chen@dendrite.pharmacy.ohio-state.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009851200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FcRI, the high
affinity receptor for IgE;
PI3K, phosphoinositide 3-kinase;
PLC, phospholipase C;
PI(3, 4,5)P3, phosphatidylinositol
3,4,5-trisphosphate;
PI(3, 4)P2, phosphatidylinositol
3,4-bisphosphate;
PI(4, 5)P2, phosphatidylinositol
4,5-bisphosphate;
PI(3)P, phosphatidylinositol 3-monophosphate;
Ins(1, 4,5)P3, D-myo-inositol
1,4,5-trisphosphate;
Ins(1, 3,4,5)P4,
D- myo-inositol
1,3,4,5-tetrakisphosphate;
ITAM, immunoreceptor
tyrosine-based activation motif;
PTK, protein-tyrosine
kinase;
SH2, Src homology-2;
Btk, Bruton's tyrosine kinase;
DNP-HSA, dinitrophenol-conjugated human serum albumin;
2-APB, 2-aminoethoxy
diphenylborate;
HA·
p85, hemagglutinin epitope-tagged
p85;
PH, pleckstrin homology;
fura-2 AM, fura-2 acetoxymethyl ester;
fluo-3
AM, fluo-3 acetoxymethyl ester;
CMV, cytomegalovirus;
di-C8-PI(3, 4,5)P3,
1-O-(1,2-di-O-octanoyl-sn-glycero-3-O-phosphoryl)-D-myo-inositol
3,4,5- trisphosphate.
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