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Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R1 on the surface of
mast cells (1, 2). Recently, it has been shown that mast
cell degranulation is responsible for the coating of nonimmunogenic
surgically implanted biomaterials (3). Specifically,
release of histamine at the site of the implant appears to act as a
chemoattractant for phagocytic cells that encapsulate the implant and
can lead to its failure (3). It is therefore important to
elucidate the intracellular pathways that regulate mast cell
degranulation. Studies to date indicate that this degranulation process
is strictly dependent on the influx of extracellular calcium. The
depletion of extracellular calcium by the chelator EGTA results in
the complete inhibition of Ag-induced degranulation (4).
In general, receptors that are capable of stimulating the release of
intracellular calcium induce the tyrosine phosphorylation of
phospholipase C-
1
(PLC-1)4 and
PLC-2 (5), resulting in the generation of the two
second messengers inositol-1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG) (6).
IP3 binds to its receptor in the membrane of the
endoplasmic reticulum and induces the release of intracellular calcium,
whereas DAG associates with certain isoforms of the serine/threonine
protein kinase C (PKC), thereby promoting their activation
(6). The IP3-induced emptying of
intracellular calcium stores then triggers the entry of extracellular
calcium through store-operated calcium channels in the plasma membrane
(7).
We recently generated a mouse containing a targeted disruption of the
gene encoding the hemopoietic specific src homology
2-containing inositol phosphatase, SHIP. Bone marrow-derived mast cells
(BMMCs) from these mice were found to readily degranulate with either
IgE (4) or steel factor (SF) (8), two
proteins that do not by themselves stimulate degranulation in normal
murine BMMCs. This hyperresponsiveness was due in large part to
markedly elevated levels of PI-3 kinase-generated
phosphatidylinositol-3,4,5-trisphosphate (PIP3),
because SHIP was not present to hydrolyze PIP3 to
PI-3,4-P2. Specifically, in SF-induced
degranulation of SHIP-/-
BMMCs, we found that PI-3 kinase-generated PIP3
was critical both for a step upstream of intracellular calcium release
and between intracellular calcium release and extracellular calcium
entry (8). Our working hypothesis based on these results
was that the markedly increased, membrane-anchored
PIP3 in
SHIP-/- BMMCs attracted
substantially more pleckstrin homology (PH) domain-containing proteins,
such as PLC- (9) and Btk (10), to the
plasma membrane to mediate these calcium fluxes. Thus, SHIP appears to
function in normal BMMCs to restrict the entry of extracellular calcium
by reducing the level of PI-3 kinase-generated
PIP3 (4, 8).
One approach to studying the regulation of mast cell degranulation downstream of extracellular calcium entry is by bypassing the activation of plasma membrane receptors by using calcium ionophores that transport calcium ions across the plasma membrane or via chemicals that induce the release of calcium from intracellular stores. In this study, the effect of the tumor promoter thapsigargin on mast cell degranulation was evaluated in SHIP+/+ and SHIP-/- BMMCs. Thapsigargin is a specific inhibitor of the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, which pumps calcium that leaks from the endoplasmic reticulum back into this organelle (11). Adding thapsigargin to mast cells thus results in the draining of calcium ions from the endoplasmic reticulum, capacitative entry of extracellular calcium through store-operated calcium surface channels, and subsequent degranulation (12).
The results presented in this work demonstrate that although
thapsigargin induces degranulation in a cell surface
receptor-independent fashion, this process is still dependent on the
activation of PI-3 kinase in normal BMMCs and is strongly enhanced in
SHIP-/- BMMCs. In fact,
the independence of thapsigargin-mediated degranulation from surface
receptors as well as PLC- enabled us to identify an additional PI-3
kinase-dependent step within the mast cell degranulation process
downstream from intracellular calcium release and influx of
extracellular calcium. We also show that thapsigargin stimulation of
BMMCs leads to the activation of protein kinase B (PKB) and survival of
cells in the absence of cytokines, thereby providing a possible
mechanism for the tumor-promoting ability of this molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow cells from 4- to 8-wk-old
SHIP+/+ and
SHIP-/- littermates were
plated in methylcellulose (Methocult M3234; StemCell Technologies,
Vancouver, Canada) supplemented with 30 ng/ml murine IL-3, 50 ng/ml
murine SF, and 10 ng/ml human IL-6 for 10–14 days. They were then
harvested and grown in suspension in IMDM containing 15% FCS (StemCell
Technologies), 150 µM monothioglycerol (Sigma, Oakville, Canada), and
30 ng/ml IL-3. By 8 wk in culture, greater than 99% of the cells were
c-kit and FcR1 positive, as assessed by FITC-labeled
anti-c-kit Abs (PharMingen, Mississauga, Canada) and
FITC-labeled IgE (anti-erythropoietin) (26),
respectively (4, 8).
Reagents
Polyclonal Abs against S473-phosphorylated PKB (P-PKB), PKB, and T202/Y204-phosphorylated MAPK (P-MAPK) were obtained from New England Biolabs (Mississauga, Canada). The polyclonal anti-ERK-1 Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Compound 3 (bisindolylmaleimide) (catalog no. 203290), thapsigargin (catalog 586005), and LY294002 (catalog no. 440202) were obtained from Calbiochem (San Diego, CA). Wortmannin (catalog no. W1628) and PMA (catalog no. P8139) were purchased from Sigma.
Measurement of intracellular calcium
Calcium fluxes were measured according to Huber et al. (8). In brief, SHIP+/+ and SHIP-/- BMMCs were incubated with 2 µM fura 2-acetoxymethyl ester (Molecular Probes, Eugene, OR) in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA in 10 mM HEPES, pH 7.4) at 23°C for 45 min. The cells were then washed, resuspended in 1 ml of the same buffer at 5 x 105 cells/ml in a stirring cuvette. Following stimulation with thapsigargin or SF, cytosolic calcium was measured by monitoring fluorescence intensity at 510 nm, after excitation of the sample with two different wavelengths (340 and 380 nm) using an MC200 monochromator from SLM AMINCO with a 8100 V3.0 software program.
Degranulation assay
For degranulation studies, 5 x 105 cells/sample were washed with IMDM and starved overnight in IMDM, containing 10% FCS and 150 µM monothioglycerol. The cells were then resuspended in Tyrode’s buffer and treated for 15 min at 37°C with or without 1 µg/ml thapsigargin. The degree of degranulation was determined by measuring release of ß-hexosaminidase (13).
Plasma membrane preparation, p85 immunoblotting, and PI-3 kinase assay
SHIP-/- and SHIP+/+ BMMCs were starved as above and incubated for 1 min at 37°C with control buffer,1 µg/ml thapsigargin, or 300 ng/ml SF. Plasma membrane-enriched membrane fractions were prepared as described by Miura et al. (14). Briefly, the cells were then pelleted, resuspended at 1.5 x 107 cells/ml in 4°C hypotonic lysis buffer (20 mM Tris-Cl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 5 mM DTT, 5 mM Na3VO4, 0.5 mM PMSF, 2 µg/ml leupeptin, and 10 µg/ml aprotinin), allowed to swell for 5 min on ice, and sonicated for 15 x 1-s bursts on ice using an ultrasonic cell disruptor (Heat Systems Ultrasonics, Faimingdale, NY). After centrifugation at 2000 x g for 5 min at 4°C, the supernatant was centrifuged at 100,000 x g for 10 min at 4°C in an airfuge (Beckman Instruments, Fullerton, CA). The pellet was resuspended in 400 µl of hypotonic lysis buffer containing 1% Nonidet P-40 by repeated vortex mixing. After a 60-min incubation at 4°C, the suspension was centrifuged at 100,000 x g for 10 min, and the supernatant was collected as the membrane fraction. This solubilized membrane fraction was then subjected either to Western blot analysis with anti-p85 (Upstate Biotechnology), and the blot reprobed with anti-c-kit as a loading control or to immunoprecipitation by first incubating at 4°C for 1 h with anti-p85 Ab (Upstate Biotechnology) and then with protein A-Sepharose beads at 4°C for 1 h. Beads were then washed and PI-3 kinase assays were performed, as described previously (15).
Immunoblotting
SHIP+/+ and SHIP-/- BMMCs were starved as above and incubated for various times at 37°C with 1 µg/ml thapsigargin. The cells were then solubilized with 0.5% Nonidet P-40 in 4°C phosphorylation solubilization buffer (16), and subjected to Western blot analysis, as described previously (16).
Survival studies
SHIP+/+ and SHIP-/- BMMCs were washed with IMDM and incubated at 5 x 105 cells/ml in IMDM containing 10% FCS and vehicle (DMSO) or various concentrations (0.015–0.06 µg/ml) of thapsigargin at 0.4 ml/well in Falcon 3047 24-well flat-bottom plates. Viability was assessed by trypan blue exclusion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently demonstrated that both IgE and SF stimulate a much
more profound calcium influx into
SHIP-/- than
SHIP+/+ BMMCs, and this leads to a
substantially greater degranulation of the
SHIP-/- cells (4, 8). Moreover, the enhanced calcium entry and degranulation of
the SHIP-/- BMMCs by
these two agonists were shown to be dependent on elevated
PIP3 levels in these cells (8). This
is consistent with our finding that SHIP is the primary enzyme
responsible for hydrolyzing SF-induced PIP3 in
these BMMCs and that PIP3 reaches substantially
higher levels in response to SF or IgE when SHIP is absent (8, 17). To gain some insight into the regulation of the
degranulation process downstream of calcium release from intracellular
stores, we investigated the degranulation potential of thapsigargin in
SHIP+/+ and
SHIP-/- BMMCs. We found
that while SHIP+/+ BMMCs only degranulated to
10% in response to this agent, stimulation of
SHIP-/- BMMCs resulted in
about 55% degranulation (Fig. 1
A). Based on our previous
findings with IgE- and SF-stimulated BMMCs (4, 8), we
anticipated an increased thapsigargin-induced extracellular calcium
influx in SHIP-/- BMMCs.
However, the calcium entry induced by thapsigargin in
SHIP+/+ and
SHIP-/- BMMCs was
identical (Fig. 1B). For comparison, a SF-induced calcium
influx was conducted with the same cells and, as reported previously
(8), SF stimulated a far greater influx of calcium into
SHIP-/- than into
SHIP+/+ BMMCs (Fig. 1B). These results
suggested that there was at least one additional, calcium-independent
pathway present in thapsigargin-stimulated mast cells that contributes
to the enhanced degranulation in
SHIP-/- BMMCs.
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Since one characteristic of
SHIP-/- BMMCs is their
augmented, PI-3 kinase-generated PIP3 levels in
response to various stimuli (8, 17), we asked whether PI-3
kinase activation might be involved in the thapsigargin-induced
degranulation process. To address this, SHIP+/+
and SHIP-/- BMMCs were
preincubated with various concentrations of the PI-3 kinase inhibitors,
LY294002 and wortmannin, and thapsigargin-stimulated degranulation was
assessed. As shown in Fig. 2A,
thapsigargin-induced degranulation of SHIP+/+
BMMCs was significantly inhibited by LY294002, with 10 µM LY294002
giving a maximal reduction of 75% (Fig. 2A, left
panel). In SHIP-/-
BMMCs, thapsigargin-induced degranulation was even more inhibited by
LY294002. However, higher concentrations of this PI-3 kinase inhibitor
were required to achieve maximal inhibition, i.e., 97% at 100 µM
LY294002 (Fig. 2A, right panel). Similar results
were obtained with wortmannin (Fig. 2B). Both LY294002 and
wortmannin reduced the thapsigargin-induced degranulation of the two
cell types to 3%, suggesting that this residual degranulation may
be PI-3 kinase independent. These results suggested that thapsigargin
was capable of stimulating PI-3 kinase activity in both
SHIP+/+ and
SHIP-/- BMMCs, and that
this activation plays a critical role within the thapsigargin-mediated
degranulation process. Importantly, as was found previously with IgE-
or SF-stimulated SHIP+/+ and
SHIP-/- BMMCs (4, 8), thapsigargin-induced PI-3 kinase activity in
SHIP+/+ and
SHIP-/- BMMCs was
similar, as assessed both by the level of p85 associated with plasma
membrane-enriched membrane preparations (Fig. 3A) and by PI-3 kinase assays
with p85 immunoprecipitates from these membrane preparations (Fig. 3B). Densitometric analysis of our Western blots (Fig. 3A) revealed that unstimulated levels of p85 were similar in
the two cell types and that thapsigargin recruited p85 to a similar
degree in SHIP+/+ and
SHIP-/- cells
(1.4-fold over unstimulated levels). Moreover, this p85 recruitment
was substantially less than that recruited by SF (2.9-fold over
unstimulated levels), consistent with the greater effect of SF on PKB
activation in SHIP+/+ BMMCs (see below, Fig. 4). Because of the modest PI-3 kinase
activation induced by thapsigargin and the many washing steps involved
in the PI-3 kinase assay (15), we obtained some variation
in our assay results (e.g., in the top panel of Fig. 3B, there appears to be more PI-3 kinase activity in
thapsigargin-stimulated SHIP+/+ cells, while in
the bottom panel the activation levels look similar in the
two cell types). Averaging the densitometric results of five separate
experiments revealed no significant difference in thapsigargin-induced
PI-3 kinase activity. We also conducted PI-3 kinase assays with
anti-phosphotyrosine (4G10) (four separate experiments) and
anti-p85 (Upstate Biotechnology) (four separate experiments)
immunoprecipitates from whole cell lysates and obtained a similar
degree of variation and no significant difference in the
SHIP+/+ and
SHIP-/- BMMCs (data not
shown). Thus, our finding that higher concentrations of LY294002 and
wortmannin are required to inhibit thapsigargin-induced degranulation
in SHIP-/- BMMCs is
consistent with there being higher levels of PIP3
in SHIP-/- BMMCs
following thapsigargin exposure due to the loss of SHIP rather than
higher PI-3 kinase activity.
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C (left panel),
preincubation of SHIP-/-
BMMCs with 50 µM LY294002 had no effect on the initiation of calcium
mobilization by thapsigargin and only a minor effect on the overall
intracellular calcium level after stimulation. The same concentration
of this inhibitor, however, was capable of almost completely abolishing
SF-stimulated calcium entry into the same cells (Fig. 3C,
right panel). The same result was obtained in
SHIP+/+ BMMCs (data not shown). These data
suggested that in thapsigargin-treated mast cells, PI-3 kinase plays a
role in mediating the degranulation process. However, unlike in
SF-stimulated mast cells, this enzyme appears to be acting downstream
of calcium entry into thapsigargin-treated cells. Transient PKB activation occurs in thapsigargin-stimulated BMMCs
One of the major targets of PI-3 kinase activation is PKB
(18, 19). Because optimal thapsigargin-induced
degranulation was dependent on PI-3 kinase (Fig. 2), we asked whether
thapsigargin stimulated PKB activation. To study this,
SHIP+/+ BMMCs were stimulated with thapsigargin
for different times, and the activation of PKB was assessed in total
cell lysates using a phospho-specific Ab recognizing PKB phosphorylated
at Ser473 (P-PKB). Intriguingly, a very transient
phosphorylation of PKB was detected only at 1 and 2 min after
stimulation (Fig. 4A, upper panel), a PKB
activation pattern totally distinct from that elicited by SF with these
cells (Fig. 4B, upper panel). To verify equal
loading, the blots were reprobed with anti-PKB Abs (Fig. 4, A and B, lower panels). Because
thapsigargin has been shown previously to activate MAPK in the rat
hippocampal cell line, H19-7 (20), we asked whether MAPK
was also activated by thapsigargin in murine BMMCs and
whether this activation followed the same transient pattern. Total cell
lysates from thapsigargin-treated SHIP+/+
BMMCs were assessed by Western blot analysis with a
phospho-specific Ab recognizing doubly phosphorylated
(Thr202/Tyr204) ERK-1 and
ERK-2 (P-ERK-1 and P-ERK-2). Interestingly, a sustained MAPK
phosphorylation/activation was observed in thapsigargin-stimulated
SHIP+/+ BMMCs (Fig. 4C), paralleling
the prolonged calcium mobilization induced by this drug (Fig. 1B, left panel). To verify equal loading, the
blot was reprobed with anti-ERK-1 Abs (Fig. 4C,
lower panel).
Thapsigargin induces PKB activation via PI-3 kinase
Recently, Yano et al. identified a calcium-triggered signaling
cascade in which calcium/calmodulin-dependent kinase kinase activates
PKB in a PI-3 kinase-independent fashion (21). We
therefore investigated whether the transient PKB activation observed in
BMMCs in response to thapsigargin was dependent on PI-3 kinase
activation. Specifically, SHIP+/+ BMMCs were
stimulated with 1 µg/ml thapsigargin for 1 or 5 min in the presence
or absence of the two PI-3 kinase inhibitors, LY294002 (50 µM) and
wortmannin (50 nM), and cell lysates were examined for P-PKB
expression. As shown in Fig. 5
(upper panel), both inhibitors were capable of inhibiting
thapsigargin-induced PKB phosphorylation, indicating PI-3 kinase
dependence. Equal loading was verified by reprobing with anti-PKB
Abs (Fig. 5, lower panel). Interestingly, thapsigargin
stimulation in the presence of the PI-3 kinase inhibitors had no effect
on the activation of MAPK (data not shown).
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In previous reports, we demonstrated that SHIP deficiency in
murine BMMCs results in the potentiation of PI-3 kinase-mediated
effects, such as IgE- and SF-stimulated calcium mobilization and
degranulation (4, 8). Since we have shown in this study
that PI-3 kinase-dependent PKB activation takes place in
SHIP+/+ BMMCs in response to thapsigargin (Figs. 4A and 5), we asked whether thapsigargin treatment of
SHIP-/- BMMCs might
result in a more pronounced activation of PKB. To investigate this,
SHIP+/+ and
SHIP-/- BMMCs were
stimulated with 1 µg/ml thapsigargin for various times and cell
lysates subjected to Western blot analysis using anti-P-PKB Abs. As
shown in Fig. 6A, PKB
phosphorylation in
SHIP-/- BMMCs was easily
detected after a 5-s exposure (upper panel), whereas 3 min
of exposure was required to barely see phosphorylation of PKB in
SHIP+/+ BMMCs (middle panel). This
demonstrates that PKB is much more activated in
SHIP-/- BMMCs in
response to thapsigargin. Interestingly, even in the absence of
thapsigargin, the level of PKB phosphorylation was significantly higher
in SHIP-/- BMMCs (see
Fig. 6A, middle panel), most likely because the
cells were starved in the presence of 10% FCS, which contains
substantial levels of SF (22). Also of interest, PKB
phosphorylation was lower following 30 and 60 min of thapsigargin
treatment than in unstimulated
SHIP-/- BMMCs (see Fig. 6A, middle panel), and this could be due to
activation of the serine/threonine phosphatase, PP2A, which has been
implicated in the in vivo dephosphorylation/inactivation of PKB
(23). Equal loading of the gels shown in the
upper and middle panels of Fig. 6A was
confirmed by reprobing with anti-PKB Abs (Fig. 6A,
lower panel). Thapsigargin stimulation in the presence of
the PI-3 kinase inhibitor LY294002 resulted in the inhibition of PKB
phosphorylation in SHIP+/+ (Fig. 5) as well as in
SHIP-/- BMMCs (data not
shown). To determine whether all signaling pathways were activated more
strongly in SHIP-/-
BMMCs, the same cell lysates were analyzed by Western blotting with
anti-P-MAPK Abs. As shown in Fig. 6B (upper
panel), phosphorylation of the MAPKs, ERK-1 and ERK-2, was the
same in the two cell types, consistent with the notion that the loss of
SHIP primarily enhances PIP3-mediated pathways.
The membrane was reprobed with anti-ERK-1 Abs to demonstrate equal
loading (Fig. 6B, lower panel).
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Having established, using thapsigargin as a probe, that a PI-3
kinase-regulated step in degranulation is present downstream of
extracellular calcium entry, we asked what
PIP3-binding protein(s) could be mediating this
process. Recently, certain conventional (PKC 
and ßII), novel (PKC
), and atypical (PKC 
) PKC isotypes have been reported to be
regulated by PI-3 kinase via the downstream
3-phosphoinositide-dependent protein kinase, PDK-1, which binds
PIP3 in the plasma membrane
(24, 25, 26, 27, 28). Because PDK-1 is involved in the
phosphorylation/activation of PKB (26, 29) and because we
found that thapsigargin increased PKB phosphorylation more in
SHIP-/- than in
SHIP+/+ BMMCs, we investigated whether a
pan-specific PKC inhibitor, compound 3, could inhibit
thapsigargin-induced degranulation. As shown in Fig. 7A, compound 3 markedly
inhibited thapsigargin-induced degranulation in both
SHIP+/+ and
SHIP-/- BMMCs. To gain
some insight into which PKC might be involved in this process, we
assessed degranulation in the presence of the phorbol ester, PMA, which
specifically activates DAG-dependent PKC isoforms. Although 50 nM PMA
had no statistically significant effect on degranulation by itself in
SHIP+/+ or in
SHIP-/- BMMCs, it
slightly enhanced thapsigargin-induced degranulation (Fig. 7B). It did not, however, increase thapsigargin-induced
degranulation in SHIP+/+ cells to the same level
observed in SHIP-/-
BMMCs. This suggests that a PDK-1-dependent DAG-independent PKC, such
as PKC (28), might be important in this step.
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Because we found that thapsigargin stimulated PKB, a kinase shown
to enhance survival of many cell types (29), we asked
whether thapsigargin might promote the survival of our
SHIP+/+ and
SHIP-/- BMMCs following
withdrawal of IL-3. As can be seen in Fig. 8A, thapsigargin at
concentrations between 0.02 and 0.06 µg/ml dramatically enhanced the
survival of both SHIP+/+ and
SHIP-/- BMMCs in the
absence of IL-3. Also, as predicted based on the greater activation of
PKB in SHIP-/- BMMCs,
thapsigargin was more effective at promoting survival of
SHIP-/- BMMCs at low
concentrations of the tumor promoter. This is also shown in a time
course study with SHIP+/+ and
SHIP-/- BMMCs using 0.02
µg/ml thapsigargin (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). However, unlike IgE and SF, this thaspigargin-induced
increased degranulation did not correlate with an elevated calcium
influx into SHIP-/- BMMCs
(Fig. 1B), suggesting that calcium entry is a crucial
trigger, but not the only factor responsible for degranulation.
Consistent with this observation, Ludowyke et al. (30)
reported that the calcium ionophore, A23187, caused degranulation of
the rat mast cell line RBL-2H3, but far less than that elicited with
IgE plus cross-linking Ag. However, they found that A23187 plus the
phorbol ester PMA (which did not stimulate degranulation by itself)
induced degranulation to the same extent as IgE plus cross-linking Ag
(30) and proposed that in addition to calcium, other
signals have to be generated for optimal degranulation. Related to
this, we found in the present study that LY294002 and wortmannin
inhibited thapsigargin-induced degranulation in both
SHIP+/+ and
SHIP-/- BMMCs (Fig. 2),
suggesting that activation of PI-3 kinase by thapsigargin provides a
signal in addition to calcium mobilization to attain maximal
degranulation. This is consistent with a report by Marquardt et al.
(31), who showed that A23187-induced degranulation of
BMMCs could be attenuated using the PI-3 kinase inhibitor, wortmannin.
Intriguingly, we found that LY294002 had no significant effect on the
calcium influx induced by thapsigargin (Fig. 3B), suggesting
a PI-3 kinase-dependent step downstream of calcium mobilization. This
downstream step could not be detected with PI-3 kinase inhibitors in
our earlier studies employing IgE or SF to trigger mast cell
degranulation because the degranulation pathway initiated by these two
agonists was blocked by PI-3 kinase inhibitors at two much earlier
steps (intracellular calcium release and extracellular calcium entry)
(4, 8). Thus, thapsigargin has proven extremely useful, by
bypassing PLC--induced calcium release and PI-3 kinase-mediated
extracellular calcium entry, in revealing this third PI-3
kinase-regulated step in degranulation.
PI-3 kinase phosphorylates PI-4,5-P2 at the 3'
position of the inositol ring to generate PIP3,
which then serves as a substrate for SHIP, yielding
PI-3,4-P2 (32). We demonstrated
recently that the loss of SHIP enhances PI-3 kinase-induced cellular
responses by elevating PIP3 levels (4, 8). Because several signaling proteins containing PH domains,
such as PLC- (9), the tyrosine kinase Btk
(10), and the serine/threonine kinase PKB (33, 34), are capable of binding to and becoming activated by PI-3
kinase-generated phosphoinositides, PI-3 kinase is an important switch
for the initiation of various pathways. Related to this, we observed
transient PI-3 kinase-mediated activation of PKB in response to
thapsigargin. This is especially interesting given that PKB activation
requires colocalization of the PH-containing kinase PDK-1 (which
phosphorylates PKB at Thr308) at the plasma
membrane. PDK-1 has been shown to phosphorylate/activate various PKC
isoforms (24, 25, 26, 27, 28). This, coupled with our data showing
that the pan-specific PKC inhibitor, compound 3, prevents
thapsigargin-induced degranulation and that PMA doesn’t bring
thapsigargin-induced degranulation in SHIP+/+
BMMCs to levels obtained with
SHIP-/- BMMCs suggests
that a PIP3-binding DAG-independent PKC isotype,
such as PKC , might be connecting the activation of PI-3 kinase with
the degranulation machinery (Fig. 7). Consistent with this model,
Cissel et al. (35) have demonstrated in the rat RBL-2H3
mast cell line, using a variety of pharmacological activators and
inhibitors of signaling molecules, that thapsigargin-induced
degranulation is regulated by both phospholipase D and PKC in a PI-3
kinase-dependent manner. As well, it has been known for some time that
activation of PKC is a critical event for effective degranulation to
occur (30). Moreover, as mentioned earlier, the
combination of calcium-mobilizing probes and pharmacological PKC
activators has been shown to lead to a synergistic increase in mast
cell degranulation (30).
With respect to thapsigargin being a tumor promoter, the activation of
PKB and the enhanced survival in the absence of exogenous cytokines
offer an interesting new possibility for the tumorigenicity of
thapsigargin. PKB is known to be a key survival kinase required for the
inhibition of apoptosis in both hemopoietic cells and other cell types
(36, 37). Seven targets of PKB have been identified to
date, and they are the Bcl-2 family member, Bad (38),
glycogen synthase kinase-3 (39), caspase-9
(40), a forkhead transcription factor (FKHRL1)
(41), I
(42), endothelial NO
synthase (eNOS) (43, 44), and Raf (45).
Phosphorylation of these proteins by PKB inactivates them, thus
promoting survival in certain cell types. Because we also found that
thapsigargin activates MAPK (Fig. 4), which has been shown to promote
survival in some cell types and proliferation in others (46, 47), the initiation of antiapoptotic (and/or cell proliferation)
pathways might be the main mechanism(s) by which thapsigargin mediates
its tumor-promoting activity. However, it is worth noting that
thapsigargin has been reported to activate both a tyrosine as well as a
serine/threonine kinase, leading to transcriptional activation of the
glucose-regulated protein GRP78 promoter (48). Consistent
with this, Chao et al. (20, 49, 50) have reported the
activation of Src tyrosine kinase as well as Raf-1 and MAPK
serine/threonine kinases in response to thapsigargin in H19-7 cells.
Thus, we cannot rule out at this time that activation of a tyrosine
kinase such as Src is also involved in the tumor-promoting activity of
thapsigargin. In contrast to our studies, Conus et al., who assessed
the role of calcium in the regulation of PKB and
p70S6K in BALB/c-3T3 fibroblasts, found that
thapsigargin stimulated full activation of
p70S6K, whereas little or no activation of PKB
was observed (51). However, because a different source of
cells was used and the time point assessed was 5 min (51),
a time in which PKB activation is almost back to baseline in BMMCs
(Figs. 4 and 5), their results do not necessarily contradict
ours.
In summary, we have shown that the tumor promoter thapsigargin activates a PI-3 kinase-dependent survival pathway, thereby providing a new model for its tumor promotion. Moreover, we have identified a PI-3 kinase-dependent pathway important for primary mast cell degranulation that is downstream of intracellular calcium release and extracellular calcium entry. This pathway might involve PDK-1 and a DAG-independent PKC isoform. Further studies are currently underway to identify the PKC isoform(s) involved in this pathway.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck-Institute for Immunobiology, 79108 Freiburg, Germany.
3 Address correspondence and reprint requests to Dr. Gerald Krystal, Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3.
4 Abbreviations used in this paper: PLC, phospholipase C; BMMC, bone marrow-derived mast cell; DAG, diacylglycerol; ERK, extracellular signal-related kinase; IP3, inositol-1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; P-ERK, phospho-ERK; P-MAPK, T202/Y204-phosphorylated MAPK; P-PKB, S473-phosphorylated PKB; PDK, 3-phosphoinositide-dependent protein kinase; PH, pleckstrin homology; PI, phosphatidylinositol; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKB, protein kinase B; PKC, protein kinase C; SF, steel factor; SHIP, src homology 2-containing inositol phosphatase.
Received for publication May 27, 1999. Accepted for publication April 17, 2000.
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)
BMMCs were incubated at 5 x 105 cells/ml with the
indicated concentrations of thapsigargin, in the absence of IL-3, and
viable cells were counted on day 3. B,
SHIP+/+ (•) and SHIP-/- (