1 Departments of Pediatrics and Biochemistry/Biophysics, University of Pennsylvania School of Medicine, The Joseph Stokes Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; and 2 Obesity Research Unit, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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In phagocytic cells,
fMet-Leu-Phe triggers phosphoinositide remodeling, activation of
protein kinase C (PKC), release of intracellular Ca2+ and
uptake of extracellular Ca2+. Uptake of extracellular
Ca2+ can be triggered by store-operated Ca2+
channels (SOCC) and via a receptor-operated nonselective cation channel(s). In neutrophilic HL60 cells, the PKC activator phorbol myristate acetate (PMA) activates multiple PKC isotypes, PKC-, PKC-
, and PKC-
, and inhibits ligand-initiated
mobilization of intracellular Ca2+ and uptake of
extracellular Ca2+. Therefore PKC is a negative regulator
at several points in Ca2+ mobilization. In contrast,
selective depletion of PKC-
in HL60 cells by an antisense strategy
enhanced fMet-Leu-Phe-initiated Ca2+ uptake but not
mobilization of intracellular Ca2+. Thapsigargin-induced
Ca2+ uptake through SOCC was not affected by PKC-
II
depletion. Thus PKC-
II is a selective negative regulator of
Ca2+ uptake but not release of intracellular
Ca2+ stores. PKC-
II inhibits a receptor-operated cation
or Ca2+ channel, thus inhibiting ligand-initiated
Ca2+ uptake.
calcium mobilization; protein kinase C isotypes; inositol 1,4,5-trisphosphate; signal transduction
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INTRODUCTION |
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MOBILIZATION
of intracellular Ca2+ and uptake of extracellular
Ca2+ plays an important role in signal transduction
for multiple cell responses (1, 33, 35). In
neutrophils and HL60 cells differentiated to a neutrophil-like
phenotype (dHL60 cells), ligands such as fMet-Leu-Phe elicit the twin
signals of elevated cytosolic Ca2+ concentration and
activation of protein kinase C (PKC). fMet-Leu-Phe triggers activation
of a phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol
1,4,5-trisphosphate (IP3), a trigger for release of
intracellular Ca2+ stores, and diglyceride (DG), an
activator of DG-dependent PKC (3, 15, 16, 18). Elevation
of cytosolic Ca2+ is essential for optimal
fMet-Leu-Phe-induced O
Non-excitable cells such as HL60 cells and neutrophils lack voltage-gated Ca2+ channels but possess receptor-operated Ca2+ channels. Ligands such as fMet-Leu-Phe trigger Ca2+ entry through receptor-operated nonselective cation channels that conduct Ca2+, Na+, and K+ (26). Ca2+-activated cation nonselective channels have been demonstrated in dHL60 cells and in neutrophils (6, 19, 32, 42). In addition, IP3-initiated depletion of the endoplasmic reticulum (ER) Ca2+ stores can trigger uptake of extracellular Ca2+ via store-operated calcium channels (SOCC) (30). The SOCC can also be directly activated by the sarco(endo)plasmic Ca2+-ATPase (SERCA) inhibitor thapsigargin (40). The Ca2+ channels in HL60 cells and neutrophils have not been fully characterized.
A role for PKC as a negative regulator of Ca2+
signaling has been demonstrated in multiple cell types, including dHL60
cells and neutrophils (12). Activation of PKC inhibits
ligand-induced increases in cytosolic Ca2+ and
ligand-induced IP3 generation via inhibition of
phospholipase C and may inhibit Ca2+ uptake (5, 9,
20, 22, 27, 39). These effects of PKC on Ca2+
mobilization were demonstrated in response to the phorbol ester phorbol
myristate acetate (PMA), which activates multiple DG-dependent PKC
isotypes including ,
,
, and
. dHL60 cells and neutrophils contain multiple isotypes of PKC, including
Ca2+/DG-dependent isotypes PKC-
and PKC-
,
Ca2+-independent DG-dependent isotype PKC-
, and atypical
phosphatidylserine-dependent, Ca2+/DG-independent
PKC-
(17). Activation of PKC can act as a
positive signal in triggering cell responses such as adherence and
O
is specifically required in
positive signaling for activation of the NADPH oxidase for generation
of O
In this study, an HL60 cell clone, which is protein null for
PKC-I but positive for PKC-
II, was used to probe a role for PKC-
II in regulation of ligand-initiated Ca2+
mobilization (11, 13). An antisense approach was used to selectively deplete PKC-
II but not PKC-
, PKC-
, or PKC-
.
Ca2+ uptake triggered by fMet-Leu-Phe, but not mobilization
of intracellular Ca2+, was enhanced in PKC-
-depleted
cells. Thus PKC-
II is a selective, negative regulator of
ligand-initiated Ca2+ uptake but, unlike the PKC activator
PMA, was not an inhibitor of intracellular Ca2+ release.
Ligand-initiated Ca2+ uptake by neutrophils and HL60 cells
is mediated by a receptor-operated Ca2+ channel(s) and by
an SOCC, which can also be activated by thapsigargin (40).
Ca2+ uptake triggered by thapsigargin was not modulated by
depletion of PKC-
. These results indicate that PKC-
II selectively
inhibits Ca2+ uptake via a thapsigargin-insensitive,
receptor-operated Ca2+ channel.
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MATERIALS AND METHODS |
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HL60 cell culture.
A subclone of human promyelocytic HL60 leukemic cells, originally
obtained from the American Type Culture Collection (Rockville, MD)
expressed PKC-, PKC-
II, PKC-
, and PKC-
but was protein null
for PKC-
I (11, 13). These HL60 cells were grown in
suspension culture in RPMI 1640 medium supplemented with 2 mM
L-glutamine, 1% nonessential amino acids, 1% MEM vitamin
solution, 0.1% gentamicin, and 10% heat-inactivated fetal bovine
serum (FBS). The cell cultures were maintained at 37°C in a 5%
CO2 humidified atmosphere. HL60 cells were cultured in the
presence of 1.3% DMSO for 6 days to initiate differentiation to a
neutrophil-like phenotype (dHL60 cells).
Oligonucleotide synthesis and sequences.
A 19-mer antisense oligonucleotide against the translation start site
of human PKC- was used for depletion of PKC-
in dHL60 cells as
previously described (17). The 19-mer oligonucleotides had
the following sequences: PKC-
antisense (
AS), 5' AGC CGG GTC AGC
CAT CTT G-3'; PKC-
sense (
SS), 5' C AAG ATG GCT GAC CCG GCT 3'.
Antisense and scrambled control oligonucleotides were synthesized as
the phosphorothioate derivatives and purified by HPLC.
Treatment of cells with oligonucleotides.
HL60 cells were cultured in the presence of 1.3% DMSO for 4 days to
initiate differentiation before treatment with the oligonucleotide. On
day 4, the cells were washed and resuspended in Opti-MEM I reduced serum medium (GIBCO/BRL) at a cell concentration of 25 × 106 cells/well. Oligonucleotides AS or
MS (a
scrambled AS oligonucleotide) were suspended in Opti-MEM, at a final
concentration of 400 nM. Delivery of the oligonucleotides was enhanced
with the cationic lipid 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl
ammonium bromide/cholesterol [DMRIE-C, 1:1 (M/M)] at 4 µg/ml. The
cationic lipid/oligonucleotide mixture was added to the cells and
incubated at 37°C for 4 h. An equal volume of RPMI 1640 medium
containing 20% heat-inactivated FBS plus DMSO (1.3% final
concentration) was then added, and the cells were cultured for 20 h. On day 5, the cells were washed and resuspended in fresh
Opti-MEM medium and treated again with the cationic
lipid/oligonucleotide mixture. Following a 4-h incubation, an equal
volume of RPMI 1640 medium containing 20% heat-inactivated FBS plus
DMSO (1.3% final concentration) was then added, and the cells were
cultured for an additional 24 h. The cells were harvested and
suspended in HEPES buffer (pH 7.5) having the composition 150 mM
Na+, 5 mM K+, 1.29 mM Ca2+, 1.2 mM
Mg2+, 155 Cl
mM, and 10 mM HEPES
(17).
Western blots. Differentiated HL60 cell lysates (1 × 106 cells/sample) were prepared by heating the cells at 95°C for 5 min in 2× SDS-PAGE sample buffer. The samples were briefly sonicated (12 s) to reduce viscosity. The dHL60 cell lysates were run on a 4-12% gradient SDS-PAGE, transferred to polyvinylidene difluoride membrane, and blocked for 1 h at room temperature with Tris-buffered saline, pH 7.5, containing 0.1% Tween 20 and 1% BSA/3% casein. To identify the different PKC isotypes, we incubated the membrane with a panel of PKC antibodies, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive bands were visualized by Pierce SuperSignal ULTRA chemiluminescence substrate.
Measurement of cytosolic Ca2+ in
fluo-3-loaded dHL60 cells.
HL60 cells were incubated with 10 µM of the acetoxymethyl ester
of fluo-3 (fluo-3/AM) in HEPES buffer at 37°C for 5 min and then
diluted 10 times to 1 ml with HEPES buffer at 37°C and incubated for
a further 20 min. Suspensions were centrifuged (800 rpm, 10 min), and
cells were resuspended in buffer at a concentration of 5 × 106 cells/ml. Immediately prior to use, aliquots of 1.0 ml
were microcentrifuged, and the cells resuspended in fresh 30°C buffer
at a cell concentration of 2 × 106 cells/200 µl.
The kinetics of fluorescence changes were monitored at 30°C in an
unstirred suspension of preloaded cells, excitation 485 nm and emission
530 nm. Triton X-100 was added to measure Fmax (to
calculate maximal Ca2+ concentration), and excess EGTA was
added to measure F0 (to calculate minimal
Ca2+). Cytosolic Ca2+
([Ca]in, in nM) was calculated as
[Ca]in = 390(F F0)/(Fmax
F) (23, 24).
Measurement of Ca2+ uptake as Mn2+-induced quenching of fura-2. dHL60 cells were loaded with fura-2 using the same protocol as fluo-3 loading. Immediately prior to use, aliquots of cells were microcentrifuged, and the cells were resuspended in fresh 30°C buffer, in the presence or absence of 100 µM MnCl2, at a cell concentration of 10 × 106 cells/ml. Leak of dye from the cells was not observed over the time course of the experiment. The kinetics of fluorescence quenching triggered by fMet-Leu-Phe or thapsigargin, a SERCA inhibitor that triggers opening of SOCC (40), were monitored using a 360-nm excitation filter and a 530-nm emission filter. Fluorescence was corrected for nonspecific changes such as cell shape change, by subtraction of the values obtained in the absence of MnCl2 from values obtained in the presence of MnCl2. Excitation at 360 nm is the calcium-insensitive isosbestic point for fura-2; uptake of Mn2+ through a Ca2+ channel quenches fura-2 fluorescence (27).
Measurement of IP3. Generation of IP3 was measured by a radioreceptor assay kit (model TRK1000, Amersham) (31). dHL60 cells (2 × 106) were suspended in 200 µl HEPES buffer at 37°C and activated by 1 µM fMet-Leu-Phe for 0, 5, 15, 30, and 60 s. The reaction was stopped by the addition of 40 µl ice-cold 20% perchloric acid and kept on ice for 20 min. After centrifugation at 2,000 g for 15 min at 4°C, the supernatant was adjusted to pH 7.5 with ice-cold KOH. After centrifugation to remove KClO4, IP3 was measured in the supernatant using the Amersham IP3 assay system (model TRK1000). This assay is based on competition between unlabeled IP3 in the sample and a fixed amount of [3H]IP3 for binding sites on an IP3 binding protein (31). Determinations were made in duplicate, and the results expressed as picomoles per 106 cells.
Statistical analysis. Results are means ± SE (n = number of observations). Data were analyzed by Student's t-test.
Reagents.
Cytochalasin B, BSA, PMA, and fMet-Leu-Phe were purchased from Sigma.
Fluo-3/AM and fura-2/AM were obtained from Molecular Probes, and
thapsigargin was from Biomol. PMA was stored as a concentrated stock
solution in DMSO and diluted with HEPES buffer before use. fMet-Leu-Phe
was stored as a stock solution in ethanol and diluted in buffer prior
to use. Anti-peptide polyclonal antibodies, to PKC-, PKC-
I,
PKC-
II, and PKC-
, and peroxidase-conjugated goat anti-rabbit IgG
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse
monoclonal antibody to PKC-
was purchased from Transduction Laboratories.
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RESULTS |
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Ligand-initiated increases in cytosolic
Ca2+ in dHL60 cells: regulation by PKC.
The kinetics of fMet-Leu-Phe-triggered increases in cytosolic
Ca2+ were monitored in fluo-3-loaded dHL60 cells. Resting
concentration of cytosolic Ca2+ was 88.9 ± 5.6 (n = 12) nM (Table 1).
The addition of 1 µM fMet-Leu-Phe triggered a rapid increase in
cytosolic Ca2+ that peaked by 18 s at a level of
264.4 ± 15.3 nM (Table 1, Fig.
1A). The stimulated increase
in cytosolic Ca2+ subsequently declined toward resting
levels by 2 min after addition of the stimulus. In the presence of
EGTA, the peak concentration of cytosolic Ca2+ elicited by
fMet-Leu-Phe was reduced to 123.5 ± 8.1 nM, a level that was
significantly less than the peak attained in the presence of
extracellular Ca2+ (46.7% control, P < 0.00002) (Table 1). Therefore, the ligand-induced increase in cytosolic
Ca2+ in dHL60 cells is mobilized from both intracellular
stores and by uptake from extracellular sources. PKC stimulated by the
phorbol ester, PMA, has been shown to modulate ligand-induced
Ca2+ movements in neutrophils and in differentiated HL60
cells (5, 9, 20, 22, 27, 39). Resting cytosolic
Ca2+ was 88.9 nM (see above) in control cells and 85.1 ± 7.5 (n = 11) nM Ca2+ after preincubation
with 1 µg/ml PMA for 5 min, a difference that was not statistically
significant (Table 1, Fig. 1A). However, after activation by
1 µM fMet-Leu-Phe, the peak cytosolic Ca2+ was
significantly reduced to 180.0 ± 15.3 nM (n = 11)
in cells pretreated for 5 min in the presence of 1 µg/ml PMA compared
with a level of 264.4 ± 15.3 nM in control cells (68.1% control,
P < 0.01) (Fig. 1A, and Table 1).
Furthermore, pretreatment with PMA decreased the peak cytosolic
Ca2+ levels attained in the presence of EGTA from
123.5 ± 7.5 nM Ca2+ in control cells exposed to 1 µM fMet-Leu-Phe, to 69.4 ± 11.0 nM Ca2+ in
PMA-pretreated cells (P = 0.05) (Table 1). The
difference in ligand-induced increase in cytosolic Ca2+ in
the presence and absence of EGTA represents uptake of Ca2+
from the medium. The difference in peak Ca2+ triggered by
fMet-Leu-Phe in the presence and absence of extracellular Ca2+ was 140.9 nM in control cells but only 110.6 nM in
dHL60 cells pretreated with PMA. These findings suggest that PMA
pretreatment inhibits both mobilization of intracellular
Ca2+ stores and uptake of extracellular Ca2+.
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Depletion of PKC- by an antisense strategy.
To test a role for PKC-
in regulating ligand-initiated
Ca2+ mobilization, dHL60 cells were treated with
-antisense (
AS) and control
-missense (
MS) oligonucleotides
for 48 h (see MATERIALS AND METHODS). The HL60
I-null cells were shown to express PKC-
II but not PKC-
I (Fig.
2A). Previous work
demonstrated that these cells also contained
-,
-, and
-isotypes of PKC (13). Pretreatment with 400 nM
AS selectively depleted PKC-
II (Fig. 2A) but not PKC-
, PKC-
, or PKC-
as previously shown (13), in
these differentiated HL60 cells. Densitometry demonstrated that the
level of PKC-
II declined from a level of 600 ± 64 density
units (DU) (n = 6) in control
MS treated cells, to a
level of 383 ± 87 DU (n = 6) (52.1 ± 11.6%
of control, P < 0.001) (Fig. 2B).
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Depletion of PKC- and Ca2+
mobilization.
Resting cytosolic Ca2+ levels were not affected by
depletion of PKC-
by
AS pretreatment (Table
2). Cytosolic Ca2+
concentration was 119.8 ± 14.4 nM (n = 6) in
cells treated with
AS, compared with a level of 99.9 ± 7.4 nM
in control cells pretreated with
MS (Fig.
3A), and not significantly
different from resting cytosolic Ca2+ levels in untreated
cells (Fig. 1A). In contrast, antisense depletion of PKC-
enhanced the fMet-Leu-Phe-triggered increase in cytosolic Ca2+. In cells pretreated with
MS and activated with 1 µM fMet-Leu-Phe, cytosolic Ca2+ peaked at 270.2 ± 15.9 nM (Fig. 3A). In contrast, cells pretreated with
AS
underwent an enhanced peak increase in cytosolic Ca2+ of
374.0 ± 16.5 nM (n = 6) (Fig. 3A,
Table 2), a difference that was significantly different from the
response in control
MS-treated cells (P < 0.002).
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Ligand initiated generation of IP3 and PKC-.
Ligands such as fMet-Leu-Phe trigger the activation of a phospholipase
C, which catalyzes cleavage of PIP2 to generate DG and
IP3, a trigger for release of intracellular
Ca2+ stores. In control dHL60 cells pretreated with
MS,
addition of 1 µM fMet-Leu-Phe triggered an elevation in cellular
IP3 from 1.22 ± 0.26 (n = 4) to
2.57 ± 0.61 pmol IP3/106 cells by 5 s after addition of the stimulus and to 2.50 ± 0.34 pmol
IP3/106 cells by 15 s (Fig.
4). When IP3 was measured in
PKC-
-depleted cells (pretreated with
AS), addition of 1 µM
fMet-Leu-Phe triggered an elevation in cellular IP3, which
increased from 1.50 ± 0.25 pmol IP3/106
cells (n = 4) in resting cells to peak at 3.09 ± 1.01 pmol IP3/106 cells by 15 s (Fig. 4).
Although the peak value of IP3 generated in
AS-pretreated cells was higher than in control cells, the difference
was not statistically significant. Thus PKC-
does not regulate
ligand-initiated activation of phospholipase C
and generation of
IP3, a finding that is concordant with the demonstration that PKC-
does not regulate release of intracellular
Ca2+ stores.
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SOCC opening triggered by thapsigargin is regulated by PKC.
A role for PKC has been suggested in the regulation of SOCC and
receptor-operated Ca2+ or cation nonselective channels in
HL 60 cells (26, 30). The SERCA inhibitor, thapsigargin,
inhibits the Ca2+-ATPase responsible for pumping
Ca2+ into the ER and induces Ca2+ uptake
through the SOCC without inducing hydrolysis of phosphoinositides. Thapsigargin triggered Ca2+ uptake in dHL60 cells,
concordant with activation of SOCC (30). Addition of 100 nM thapsigargin to fluo-3-loaded dHL60 cells in the presence of
extracellular Ca2+, triggered an increase in cytosolic
Ca2+ from 90.9 ± 5.9 nM (n = 5) to
235.9 ± 24.8 nM (Fig.
5A). This increase in
cytosolic Ca2+ was due to uptake of extracellular
Ca2+, since only a small increase in cytosolic
Ca2+ was observed in the absence of extracellular
Ca2+ (results not shown). When cells were pretreated with 1 µg/ml PMA for 5 min, thapsigargin triggered an increase of cytosolic Ca2+ from 92.7 ± 8.1 to only 142.5 ± 17.0 nM
(n = 5), a difference that was significantly different
from control (P < 0.025) (Fig. 5A).
Therefore, PMA reduced Ca2+ uptake through SOCC upon
addition of thapsigargin implicating PKC as a regulator of SOCC.
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Depletion of PKC- and Ca2+ uptake
triggered by thapsigargin.
The role of PKC-
in regulation of thapsigargin-induced
Ca2+ uptake was investigated using dHL60 cells pretreated
with
AS and
MS. Thapsigargin-induced Ca2+ uptake was
measured in fluo-3-loaded cells (Fig.
6A). In control dHL60 cells
treated with
MS, thapsigargin triggered an increase in cytosolic
Ca2+ from 103.6 ± 8.1 nM (n = 4) in
resting cells to 153.7 ± 9.5 nM in cells treated for 5 min with
thapsigargin (Fig. 6A), representing uptake of
Ca2+ through the SOCC. Similarly, in PKC-
-depleted dHL60
cells treated with
AS, thapsigargin triggered an initial increase in
cytosolic Ca2+ levels from 90.6 ± 11.1 nM
(n = 4) in resting cells to 147.8 ± 11.1 nM after
addition of thapsigargin (Fig. 6A). Thus no significance difference in thapsigargin-induced uptake of extracellular
Ca2+ was noted between cells depleted of PKC-
by
AS
and the control cells treated with
MS.
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DISCUSSION |
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Alterations in intracellular Ca2+ homeostasis has
profound effects on many cell functions. A role for PKC as a negative
regulator of Ca2+ homeostasis has been proposed in
differentiated HL60 cells, neutrophils (5, 9, 12, 20, 22, 27,
39), and in other cell types (21, 28, 34, 36, 37).
Three steps in Ca2+ signaling have been implicated as
targets for negative regulation by PKC. 1) PKC-dependent
phosphorylation inhibits activation of phospholipase C and reduces
the subsequent production of IP3 and release of
intracellular Ca2+ stores. 2) Phosphorylation of
the plasmalemmal Ca2+-ATPase by PKC activates
Ca2+ efflux, which is responsible for the return of
cytosolic Ca2+ to resting levels (4, 14, 41).
3) Activation of PKC inhibits the rate of Ca2+
uptake from the extracellular milieu. The chemotactic peptide fMet-Leu-Phe triggers mobilization of intracellular Ca2+
stores and uptake of extracellular Ca2+ by dHL60 cells.
Pretreatment of dHL60 cells with the PKC activator PMA elicits
inhibition of both ligand-triggered release of intracellular Ca2+ stores and uptake of extracellular Ca2+.
Since PMA activates multiple isotypes of PKC, including PKC-
, PKC-
, and PKC-
, these studies do not demonstrate isotype
specificity. We questioned whether the inhibitory effect of PMA on both
release of intracellular Ca2+ stores and uptake of
extracellular Ca2+ was due to the activation of a single
PKC isotype or, alternatively, whether different PKC isotypes regulated
discrete Ca2+ movements.
Phagocytic cells such as HL60 cells and neutrophils possess a number of
PKC isotypes, PKC-, PKC-
I, PKC-
II, PKC-
, and PKC-
. In
this study, we have demonstrated a role for PKC-
II in negative regulation of ligand-initiated uptake of extracellular
Ca2+, but not of intracellular Ca2+ release. An
antisense strategy was used to selectively deplete PKC-
II from a
clone of HL60 cells, which is protein null for PKC-
I but
positive for
-,
II-,
-, and
-isotypes of PKC.
Selective depletion of PKC-
, i.e., PKC-
II, enhanced the
fMet-Leu-Phe-triggered increase in cytosolic Ca2+ in the
presence of extracellular Ca2+. In contrast, depletion of
PKC-
II had no effect on fMet-Leu-Phe-elicited increase in cytosolic
Ca2+ in the absence of extracellular Ca2+,
indicating that PKC-
II was a negative regulator of
fMet-Leu-Phe-induced Ca2+ uptake but not of ligand-induced
release of intracellular Ca2+ stores.
Signaling for release of intracellular Ca2+ stores involves
fMet-Leu-Phe-induced activation of phospholipase C (2,
39) and cleavage of PIP2 to generate
IP3. Phospholipase C
2 is a substrate for PKC
(5). The finding that generation of IP3 was
not affected by PKC-
II depletion is concordant with our findings
that ligand-initiated release of intracellular Ca2+ stores
was not affected by PKC-
II depletion. This finding indicates that
any effect of PKC-
II must occur independently of phospholipase C
activation. Furthermore, the PMA-induced inhibition of release of
intracellular Ca2+ stores still occurred in
PKC-
-depleted cells, demonstrating that the negative effect of PMA
on Ca2+ mobilization was not solely dependent on PKC-
and may be due to another PKC isotype such as PKC-
or PKC-
or to
a non-PKC-dependent mechanism. Indeed, a role for PKC-
has been
demonstrated in negative regulation of phospholipase C in visual signal
transduction in Drosophila (25).
A role for PKC-II in negative regulation of Ca2+ uptake
was confirmed by the finding that PKC-
II depletion also enhanced
fMet-Leu-Phe-induced uptake of Mn2+, which acts as a
surrogate for Ca2+ during opening of the ligand-operated
Ca2+ channels in HL60 cells (27) and of SOCC
in numerous cell types (1). A similar role for
PKC-
in the regulation of Ca2+ uptake has been
demonstrated in platelets and lymphocytes (10, 43).
Two distinct channels for Ca2+ uptake are potential targets
for PKC. fMet-Leu-Phe triggers Ca2+ uptake via a
nonselective cation channel(s) that is activated by elevation of
cytosolic Ca2+ (6).
Ca2+-mobilizing ligands such as fMet-Leu-Phe also activate
SOCC, initiated by IP3-triggered emptying of ER
Ca2+ stores. The SERCA inhibitor thapsigargin can bypass
ligand-initiated depletion of Ca2+ stores and directly
activate the store-operated Ca2+ entry pathway
(40). In dHL60 cells, thapsigargin triggered enhanced
Ca2+ uptake monitored as increased cytosolic
Ca2+ or alternatively as increased Mn2+ uptake.
Thapsigargin-induced Ca2+ uptake was inhibited by
pretreatment of the cells with PMA and activation of PKC, in agreement
with other workers (9, 20, 22, 27, 39). However, the
Ca2+ uptake through SOCC activated by thapsigargin was not
affected by depletion of PKC-II. Therefore, the SOCC per se is not
regulated by PKC-
II. Thus PKC-
II could regulate a non-SOCC
Ca2+ channel such as the receptor-operated nonselective
cation channel. Indeed, thapsigargin and fMet-Leu-Phe triggered an
additive Ca2+(Mn2+) uptake (results
not shown), indicating that fMet-Leu-Phe and thapsigargin activate
different channels. Alternatively, PKC-
II might not act directly on
SOCC but might play a role in the ligand-initiated signaling for the
opening of SOCC.
These studies demonstrate a selective role for PKC-II in the
negative regulation of ligand-initiated Ca2+ uptake but not
mobilization of intracellular Ca2+ stores. Since modulation
of PKC-
II affected receptor-operated Ca2+ uptake but had
no effect on thapsigargin-initiated Ca2+ uptake, these
findings suggest a role for PKC-
II in the signaling for activation
of Ca2+ uptake through a receptor-operated nonselective
cation channel rather than the SOCC. PKC-
II is involved in positive
signaling for O
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant AI-24840.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. M. Korchak, Immunology Section, Rm. 1208C Abramson Bldg., Children's Hospital of Philadelphia, 3516 Civic Center Boulevard, Philadelphia, PA 19104 (E-mail: korchak{at}emailchop.edu).
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.
Received 17 October 2000; accepted in final form 27 March 2001.
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