Negative regulation of ligand-initiated Ca2+ uptake by PKC-beta II in differentiated HL60 cells

Helen M. Korchak1, Barbara E. Corkey2, Gordon C. Yaney2, and Laurie E. Kilpatrick1

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


    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-alpha , PKC-beta , and PKC-delta , 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-beta 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-beta II depletion. Thus PKC-beta II is a selective negative regulator of Ca2+ uptake but not release of intracellular Ca2+ stores. PKC-beta 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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ABSTRACT
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MATERIALS AND METHODS
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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 Cbeta , 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<UP><SUB>2</SUB><SUP>−</SUP></UP> generation and degranulation (16, 30).

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 alpha , beta , delta , and epsilon . dHL60 cells and neutrophils contain multiple isotypes of PKC, including Ca2+/DG-dependent isotypes PKC-alpha and PKC-beta , Ca2+-independent DG-dependent isotype PKC-delta , and atypical phosphatidylserine-dependent, Ca2+/DG-independent PKC-zeta (17). Activation of PKC can act as a positive signal in triggering cell responses such as adherence and O<UP><SUB>2</SUB><SUP>−</SUP></UP> generation. PKC-beta is specifically required in positive signaling for activation of the NADPH oxidase for generation of O<UP><SUB>2</SUB><SUP>−</SUP></UP> but not for adherence by HL60 cells (17). However, isotype specificity has not been determined for the negative effects of PKC on Ca2+ signaling. It is not known whether a single PKC isotype is responsible for the inhibition of both intracellular Ca2+ mobilization and uptake of extracellular Ca2+ triggered by PMA or whether different PKC isotypes target different points in Ca2+ signaling.

In this study, an HL60 cell clone, which is protein null for PKC-beta I but positive for PKC-beta II, was used to probe a role for PKC-beta II in regulation of ligand-initiated Ca2+ mobilization (11, 13). An antisense approach was used to selectively deplete PKC-beta II but not PKC-alpha , PKC-delta , or PKC-zeta . Ca2+ uptake triggered by fMet-Leu-Phe, but not mobilization of intracellular Ca2+, was enhanced in PKC-beta -depleted cells. Thus PKC-beta 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-beta . These results indicate that PKC-beta II selectively inhibits Ca2+ uptake via a thapsigargin-insensitive, receptor-operated Ca2+ channel.


    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-alpha , PKC-beta II, PKC-delta , and PKC-zeta but was protein null for PKC-beta 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-beta was used for depletion of PKC-beta in dHL60 cells as previously described (17). The 19-mer oligonucleotides had the following sequences: PKC-beta antisense (beta AS), 5' AGC CGG GTC AGC CAT CTT G-3'; PKC-beta sense (beta 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 beta AS or beta 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-alpha , PKC-beta I, PKC-beta II, and PKC-delta , and peroxidase-conjugated goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal antibody to PKC-zeta was purchased from Transduction Laboratories.


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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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Table 1.   Effect of pretreatment with 1 µg/ml PMA on fMet-Leu-Phe-induced changes in cytosolic Ca2+ in the presence or absence of extracellular Ca2+



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Fig. 1.   Effect of phorbol myristate acetate (PMA) on Ca2+ mobilization. A: effect of the protein kinase C (PKC) activator PMA on Ca2+ mobilization triggered by 1 µM fMet-Leu-Phe (fMLP). Time course of changes in cytosolic Ca2+ in dHL60 cells in response to fMet-Leu-Phe. Cells were preincubated for 5 min at 30°C in the presence of buffer, or 1 µg/ml PMA, before the addition of the stimulus (arrow). Changes in cytosolic calcium triggered by 1 µM fMet-Leu-Phe were monitored as changes in fluorescence of fluo-3-loaded dHL60 cells (see MATERIALS AND METHODS). B: effect of PMA on Ca2+ uptake measured as Mn2+ quenching of fura-2. HL60 Cells preloaded with fura-2 were incubated for 5 min at 30°C in the presence of buffer, or 1 µg/ml PMA, before the addition of buffer or 100 µM MnCl2 and then 1 µM fMet-Leu-Phe (arrow). Changes in fura-2 quenching were monitored as quenching of fluorescence of fura-2 (see MATERIALS AND METHODS). This experiment is representative of n = 5. AFU, arbitrary fluorescence units.

Ca2+ uptake pathways were more directly assessed as fMet-Leu-Phe-induced uptake of Mn2+ into fura-2-loaded cells and quenching of fura-2 fluorescence, measured at the isosbestic point, which is not Ca2+ sensitive. Mn2+ is a good surrogate for Ca2+, since it is not pumped out of the cells and thus serves as a relatively selective monitor for Ca2+ entry (20, 27). Addition of 1 µM fMet-Leu-Phe triggered a prompt increase in Mn2+ influx and thus a decrease in fluorescence of fura-2, which was most rapid in the first minute but which continued over the 5 min monitored after addition of the stimulus (Fig. 1B). fMet-Leu-Phe triggered a loss of fluorescence of 215.6 ± 71.9 arbitrary fluorescence units (AFU) (n = 8) by 1 min in control cells and 602.3 ± 83.5 AFU (n = 8) by 5 min after addition of the stimulus. When the dHL60 cells were pretreated for 5 min with 1 µg/ml PMA, the extent of Mn2+ influx and thus of fluorescence quenching was reduced to only 64.2 ± 30.0 AFU (n = 7) by 1 min and 207.8 ± 70.8 AFU (n = 7) by 5 min after addition of the stimulus, which is significantly less than the quenching attained in control cells (P < 0.025 for 1 min and P < 0.01 for 5 min, paired Student's t-test) (Fig. 1B). Thus PMA, an activator of PKC, inhibited ligand-induced Ca2+(Mn2+) uptake. Therefore, PMA, which is an activator of DG-dependent alpha -, beta -, and delta -isotypes of PKC, but not DG-independent PKC-zeta , inhibited ligand-initiated increases in cytosolic Ca2+ in the presence and in the absence of extracellular Ca2+. These findings implicate a DG-dependent PKC isotype(s) as a negative regulator of ligand-initiated mobilization of intracellular Ca2+ and uptake of Ca2+ from the extracellular milieu. We questioned whether one PKC isotype was responsible for regulation of Ca2+ mobilization, or whether different PKC isotypes selectively regulated specific pathways of Ca2+ mobilization.

Depletion of PKC-beta by an antisense strategy. To test a role for PKC-beta in regulating ligand-initiated Ca2+ mobilization, dHL60 cells were treated with beta -antisense (beta AS) and control beta -missense (beta MS) oligonucleotides for 48 h (see MATERIALS AND METHODS). The HL60 beta I-null cells were shown to express PKC-beta II but not PKC-beta I (Fig. 2A). Previous work demonstrated that these cells also contained alpha -, delta -, and zeta -isotypes of PKC (13). Pretreatment with 400 nM beta AS selectively depleted PKC-beta II (Fig. 2A) but not PKC-alpha , PKC-delta , or PKC-zeta as previously shown (13), in these differentiated HL60 cells. Densitometry demonstrated that the level of PKC-beta II declined from a level of 600 ± 64 density units (DU) (n = 6) in control beta 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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Fig. 2.   Selective depletion of PKC-beta by a PKC-beta antisense oligonucleotide. Densitometry of Western Blots of PKC-beta -depleted, beta AS-treated cells and control, beta MS-treated dHL60 cells. Cells were treated with 400 nM PKC-beta antisense oligonucleotide (AS) or PKC-beta missense oligonucleotide (MS) in the presence of 4 µg/ml 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide/cholesterol (DMRIE-C; see MATERIALS AND METHODS). Cell lysates were prepared by adding Laemmli buffer, and the samples were subjected to 4-12% SDS-PAGE. Western blotting was performed using polyclonal antibodies to PKC-beta I and PKC-beta II (representative experiment of 6). A: Western blots of beta I- and PKC-beta II isotypes in AS- and MS-pretreated dHL60 cells. PKC isotypes are indicated on the right. B: scanning densitometry of the Western Blots of PKC isotypes in beta AS- and beta MS-pretreated dHL60 cells was analyzed by ScanPro and plotted as density units.

Depletion of PKC-beta and Ca2+ mobilization. Resting cytosolic Ca2+ levels were not affected by depletion of PKC-beta by beta AS pretreatment (Table 2). Cytosolic Ca2+ concentration was 119.8 ± 14.4 nM (n = 6) in cells treated with beta AS, compared with a level of 99.9 ± 7.4 nM in control cells pretreated with beta MS (Fig. 3A), and not significantly different from resting cytosolic Ca2+ levels in untreated cells (Fig. 1A). In contrast, antisense depletion of PKC-beta enhanced the fMet-Leu-Phe-triggered increase in cytosolic Ca2+. In cells pretreated with beta 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 beta 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 beta MS-treated cells (P < 0.002).

                              
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Table 2.   Changes in cytosolic Ca2+ triggered by 1 µM fMet-Leu-Phe in control and in beta -PKC-depleted dHL60 cells



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Fig. 3.   Depletion of PKC-beta enhances increases in cytosolic Ca2+ and Ca2+ uptake triggered by 1 µM fMet-Leu-Phe. Cells pretreated with PKC-beta antisense oligonucleotide (beta AS) or PKC-beta missense oligonucleotide (beta MS) were loaded with fluo-3 and incubated for 5 min at 30°C in the presence of buffer containing 1.3 mM Ca2+ or 1.25 mM EGTA, before the addition of fMet-Leu-Phe (arrow). Changes in cytosolic Ca2+ were monitored as fluorescence of fluo-3 (see MATERIALS AND METHODS). A: beta AS- and beta MS-pretreated cells activated by 1 µM fMet-Leu-Phe in the presence of 1.3 mM Ca2+ (representative experiment of n = 6). B: beta AS- and beta MS-pretreated cells activated by 1 µM fMet-Leu-Phe in the presence of 1.25 mM EGTA (representative experiment of n = 4). C: dHL60 cells pretreated with beta AS or beta MS were loaded with fura-2 and incubated in the presence of buffer or 100 µM MnCl2 for 5 min at 30°C before the addition of 1 µM fMet-Leu-Phe (arrow). Changes in Ca2+ were monitored as Mn2+-induced quenching of fura-2 (see MATERIALS AND METHODS). This experiment is representative of 7.

In the presence of 1.25 mM EGTA, where cytosolic Ca2+ changes were only from intracellular stores, resting cytosolic Ca2+ was 69.0 ± 3.7 nM (n = 5) and 65.6 ± 7.3 nM (n = 5) in beta AS- and beta MS-pretreated cells, respectively (Table 2). When the cells were activated by addition of 1 µM fMet-Leu-Phe (Fig. 3B), cytosolic Ca2+ rose to peak values of 121.0 ± 6.0 nM (n = 5) and 113.4 ± 5.6 nM (n = 5) in beta AS- and beta MS-pretreated cells, respectively, a difference that was not statistically significant. Thus no alteration in ligand-initiated mobilization of intracellular Ca2+ stores was observed in PKC-beta -depleted cells. The increase in peak cytosolic Ca2+ observed in the presence of extracellular Ca2+ in PKC-beta -depleted cells may reflect an increase in Ca2+ uptake from extracellular sources.

We next determined whether PMA still retained the ability to downregulate Ca2+ mobilization in PKC-beta -depleted dHL60 cells. Pretreatment with PMA had no effect on resting cytosolic Ca2+ concentration, but in control beta MS-pretreated cells the peak cytosolic Ca2+ level was 198.8 ± 25.3 nM (n = 5) when cells were pretreated for 5 min with PMA (Table 2), compared with a peak level of 270.2 ± 15.9 nM (n = 6) in beta MS cells not exposed to PMA [72.2 ± 8.5% (n = 5) of control beta MS-treated cells, P < 0.02]. When dHL60 cells were depleted of PKC-beta by treatment with beta AS, pretreatment with PMA for 5 min significantly reduced the fMet-Leu-Phe-induced peak cytosolic Ca2+ to 202.0 ± 24.8 nM (n = 5), a level that was similar to the level of 198.8 ± 25.3 nM observed in PMA-treated beta MS-treated cells (Table 2) but that was significantly reduced from the peak level of 374.0 ± 16.5 nM observed in beta AS-treated cells in the absence of PMA [54.1 ± 7.4% (n = 5) of control beta AS-treated cells, P < 0.0002] (Table 2). These findings suggest that alterations in cytosolic Ca2+ in PKC-beta -depleted cells were due to changes in ligand-initiated uptake of extracellular Ca2+ and not due to alterations in mobilization of intracellular Ca2+ stores. Furthermore, the ability of PMA to inhibit ligand-initiated release of intracellular Ca2+ stores was retained in PKC-beta -depleted cells. Peak cytosolic Ca2+ in the presence of EGTA was reduced from 121.0 ± 6.0 to 89.3 ± 12.0 nM after pretreatment by PMA (Table 2). These findings indicate a possible role for PKC-alpha or PKC-delta in the regulation of fMet-Leu-Phe-induced release of Ca2+ from intracellular Ca2+ stores.

The role of PKC-beta in regulation of ligand-induced Ca2+ uptake pathways was more directly investigated measuring Ca2+ uptake as Mn2+-induced fluorescence quenching in fura-2-loaded dHL60 cells. Greater quenching of fura-2 fluorescence in response to fMet-Leu-Phe was observed in PKC-beta -depleted (beta AS) cells compared with control (beta MS) cells (Fig. 3C). fMet-Leu-Phe triggered a loss of fluorescence by 1 min after addition of the stimulus of 113.0 ± 49.9 AFU (n = 6) in control beta MS-pretreated cells, while in PKC-beta -depleted cells (beta AS) the decrease in fluorescence was 219.7 ± 51.8 AFU (n = 6) (Fig. 3C). The loss of fluorescence by 5 min after addition of fMet-Leu-Phe was 507.7 ± 73.3 AFU (n = 6) in control beta MS-pretreated cells, whereas in PKC-beta -depleted cells (beta AS) the decrease in fluorescence was increased to 850 ± 136.0 AFU (n = 6), an increase that was significantly increased compared with the response in control cells (P < 0.04 paired t-test) (Fig. 3C). Therefore, fMet-Leu-Phe-induced Ca2+ uptake was enhanced in PKC-beta -depleted dHL60 cells, further implicating PKC-beta in negative regulation of ligand-induced Ca2+ uptake.

Ligand initiated generation of IP3 and PKC-beta . 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 beta 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-beta -depleted cells (pretreated with beta 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 beta AS-pretreated cells was higher than in control cells, the difference was not statistically significant. Thus PKC-beta does not regulate ligand-initiated activation of phospholipase Cbeta and generation of IP3, a finding that is concordant with the demonstration that PKC-beta does not regulate release of intracellular Ca2+ stores.


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Fig. 4.   Effect of depletion of PKC-beta on generation of inositol 1,4,5-trisphosphate (IP3) triggered by 1 µM fMet-Leu-Phe in dHL60 cells. dHL60 cells were treated with beta AS or beta MS oligonucleotides (see MATERIALS AND METHODS). dHL60 cells were treated with 1 µM fMet-Leu-Phe for 0, 5, 15, 30, or 60 s. Levels of IP3 were measured by an IP3 binding assay (see MATERIALS AND METHODS). Results are expressed as pmol IP3/106 cells and are means ± SE (n = 4).

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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Fig. 5.   Effect of PMA on thapsigargin-induced changes in cytosolic Ca2+ and in Ca2+ uptake. A: dHL60 cells were loaded with fluo-3 and incubated for 5 min at 30°C in the presence or absence of 1 µg/ml PMA in Ca2+-free buffer, before the addition of 100 nM thapsigargin (arrow), and after a further 4 min of 1.25 mM Ca2+. Changes in cytosolic Ca2+ were monitored as fluorescence of fluo-3. This experiment is representative of 3. B: dHL60 cells preloaded with fura-2 were incubated for 5 min at 30°C in the presence of buffer or 1 µg/ml PMA, before the addition 100 µM MnCl2 and then 100 nM thapsigargin (arrow). Changes in Ca2+ uptake were monitored as quenching of fluorescence of fura-2 (see MATERIALS AND METHODS). This experiment is representative of 3.

When Ca2+ uptake was measured as Mn2+ uptake in fura-2-loaded cells, a slow but persistent Ca2+ uptake of 2,255 ± 159 AFU/5 min (n = 5) was triggered in dHL60 cells by 100 nM thapsigargin (Fig. 5B). When the cells were preincubated for 5 min with 1 µg/ml PMA, the thapsigargin-initiated Ca2+ uptake was reduced to 1,328 ± 287 AFU/5 min (P < 0.02 paired Student's t-test), indicating that a PKC isotype(s) activated by PMA could act as a negative regulator of SOCC.

Depletion of PKC-beta and Ca2+ uptake triggered by thapsigargin. The role of PKC-beta in regulation of thapsigargin-induced Ca2+ uptake was investigated using dHL60 cells pretreated with beta AS and beta MS. Thapsigargin-induced Ca2+ uptake was measured in fluo-3-loaded cells (Fig. 6A). In control dHL60 cells treated with beta 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-beta -depleted dHL60 cells treated with beta 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-beta by beta AS and the control cells treated with beta MS.


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Fig. 6.   Effect of depletion of PKC-beta on Ca2+ uptake triggered by thapsigargin. A: dHL60 cells were pretreated with beta AS or beta MS and then loaded with fluo-3 and monitored for 5 min at 30°C in buffer, followed by addition of 100 nM thapsigargin (arrow). Changes in cytosolic Ca2+ were monitored as fluorescence of fluo-3. This experiment is representative of 4. B: dHL60 cells were pretreated with beta AS or beta MS, were loaded with fura-2, and incubated in the presence of 100 µM MnCl2 for 5 min at 30°C before the addition of 100 nM thapsigargin (arrow). Changes in Ca2+ uptake were monitored as Mn2+-induced quenching of fura-2 (see MATERIALS AND METHODS). This experiment is representative of 4.

Similarly, when Ca2+ uptake triggered by thapsigargin-induced opening of the SOCC was measured as Mn2+ uptake into fura-2-loaded cells, an equivalent decrease in fluorescence triggered by 100 nM thapsigargin was observed in PKC-beta -depleted (beta AS) cells compared with control (beta MS) cells (Fig. 6B). Thapsigargin triggered a loss of fluorescence of 1,736 ± 371 AFU/5 min in control beta MS-treated cells, whereas in PKC-beta -depleted cells (beta AS) the decrease in fluorescence was 1,896 ± 371 AFU/ 5 min (113.6 ± 8.1% control, n = 5), a difference that was not statistically significant. Therefore, PKC-beta is a negative regulator of ligand-initiated Ca2+ uptake. However, PKC-beta has no direct effect on SOCC, since thapsigargin-induced Ca2+ uptake through SOCC was not affected in PKC-beta -depleted dHL60 cells. Thus PKC-beta was not responsible for the PMA-induced inhibition of SOCC. The increase in Ca2+ uptake observed in PKC-beta -depleted dHL60 cells occurs through a thapsigargin-insensitive channel such as the receptor-operated nonselective cation channel.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cbeta 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-alpha , PKC-beta , and PKC-delta , 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-alpha , PKC-beta I, PKC-beta II, PKC-delta , and PKC-zeta . In this study, we have demonstrated a role for PKC-beta 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-beta II from a clone of HL60 cells, which is protein null for PKC-beta I but positive for alpha -, beta II-, delta -, and zeta -isotypes of PKC. Selective depletion of PKC-beta , i.e., PKC-beta II, enhanced the fMet-Leu-Phe-triggered increase in cytosolic Ca2+ in the presence of extracellular Ca2+. In contrast, depletion of PKC-beta II had no effect on fMet-Leu-Phe-elicited increase in cytosolic Ca2+ in the absence of extracellular Ca2+, indicating that PKC-beta 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 Cbeta (2, 39) and cleavage of PIP2 to generate IP3. Phospholipase Cbeta 2 is a substrate for PKC (5). The finding that generation of IP3 was not affected by PKC-beta II depletion is concordant with our findings that ligand-initiated release of intracellular Ca2+ stores was not affected by PKC-beta II depletion. This finding indicates that any effect of PKC-beta II must occur independently of phospholipase Cbeta activation. Furthermore, the PMA-induced inhibition of release of intracellular Ca2+ stores still occurred in PKC-beta -depleted cells, demonstrating that the negative effect of PMA on Ca2+ mobilization was not solely dependent on PKC-beta and may be due to another PKC isotype such as PKC-alpha or PKC-delta or to a non-PKC-dependent mechanism. Indeed, a role for PKC-alpha has been demonstrated in negative regulation of phospholipase C in visual signal transduction in Drosophila (25).

A role for PKC-beta II in negative regulation of Ca2+ uptake was confirmed by the finding that PKC-beta 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-beta 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-beta II. Therefore, the SOCC per se is not regulated by PKC-beta II. Thus PKC-beta 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-beta 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-beta II in the negative regulation of ligand-initiated Ca2+ uptake but not mobilization of intracellular Ca2+ stores. Since modulation of PKC-beta II affected receptor-operated Ca2+ uptake but had no effect on thapsigargin-initiated Ca2+ uptake, these findings suggest a role for PKC-beta II in the signaling for activation of Ca2+ uptake through a receptor-operated nonselective cation channel rather than the SOCC. PKC-beta II is involved in positive signaling for O<UP><SUB>2</SUB><SUP>−</SUP></UP> generation (17) but also plays a role in signaling crosstalk in which activation of PKC also downregulates Ca2+ uptake. Ca2+ uptake leads to localized changes in Ca2+ concentration, so that modulation of Ca2+ uptake could regulate cell function at the membrane. Downregulation of Ca2+ uptake could serve as a turn-off mechanism for Ca2+-dependent responses such as O<UP><SUB>2</SUB><SUP>−</SUP></UP> generation or serve as an essential element in signaling for functions such as cell shape changes and chemotaxis (8).


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant AI-24840.


    FOOTNOTES

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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