INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Protein kinase C (PKC)¹ mediates signaling for multiple functions of phagocytic cells, neutrophils, monocytes, macrophages, and differentiated HL60 (dHL60) cells. Ligands such as the chemotactic peptide fMet-Leu-Phe and phagocytic stimuli (immune complexes) trigger responses that include O₂ generation, degranulation, adherence, and actin filament assembly (1-8). These functions are essential for the microbicidal activity of phagocytic cells and are also proinflammatory.

PKC, a phospholipid-dependent family of serine/threonine kinases, acts in multiple signal transduction pathways. The cofactor requirements differ between different classes of PKC isotypes. Classical -, -, and -PKC are acidic phospholipid, diglyceride (DG), and Ca²⁺-dependent; novel forms -, -, -, and -PKC also require acidic phospholipid and DG, but are Ca²⁺ independent. The atypical PKC isotypes, - and -PKC, require PS but are DG and Ca²⁺ independent (9-14). PKC isotypes differ in their tissue distribution and localization within the cell, suggesting that each isotype plays a specific role in signal transduction.

Neutrophils, monocytes/macrophages, and dHL60 cells contain multiple isotypes of PKC, including Ca²⁺-dependent isotypes -PKC, I-PKC, and II-PKC, Ca²⁺-independent DG-dependent isotype, -PKC, and atypical PS-dependent, Ca²⁺/DG-independent -PKC (3, 14-16). PKC has been implicated in the signaling for several different responses of phagocytic cells because PMA triggers O₂ generation and adherence but not the release of azurophil granules (1, 17). The PKC substrates involved in these processes are largely unknown. The cytoskeleton and integrins are involved in cell adherence, and several cytoskeletal proteins, including MARCKS, are PKC substrates (8). Assembly of an active NADPH oxidase for generation of O₂ requires translocation of cytosolic factors p47^phox, p67^phox, and rac2 to the plasma membrane, where they interact with the integral membrane protein cytochrome b₅₅₈ (18-25). p47^phox is phosphorylated in ligand-activated phagocytic cells. p47^phox contains multiple phosphorylation sites, including a number of classical PKC substrate sites (RXXS/TXRX) and is phosphorylated by -PKC in vitro (3). In addition, p47^phox phosphorylated in vitro by PKC, but not cAMP-activated kinase or mitogen-activated kinase, was active in a cell-free system for activation of the NADPH oxidase (4-7). However, in vitro activity does not necessarily predict a role for a particular PKC isotype in the intact cell, where access to both substrate and cofactors is critical in controlling signaling specificity. Specific functions for each PKC isotype remain to be established.

In the present study, an antisense strategy was used to probe a role for a specific PKC isotype in signaling for the ligand-activated responses of O₂ generation and cell adherence in dHL60 cells. -PKC was selectively depleted by an antisense strategy in dHL60 cells. Selective depletion of -PKC in dHL60 cells decreased O₂ generation but not degranulation or adherence. Depletion of -PKC reduced ligand-induced phosphorylation of a subset of proteins including p47^phox and reduced translocation of p47^phox to the membrane, concordant with a selective role for -PKC in signaling for assembly of an active NADPH oxidase.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Reagents-- Cytochalasin B, cytochrome c, phenolphthalein glucuronidate, protease inhibitors (leupeptin and aprotinin), BSA, PMA, fMet-Leu-Phe, and PMSF were purchased from Sigma. PMA was stored as a concentrated stock solution in Me₂SO and diluted with Krebs-Ringer buffer before use. fMet-Leu-Phe was stored as a stock solution in ethanol and diluted in buffer prior to use. BSA and anti-BSA IgG (Cappel, Durham, NC) were used to form an immune complex by the method of Ward and Zvaifler (26).

HL60 Cell Culture-- Human promyelocytic HL60 leukemic cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in suspension culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% minimal essential medium vitamin solution, 0.1% gentamicin, and 10% heat-inactivated fetal bovine serum. The cell cultures were maintained at 37 °C in a 5% CO₂ humidified atmosphere.

Oligonucleotide Synthesis and Sequences-- An antisense oligonucleotide was designed against the translation start site of human -PKC using the commercial primer analysis software Oligo (National Biosciences). Since I-PKC and II-PKC differ only in the 3' terminus, this oligonucleotide should target both forms of -PKC. A 19-mer sequence was chosen, which was without significant self-complimentarity and did not dimerize. The chosen sequence was also optimized for maximal Tm to promote high affinity binding to mRNA; a T_m of 70.0 °C was calculated at 150 mM salt and 37 °C. 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'. The unique nature of these sequences was confirmed by searching the GenBank data base. Antisense, sense, and scrambled control oligonucleotides were synthesized by the PENN Nucleic Acid Facility as the phosphorothioate derivatives and purified by high pressure liquid chromatography. In all oligonucleotides, the internucleoside linkages were completely phosphorothioate-modified.

Treatment of Cells with Oligonucleotides-- Delivery of the oligonucleotides was enhanced equally with cationic lipids DMRIE-C (1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/cholesterol (1:1 (M/M))) and Lipofectin (Life Technologies, Inc.); however, DMRIE-C was chosen since this lipid lacks dioctanoyl phosphatidylethanolamine, which can be toxic to phagocytic cells, and allowed optimal retention of cell functions (27). HL60 cells were cultured in the presence of 1.3% Me₂SO 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 (Life Technologies, Inc.) at a cell concentration of 25 × 10⁶ cells/well. Oligonucleotides AS, SS, or MS (a scrambled AS oligonucleotide) were suspended in Opti-MEM at a final concentration of 100-1000 nM and mixed with the cationic lipid DMRIE-C (2.5 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 fetal bovine serum plus Me₂SO (1.3% final concentration) was then added, and the cells cultured for 20 h. On day 5, the cells were washed and resuspended in fresh Opti-MEM and treated again with the cationic lipid/oligonucleotide mixture. Following 4 h of incubation, an equal volume of RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum plus Me₂SO (1.3% final concentration) was then added, and the cells were cultured for an additional 24 h. The cells were harvested and suspended in Krebs-Ringer phosphate-buffered saline, pH 7.4, containing 1.2 mM Mg²⁺ and 1.3 mM Ca²⁺.

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 4-16% gradient SDS-PAGE, transferred to PVDF 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, the membrane was incubated 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. The software SigmaProscan (Jandel/SPSS) was used for densitometric analysis; rhPKC isotypes (Pan Vera) were used as standards for quantitation.

Protein Kinase C Assay-- Cytosol fractions were prepared by sonicating dHL60 cells in 50 mM Tris-HCl, pH 7.5, containing 2 mM PMSF, 33 µM leupeptin, 35 µM antipain, 24 µg/ml chymostatin, 35 µM pepstatin, and 0.48 TIU/ml aprotinin, 10 mM EGTA, and 50 mM mercaptoethanol, as described previously (2), and by centrifuging for 20 min at 115,000 × g at 4 °C. PKC activity of cytosol fractions was assayed in the presence of Ca²⁺, PS, and DG by measuring the incorporation of ³²P into histone type IIIS or into PKC substrate peptide [Ser²⁵]PKC(19-31) (2). The net Ca²⁺-dependent activity was obtained by subtraction of the Ca²⁺-independent, PS/DG activity from the Ca²⁺/PS/DG-dependent activity.

Superoxide Anion Generation-- The generation of superoxide anion (O₂) by dHL60 cells was measured as superoxide dismutase-inhibitable cytochrome c reduction by either a continuous recording method (28) or end point analysis. Cells were activated by 1 µM fMet-Leu-Phe in the presence of 5 µg/ml cytochalasin B, or by 1 µg/ml PMA, or 300 µg/ml BSA/anti-BSA immune complex in the absence of cytochalasin B.

Degranulation-- The release of the azurophil granule-associated enzyme -glucuronidase triggered by fMet-Leu-Phe from dHL60 cells was measured in the presence of 5 µg/ml cytochalasin B to allow extracellular release of granule contents. Degranulation triggered by insoluble immune complex, BSA/anti-BSA, was measured in the absence of cytochalasin B. dHL60 cells (5 × 10⁶ cells/ml) were incubated for 5 min at 37 °C with buffer, fMet-Leu-Phe (1 µM) or BSA/anti-BSA (300 µg/ml). -Glucuronidase was determined by a microplate assay using phenolphthalein glucuronidate as substrate (29, 30). Total enzyme activities were determined simultaneously in reaction mixtures containing the detergent Triton X-100 (0.2%) and using phenolphthalein to calibrate the nmoles of cleaved substrate.

Adherence-- Adherence of dHL60 cells to fibronectin-coated plates was determined by a colorimetric assay according to the method of Aumailley et al. (31). 96-well microtiter plates were coated with fibronectin as described by Nathan et al. (32). dHL60 cells (4 × 10⁵ cells/well) were incubated for 30 min at 37 °C with either buffer, fMet-Leu-Phe (1 µM) or PMA (1 µg/ml). Nonadherent cells were removed by washing with Krebs-Ringer phosphate-buffered saline buffer, and the adherent cells were fixed with 70% ethanol. The bound cells were stained with crystal violet (0.1% in dH₂O). The plates were washed extensively to remove excess stain, and the cells were solubilized with 1% Nonidet P-40 in dH₂O. Optical density was read at 550 nm.

Protein Phosphorylation in Activated dHL60 Cells-- dHL60 cells (50 × 10⁶ cells/ml) were incubated for 60 min at 37 °C with ³²P-P_i (250 µCi of [³²P]orthophosphoric acid/ml). The ³²P-labeled cells were stimulated with either buffer alone or fMet-Leu-Phe (1 µM) for 1 min in the presence of 5 µg/ml cytochalasin B. The reaction was stopped by placing the samples on ice. Cell lysates were prepared and run on 4-12% gradient SDS-PAGE. The gel was dried and subjected to autoradiography.

Immunoprecipitation of p47^phox-- dHL60 cells (10 × 10⁶ cells/ml) were incubated for 60 min at 37 °C with ³²P-P_i (250 µCi of [³²P]orthophosphoric acid/ml). The ³²P-labeled cells were stimulated with either buffer alone, PMA (1 µg/ml) or fMet-Leu-Phe (1 µM), for 10 min. The reaction was stopped by the addition of cold immunoprecipitation (IP) buffer. IP buffer consisted of 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 2 mM PMSF, 0.2% Nonidet P-40, 0.027 TIU/ml aprotinin, 2 µg/ml leupeptin, and 5 mg/ml BSA. The samples were then vortexed for 20 min to solubilize the membrane fraction, and the supernatant was collected after microfuging for 5 min. A rabbit polyclonal antibody to p47^phox was added and incubated overnight at 4 °C. Protein A-agarose was added and incubated for 1 h at 4 °C with shaking. The reaction tubes were then microfuged for 30 s, and the supernatants were discarded. The protein A-agarose pellet was washed four times with IP buffer, and the sample was eluted by incubation for 20 min at 65 °C in 2 × SDS-PAGE sample buffer.

Preparation of Particulate Fractions from fMet-Leu-Phe-stimulated dHL60 Cells and Translocation of p47^phox and PKC Isotypes to Cell Membranes-- dHL60 cells (2.5 × 10⁷ cells/ml) were incubated at 37 °C in the presence of buffer, 1 µM fMet-Leu-Phe (1 min) or 1 µg/ml PMA (5 min). At the end of the incubation period, the cell suspension was transferred to an ice bath. The suspension was then centrifuged for 10 min at 300 × g, and the cell pellet was resuspended in buffer containing 131 mM NaCl, 1 mM EGTA, 100 mM potassium phosphate buffer, pH 7.0, 2 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 20 µg/ml pepstatin (buffer A). (24). The cells were disrupted using three 5-s bursts of a microprobe sonicator at low power at 4 °C and centrifuged at low speed (500 × g, 5 min) to remove unbroken cells and nuclei. The supernatant was layered over a 15% sucrose cushion made up in buffer A and centrifuged at 4 °C for 20 min at 115,000 × g. The supernatant (cytosol) was mixed 4:1 with 4 × SDS sample buffer. The pellet was solubilized in hot Laemmli buffer; pellet (10 × 10⁶ cell equivalents/lane) and cytosol (3 × 10⁶ cell equivalents/lane) were run on 4-12% SDS-PAGE and probed by Western blotting.

Statistical Analysis-- Results are expressed as mean ± S.E. (n). Data were analyzed by Student's one-tailed t test for paired samples.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Depletion of -PKC Isotypes by Antisense Treatment-- To deplete -PKC, dHL60 cells were first treated with 1.3% Me₂SO for 4 days to initiate differentiation, followed by two treatments with -PKC antisense oligonucleotide (AS) and the cationic lipid DMRIE-C (2.5 µg/ml) at 24-h intervals (see "Materials and Methods"). Preliminary experiments demonstrated that cationic lipids were essential to enhance the potency of the oligonucleotides (33, 34). Phosphorothioate oligonucleotides were synthesized since these have a greater intracellular half-life than the native oligonucleotides. Since the half-life of PKC is long, reported to vary from 6.7 h to over 24 h, prolonged treatment with AS was used (35). The cells were treated twice with the AS/DMRIE-C mixture to prolong the exposure to AS and to achieve decreased protein expression of -PKC (35); a single treatment with AS/DMRIE-C was not effective. The effect of antisense treatment on dHL60 cells following differentiation was examined over a concentration range of 0-1000 nM AS (Fig. 1). Treatment with AS resulted in decreased expression of -PKC (Fig. 1). Depletion of -PKC followed a bell-shaped concentration response curve; maximal depletion of -PKC immunoreactivity occurred at 250 and 500 nM AS (Fig. 1). For this experiment, -PKC was reduced to 23 and 31% of control levels by 250 and 500 nM AS, respectively. At higher concentrations of AS oligonucleotide, there was increased -PKC expression. Attempts to enhance the depletion of -PKC by more prolonged incubation or by increases in cationic lipid levels were not successful due to a loss of cell viability.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of -PKC antisense oligonucleotide concentration on depletion of -PKC in neutrophilic HL60 cells. dHL60 cells were treated with 0, 100, 250, 500, or 1000 nM -PKC AS oligonucleotide (AS) for 48 h (see "Materials and Methods"). Cell lysates were prepared by adding Laemmli buffer, and the samples were subjected to 4-16% SDS-PAGE followed by Western blotting (representative experiment of three). -PKC is indicated by an arrow on the right.

Selective Depletion of -PKC and Not -PKC, -PKC, or -PKC-- dHL60 contained numerous PKC isotypes, -PKC, _I-PKC, _II-PKC, -PKC, and -PKC, in agreement with other workers (Fig. 2A) (15). The predominant isotypes were -, II-, and -PKC, whereas I-PKC and -PKC were present at lower levels (Fig. 2A). No evidence for -PKC (Fig. 2A), -PKC, -PKC, or -PKC (results not shown) was found in dHL60 cells. dHL60 cells were treated for 48 h with 300 nM -PKC antisense (AS) and -PKC missense oligonucleotide (MS) in the presence of DMRIE-C (2.5 µg/ml). Selectivity of depletion of -PKC was assessed by probing the blot with antibodies to -PKC, I-PKC, II-PKC, -PKC, and -PKC. Both -PKC isotypes, _I-PKC and _II-PKC, were effectively depleted by AS treatment but not by MS treatment (Fig. 2B) or in sense oligonucleotide-treated cells (results not shown). I-PKC was reduced to 45.6 ± 5.3% (n = 10) of control (MS) levels, and II-PKC was reduced to 60.8 ± 4.4% (n = 10) of control levels. In contrast, a concentration of AS, which was effective in depleting -PKC, had no effect on the expression of other PKC isotypes including -PKC, -PKC, and -PKC (Fig. 2B). Calcium/PS/DG-dependent histone IIIs phosphorylating activity was 21.6 pmol/10⁷ cell equivalents/min in cytosol from control MS-treated cells; calcium/PS/DG-dependent phosphorylating activity in -PKC-depleted cells was reduced to 73.6 ± 1.7% (n = 3, p < 0.001) of control activity. This finding demonstrates that AS depleted, but did not abolish, calcium-dependent PKC activity. Thus, treatment with the -PKC antisense oligonucleotide selectively depleted dHL60 cells of -PKC but not calcium-dependent -PKC or calcium-independent -PKC and -PKC.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   An antisense -PKC oligonucleotide selectively decreases -PKC expression. A, PKC isotypes in neutrophilic HL60 cells. Cell lysates were prepared from dHL60 cells, run on a 4-15% gradient SDS-PAGE, and blotted to PVDF membrane. Western blotting was performed using polyclonal antibodies to -, I-, II-, -, and -PKC and a monoclonal antibody to -PKC. B, selective depletion of -PKC by a -PKC antisense oligonucleotide. dHL60 cells were treated with 300 nM -PKC antisense oligonucleotide (AS) or -PKC missense oligonucleotide (MS) in the presence of 2.5 µg/ml DMRIE-C (see "Materials and Methods"). Cell lysates were prepared by adding Laemmli buffer, and the samples were subjected to 4-16% SDS-PAGE followed by Western blotting (representative experiment of four). Molecular weight markers are indicated on the left, and PKC is indicated by an arrow on the right.

Effect of -PKC Depletion on Superoxide Anion Generation by dHL60 Cells-- Activation of dHL60 cells by a variety of stimuli elicits the assembly of an active NADPH oxidase enzyme complex, which generates superoxide anion (O₂). Previous studies suggested that -PKC may play an important role in the activation of the NADPH oxidase (3, 20). Therefore, the effect of -PKC depletion on O₂ generation triggered by different ligands was evaluated. The PKC activator PMA (1 µg/ml) triggered continuous generation of O₂ by dHL60 cells following a lag period of approximately 1-3 min (Fig. 3A). The mean lag period for control MS-treated cells was 205 ± 55 s (n = 5); the lag period was significantly increased to 222 ± 43% (n = 5) of control (p < 0.05) in AS-treated cells. Thus, depletion of -PKC delayed the onset of O₂ generation in PMA-activated cells. Control MS-treated cells generated 33.5 ± 6.2 nmol (n = 5) O₂/10⁶ cells/15 min (Fig. 3C). Treatment of cells with DMRIE-C/oligonucleotide was not toxic to the cells since the rate of O₂ generation in the absence of DMRIE-C/oligonucleotide was 39.14 ± 5.7 (n = 4) nmol O₂/10⁶ cells/15 min as compared with the rate of 33.50 ± 6.2 (n = 5) nmol O₂/10⁶ cells/15 min in DMRIE-C/MS-treated cells, a difference that is not significant. When dHL60 cells were treated with AS (Fig. 2B), PMA-elicited O₂ production was significantly decreased to a level of 25.1 ± 6.2 (n = 5) nmol O₂/10⁶ cells/15 min (p < 0.01), a level that was 71.6 ± 7.8% of control (p < 0.02) (Fig. 3C). In addition, the V_max of PMA-induced O₂ generation, defined as the maximal rate of O₂ generation, was reduced in cells depleted of -PKC. Calculation of the V_max demonstrated that in MS-treated cells activated by 1 µg/ml PMA, the V_max was 2.51 ± 0.80 (n = 5) nmol/min/10⁶ cells; the V_max of -PKC-depleted cells was significantly reduced to 2.04 ± 0.70 nmol/min/10⁶ cells (n = 5), which was 75.8 ± 7.8% of control MS-treated cells (p < 0.04).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Depletion of -PKC inhibits O2 generation by dHL60 cells. Characterization of O2 generation by dHL60 cells depleted of -PKC. O2 generation measured as the superoxide dismutase-inhibitable reduction of cytochrome c (see "Materials and Methods") in missense (MS) and antisense (AS) pretreated dHL60 cells. A, time course of O2 generation triggered by 1 µg/ml PMA. B, time course of O2 generation triggered by 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B. C, O2 generation triggered by 1 µg/ml PMA, 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B, and 300 µg/ml BSA/anti-BSA immune complex. Data shown are representative of five experiments (PMA), eight experiments (fMLP), and three experiments (BSA/anti-BSA) and are expressed as nmol O2/106 cells/15 min.

Depletion of -PKC Isotypes Does Not Inhibit fMet-Leu-Phe-elicited Degranulation by dHL60 Cells-- Activation of dHL60 cells with ligands such as fMet-Leu-Phe triggers release of -glucuronidase, an azurophil granule marker, simultaneously with the generation of O₂. The effect of depletion of -PKC on the extracellular release of -glucuronidase was monitored in dHL60 cells following stimulation with 1 µM fMet-Leu-Phe. Treatment of dHL60 cells with AS plus DMRIE-C, at a concentration that significantly depleted levels of -PKC and inhibited O₂ production in response to fMet-Leu-Phe, had no significant effect on degranulation (Fig. 4); 10.5 ± 2.7% (n = 4) of total -glucuronidase was released in response to fMet-Leu-Phe in control (MS-treated) cells, whereas 12.2 ± 2.5% (n = 4) of total -glucuronidase was released from -PKC-depleted cells. Similarly, degranulation triggered by BSA/anti-BSA in the absence of cytochalasin B was not affected by depletion of -PKC; 7.5 ± 0.2% (n = 4) of total -glucuronidase was released in response to BSA/anti-BSA in control (MS-treated) cells, whereas 8.3 ± 0.6% (n = 4) of total -glucuronidase was released from -PKC-depleted cells. Furthermore, total cell content of -glucuronidase, a marker for HL60 cell differentiation, was not altered in -PKC-depleted cells (Fig. 4). Therefore, depletion of -PKC with a -PKC antisense oligonucleotide selectively inhibits ligand-elicited O₂ generation but not -glucuronidase release.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Release of -glucuronidase by dHL60 cells depleted of -PKC. The effect of depletion of -PKC on the release of -glucuronidase was monitored in missense (MS) and antisense (AS) pretreated dHL60 cells (see "Materials and Methods"). dHL60 cells were preincubated for 5 min at 37 °C before addition of 1 µM fMet-Leu-Phe; cytochalasin B was present at 5 µg/ml in all tubes. Results are expressed as nmol of glucuronate released/106 cells, mean ± S.E. (n = 4).

Effect of PKC Depletion on Adherence of dHL60 Cells to Fibronectin-- A role for PKC in integrin-mediated adherence to fibronectin has been suggested since phorbol esters activate adherence mechanisms in phagocytic cells (7, 36). To ascertain whether -PKC plays a role in integrin-mediated adherence in dHL60 cells, the effect of -PKC depletion on basal and stimulated adherence to fibronectin was examined in dHL60 cells. Activation of control (MS-treated) cells by PMA (1 µg/ml) or fMet-Leu-Phe (1 µM) triggered increased cell adherence to fibronectin-coated wells (Fig. 5). PMA produced the greatest increase in adherence, to 242.1 ± 64.0% (n = 4) of resting levels in control (MS-treated) cells, whereas in -PKC-depleted cells, a similar level of PMA-induced adherence, 246.8 ± 40.0% (n = 4) of resting levels, was observed. fMet-Leu-Phe (1 µM) triggered increased adherence to 175.1 ± 41.5% (n = 3) of resting levels in control (MS-treated) cells, whereas in -PKC-depleted cells, a similar level of fMet-Leu-Phe-induced adherence, 214.38 ± 62.5% (n = 4) of resting levels, was observed. As shown in Fig. 5, AS treatment under conditions that significantly altered fMet-Leu-Phe and PMA-induced O₂ production had no significant effect on cell adherence to fibronectin in unstimulated or stimulated cells. Thus, depletion of -PKC in dHL60 cells elicits selective inhibition of O₂ generation but not -glucuronidase release or adherence to fibronectin.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effect of depletion of -PKC on adherence of dHL60 cells to fibronectin. Adherence of dHL60 cells to fibronectin-coated plates was determined in missense (MS) and antisense (AS) pretreated dHL60 cells (see "Materials and Methods"). dHL60 cells were preincubated for 5 min at 37 °C before addition of buffer, 1 µg/ml PMA or 1 µM fMet-Leu-Phe, followed by incubation for 30 min at 37 °C. Data are representative of six experiments for PMA and three experiments for fMet-Leu-Phe.

Effect of -PKC Depletion on Protein Phosphorylation Triggered by 1 µM fMet-Leu-Phe-- AS- and MS-treated dHL60 cells were labeled with ³²P inorganic phosphate for 1 h at 37 °C and treated with buffer or fMet-Leu-Phe. Cell lysates were prepared, the phosphorylated proteins were separated on SDS-PAGE, and the gels were subjected to autoradiography. Activation of the cells for 1 min with 1 µM fMet-Leu-Phe triggered phosphorylation of multiple proteins, including 18-, 24-, 38-, 43-, 47-, 54-, 68-, and 80-kDa proteins (Fig. 6A). Phosphorylation of prominent 14-, 97-, and 105-kDa bands was not significantly altered in fMet-Leu-Phe-activated cells (Fig. 6A). Densitometry of the autoradiograph demonstrated that treatment of cells with antisense to -PKC reduced the fMet-Leu-Phe-induced phosphorylation of the 18-, 38-, 43-, 47-, 54-, and 68-kDa proteins (Fig. 6B); the 47-kDa band is a candidate for p47^phox. In contrast, phosphorylation of a band at 105 kDa was enhanced in -PKC-depleted cells. The effect of -PKC depletion on fMet-Leu-Phe-induced protein phosphorylation was selective, since treatment with AS had no significant effect on the fMet-Leu-Phe-induced phosphorylation of the 24- or 80-kDa bands. These results suggest that depletion of -PKC inhibits fMet-Leu-Phe-induced phosphorylation of a discrete number of proteins including a 47-kDa band, which is a candidate for p47^phox, an important component of the NADPH oxidase.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of depletion of -PKC on protein phosphorylation in activated dHL60 cells. dHL60 cells were treated with -PKC antisense (AS) or missense (MS) oligonucleotides (see "Materials and Methods") and then labeled with 250 µCi 32Pi/ml for 1 h at 37 °C. Cells were pretreated with 5 µg/ml cytochalasin B for 1 min and then stimulated with buffer (control) or 1 µM fMLP for 1 min. The cells were solubilized in Laemmli buffer and subjected to 4-12% SDS-PAGE followed by autoradiography (representative of three experiments). A, autoradiography of gel. Lane 1, AS-pretreated control cells; lane 2, MS-pretreated control cells; lane 3, AS-pretreated fMLP-stimulated cells; and lane 4, MS-pretreated fMLP-stimulated cells. Molecular weight markers are indicated on the left margin, and phosphorylated bands are indicated on the right margin. B, scanning densitometry of the autoradiograph was analyzed by ScanPro and plotted as density versus molecular weight. AS-pretreated control cells and MS-pretreated control cells are shown in the upper panel, and AS-pretreated fMLP-stimulated cells and MS-pretreated fMLP-stimulated cells are shown in the lower panel.

Depletion of -PKC and Reduced Ligand-initiated Phosphorylation of p47^phox-- Phosphorylation of p47^phox and its translocation to the membrane-associated cytochrome b₅₅₈ is essential for assembly of the NADPH oxidase. -PKC-depleted dHL60 cells were used to determine whether -PKC was essential for phosphorylation of p47^phox in cells activated by fMet-Leu-Phe. dHL60 cells were pretreated with AS or MS and stimulated for 1 min with 1 µM fMet-Leu-Phe; the p47^phox was then immunoprecipitated. Western blotting and densitometry analysis demonstrated that control (MS) and -PKC-depleted (AS) dHL60 cells contained equivalent amounts of p47^phox immunoreactivity (Fig. 7A). However, phosphorylation of the p47^phox band was reduced in the AS-treated dHL60 cells as compared with the control (MS-treated) cells; ³²P counts eluted from the AS band were 56% of control levels measured in the MS band (Fig. 7B). -PKC-depleted cells contained equivalent protein levels of p47^phox as compared with control cells. However, fMet-Leu-Phe-induced phosphorylation of p47^phox was decreased in the -PKC-depleted cells, concordant with a role for -PKC in phosphorylation of p47^phox in ligand-initiated signaling and in the assembly of an active NADPH oxidase.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Effect of depletion of -PKC on phosphorylation of p47phox in dHL60 cells activated by fMet-Leu-Phe. dHL60 cells treated with AS or MS oligonucleotides were prelabeled with 32P-Pi (300 µCi) for 60 min at 37 °C. fMLP (1 µM) was added (zero time), and the reaction was stopped after 1 min. p47phox was immunoprecipitated from each sample, and the immune complexes were run on 4-12% gradient SDS-PAGE and blotted to PVDF membrane. Representative experiment of two. A, Western blot using anti p47phox. B, autoradiogram of membrane. Molecular weight markers are indicated on the left margin and p47phox on the right margin.

Effect of PKC Depletion on p47^phox Translocation-- Phosphorylation and translocation of p47^phox to the membrane and association of phosphorylated p47^phox with cytochrome b₅₅₈ are essential steps in the assembly of an active NADPH oxidase complex. Activation of dHL60 cells by fMet-Leu-Phe or PMA elicited translocation of p47^phox from the cytosol to the membrane fraction (Fig. 8). Depletion of -PKC by AS treatment was associated with decreased translocation of p47^phox from the cytosol to the membrane in both fMet-Leu-Phe- and PMA-activated cells as compared with MS-treated controls (Fig. 8). In contrast, the membrane-associated gp91^phox subunit of cytochrome b₅₅₈ was not affected by depletion of -PKC (Fig. 8). In resting cells, I-PKC and II-PKC were predominantly in the cytosol (Fig. 8). Activation of dHL60 cells by 1 µM fMet-Leu-Phe for 1 min elicited translocation of both I-PKC and II-PKC from the cytosol to the membrane (Fig. 8). Activation of the cells with 1 µg/ml PMA for 5 min triggered an almost total disappearance of I-PKC and II-PKC from the cytosol and translocation of both isotypes to the membrane (Fig. 8). In the -PKC-depleted cells, less I and II-PKC was associated with the membrane in fMet-Leu-Phe- and PMA-treated cells (Fig. 8). Densitometry demonstrated that in fMet-Leu-Phe-activated cells, 606 ± 121 DU (density units) (n = 5) of II-PKC was associated with the membrane in -PKC-depleted cells as compared with 999 ± 162 DU (n = 5) in MS-treated cells (60.1 ± 9.5% control, p < 0.03). In PMA-activated cells, 1125 ± 162 DU (n = 4) of II-PKC was associated with the membrane in -PKC-depleted cells as compared with 1483 ± 181 DU (n = 4) in MS-treated cells (75.0 ± 3.9% control, p < 0.02). Similarly, membrane-associated I-PKC was significantly reduced to 125 ± 17 DU (n = 5) in -PKC-depleted cells activated with fMet-Leu-Phe as compared with 481 ± 144 DU (n = 5) in MS-treated control cells (39.0 ± 3.7% control, p < 0.01). In PMA-activated cells, membrane-associated I-PKC was significantly reduced to 596 ± 113 DU (n = 5) in -PKC-depleted cells activated as compared with 991 ± 98 DU (n = 5) in MS-treated control cells (58.4 ± 6.7% control, p < 0.01). These results are concordant with a role for -PKC-dependent phosphorylation of p47^phox in translocation of p47^phox to the membrane and assembly of an active NADPH oxidase.

View larger version (85K): [in this window] [in a new window]   Fig. 8.   Effect of depletion of -PKC on translocation of cytosolic p47phox and -PKC isotypes to cell membranes in resting, fMet-Leu-Phe-, and PMA-activated dHL60 cells. dHL60 cells were treated with AS or MS oligonucleotides (see "Materials and Methods"). dHL60 cells were treated with buffer, 1 µM fMet-Leu-Phe (1 min) or 1 µg/ml PMA (5 min). Membrane (10 × 106 cell equivalents/lane) and cytosol (3 × 106 cell equivalents/lane) fractions were prepared, run on 4-12% gradient SDS-PAGE, blotted to PVDF membrane, and probed with antibodies to p47phox, I-PKC, II-PKC, and gp91phox. Representative experiment of five.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

A role for PKC is implicated in the signaling for proinflammatory responses of phagocytic cells such as generation of O₂, actin assembly, and cell adherence. PKC is a family of structurally related isotypes that have different cofactor and substrate specificities (9, 10). It has therefore been suggested that different isotypes of PKC transduce different signals, although there is little evidence to date to indicate specific functions for each isotype of PKC.

Multiple isotypes of PKC were demonstrated in dHL60 cells including -, I-, II-, -, and -PKC, in agreement with other studies (15); the expression of these PKC isotypes is similar to the PKC isotypes observed in neutrophils (3, 14). In addition, the cofactors required for activation of PKC, calcium, DG and PIP₃, are elevated in ligand-activated phagocytic cells, indicating a potential role for these PKC isotypes in activating responses such as O₂ generation and adherence (37-40).

A role for -PKC in ligand-initiated responses was probed by selective depletion of the -PKC isotypes. Selective depletion by antisense was used since difficulty in distinguishing primary and secondary effects may be seen in overexpression studies either of the wild-type isotype or of a dominant negative mutant (41). Selective depletion of -PKC but not depletion of -, -, or -PKC was achieved using an antisense oligonucleotide designed against the translocation start site of -PKC. This oligonucleotide depleted both I-PKC and II-PKC, which are derived by alternate splicing and only differ at the 3' terminus. Depletion of -PKC by 45-60% was associated with decreased O₂ generation triggered by PMA, fMet-Leu-Phe, or immune complexes. The extent of inhibition of O₂ generation in -PKC-depleted cells was ligand dependent; the greatest inhibition was observed in fMet-Leu-Phe-activated cells and the smallest inhibition in PMA-activated cells. The greater degree of inhibition of O₂ generation observed in response to fMet-Leu-Phe and immune complexes, as compared with PMA, may reflect differences in signaling initiated by these ligands. fMet-Leu-Phe may use -PKC at more than one step in the signaling pathway. There is a noteworthy difference in the kinetics of O₂ generation triggered by PMA, which is continuous, as compared with the fMet-Leu-Phe-induced response, which ceases after approximately 3 min (1, 17). In addition, continuous receptor occupancy is required to maintain fMet-Leu-Phe-induced O₂ generation (42). Thus, a role for -PKC might be involved in maintaining fMet-Leu-Phe-induced O₂ generation. Finally, PMA is promiscuous and might recruit other PKC isotypes, whereas fMet-Leu-Phe may be constrained to recruit only -PKC for a key step in activation of the NADPH oxidase.

The inhibitory effect of -PKC depletion on O₂ generation was functionally selective. The simultaneously elicited release of the azurophil granule markers, -glucuronidase and elastase (results not shown), from fMet-Leu-Phe-activated cells was not decreased in -PKC-depleted cells. This finding is concordant with previous findings that activators of PKC, such as PMA, do not trigger azurophil degranulation and that the kinase inhibitor staurosporine does not inhibit azurophil degranulation (17). These findings also demonstrate that -PKC depletion did not have a nonspecific effect on signaling for cell activation.

dHL60 cells adhere to fibronectin by a 1 integrin (36); adherence of dHL60 cells to a fibronectin-coated surface was triggered by PMA and fMet-Leu-Phe. Depletion of -PKC in dHL60 cells did not inhibit either PMA or fMet-Leu-Phe-induced adherence to fibronectin. Thus, signaling for adherence must use other isotypes of PKC such as - or -PKC that, like -PKC, are also activated by PMA.

Identifying key substrate(s) is important in defining a role for -PKC in signal transduction. Addition of fMet-Leu-Phe to dHL60 cells triggered phosphorylation of numerous proteins. In -PKC-depleted cells, phosphorylation of a subset of proteins was reduced in response to fMLP, indicating a selective effect of -PKC depletion on ligand-induced phosphorylation. Phosphorylation of a cytosolic component of the NADPH oxidase, p47^phox, and translocation and binding of p47^phox to cytochrome b₅₅₈ are essential steps in ligand-initiated activation of the NADPH oxidase. Indeed, depletion of -PKC reduced the fMet-Leu-Phe-induced phosphorylation of a 47-kDa band and phosphorylation of immunoprecipitated p47^phox. However, the level of p47^phox, which is a differentiation marker in HL60 cells, was not altered by depletion of -PKC. Thus, phosphorylation of p47^phox is triggered by -PKC in activated dHL60 cells.

Phosphorylation of p47^phox is required for a conformational change in p47^phox, which releases binding of p47^phox to itself and to p40^phox (43), and allows translocation and binding of p47^phox to membrane-associated cytochrome b₅₅₈ (43-46). In the present study, fMet-Leu-Phe and PMA triggered phosphorylation and translocation of p47^phox from the cytosol to the membrane in dHL60 cells. In -PKC-depleted dHL60 cells, the ligand-induced translocation of p47^phox to the membrane was reduced, concordant with a role for -PKC in the phosphorylation and translocation of p47^phox to the membrane-associated cytochrome b₅₅₈ and activation of the NADPH oxidase. Several serines in the C terminus of p47^phox, including Ser-303, Ser-304, and Ser-379, are consensus sequences for phosphorylation by PKC (47), and we have demonstrated that in vitro -PKC phosphorylates p47^phox (3). The importance of a role for phosphorylation of p47^phox by PKC for assembly of an active NADPH oxidase was demonstrated in a neutrophil cell-free system (4). Phosphorylation of Ser-379 was essential for translocation of p47^phox to the membrane and activation of the NADPH oxidase, whereas a double mutatio