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Address correspondence to Rachel Levy, Infectious Diseases Laboratory, Dept. of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. Tel.: 972-8-6403186. Fax: 972-8-6467477. email: ral{at}bgumail.bgu.ac.il
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Abstract |
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Key Words: neutrophils; granulocyte-like PCB cells; superoxide production; arachidonic acid; PGE2
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Introduction |
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Cytosolic phospholipase A2 (cPLA2) which hydrolyzes phospholipids containing arachidonate at the sn-2 position (Clark et al., 1990; Kramer et al., 1991), has been implicated as the major enzyme in the formation of proinflammatory lipid mediators. Recently, we have created in the human phagocyte myeloid cell line, PLB-985, a p85 cPLA2-deficient model cell (Dana et al., 1998) and demonstrated an essential requirement for arachidonic acid (AA) in activation of the assembled phagocyte NADPH oxidase, the oxidase-associated H+ channel (Lowenthal and Levy, 1999), and the oxidase-associated diaphorase activity (Pessach et al., 2001) induced with a variety of agonists. The normal translocation of the oxidase cytosolic components in activated differentiated PLB-985 cells lacking cPLA2 or in stimulated neutrophils in the presence of PLA2 inhibitors (Levy et al., 1994; Dana et al., 1998; Pessach et al., 2001) and the effect of PLA2 inhibitors even when added after oxidase activation (Henderson et al., 1993) indicate that cPLA2 is not required for translocation of the cytosolic factors to the membranes but rather serves a critical role in oxidase activation after the assembly of the oxidase complex. Addition of free AA but not other free fatty acids nor AA metabolites restored the activity, indicating the specificity of AA for the process. The requirement of cPLA2 for oxidase activation is consistent with our and other previous studies in human neutrophils and monocytes (Henderson et al., 1993; Dana et al., 1994; Li and Cathcart, 1997; Bae et al., 2000). In addition, a recent study (Zhao et al., 2002) has demonstrated by the use of antisense molecules that cPLA2 is absolutely required for superoxide generation in monocytes stimulated with opsonized zymosan. However, that study suggests that cPLA2 is required for translocation of oxidase cytosolic components to the membranes and not to activation of the assembled oxidase. The discrepancy may be due to the different roles of cPLA2 in the different cell types, as proposed by the authors. In contrast to the findings demonstrating requirement of the cPLA2 for oxidase activation, resident peritoneal macrophages from cPLA2-deficient mice exhibited normal stimulated superoxide release (Gijon et al., 2000), which may be attributed to either compensatory effects of isoenzymes frequently observed in models of knockout animals or to the known differences of the PLA2 isotypes between mice and human/rat (Suzuki et al., 2000). In an earlier study, we showed that AA increases the affinity of the assembled oxidase in purified membranes for NADPH (Rubinek and Levy, 1993). The mechanism by which cPLA2 regulates the NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membrane in stimulated cells, whereas cPLA2 has been shown to translocate to the nuclear and ER membranes in a variety of cells (for review see Hirabayashi and Shimizu, 2000). To elucidate the mechanism by which cPLA2 regulates the activation of NADPH oxidase, the present study investigated the cellular localization of cPLA2 upon stimulation and whether there is any association between cPLA2 and the NADPH oxidase complex during activation of phagocytic cells.
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Results |
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Discussion |
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Recent studies have suggested that cPLA2 has three functionally distinct domains: an NH2-terminal C2 domain necessary for Ca2+-dependent phospholipid binding, a COOH-terminal Ca2+-independent catalytic region (Nalefski et al., 1994), and a putative pleckstrin homology domain within this region that is responsible for the interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2; Mosior et al., 1998). Subcellular localization of the various C2 domains upon cytoplasmic [Ca2+] elevation has been shown to correlate with their phospholipid binding specificity. It has also been shown that the cPLA2-C2 domain has specificity to the phosphatidylcholine-rich nuclear envelope and ER (Gijon et al., 1999; Perisic et al., 1999; Hurley and Misra, 2000) and that aromatic and hydrophobic residues in the calcium binding loop of the cPLA2-C2 domain are important for its lipid specificity (Stahelin et al., 2003b). In contrast, the PKC-C2 domain has specificity to the phosphatidylserine-rich plasma membranes (Oancea and Meyer, 1998; Corbalan-Garcia et al., 1999) and ASn189 plays a key role in this specificity (Stahelin et al., 2003b). Several studies have shown that cPLA2 translocates from the cytosol to the nuclear membrane and to the ER by calcium ionophores or agonists such as histamine or IgE/antigen, which increase cytoplasmic [Ca2+] in a variety of cells (Peters-Golden and McNish, 1993; Glover et al., 1995; Schievella et al., 1995; Sierra-Honigmann et al., 1996). However, PMA, which does not induce an increase in cytoplasmic [Ca2+], caused activation and translocation of cPLA2 (Hazan et al., 1997; Qiu et al., 1998), suggesting the existence of alternative pathways that induce translocation of cPLA2 not involving elevation of cytoplasmic [Ca2+], which is necessary for the C2 domain phospholipid binding. The results of our present study suggest that the cPLA2-C2 domain does not participate in the translocation of cPLA2 to the plasma membranes, since cPLA2 translocation is inconsistent with its C2 domain phospholipid binding specificity. In addition, cPLA2 translocation to the cell periphery did not occur in the absence of a functional NADPH oxidase (Fig. 7), and it can be induced with PMA (Fig. 3 and Fig. 6 A), which does not cause elevation of cytoplasmic [Ca2+]. However, we cannot rule out the possibility that the binding of cPLA2 to the assembled oxidase is mediated through the cPLA2-C2 domain. Similar to our observation that the C2 domaincontaining cPLA2 binds NADPH oxidase, a recent study (McAdara Berkowitz et al., 2001) has shown that C2 domaincontaining protein JFC1, which is restricted to the plasma membranes/secretory vesicles, binds p67phox without affecting the interaction between p47phox and p67phox. High levels of PtdIns(4,5)P2 have been detected in neutrophil plasma membranes upon stimulation (Botelho et al., 2000; Martin, 2001), and PtdIns(4,5)P2 has been shown to display a particularly dramatic effect on the activity of cPLA2 (Mosior et al., 1998). However, it seems that the cPLA2 pleckstrin homology domain by itself does not play a critical role in targeting cPLA2 to the plasma membranes, since in the absence of the functional oxidase cPLA2 did not translocate to this compartment, as shown in gp91phox-deficient granulocytic PLB-985 cells (Fig. 7). Thus, the assembled NADPH oxidase appears to be the major determinant in directing cPLA2 to the plasma membranes, although the interaction sites among cPLA2, NADPH oxidase, and the plasma membranes are not yet defined.
Eicosanoid generation has been shown to be regulated in part by perinuclear envelope localization or translocation of individual enzymes of leukotriene and prostaglandin biosynthesis (Ueno et al., 2001). The perinuclear translocation of cPLA2, shown in a variety of cells, is in agreement with its role in leukotriene and prostaglandin formation. Similarly, our present study demonstrates a correlation between the kinetics of cPLA2 translocation to nuclear membranes and PGE2 production in stimulated granulocyte-like PLB-985 cells (Fig. 6, C and D). The differences in the kinetics of superoxide production and PGE2 formation in PLB-985 cells differentiated with DMSO enabled us to efficiently follow the distribution of cPLA2 during stimulation, from early translocation to the cell periphery to later translocation to the nuclear envelope. Thus, the mechanism which permits the participation of cPLA2 in two different processes in the same cell (regulation of NADPH oxidase and eicosanoid production) is controlled by localization of the enzyme in different subcellular compartments.
In conclusion, the use of combined biochemical and microscopical approaches enhanced our ability to gain better insight into cellular processes and to clearly demonstrate that upon activation of peripheral blood neutrophils and granulocyte-like PLB-985 cells, both of which contain abundant levels of NADPH oxidase, cPLA2 translocates to the plasma membranes where it binds the assembled oxidase complex and releases AA, which promotes oxidase activity. The absence of cPLA2 in the cell periphery of stimulated granulocyte-like gp91phox-deficient PLB-985 cells and its translocation after the expression of retroviral gp91phox protein in these cells indicate that cPLA2 is anchored to the plasma membranes by the assembled oxidase. The physical association between these two enzymes enables the regulation of NADPH oxidase by cPLA2-generating AA. The novel dual subcellular localization of cPLA2 in different compartments, first in the plasma membranes and then in the nucleus, provides a molecular mechanism for the participation of cPLA2 in different processes in the same cells. The exact binding sites between these two enzymes and the target for AA action are currently under investigation.
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Materials and methods |
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Cell culture and differentiation
PLB-985 and gp91phox-deficient PLB-985 cells lacking the expression of normal gp91phox (provided by M.C. Dinauer, James Whitcomb Riley Hospital for Children, Indianapolis, IN) were grown in stationary suspension culture in RPMI-1640 as described earlier (Dana et al., 1998). The optimal concentration of 1.25% of DMSO was added to 2 x 105 PLB-985 cells/ml at their logarithm growth phase to induce differentiation toward the granulocyte phenotype. Mac-1 antigen determination was detected by indirect immunofluorescence as described previously (Hazav et al., 1989).
Isolation of membrane and cytosol fractions
Neutrophils were suspended at 107 cells/ml in phosphate buffer saline and treated with 5 mM diisopropylfluorophosphate for 30 min at room temperature before stimulation. Membrane and cytosol fractions were prepared as described previously (Levy et al., 1990).
Immunoprecipitation
Goat antiserum raised against recombinant p47phox or p67phox (Leto et al., 1991) or rabbit antiserum raised against cPLA2 (Hazan et al., 1997) was added to 3 x 107 membranes or cytosol cell equivalents in 500 µl solubilization buffer (150 mM NaCl, 5 mM EDTA, 10 mM Tris, pH 7.5, 1% sodium deoxycholate, and 1% NP-40), and they were incubated on ice overnight. The extracts were brought to a volume of 1 ml in solubilization buffer containing 30 µl of 50% slurry of recombinant protein Gsepharose. The samples were tumbled end-over-end for 1 h and washed twice with 1 ml solubilization buffer containing 20% (wt/vol) sucrose and 0.15% (wt/vol) BSA and twice with 1 ml solubilization buffer containing 20% sucrose. The samples were boiled in SDS sample buffer and electrophoresed on a 10 or 7% SDS-PAGE. For detection of protein translocation, cell membranes (2 x 106 cell equivalent) were separated by SDS-PAGE electrophoresis. The resolved proteins were electrophoretically transferred to nitrocellulose, and the detection of cPLA2 or the oxidase components was analyzed as described previously (Hazan et al., 1997).
Subcellular fractionation
Subcellular fractionation was performed as described by others (Kjeldsen et al., 1999). Neutrophils (5 x 108) treated with diisopropylfluorophosphate were suspended in relaxation buffer (as described earlier; Levy et al., 1990) and disrupted by nitrogen cavitation at 400 pounds per square inch. Nuclei and unbroken cells were pelleted by centrifugation at 500 g for 10 min at 4°C. The supernatant was decanted and loaded onto a precooled discontinuous density gradient Percoll, and 10x concentrated relaxation buffer and distilled water were mixed to give solutions of densities of 1.05, 1.09, and 1.12 g/ml. The gradients were centrifuged at 32,800 g for 35 min at 4°C using a fixed angle Beckman Coulter JA20 rotor. Four visible bands were collected, and the markers for azurophil granules (), specific granules (ß), and plasma membranes (
) were analyzed as described previously (Kaufman et al., 1996).
Immunofluorescence microscopy
Preparation of labeled cells was done as described by others (Bingham et al., 1999) with some modification. Cells were adhered on coverslips for 30 min at 37°C. The cells were stimulated with various agonists for the desired duration and fixed with 3% (wt/vol) formaldehyde. The first antibodies against cPLA2, gp91phox, and lamin B (Santa Cruz Biotechnoloy) dissolved in PBS containing 0.2% saponin were added for 1 h at room temperature. cy2- or cy3-conjugated antibodies (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. The fluorescence was visualized using a four channel Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc.). The LSM 510 software was used for imaging cPLA2, gp91phox, and lamin B localization.
Retroviral transduction of gp91phox-deficient PLB-985 cells
Retroviaral gp91phox was expressed in gp91phox-deficient PLB-985 cells (Zhen et al., 1993) as done in our previous study (Pessach et al., 2001).
Affinity-binding assay
The GST fusion proteins were affinity purified on glutathione-sepharose as described previously (Leto et al., 1994). 50 µl (1 µg) of GST-p47phox or GST-p67phox bound to glutathione-sepharose beads was added to lysates of 5 x 107 resting or stimulated neutrophils prepared as described before (Hazan-Halevy et al., 2000) and was tumbled end-over-end for 2 h at 4°C. Bound proteins were washed three times in 15 vol of ice cold 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 0.15 mM PMSF, and 10 mM Pipes (pH 7.5), eluted with 2% SDS, and analyzed by SDS-PAGE followed by immunoblotting with the appropriate antibodies.
Superoxide anion measurements
The production of superoxide anion (O2-) by intact cells was measured as the superoxide dismutase inhibitable reduction of ferricytochrome c (Dana et al., 1998).
Preparation of nuclei
Nuclei were separated from granulocyte-like PLB cells (2 x 107 cells) before and after stimulation as described by others in neutrophils (Surette et al., 1998). Stimulated cells were pelleted and resuspended in 600 µl of ice-cold NP-40 lysis buffer (0.1% NP-40, 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotonin, and 1 mM PMSF). The cells were vortexed for 15 s, kept on ice for 5 min, and centrifuged at 300 g for 10 min at 4°C. The resulting pellets (the nuclei containing fractions) were then immediately solubilized in electrophoresis sample buffer and processed for SDS-PAGE and immunoblot determination of cPLA2 and the nuclear lamin B. Nuclear integrity was verified directly by light microscopy, which also revealed that intact cells were rarely observed in nuclei-containing fraction (<2%).
PGE2 determination
PGE2 levels were determined in the supernatant of stimulated cells by RIA using commercial kits (NEN Life Science Products). The samples were immediately stored at -70°C and analyzed during 1 wk of the experiments.
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Acknowledgments |
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This research was supported by a grant from The Israel Science Foundation founded by The Academy of Sciences and Humanities (grant no. 619/98-3).
Submitted: 15 November 2002
Accepted: 9 May 2003
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References |
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