Protein Phosphatase 2A and Protein Kinase Cα Are Physically Associated and Are Involved in Pseudomonas aeruginosa-induced Interleukin 6 Production by Mast Cells*

Pulmonary infection with Pseudomonas aeruginosa is characterized by massive airway inflammation, which comprises significant cytokine production. Although mast cells are abundant in the lung and are potent sources of various cytokines, a role of mast cells in P. aeruginosa infection remains undefined, and P. aeruginosa-induced signaling mechanisms in mast cells have not been studied previously. Here we demonstrate that human cord blood-derived mast cells, mouse bone marrow-derived mast cells, and the mouse mast cell line MC/9 produce significant amounts of interleukin 6 (IL-6) in response to P. aeruginosa. This response was accompanied by a stimulation of protein kinase Cα (PKCα) phosphorylation and PKC activity and was significantly blocked by the PKC inhibitors Ro 31-8220 and PKCα pseudosubstrate. Interestingly, mast cells treated with P. aeruginosa had reduced protein levels of phosphatase 2A catalytic unit (PP2Ac), which prompted us to determine whether a direct association between PKCα and PP2A occurs in mast cells. In mouse bone marrow-derived mast cells and MC/9 cells, as well as in the human mast cell line HMC-1, PP2A coimmunoprecipitated with PKCα either using PKCα- or PP2Ac-specific antibodies, suggesting that PKCα and PP2Ac are physically associated in mast cells. The PP2A inhibitor okadaic acid induced P. aeruginosa-like responses in mast cells including increased PKCα phosphorylation, stimulated PKC activity, and augmented IL-6 production, the last being blocked by the PKC inhibitor Ro 31-8220. Finally, okadaic acid potentiated the P. aeruginosa-induced IL-6 production. Collectively, these data provide, to our knowledge, the first evidence of both a direct physical association of PP2A and PKCα in mammalian cells and their coinvolvement in regulating mast cell activation in response toP. aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that often colonizes the lungs of patients with cystic fibrosis or immune compromised individuals. The chronically overactive inflammatory response associated with persistent P. aeruginosa lung infections is believed to be caused by the continuous stimulation of host cells to produce cytokines (1)(2)(3). Indeed, high levels of cytokines such as interleukin 6 (IL-6) 1 have been found in blood and sputa of cystic fibrosis patients with P. aeruginosa infection (1)(2)(3). Some studies suggest that impairment of IL-6 regulation may represent an important component of the excessive inflammatory response observed during P. aeruginosa infection (1,4). Mast cells are recognized as sentinels in host defense against bacterial infection (5)(6)(7). Although mast cells are found in large numbers in airways and are potent sources of cytokines and chemokines, a role for mast cells in P. aeruginosa-induced dysregulation of cytokine production has not been studied previously.
Mast cells contain a series of protein serine/threonine phosphatases including protein phosphatase 2A (PP2A) (8). One recent study demonstrated that stimulation of RBL 2H3 cells, a rat mast cell line, with antigen leads to a transient translocation and activation of PP2A (9). The rate of translocation of PP2A to the membrane coincides with the kinetic pattern of degranulation (9), suggesting a link between mast cell PP2A and granule-bound mediator secretion. In addition, several studies have described in human and rodent mast cells that okadaic acid blocks IgE-dependent and IgE-independent degranulation (10 -14), implicating a role for PP2A in the regulation of mast cell mediator secretion. However, the molecular target of PP2A in the regulation of mast cell function or the role that PP2A plays during cytokine production remains to be determined.
Protein kinase C (PKC) is a family of serine/threonine kinases comprising at least 12 different isoforms that have been grouped into three categories: conventional PKCs (␣, ␤I, ␤II, and ␥), novel PKCs (␦, ⑀, , and ) and atypical PKCs (, , , and ). PKC isoform expression appears to be cell type-specific (15). PKC isoforms that have been characterized in mast cells include PKC␣, ␤I, ␥, ␦, ⑀, , , and (15)(16)(17)(18)(19)(20). PKC isoforms participate in signal transduction in many cell types and mediate a wide range of intracellular functions. Compared with other PKC isoforms, PKC␣ has distinct roles in a number of processes such as cell proliferation, apoptosis (21,22), and bacteria-or cytokine-induced inflammatory responses (22,23). In vivo, overexpression of PKC␣ in transgenic mice results in striking alterations of proinflammatory mediator production during inflammation (24). In vitro, Escherichia coli infection induces PKC␣ translocation from cytosol to membrane in T84 carcinoma cells (25), suggesting bacteria-induced activation of PKC␣. Bacterial lipopolysaccharide-induced mediator production is enhanced significantly by overexpression of PKC␣ (26). Overexpression of a dominant negative version of PKC␣ strongly inhibits lipopolysaccharide-induced cytokine production by macrophages (27). Impaired PKC␣ function induced by Leishmania donovani in macrophages correlates with defective phagosome maturation and survival of the parasite in host cells (28). Thus, PKC␣ appears to play an important role during pathogen-induced inflammatory responses. In mast cells, PKC␣ has been implicated in several functions (29) such as antigen-induced hydrolysis of inositol phospholipids (16) and cytokine production (30).
PKC␣ kinase activity is regulated by phosphorylation of three conserved residues in its kinase domain: the activation loop site Thr-497, the autophosphorylation site Thr-638, and the hydrophobic C-terminal site Ser-657 (31). Without phosphorylation at these sites, PKC␣ has little or no activity (31). Phosphorylation at the C-terminal site Ser-657 plays a critical role in controlling the net phosphorylation and dephosphorylation rates (32). In vitro, PKC␣ activity can be inhibited through dephosphorylation by PP2A (33). The removal of phosphate from these sites is crucial to the desensitization of PKC␣ (34). In intact cells, circumstantial evidence has implied that the dephosphorylation of PKC␣ is catalyzed by a membraneassociated PP2A (35). Consistent with a role of PP2A in the regulation of PKC␣ activity, okadaic acid, a potent PP2A inhibitor (36), induces numerous effects through mimicking or enhancing the actions of phorbol 12-myristate 13-acetate (PMA), a potent PKC activator (37,38). Moreover, activation of PKC by PMA induced PP2A translocation to the membrane. Cumulatively, this evidence suggests an intimate interaction between PP2A and PKC␣.
In this study, we demonstrate for the first time that PP2Ac and PKC␣ are physically associated in mast cells and that the associated enzymes participate in the regulation of P. aeruginosa-induced IL-6 production by mast cells.

MATERIALS AND METHODS
Reagents-Rabbit anti-PKC␣ antibodies, aprotinin, leupeptin, pepstatin, Triton X-100, sodium deoxycholate, prostaglandin E 2 , and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co. Rabbit anti-phospho-PKC␣ (Ser 657) antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse anti-PP2A catalytic subunit (PP2Ac) antibodies were purchased from Transduction Laboratories of BD Biosciences (Mississauga, Ontario, Canada). Protein A/G PLUS-agarose immunoprecipitation beads, donkey anti-mouse IgGhorseradish peroxidase and donkey anti-rabbit IgG-horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture media, okadaic acid, antibiotics, and fetal bovine serum were from Invitrogen. Ro 31-8220 and cell-permeable myristoylated PKC inhibitor peptide 19 -27 were purchased from Calbiochem-Novabiochem Co. All other chemicals and reagents were of analytical grade.
Mast Cells-The HMC-1 5C6 human mast cells were maintained in Iscove's modified Dulbecco's medium in a 5% CO 2 , humidified atmosphere at 37°C. Culture medium was supplemented with 10% fetal calf serum and 50 units/ml each of penicillin and streptomycin. Prior to experimental treatment, HMC-1 5C6 cells were starved overnight (18 -24 h) in Iscove's modified Dulbecco's medium alone at a density of 0.5 ϫ 10 6 cells/ml. For treatment, cells were resuspended in complete medium at a higher density, typically 2 ϫ 10 6 cells/ml. After treatment cells were harvested in 50-ml conical centrifuge tubes and pelleted at 300 ϫ g for 10 min at 4°C.
Mouse bone marrow-derived mast cells (BMMC) were harvested from the femurs and tibias of C57-black mice (Charles River Laboratories, Montreal, Quebec, Canada). Briefly, two or three mice were sacrificed and dissected, and the legs were cleaned of fur and harvested. Tissue was then removed in a sterile environment, and the cleaned bones were kept moist in a dish containing RPMI 1640 medium. The ends were then cut off with sterile surgical scissors, and RPMI medium was run through the shaft using a 30-ml syringe and a 31.5-gauge needle. Cells were collected, centrifuged at 500 ϫ g for 5 min at 4°C, and resuspended at a density of 0.5 ϫ 10 6 cells/ml (disregarding erythrocytes) in BMMC complete medium (RPMI 1640 medium containing 10% fetal bovine serum, 10% WEHI-3B conditioned medium, 50 units/ml each of penicillin and streptomycin, 50 M 2-mercaptoethanol, and 200 nM prostaglandin E 2 ). Nonadherent cells were resuspended in fresh complete medium twice/week and transferred to a fresh flask once/week. After 4 -6 weeks, mast cell purity of Ͼ98% was achieved as assessed by alcian blue or toluidine blue staining of fixed cytocentrifuge preparations. Before experimental treatment BMMC were starved for 6 h in nonsupplemented RPMI 1640 at a density of 0.5 ϫ 10 6 cells/ml. For treatment, cells were resuspended in complete medium at a higher density; typically 2 ϫ 10 6 cells/ml. After treatment cells were harvested in 50-ml conical centrifuge tubes and pelleted at 300 ϫ g for 10 min at 4°C.
Highly purified cord blood-derived mast cells (CBMC, Ͼ95% purity) were obtained by long term culture of cord blood progenitor cells as described previously (39). The percentage of mast cells in the cultures was assessed by toluidine blue staining (pH 1.0) of cytocentrifuged samples. After Ͼ8 weeks in culture, mature mast cells were identified by their morphological features and the presence of metachromatic granules and used in our study.
Bacterial Treatment-P. aeruginosa strain 8821 (a kind gift from Dr. A. Chakrabarty, University of Illinois, Chicago) is a mucoid strain isolated from a cystic fibrosis patient (40). P. aeruginosa was cultured in Luria-Bertani broth and harvested when the culture reached an optical density at 640 nm of 2 units (early stationary phase). Bacteria were washed in phosphate buffer and their density adjusted to 1 optical density unit before treatment with 100 g/ml gentamycin for 2 h. Mast cells were typically treated with P. aeruginosa, for the indicated times, at a mast cell:bacteria ratio of 1:50.
Coimmunoprecipitation Studies-To 1 ml of clarified total cell lysate was added at least 1 g of primary antibody, and the sample was incubated for 1 h at 4°C with end-over-end mixing. Immunoreactive proteins and protein complexes were then precipitated with the addition of 20 l of protein A/G PLUS-agarose beads and incubated at 4°C overnight with end-over-end mixing. The beads were pelleted by centrifugation at 1,200 ϫ g for 5 min at 4°C, and the supernatant was discarded. The pellet was washed four times with ice-cold PBS (NaCl concentration adjusted to 1.0 M) before the addition of 40 l of 3ϫ SDS-PAGE sample buffer and storage at Ϫ20°C until further SDS-PAGE and Western analysis.
Measurement of IL-6 by ELISA-Human and mouse IL-6 levels in supernatants were measured using an "in-house" ELISA assay. Briefly, 96-well plates were coated with anti-human IL-6 (R & D Systems, Minneapolis) or anti-mouse IL-6 (Endogen, Woburn, MA) at 1 g/ml for 16 -20 h at 4°C. Nonspecific binding to the plates was blocked using a 1% bovine serum albumin, 0.1% Tween 20 solution in PBS for 1 h at 37°C. A total of 50 l/well IL-6 standard (human rIL-6, R & D Systems; murine rIL-6, Endogen) and samples were added to the plate and incubated for 18 -20 h at 4°C. Biotinylated anti-human IL-6 (R & D Systems) and anti-murine IL-6 (Endogen) (0.2 g/ml) were added to each well and incubated for 1 h at 37°C. After washing, 50 l/well of a 1/2,000 dilution of streptavidin-alkaline phosphatase (Invitrogen) was added according to the manufacturer's instructions. The minimal detectable dose was 3 pg/ml for human IL-6 and 10 pg/ml for murine IL-6 using this system.
Measurement of PKC Activity-PKC activity was measured based on the phosphorylation of a PKC substrate peptide using a radioactive PKC assay kit or a nonradioactive protein kinase assay kit according to the manufacturer's protocol (both from Calbiochem-Novabiochem Co.).
Confocal Microscopy Imaging of PKC␣ and PP2Ac-Confocal microscopy was used to demonstrate the colocalization of PP2Ac and PKC␣ in mast cells. HMC-1 cells (5ϫ10 5 cells/test) were washed with cold PBS and fixed with 4% paraformaldehyde for 5 min. After washing, cells were resuspended in 10% dimethyl sulfoxide in PBS and stored at Ϫ80°C. Thawed cells were washed and incubated with 0.1% saponin and 3% bovine serum albumin in PBS for 1 h at room temperature. After washing, cells were incubated with mouse anti-PP2Ac IgG1 and rabbit anti-PKC␣ IgG for 1 h at 4°C. Then cells were incubated further for 45 min with Alexa Fluor®-594 conjugated goat anti-mouse IgG, F(abЈ) 2 , and Alexa Fluor®-488 conjugated goat anti-rabbit IgG, F(abЈ) 2 (Molecular Probes Inc.). Cells were washed three times and resuspended in 1% formalin. Cytospins of fluorescence-labeled mast cells were made by vortexing slides in a Cytospin 3 (Shandon, U. K.) at 600 rpm for 3 min. Antibleaching solution (10 mM n-propyl gallate (Sigma), 8.1 M glycerol in Tris-buffered saline) was dropped onto slides before coverslip attachment. Cells were examined with a Zeiss LSM410 confocal laser scanning microscope (Jena, Germany). PP2Ac would then be tagged in red and PKC␣ in green. A yellow color indicates the colocalization of these two enzymes (overlay of red and green).

P. aeruginosa Stimulates IL-6 Production by Mast Cells-
IL-6 is a pleiotropic cytokine that is produced during the course of infectious and inflammatory disorders and plays a crucial role in both local and systemic inflammatory responses (41)(42)(43). To test whether mast cells produce the cytokine IL-6 after P. aeruginosa stimulation, the mouse mast cell line, MC/9 cells, and primary cultured mouse and human mast cells, BMMC and CBMC, were employed in this study. Mast cells at a concentration of 5 ϫ 10 5 cells/ml were treated with cystic fibrosisassociated P. aeruginosa strain 8821 (mast cell:bacteria ratio of 1:50) for 3-48 h. IL-6 levels in cell free supernatants were determined by ELISA. P. aeruginosa treatment for 24 h stimulated IL-6 production by BMMC and MC/9 significantly (Fig.  1, a and b). In CBMC, significant IL-6 production was observed as early as 6 h after P. aeruginosa treatment (Fig. 1, c and d).
A Role of PKC in P. aeruginosa-induced IL-6 Production by Mast Cells-To determine the role of PKC␣ in P. aeruginosainduced mast cell activation, PKC␣ phosphorylation and PKC activity were determined in MC/9 cells after P. aeruginosa treatment. MC/9 cells were treated with P. aeruginosa strain 8821 for 3 h or 12 h and lysed in extraction buffer. Cell lysates were subjected to SDS-PAGE and probed with Ab to phosphorylated PKC␣ on serine 657. Increased phosphorylation of PKC␣ on serine 657 was seen in mast cells after treatment with P. aeruginosa (Fig. 2a). Interestingly, significant PKC␣ phosphorylation was seen in both the shorter (3 h) and longer (12 h) exposures to P. aeruginosa, suggesting a sustained stimulation of PKC␣. Treatment of mast cells with P. aeruginosa did not affect the total PKC␣ levels, suggesting that the increase of phosphorylated PKC␣ is not the result of the increase of total PKC␣ levels. It is noteworthy that no degradation of PKC␣ protein was observed after sustained stimulation of mast cells with P. aeruginosa for 12 h.
To determine the effect of P. aeruginosa treatment on mast cell PKC activity, MC/9 cells were incubated with P. aeruginosa for 1, 2, or 3 h and lysed in extraction buffer. PKC activity was determined in cell lysates. As shown in Fig. 2b, treatment with P. aeruginosa for 1 h stimulated PKC activity in mast cells significantly. Similar stimulatory effects on PKC activities were observed when mast cells were treated with P. aeruginosa for 2 or 3 h, suggesting a sustained PKC activation. No PKC activity was observed in P. aeruginosa lysates (data not shown).
The involvement of PKC in P. aeruginosa-induced mast cell activation was confirmed further by using PKC inhibitors, Ro 31-8220 and PKC inhibitor peptide. BMMC and CBMC were treated with Ro 31-8220 at a dose of 10 M during the course of P. aeruginosa stimulation. Treatment of mast cells with Ro 31-8220 dramatically blocked P. aeruginosa-induced IL-6 production by BMMC (Fig. 2c) and CBMC (Fig. 2d). To confirm further the specific effect of PKC␣ on IL-6 production, a cellpermeable PKC pseudosubstrate sequence from PKC␣ (IC 50 ϭ 8 M in fibroblasts according to the manufacturer) was incubated with CBMC during P. aeruginosa stimulation. P. aeruginosa-induced IL-6 production by CBMC was inhibited significantly by PKC peptide at the dose of 20 M (Fig. 2e).
P. aeruginosa Treatment Decreased PP2Ac Levels-Given that PP2A has been shown in vitro to regulate PKC␣ activity and phosphorylation (33), the effect of P. aeruginosa treatment on mast cell PP2Ac was assessed. MC/9 cell and BMMC were treated with P. aeruginosa strain 8821 for 18 h and lysed in RIPA buffer. Total cell lysates were used for Western blot analysis and probed with Ab to PP2Ac. P. aeruginosa treatment induced a decrease of PP2Ac protein in both BMMC (Fig.  3a) and MC/9 cells (Fig. 3b).

PP2Ac and PKC␣ Are Physically Associated in Human and
Mouse Mast Cells-Based on circumstantial evidence, it has been proposed that activation of PP2A by stimuli will lead to dephosphorylation and inactivation of PKC␣ and subsequent responses in smooth muscle cells and Molt-4 human leukemia cells (44,45). The stimulation of PKC␣ phosphorylation and PKC activity and reduction of PP2Ac protein by P. aeruginosa treatment of mast cells, together with the in vitro functional inter-regulation between PKC␣ and PP2Ac (33), suggest that PKC␣ and PP2Ac may interact closely in the regulation of P. aeruginosa-induced mast cell responses. However, interactions between these two enzymes in mast cells have not been previously described. To determine whether PP2Ac and PKC␣ are physically associated in mast cells, human mast cell line HMC-1 5C6, mouse primary cultured BMMC, and mouse mast cell line MC/9 were used in our study. Immunoprecipitation and Western blot analysis showed the constitutive expression of both PKC␣ and PP2Ac in unstimulated mast cells (Fig. 4, b  and d). As seen in Fig. 4a, immunoprecipitates of PKC␣, when probed with anti-PP2Ac Ab, demonstrated the presence of PP2Ac. To confirm further the association of PP2Ac and PKC␣, p Ͻ 0.05 compared with group without bacterial treatment). c, BMMC (5 ϫ 10 5 cells/ml) were treated with P. aeruginosa (Ps.a) for 24 h in the presence or absence of PKC inhibitor Ro 31-8220 (Ro). Cell-free supernatants were used to determine IL-6 production using ELISA. P. aeruginosa-induced IL-6 production by mast cells was completely abrogated by Ro 31-8220. Results are the means Ϯ S.E. for four independent experiments. d, CBMC (5 ϫ 10 5 cells/ml) were treated with P. aeruginosa for 24 h in the presence or absence of Ro 31-8220. IL-6 protein was determined in cell-free supernatants using ELISA. Ro 31-8220 dramatically blocked P. aeruginosa-induced IL-6 production by human mast cells. Results are representative of three similar experiments. Values are the means Ϯ S.E. of triplicate determinations (*, p Ͻ 0.05 compared with bacterial treatment alone). e, CBMC (5 ϫ 10 5 cells/ml) were treated with P. aeruginosa for 24 h in the presence or absence of a cell-permeable PKC pseudosubstrate, a sequence derived from PKC␣ (PKC peptide). Treatment of mast cells with PKC peptide significantly inhibited P. aeruginosa-induced IL-6 production. Results are the means Ϯ S.E. of triplicate determinations (*, p Ͻ 0.05 compared with bacterial treatment alone). mast cell lysates were immunoprecipitated with Ab to PP2Ac and then blotted with Ab to PKC␣. The presence of PKC␣ was observed in the immunoprecipitates of PP2Ac (Fig. 4c). Thus, PKC␣ and PP2Ac are physically associated in mast cells. To exclude the possibility of nonspecific association between PP2Ac and PKC␣, an anti-focal adhesion kinase (FAK) Ab (Santa Cruz Biotechnology) was used for immunoprecipitation and Western blot. No PP2Ac can be found in FAK immunoprecipitates (Fig. 4e). Similarly, no FAK can be seen in PP2Ac immunoprecipitates, although mast cells express a substantial amount of FAK proteins (Fig. 4e).
Confocal microscopy was used to demonstrate the colocalization of PP2Ac and PKC␣ in mast cells. Unstimulated HMC-1 cells were permeabilized and stained with Abs to PP2Ac (mouse IgG1) and PKC␣ (rabbit IgG). Fluorescence-labeled second Abs to mouse IgG (Alexa Fluor® 594, red) and to rabbit IgG (Alexa Fluor® 488, green) were used to visualize the distribution of PP2Ac (red) and PKC␣ (green) in mast cells. The yellow color indicates the colocalization of these two enzymes (overlay of red and green). As shown in Fig. 4, f-h, PKC␣ was mainly located in the cell cytosol, whereas PP2A was distributed in both cytosol and nuclear fractions. Colocalization of PKC␣ and PP2Ac was observed in cytosols of mast cells (Fig.  4h). Although PP2A has long been considered as a predominantly cytosolic enzyme, the presence of PP2Ac in fibroblast nuclei and other cellular compartments has also been reported (46). Fig. 4h indicates that mast cells express two populations of PP2Ac, a PKC␣-associated PP2Ac distributed in the cytosol and a PKC␣-unassociated population located mainly in the nuclear fraction.
PKC␣ Phosphorylation and PKC Activity in Mast Cells Are Enhanced by the PP2A Inhibitor Okadaic Acid-The physical association between PKC␣ and PP2Ac suggests a functional interaction between these two enzymes. The phosphorylation of PKC␣ on serine 657 controls accumulation of active enzyme and contributes to the maintenance of the phosphatase-resistant conformation (32). To test whether inhibition of PP2Ac by okadaic acid modulates mast cell PKC␣ phosphorylation, BMMC and MC/9 cells were treated with okadaic acid at various doses (10, 100, and 1,000 nM) for 1 h and lysed in RIPA buffer. Total cell lysates were analyzed by SDS-PAGE and probed with an Ab that recognizes phosphorylated PKC␣ on serine 657. As seen in Fig. 5a, phosphorylation of PKC␣ in both BMMC and MC/9 was enhanced by okadaic acid, an effect FIG. 4. PP2Ac and PKC␣ are physically associated in mast cells. HMC-1, MC/9, and BMMC were lysed in RIPA buffer. a, lysates were immunoprecipitated (IP) with Ab to PKC␣ and probed with Ab to PP2Ac. b, lysates were immunoprecipitated with Ab to PKC␣ and probed with Ab to PKC␣. c, lysates were immunoprecipitated with Ab to PP2Ac and probed with Ab to PKC␣. d, lysates were immunoprecipitated with Ab to PP2Ac and probed with Ab to PP2Ac. Mast cells constitutively express PKC␣ (b) and PP2Ac (d). The presence of PP2Ac in PKC␣ immunoprecipitates (a) and the presence of PKC␣ in the PP2Ac immunoprecipitates (c) demonstrate the physical association between these two enzymes. e, PP2Ac does not associate with FAK. HMC-1 cells were immunoprecipitated with Ab to FAK and probed with Abs to FAK or PP2Ac, showing no PP2Ac in FAK immunoprecipitates. In addition, PP2Ac immunoprecipitates or total cell lysate were Western blotted (WB) with Ab to FAK, showing no FAK in PP2Ac immunoprecipitates, although mast cells express FAK proteins. f-h, HMC-1 cells were fixed, permeabilized, and incubated with Abs to PKC␣ and PP2Ac. Then cells were stained with fluorescence-labeled second Abs to visualize the distribution of PP2Ac (red) and PKC␣ (green) by confocal microscopy. h is the overlay of f and g. The yellow color indicates the colocalization of these two enzymes (overlay of red and green). Colocalization of PKC␣ and PP2Ac is seen in cell cytosols. similar to P. aeruginosa treatment. The increase of phosphorylated PKC␣ was not the result of changes in total PKC␣ levels because total cell lysates, when probed with Ab to nonphosphorylated PKC␣, showed similar PKC␣ levels after okadaic acid treatment.
To test whether okadaic acid stimulates mast cell PKC activity, MC/9 cells were treated with 500 nM okadaic acid for 3 h and suspended in extraction buffer. Similar to P. aeruginosa treatment, okadaic acid treatment stimulated mast cell PKC activity using radioactive (Fig. 5c) and nonradioactive tests (Fig. 5d), suggesting that inhibition of PP2A increases PKC activity in mast cells.
PKC Inhibitors Block Okadaic Acid-induced IL-6 Production by Mast Cells-Increased PKC␣ phosphorylation by okadaic acid treatment suggests an effect on mast cell cytokine production. As shown in Fig. 6a, treatment of BMMC with okadaic acid for 24 h induced significant IL-6 production. To test whether PKC is involved in okadaic acid-induced cytokine production by mast cells, PKC inhibitor Ro 31-8220 (47) was used to treat BMMC during okadaic acid stimulation. Okadaic acidinduced IL-6 production by BMMC was inhibited by Ro 31-8220 in a dose-dependent manner (Fig. 6b). These data suggest a role of PKC␣ in okadaic acid-induced IL-6 production by mast cells and are consistent with a model that inhibition of PP2A increases PKC␣ activation and leads to IL-6 production by mast cells. This model helps our understanding of the role of PP2Ac-PKC␣ interaction in P. aeruginosa-induced IL-6 production by mast cells.
Synergistic Effects of Okadaic Acid on P. aeruginosa-and PMA-induced IL-6 Production-The physical and functional interaction between PP2Ac and PKC␣ in the regulation of IL-6 production suggests that modulation of these two enzymes will lead to an altered production of this cytokine by mast cells. When BMMC were treated with 50 nM okadaic acid together with 10 nM PMA for 24 h, okadaic acid demonstrated a synergistic effect on PMA-induced IL-6 production (IL-6 pg/ml: 9.8 Ϯ 2.9, 395.8 Ϯ 65.2, 209.1 Ϯ 15.2, and 650.9 Ϯ 55.8 by the treatment with medium, PMA alone, okadaic acid alone, and PMA ϩ okadaic acid, respectively). Strikingly, a strong synergism on IL-6 production was observed when BMMC were treated with P. aeruginosa in the presence of okadaic acid (Fig.  7). These data together with the effects of P. aeruginosa treatment on the reduction of PP2Ac protein level and activation of PKC support a role of PP2Ac-PKC␣ interaction in P. aeruginosa-induced mast cell IL-6 production.  a and b, BMMC (a) and MC/9 (b) were treated with okadaic acid at various concentrations for 1 h and lysed in RIPA buffer. Total cell lysates were subjected to SDS-PAGE and analyzed by Western blot with Ab that recognizes phosphorylated PKC␣ on serine 657 (upper panels). Blots were then stripped and re-probed with a PKC␣-specific Ab to reveal total PKC␣ (lower panels). c and d, MC/9 cells after treatment with okadaic acid (500 nM) for 1 h were lysed in extraction buffer. PKC activity was determined using a radioactive (c) and a nonradioactive (d) protein kinase assay kit from Calbiochem-Novabiochem Co. Results are the means Ϯ S.E. of triplicate determinations (*, p Ͻ 0.05 compared with groups without okadaic acid treatment).

DISCUSSION
Previous studies have described the reciprocal regulation of PKC␣ and PP2A in vitro (33) and numerous overlapping effects between okadaic acid and PMA, suggesting an intimate interaction between these two enzymes. In the present study we have demonstrated that PKC␣ and PP2Ac are physically associated in mast cells during the resting state. Moreover, these two enzymes are functionally associated in the regulation of mast cell IL-6 production and are involved in P. aeruginosainduced IL-6 production by mast cells.
Mast cells are abundant in the tissue area where they interface with external surfaces such as airway mucosa. Recently, several elegant studies have demonstrated that these cells are critical in the host defense against bacterial infection (6,7,48,49); however, little is known about the signaling mechanisms involved. We have shown previously that two members of PKC family, PKC␤ and ␦, are involved in the internalization of E. coli by mast cells (17), suggesting that PKC plays a role in mast cell responses to bacterial pathogens. In this study, a role of PKC␣ and PP2Ac in P. aeruginosa-induced IL-6 production by mast cells is shown. Treatment of mast cells with cystic fibrosis-associated P. aeruginosa induced significant increases of PKC␣ phosphorylation and PKC activity. Moreover, PKC inhibitor Ro 31-8220 and PKC pseudosubstrate blocked P. aeruginosa-induced IL-6 production. These data suggest that PKC␣ activation is one of the mechanisms involved in P. aeruginosa-induced mast cell responses.
Interestingly, treatment of mast cells with P. aeruginosa induced a significant decrease of PP2Ac level. Treatment of mast cells with P. aeruginosa for 3 or 24 h did not affect the phosphorylation of several signaling proteins such as PKB␣, CREB, STAT1, STAT5, Jak2, and RAF1 (data not shown), suggesting a specific effect on PKC␣ and PP2Ac. Given that PP2A in vitro has the capacity to down-regulate PKC␣ activation through dephosphorylation (33), we hypothesized that decreased PP2A is one of the mechanisms involved in P. aeruginosa-induced PKC␣ activation. This hypothesis prompted us to determine the possible interactions between PP2A and PKC␣ in mast cells. Although roles of PP2A and PKC␣ have been described individually in IgE-mediated signaling events, little is known about their interactions in regulating mast cell functions. In this study, the presence of PP2Ac in PKC␣ immunoprecipitates and the presence of PKC␣ in PP2Ac immunoprecipitates provided direct evidence of physical association between PKC␣ and PP2Ac. Confocal microscopy showed that these two enzymes are colocalized in cytosols. To our knowledge, this is the first direct evidence demonstrating the physical association between PKC␣ and PP2Ac in any system. The finding of a physical association of these two enzymes in mast cells could likely be applied to other cell types because coexistence of these two enzymes in the same cellular fraction was observed in COS cells (35).
In resting mast cells, association of PKC␣ and PP2Ac was observed in the cytosol. One of the dynamic features of PKC␣ upon activation is translocation from cytosol to membrane. Although the role of PP2A in PKC␣ translocation remains to be determined, it is likely that PP2Ac is translocated along with PKC␣ because of their physical association. This is supported by a recent study by Ludowyke et al. (9) that PP2A translocation to the mast cell membrane can be induced by PKC activator PMA. In COS cells, the presence of PP2Ac correlates with PKC␣ phosphatase activity in membrane fraction (35), suggesting the coexistence of PP2Ac and PKC␣ in the same cellular compartment. Dephosphorylation of PKC␣ was found in the membrane compartment (35). Thus, it is likely that PP2A, after translocation to the membrane along with PKC␣, continues to play a role in the termination of PKC␣ activation by dephosphorylation.
The physical association of PKC␣ and PP2Ac suggests a functional interaction between these two enzymes in mast cells. Treatment of mast cells with okadaic acid induced an increase of PKC␣ phosphorylation and PKC activity and stimulated IL-6 production. Moreover, okadaic acid-induced IL-6 production was blocked by PKC inhibitors. These data support the notion that in mast cells, PP2Ac physically binds to PKC␣ and regulates its activities. This interaction is involved in the regulation of IL-6 production by mast cells. The effects of okadaic acid or PP2A on cytokine production by mast cells have not been reported previously. The interaction of PKC␣ and PP2Ac in the regulation of IL-6 production in this study suggests that PP2A may have broader roles in the regulation of mast cell functions than thought previously. However, caution should be applied when making generalizations about this mechanism to other mast cell mediators such as histamine (degranulation) because mast cells possess different mechanisms in the regulation of different mediator secretion (50). Indeed, contrary to the stimulatory effects of okadaic acid on IL-6 production observed in this study, several studies demonstrated that okadaic acid inhibits mast cell degranulation in a time-and concentration-dependent manner (10 -14).
Valuable information regarding the underlying signaling mechanisms mediated by PP2A has been obtained with the use of okadaic acid. In vitro, okadaic acid blocks both PP2A and PP1 activity at 0.1-10 nM concentrations, although it is 10-fold more effective against PP2A (36,(51)(52)(53). In intact cells, higher concentrations (up to 1 M) are required to achieve an effect similar to that seen in intro (53,54). Okadaic acid has little or no effect on PP2B or PP2C. In mast cells, okadaic acid at the dose of 1 M inhibited PP2A activity but had very little or no effect on PP1 activity (9), suggesting that okadaic acid may have more selective effects on PP2A activity in mast cells than that seen in other cell types.
The demonstration of the physical and function interaction in mast cells between PP2Ac and PKC␣ and their roles in the regulation of IL-6 production provides a basis for the understanding of the mechanisms of P. aeruginosa-induced mast cell activation. Okadaic acid demonstrated a significant synergistic effect on P. aeruginosa-induced IL-6 production. These data together with the evidence of P. aeruginosa-induced PKC␣ activation and PP2Ac depletion are consistent with a model by FIG. 7. Synergistic effects of okadaic acid on P. aeruginosainduced IL-6 production by mast cells. BMMC (5 ϫ 10 5 cells/ml) were treated with P. aeruginosa (mast cell:bacteria ratio of 1:50) with or without okadaic acid (OA) at a concentration of 100 nM for 24 h. Cell-free supernatants were used to determine IL-6 protein by ELISA. Results are the means Ϯ S.E. of five experiments (*, p Ͻ 0.05 compared with groups of P. aeruginosa alone or okadaic acid alone; #, p Ͻ 0.05 compared with sham-treated group).
which down-regulation of PP2A by P. aeruginosa causes activation of PKC␣ and leads to IL-6 production by mast cells. This model provides a potential intracellular target for the therapeutic modulation of P. aeruginosa-induced inflammation.
The significant IL-6 production by mast cells after P. aeruginosa-stimulation suggests that mast cells may serve as a cellular source for this cytokine during P. aeruginosa infection. Early studies have demonstrated that mast cells secrete histamine and leukotriene C 4 in response to P. aeruginosa stimulation (55,56). Recently, the importance of mast cell-derived cytokines in the regulation of immune response has increasingly been recognized. IL-6 is a multipotent cytokine produced in the context of inflammation and infection and is critical to the development of the acute phase response during inflammation (57)(58)(59). We chose to examine the regulation of IL-6 production by mast cells in view of the wide range of biologic activities of IL-6 which are relevant to the initiation and progression of inflammation (59) and because production of IL-6 in the airway has been implicated in P. aeruginosa-associated cystic fibrosis (1,60,61). The mast cell is a potent source of IL-6 and is able to produce this cytokine relatively rapidly compared with the more traditional sources of this cytokine, such as monocytes and macrophages (50,57). Although dysregulation of cytokine production has been recognized as one of the major pathogenic mechanisms during P. aeruginosa infection (62,63), cytokine production by mast cells after P. aeruginosa stimulation has not been examined previously. Significant IL-6 production by mast cells induced by P. aeruginosa stimulation, together with the fact that mast cells are found in large numbers in the airway, suggests that mast cells may have a previously unrecognized role in P. aeruginosa-induced inflammation.
In summary, we have made the following novel observations in this study. First, PKC␣ and PP2Ac are found physically and functionally associated in mast cells and are involved in the regulation of IL-6 production by mast cells. Second, mast cells respond to P. aeruginosa to produce cytokine IL-6, suggesting a role of mast cells in P. aeruginosa-induced inflammation. Third, interaction between PKC␣ and PP2A is one of the mechanisms involved in P. aeruginosa-induced IL-6 production by mast cells.