Regulation of Cytosolic Phosphorylation by Proteolytic Cleavage of A1 in Activated Mast Cells

This information is current as Joon Hyun Kwon, Jea Hwang Lee, Ki Soon Kim, Youn of September 28, 2021. Wook Chung and Ick Young Kim J Immunol 2012; 188:5665-5673; Prepublished online 25 April 2012; doi: 10.4049/jimmunol.1102306

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Cytosolic Phospholipase A2 Phosphorylation by Proteolytic Cleavage of in Activated Mast Cells

Joon Hyun Kwon,* Jea Hwang Lee,* Ki Soon Kim,* Youn Wook Chung,† and Ick Young Kim*

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as , , and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow- derived mast cells. This cleavage event was inhibited when intracellular Ca2+ signaling was blocked. ANXA1-knockdown RBL-

2H3 cells produced a greater amount of with simultaneous upregulation of cytosolic phospholipase A2 (cPLA2) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found Downloaded from that cPLA2 interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA2 mainly interacted with full-length ANXA1 in the and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA2 and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA2 and production of eicosanoids during mast-cell activation. The Journal of Immunology, 2012, 188: 5665–5673. http://www.jimmunol.org/

ast cells are considered central effector cells in allergic domains: an N-terminal tail and a C-terminal core domain. The C- diseases, such as asthma, allergic rhinitis, and food terminal core domain consists of four repeats of a highly con- M , and mast-cell–derived proinflammatory me- served amino acid sequence responsible for binding with Ca2+ and diators play a key role in these pathologies (1–3). Ag-mediated (9). The N-terminal peptide is known to be cleaved activation of mast cells is regulated by a complex series of in- by a number of , including elastase, , plasmin, tracellular signaling processes that are initiated by FcεRI aggre- cathepsin D, and proteinase 3 (11–14), and is also phosphorylated gation. Activated mast cells show three biological characteristics: to regulate the activity of the (15, 16). N-terminal trun- by guest on September 28, 2021 degranulation ( of mediators stored in intracellular cated ANXA1 has a proinflammatory effect and promotes neu- granules), the production of cytokines and chemokines, and the trophil transendothelial migration (17). Truncated ANXA1 has release of newly synthesized lipid mediators (4–7). Most studies also been linked to an epidermal growth factor (EGF)-triggered ε on Fc RI-mediated signaling have been carried out using rat ba- signaling pathway involving cytosolic phospholipase A2 (cPLA2) sophilic leukemia 2H3 (RBL-2H3) cells and mutated sublines (8). in normal and malignant squamous epithelial cells (14). In acti- Although a number of studies have described the mast-cell acti- vated polymorphonuclear (PMN) leukocytes, ANXA1 is cleaved vation pathway, it is still not fully understood. and then translocates to the extracellular surface (13). Mutant Annexin A1 (ANXA1) is a member of the ANX family of Ca2+ ANXA1, which is resistant to cleavage by proteases isolated from and -binding , and plays different roles under PMN cell extracts, was previously shown to have anti-inflamma- various biological conditions, for example, membrane organiza- tory activities in the inflamed microcirculation of mice and in tion, , and inflammation (9, 10). ANXA1 consists of two a skin trafficking model (18). We previously demonstrated the proteolytic cleavage of ANXA1 *Laboratory of Cellular and Molecular Biochemistry, School of Life Sciences and and activation of cPLA2 in a murine OVA-induced asthma model Biotechnology, Korea University, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Korea; (19). cPLA2 plays a key role in the release of arachidonic acid and †Cardiovascular Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 (AA) for the production of lipid inflammatory mediators, such as and PGs. In mast cells, the aggregation of IgE-loaded Received for publication August 11, 2011. Accepted for publication March 31, 2012. FcεRI induced by polyvalent Ag leads to the phosphorylation of This work was supported by the Basic Science Research Program through the Na- tional Research Foundation of Korea supported by the Ministry of Education, Science ERK and cPLA2, followed by release of AA, which is then further and Technology (KRF-2008-313-C00548). converted to leukotrienes and PGs by 5-lipoxygenase (5-LO) and Address correspondence and reprint requests to Prof. Ick Young Kim, School of Life (COX), respectively (20). Cysteinyl leukotrienes Sciences and Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-Ku, (cysLTs) are involved in the pathogenesis of asthma and are Seoul 136-701, Korea. E-mail address: [email protected] determinants of the severity of asthma (21). cPLA2 activity is The online version of this article contains supplemental material. regulated by intracellular Ca2+ concentrations and phosphoryla- Abbreviations used in this article: AA, arachidonic acid; ANXA1, annexin A1; 2- tion (22). Increased intracellular Ca2+ concentrations promote the APB, 2-aminoethoxydiphenyl borate; BMMC, bone marrow-derived mast cell; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; cysLT, cysteinyl ; translocation of cPLA2 from the cytosol to the membrane (23, 24). EGF, epidermal growth factor; 5-LO, 5-lipoxygenase; mtANXA1, triple-mutant The catalytic domain of cPLA2 is phosphorylated by MAPKs ANXA1; NC, negative control; PMN, polymorphonuclear; RBL-2H3, rat basophilic 505 727 leukemia 2H3; siRNA, small interfering RNA; TG, thapsigargin; wtANXA1, wild- (Ser ) and MAPK-interaction (Ser ) (25–27). Fur- type ANXA1. thermore, cPLA2 activity is inhibited by direct interaction with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102306 5666 CLEAVAGE OF ANNEXIN A1 REGULATES PHOSPHORYLATION OF cPLA2

ANXA1 (28, 29). However, the inhibitory mechanisms of ANXA1 RBL-2H3 cells were maintained as a monolayer in DMEM medium (Life remain unclear. In particular, the functional significance of Technologies) supplemented with 15% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37˚C and 5% CO2. ANXA1 cleavage in cPLA2 activity during mast-cell activation RBL-2H3 cells (1 3 106 cells in a 60-mm dish) and BMMCs (5 3106 remains unknown. In this study, we demonstrate that cleavage cells/ml) were sensitized with or without monoclonal anti-DNP IgE (500 of ANXA1 activates cPLA2 by phosphorylation, stimulating a ng/ml) for 12 h in cytokine-free medium and serum starved for 6 h with proinflammatory reaction in activated mast cells. DMEM or RPMI 1640 containing 0.5% FBS. Serum-starved cells were activated with various stimuli for the indicated times. Materials and Methods for b-hexosaminidase release Reagents b-Hexosaminidase release was used as a measure of degranulation activity Monoclonal anti-DNP Clone SPE-7, A23187 (a ionophore), BAPTA- as described previously (31). In brief, IgE-sensitized and serum-starved AM, 2-aminoethoxydiphenyl borate (2-APB), thapsigargin (TG), HEPES, RBL-2H3 cells and BMMCs were washed twice with Tyroid buffer (135 sodium pyruvate, sodium orthovanadate, and PMSF were obtained from mM NaCl, 5 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 1 mg/ml BSA, Sigma-Aldrich. DNP-BSA and p-nitrophenyl-2-acetyl-b-D-glucosaminide and 20 mM HEPES [pH 7.4]) and then stimulated with DNP-BSA (1 mg/ were purchased from Calbiochem. Recombinant mouse IL-3 and stem cell ml) or A23187 (1 mM) at 37˚C in Tyroid buffer for the indicated times. factor were obtained from R&D Systems. Abs against p-ERK1/2, ERK1/2, The incubation buffer and cell lysates, prepared by the addition of identical and p-cPLA2 (Ser505) were purchased from Technology. volumes of Tyroid buffer containing 1% Triton X-100, were then recov- b ANXA1 Abs were obtained from Zymed, and cPLA2 (4-4B-3C) Abs were ered for quantitation of -hexosaminidase release. purchased from Santa Cruz Biotechnology. Abs against a-tubulin were purchased from LabFrontier (Seoul, Korea). Negative control (NC) stealth Assay for cysLTs and PGE2 release small interfering RNAs (siRNAs), ANXA1 stealth siRNAs, and Neon Release of cysLTs and PGE2 was measured using an immunoassay Downloaded from transfection kit (MP100) were obtained from Invitrogen. system (Amersham), according to the manufacturer’s instructions. IgE- Mast-cell culture and activation sensitized and serum-starved RBL-2H3 cells and BMMCs were stimu- lated with DNP-BSA (1 mg/ml), A23187 (1 mM), or TG (1 mM) at 37˚C Bone marrow-derived mast cells (BMMCs) were isolated from the femurs for the indicated times. When necessary, BAPTA-AM (30 mM) and 2-APB of C57BL/6 mice (8- to 12-wk-old) as described previously (30). The (50 mM) were added to the cells for 30 min before activation with DNP- isolated BMMCs were cultured in RPMI 1640 medium (Life Technolo- BSA. Then, 100 ml culture medium was concentrated by freeze-drying gies) supplemented with 10% FBS, 2 mM L-glutamine, 1% nonessential overnight, followed by reconstitution in assay buffer. Samples and stan- http://www.jimmunol.org/ amino acids (Invitrogen), 50 mM 2-ME, 1 mM sodium pyruvate, 100 U/ml dard cysLTs or PGE2 were incubated with antiserum in a 96-well plate for penicillin, 100 mg/ml streptomycin, 25 mM HEPES, 10 ng/ml stem cell 2 or 3 h, followed by cysLT or PGE2-peroxidase conjugates for 3 or 1 h, factor, and 5 ng/ml IL-3 for 4–8 wk at 37˚C and 5% CO2. Purity, measured respectively, at 4˚C. To remove unbound ligand, we washed the wells five by flow cytometry analysis using FITC-conjugated rat anti-mouse c-Kit times with wash buffer. Then the amount of bound peroxidase-labeled mAbs (BD Biosciences) at 4 wk, was .97%. cysLTs and PGE2 was determined by the addition of substrate for 30 by guest on September 28, 2021

FIGURE 1. Time-dependent activation of RBL-2H3 cells and proteolytic cleavage of ANXA1. IgE-sensitized RBL-2H3 cells were serum starved and activated with DNP-BSA (1 mg/ml) for the indicated times. (A) The incubation buffer and cell lysates were recovered for quantitative analysis of b-hexosaminidase release as a measure of degranulation activity. (B) Total RNA isolated from activated cells was subjected to RT-PCR analysis. The expression of TNF-a, IL-4, and IL-6 mRNA was determined for measurement of cytokine production. GAPDH was used as a loading control. (C) The levels of cysLTs and PGE2 secreted from the activated cells were analyzed using an enzyme immunoassay system. (D and E) Whole-cell lysates prepared from the cells activated by DNP-BSA for the indicated times were subjected to immunoblotting with Abs against (D) ERK1/2, p-ERK1/2, cPLA2, p-cPLA2,(E) ANXA1, and b-actin. (F) Total RNA isolated from activated cells was subjected to RT-PCR analysis for measurement of ANXA1 and GAPDH mRNA. (A and C) Data are expressed as the means 6 SE of three independent experiments. (B, D–F) Results are representative of at least three independent experiments. The Journal of Immunology 5667 min at room temperature. The reaction was stopped by the addition of 100 in R buffer containing 100 pmol ANXA1 siRNAs for RBL-2H3 cells ml of 1 M sulfuric acid, and OD was determined at 450 nm. (siRNA-1, 59-CCA GCA GAT CAA GGC AGC AUA CUU A-39; siRNA-2, 59-CAG AUG AAG ACA CUC UCA UUG AGA U-39)or5mgplasmid RNA isolation and RT-PCR DNA for RBL-2H3 cells and BMMCs. Resuspended cells were then transferred into a gold tip and electroporated by 1 pulse at 1600 V for 30 Total RNA was isolated from RBL-2H3 cells with TRIzol reagent (Invi- ms, followed by incubation overnight in growth media without antibiotics. trogen), and first-strand cDNA was synthesized by reverse transcription using oligodT and SuperScript III reverse transcriptase (Invitrogen). PCR Data analysis and statistics analysis of the resulting cDNA was performed using a PCR premix, Sapphire (Super Bio, Seoul, Korea), and the following forward and reverse All data are represented in this study as the means 6 SE of the control primers: ANXA1, 59-GCG GCG GAT CCA TGG TCA ATG GTA TCA value. Statistical comparisons from at least three independent experiments GAA TTC CTC-39 and 59-GCG GCG TCG ACT TAG TTT CCT CCA were determined using Student t tests. The p values ,0.05 were considered CAA AGA GCC AC-39; TNF-a,59-CAA GGA GGA GAA GTT CCC AA- significant. 39 and 59-CGG ACT CCG TGA TGT CTA AG-39; IL-4, 59-ACC TTG CTG TCA CCC TGT TC-39 and 59-TTG TGA GCG TGG ACT CAT TC- Results 39; IL-6, 59-GAA ATG ATG GAT GCT TCC AAA CTG G-39 and 59-GGA TAT ATT TTC TGA CCA CAG TGA GG-39; GAPDH, 59-AGG TCG Proteolytic cleavage of ANXA1 in activated mast cells GAG TCA ACG GAT TT-39 and 59-AGG TGG AGG AGT GGG TGT CG- We previously confirmed the proteolytic cleavage of ANXA1 in 39. The PCR products were resolved on a 1 or 1.5% agarose gel and vi- sualized by staining with ethidium bromide. lung extracts prepared from a murine asthma model (19). To further Subcellular fractionation

The subcellular fractionation of DNP-BSA or A23187-stimulated RBL-2H3 Downloaded from cells was performed using a ProteoJET extraction kit (Fermentas). In brief, activated cells were washed with cell wash solution, scraped with cell permeabilization buffer containing inhibitors, and then incubated on ice for 10 min. Permeabilized cells were centrifuged at 16,000 3 g for 15 min at 4˚C. Supernatants were collected for isolation of the cytosolic fraction. Membrane protein extraction buffer was added to the pellet, which was then incubated on ice for 30 min and centrifuged at http://www.jimmunol.org/ 16,000 3 g for 15 min at 4˚C. After centrifugation again, supernatants were collected for isolation of the membrane fraction. These cytosolic and membrane fractions were used for immunoblotting and immunoprecipi- tation assays. Immunoprecipitation and immunoblot analysis Activated RBL-2H3 cells and BMMCs were washed twice with cold PBS and lysed in lysis buffer containing 10 mM HEPES (pH 7.4), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 0.5% Nonidet P-40, 1 mM NaF, and 1 mM Na VO . After

3 4 by guest on September 28, 2021 centrifugation at 16,000 3 g for 15 min at 4˚C, supernatants containing the whole cell lysates were collected. The protein concentrations of the whole- cell lysates and subcellular fractions were determined using Bradford re- agent (Sigma). For immunoprecipitation, 500 mg protein was precleared with protein G-agarose (GE Healthcare) for 1 h at 4˚C, followed by in- cubation with 1 mg cPLA2 or GFP Abs overnight at 4˚C. Immune com- plexes were further incubated with protein G-agarose for 2 h at 4˚C and then washed with lysis buffer three times. For immunoblotting, proteins prepared by whole-cell lysis, subcellular fractionation, and immunopre- cipitation were boiled with SDS-PAGE sample buffer for 5 min and then separated by electrophoresis on 8% (for cPLA2, p-cPLA2, ERK, p-ERK, GFP, COX-2, 5-LO, , a-tubulin, and b-actin) or 10% (for ANXA1) SDS-polyacrylamide gels. The separated proteins were then transferred to a polyvinylidene fluoride membrane, and the membrane was then blocked in 5% skim milk and incubated overnight with primary Ab at 4˚C. After incubation with HRP-conjugated secondary Ab for 1 h at room temperature, immunoreactive bands were visualized using a West Pico enhanced ECL detection kit (Pierce). Band intensities were determined densitometrically using ImageJ software. Construction of ANXA1 expression vector and site-directed mutagenesis FIGURE 2. Effects of ANXA1 knockdown on RBL-2H3 cell activation. Total RNA and first-strand cDNA were obtained from HEK293 cells as NC and siANXA1 were transfected into RBL-2H3 cells by electroporation. described earlier. ANXA1 cDNA was generated by RT-PCR using specific Cells were incubated with IgE overnight, serum starved for 6 h, and then primers (forward, 59-CCG CTC GAG ATG GCA ATG GTA TCA GAA activated with DNP-BSA for 30 min. (A) Total RNAs isolated from NC- TTC C-39; reverse, 59-GGT GGA TCC TTT CCT CCA CAA AGA GCC ACC-39). PCR products were cloned into XhoI and BamHI sites of the and siANXA1-transfected cells were subjected to RT-PCR analysis for B pEGFP-N1 vector. A cleavage-resistant ANXA1 mutant (A11R/V22K/ measurement of ANXA1 and GAPDH mRNA. ( ) Degranulation activity, C D V36K) was constructed by sequential site-directed mutagenesis reactions ( ) production of cytokines, and ( ) release of cysLTs and PGE2 from NC- using QuikChange site-directed mutagenesis (Stratagene), according to the and siANXA1-transfected cells were measured as described in Fig. 1. (E) manufacturer’s instructions. Lysates from NC- and siANXA1-transfected cells were subjected to im- munoblot analysis with Abs against ANXA1, ERK1/2, p-ERK1/2, cPLA , Transfection of siRNA and plasmid DNA 2 p-cPLA2, and b-actin. (A, C, and E) Results are representative of at least Transfections were carried out using the Neon transfection system, according three experiments. (B and D) Data are expressed as the means 6 SE of to the manufacturer’s instructions. In brief, RBL-2H3 cells (1 3 107 cells/ three independent experiments. Significant differences were determined by ml) and BMMCs (2 3 107 cells/ml) were washed with PBS and resuspended comparison of siANXA1 with the NC in activated cells (*p , 0.05). 5668 CLEAVAGE OF ANNEXIN A1 REGULATES PHOSPHORYLATION OF cPLA2

elucidate the regulatory mechanisms of ANXA1 cleavage and Release of eicosanoids and phosphorylation of cPLA2 are their roles in inflammation, we activated both RBL-2H3 cells and increased in ANXA1-knockdown RBL-2H3 cells BMMCs with DNP-BSA. For activation, IgE-sensitized cells were To investigate the role of ANXA1 in mast-cell activation, we m serum starved and then stimulated with DNP-BSA (1 g/ml) for transfected ANXA1 siRNAs into RBL-2H3 cells (Fig. 2A). There the indicated times. RBL-2H3 cell activation was determined by was no difference in either degranulation activity or cytokine measuring degranulation, cytokine production, and production between the NC and ANXA1 siRNA-transfected cells generation. Release of b-hexosaminidase from cytosolic granules (Fig. 2B, 2C). However, the release of cysLTs and PGE2 was to extracellular spaces was used as a measure of degranulation significantly increased in ANXA1-knockdown cells (Fig. 2D). To activity. The activity reached a maximum at 30 min after stimu- understand the increased production of eicosanoids, we examined lation (Fig. 1A). Production of cytokines was determined by RT- the phosphorylation of ERK and cPLA . Whereas ANXA1 siRNA a 2 PCR analysis of TNF- , IL-4, and IL-6 mRNA levels. Expression transfection did not affect ERK phosphorylation, cPLA phos- a 2 of both TNF- and IL-4 was rapidly induced by DNP-BSA phorylation was significantly increased (Fig. 2E). We also found $ treatment, whereas IL-6 production was induced only after 30 that expression of COX-2 and translocation of 5-LO were not min of treatment (Fig. 1B). The generation of eicosanoids was changed in ANXA1-knockdown RBL-2H3 cells (Supplemental represented by the presence of cysLTs and PGE2 in cell-free Fig. 2). These results suggest that cPLA2 phosphorylation is as- supernatants (Fig. 1C). Like degranulation activity, the produc- sociated with ANXA1 during mast-cell activation. tion of eicosanoids also reached a maximum at 30 min. ERK and Intracellular Ca2+-dependent translocation of ANXA1 and cPLA2 were differentially phosphorylated on DNP-BSA treatment in RBL-2H3 cells (Fig. 1D). ERK was rapidly phosphorylated, cPLA2 from the cytosolic to membrane fraction in activated Downloaded from mast cells and cPLA2 phosphorylation was detected at later time points. ANXA1 was cleaved without any changes in mRNA expression ANXA1 and cPLA2 are known to translocate from the cytosol to (Fig. 1E, 1F). the membrane on increase in intracellular Ca2+ concentrations (9, BMMC activation was also determined by measuring degran- 32). Similar to DNP-BSA stimulation, both A23187, a calcium ulation and eicosanoid generation. Release of b-hexosaminidase ionophore, and TG, which increases cytosolic Ca2+ levels, acti- and production of eicosanoids were similar to those of RBL-2H3 vated RBL-2H3 cells and BMMCs (Fig. 3). Thus, ANXA1 http://www.jimmunol.org/ cells (Supplemental Fig. 1A, 1B). Similar to our results in RBL- cleavage and cPLA2 phosphorylation were measured on treatment 2H3 cells, cleavage of ANXA1 and phosphorylation of ERK and with A23187 and TG. CysLTs and PGE2 were also produced by cPLA2 were also detected in BMMCs (Supplemental Fig. 1C). A23187 and TG treatment. When the cells were pretreated with by guest on September 28, 2021

FIGURE 3. Inhibition of ANXA1 cleavage and eicosanoid production by decreasing intracellular Ca2+ concentrations. IgE-sensitized RBL-2H3 cells and BMMCs were serum starved and stimulated with DNP-BSA (1 mg/ml, 30 min), TG (1 mM, 5 min), or A23187 (1 mM, 10 min). Cells were pretreated with BAPTA-AM (30 mM) or 2-APB (50 mM) for 30 min before stimulation with DNP-BSA. (A) Lysates of activated cells were subjected to immunoblot analysis with Abs against ANXA1, cPLA2, p-cPLA2, and b-actin. Results shown are representative of at least three experiments. (B) The levels of secreted cysLTs and PGE2 were determined by an enzyme immunoassay system. Data are expressed as the means 6 SE of three independent experiments. Sig- nificant differences were determined by comparison of DNP-BSA + BAPTA-AM or 2-APB with DNP-BSA (*p , 0.05). The Journal of Immunology 5669

BAPTA-AM, a Ca2+ chelator, or 2-APB, a store-operated Ca2+ A cleavage-resistant ANXA1 mutant inhibits phosphorylation channel inhibitor, ANXA1 cleavage and the production of cysLTs of cPLA2 and PGE2 significantly decreased (Fig. 3A, 3B). We also observed It was previously reported that cleavage of ANXA1 occurred that full-length ANXA1 was localized to both the cytosolic and at three sites (Ala11,Val22,andVal36) in the N-terminal region membrane fractions of nonactivated RBL-2H3 cells. When the during the activation of neutrophils (13). The ANXA1 mutant cells were activated by DNP-BSA or A23187, ANXA1 was (A11R/V22K/V36K), which is resistant to cleavage, controls cleaved, and the amount of full-length ANXA1 in the cytosol inflammation in the microvasculature (18). In this study, we decreased. In the membrane fraction, the level of full-length investigated whether mutant ANXA1 regulates cPLA activity ANXA1 did not differ from that of control cells, although cleav- 2 during RBL-2H3 cell and BMMC activation. GFP-tagged ex- age of ANXA1 increased (Fig. 4A, 4B). Thus, in response to cell pression vectors containing wild-type ANXA1 (wtANXA1) or activation, cleaved ANXA1 translocated mainly to the membrane. triple-mutant ANXA1 (mtANXA1) were constructed; then Furthermore, both cPLA and p-cPLA translocated from the 2 2 these vectors were introduced into RBL-2H3 cells and BMMCs cytosol to the membrane in activated RBL-2H3 cells (Fig. 4A, asdescribedintheMaterials and Methods. We confirmed that 4C). However, ANXA1 knockdown did not affect the transloca- tion of either cPLA or p-cPLA (Fig. 4D). mtANXA1 was not cleaved by DNP-BSA stimulation, whereas 2 2 both endogenous ANXA1 and wtANXA1 were cleaved (Fig. Both full-length and cleaved ANXA1 interact with cPLA2 6A, Supplemental Fig. 3A). We further observed that phos-

To elucidate the interactions between ANXA1 and cPLA2 during phorylation of cPLA2 was significantly decreased in both ac- mast-cell activation, we performed coimmunoprecipitations of the tivated RBL-2H3 cells and BMMCs expressing mtANXA1, Downloaded from proteins from whole-cell lysates and subcellular fractions. In the whereas ERK phosphorylation was not affected (Fig. 6B, whole-cell lysates, cPLA2 interacted with either full-length or Supplemental Fig. 3B). We also found that although both cleaved ANXA1 (Fig. 5A). However, coimmunoprecipitation of wtANXA1 and mtANXA1 interacted with cPLA2, p-cPLA2 was proteins from the subcellular fractions showed that cPLA2 mainly not detected in mtANXA1-expressing RBL-2H3 cells and interacted with full-length ANXA1 in the cytosol and with cleaved BMMCs under activation conditions (Fig. 6C, Supplemental Fig.

ANXA1 in the membrane fraction (Fig. 5B). 3C). http://www.jimmunol.org/

FIGURE 4. Translocation of ANXA1 and cPLA2 from the cytosol to the membrane in activated RBL-2H3 cells. by guest on September 28, 2021 (A–C) DNP-BSA or A23187-stimulated RBL-2H3 cells were fractionated into cytosol and membrane fractions. (A) The subcellular fractions were subjected to immunoblot analysis with Abs against

ANXA1, cPLA2,p-cPLA2, a-tubulin, and calnexin. a-Tubulin and calnexin were used as cytosol and membrane marker proteins, respectively. Results are representative of at least three ex- periments. Relative band intensities for (B) full-length and cleaved ANXA1 and (C)cPLA2 are represented as a percentage of the control full-length

ANXA1 or control cytosolic cPLA2, re- spectively, in each fraction. Data are expressed as the means 6 SE of three independent experiments. Significant differences were determined by compar- ison of DNP-BSA or A23187 with the control (*p , 0.05). (D) NC and ANXA1 siRNA-transfected cells were stimulated with DNP-BSA and then fractionated into cytosolic and membrane fractions. Cytosolic and membrane fractions were subjected to immunoblot analysis with

Abs against cPLA2, p-cPLA2, a-tubulin, and calnexin. Results are representative of at least three experiments. 5670 CLEAVAGE OF ANNEXIN A1 REGULATES PHOSPHORYLATION OF cPLA2

ANXA1 interacted with cPLA2 in the cytosol. However, when cells were activated by DNP-BSA or A23187, both ANXA1 cleavage and cPLA2 phosphorylation were observed. Furthermore, cleaved ANXA1 and phosphorylated cPLA2 interacted and colo- calized mainly to the membrane fractions of activated mast cell. We also found that mutant ANXA1, which is resistant to cleavage in activated mast cells, exhibited inhibitory effects on both the phosphorylation of cPLA2 and production of eicosanoids. Different degrees of ANXA1 expression and susceptibility to modulation by allergic stimuli have been observed in connective tissue and mucosal mast cells (33). ANXA1-null mice have shown resistance to the anti-inflammatory actions of in several experimental models of inflammation (34–36). Mast cells from ANXA1-null mice are more prone to release of histamine and PGD2 than those of wild-type mice in response to compound 48/80 and zymosan, respectively (37). Moreover, ANXA1-derived peptide Ac2-26 and antiflammin, nonapeptide fragments of lip- ocortin I, also inhibited mast-cell degranulation (38, 39). These FIGURE 5. Interactions between ANXA1 and cPLA2 in the cytosolic and membrane fractions. IgE-sensitized mast cells were serum starved and inhibitory effects were thought to be mediated by formyl peptide Downloaded from stimulated with DNP-BSA or A23187. (A) Whole-cell lysates from RBL- receptor in mast cells. In this study, we found that ANXA1-

2H3 cells and BMMCs were immunoprecipitated with cPLA2 Abs and knockdown RBL-2H3 cells released more eicosanoids and then subjected to immunoblot analysis with Abs against ANXA1, cPLA2, exhibited increased phosphorylation of cPLA2. These effects were and p-cPLA2.(B) DNP-BSA and A23187-stimulated RBL-2H3 cells were mediated through endogenous ANXA1, not formyl peptide re- fractionated into cytosolic and membrane fractions. Each fractionated ceptor downstream signaling. The release of b-hexosaminidase sample was immunoprecipitated with cPLA Abs and then subjected to 2 from RBL-2H3 cells did not change with DNP-BSA treatment, as http://www.jimmunol.org/ immunoblot analysis with Abs against ANXA1. All data are representative has been shown with release of histamine by compound 48/80 of at least three experiments. treatment (Fig. 2). ERK phosphorylation is known to be neces- sary for the generation of inflammatory mediators in mast-cell ANXA1 cleavage regulates production of eicosanoids in activation. Activated ERK has been shown to phosphorylate mast-cell activation cPLA2 for AA release (40). To determine whether the increased Next, we determined the effects of ANXA1 cleavage on the phosphorylation of cPLA2 in ANXA1-knockdown cells was due production of proinflammatory mediators in both RBL-2H3 cells to changes in ERK phosphorylation, we measured ERK phos- and BMMCs expressing wtANXA1 or mtANX1. Compared with phorylation in RBL-2H3 cells transfected with ANXA1 siRNA. In control or wtANXA1, mtANXA1-expressing cells did not show this experiment, we found that the level of ERK phosphorylation by guest on September 28, 2021 any difference in either degranulation activity or cytokine pro- in ANXA1-knockdown RBL-2H3 cells was the same as that in duction (Fig. 7A, 7B). However, release of cysLTs and PGE2 was control cells. However, cPLA2 phosphorylation was markedly significantly decreased in mtANXA1-expressing RBL-2H3 cells increased in the knockdown cells (Fig. 2E). These data suggest and BMMCs (Fig. 7C). that ANXA1 may regulate ERK-mediated cPLA2 phosphorylation during mast-cell activation. Discussion Eicosanoids are produced predominantly by inflammatory cells, In this study, we confirmed that ANXA1 was cleaved in activated such as PMN cells, macrophages, and mast cells. The FcεRI- mast cells, and this cleavage was required for the phosphorylation mediated mast-cell activation signal pathway for eicosanoid pro- of cPLA2 and subsequent inflammatory reactions, such as the duction is regulated by a complex series of signaling molecules. 2+ production of eicosanoids. In resting RBL-2H3 cells, full-length In particular, cPLA2 activation by ERK and intracellular Ca is

FIGURE 6. Effects of ANXA1 cleavage on the phosphorylation of cPLA2. wtANXA1 or mtANXA1 constructs were prepared as described in Materials and Methods and were transfected into RBL-2H3 cells. IgE-sensitized RBL-2H3 cells were serum starved and then stimulated with DNP-BSA for 30 min. (A and B) Lysates prepared from DNP-BSA–stimulated cells were subjected to immunoblot analysis with Abs against (A) ANXA1, GFP, and b-actin and

(B) ERK1/2, p-ERK1/2, cPLA2, and p-cPLA2.(C) Cell lysates were immunoprecipitated with GFP Abs and then subjected to immunoblot analysis with Abs against cPLA2 and p-cPLA2. All data are representative of at least three experiments. The Journal of Immunology 5671 Downloaded from

FIGURE 7. A cleavage-resistant ANXA1 mutant inhibited the production of eicosanoids in DNP-BSA–stimulated mast cells. GFP-, wtANXA1-, or mtANXA1-overexpressing RBL-2H3 cells and BMMCs were activated with DNP-BSA for 30 min. (A) Incubation buffer and cell lysates were recovered for quantitative analysis of b-hexosaminidase release as a measure of RBL-2H3 cell degranulation activity. Data are expressed as means 6 SE of three independent experiments. (B) Total RNA isolated from activated RBL-2H3 cells was subjected to RT-PCR analysis. The expression of TNF-a, IL-4, and IL- http://www.jimmunol.org/ 6 mRNA was determined for measurement of cytokine production. GAPDH was used as a loading control. Results shown are representative of at least three independent experiments. (C) The levels of secreted cysLTs from activated RBL-2H3 cells and BMMCs were analyzed by an enzyme immunoassay system. Data are expressed as means 6 SE of three independent experiments. Significant differences were determined by comparison of wtANXA1 or mtANXA1 constructs with GFP (*p , 0.05).

important for the production of eicosanoids (27, 41). cPLA2 is from the cytosol to the membrane in activated RBL-2H3 cells, but located mainly in the cytosol in resting mast cells and undergoes downregulation of ANXA1, did not affect the translocation of by guest on September 28, 2021 translocation to the nuclear envelope and cPLA2. However, cPLA2 phosphorylation was increased in the in response to various stimuli (23). Phosphorylation of cPLA2 and membrane fraction of ANXA1-knockdown cells (Fig. 4D). These 2+ increased intracellular Ca synergistically promote the full acti- data indicate that ANXA1 regulates cPLA2 phosphorylation, but vation of cPLA2 for AA release (42, 43). AAs are converted to not cPLA2 translocation. leukotrienes and PGs by 5-LO and COX, respectively (20). ANXA1 is present in both the nucleus and of rat mast Membrane translocation of 5-LO and increased levels of COX-2 cells (48). In this study, we also investigated the intracellular lo- without changes in COX-1, 5-LO, and cPLA2 expression were calization of ANXA1 in activated RBL-2H3 cells. The level of observed in Ag-stimulated mast cells (44, 45). In this study, we full-length ANXA1 decreased in the cytosolic fraction, whereas demonstrated the expression of COX-2 and translocation of 5-LO ANXA1 cleavage increased in the membrane fraction on activa- in ANXA1-knockdown RBL-2H3 cells (Supplemental Fig. 2). tion of RBL-2H3 cells with DNP-BSA or A23187 (Fig. 4A, 4B, COX-2 was expressed when both control and ANXA1-knockdown 5B). However, the level of full-length ANXA1 in the membrane RBL-2H3 cells were activated. 5-LO translocated from the cytosol fraction did not change during activation. Together with the data to the membrane in activated RBL-2H3 cells and ANXA1- on cPLA2 translocation described earlier, these results indicate knockdown cells. Production of cysLTs and PGE2 also had simi- that both cleaved ANXA1 and cPLA2 translocate from the cytosol lar patterns in activated RBL-2H3 cells and BMMCs. These data to the membrane in activated mast cells. indicated that ANXA1 did not affect COX or 5-LO activity. ANXA1 is a cPLA2-binding protein that negatively regulates Previous studies have demonstrated that the phosphorylation cPLA2 activity both in vitro (28, 49) and in vivo (14, 50). Inter- 505 of cPLA2 at Ser increases its catalytic activity and phospho- actions of cPLA2 with either full-length or cleaved ANXA1 were lipid-binding affinity at low Ca2+ levels both in vitro and in vivo observed in whole-cell lysates (Fig. 5A). Using subcellular frac- (25). However, the S505A mutant cPLA2 also translocated to the tions, we found that cPLA2 mainly interacted with full-length membrane in response to a calcium ionophore, although it re- ANXA1 in the cytosol and cleaved ANXA1 in the membrane leased less AA (46). In addition, a recent study revealed that (Fig. 5B). Furthermore, phosphorylated cPLA2 was mainly ob- phosphorylation of cPLA2 acts to regulate catalytic activity, but served in the membrane fraction of activated RBL-2H3 cells. not calcium-dependent membrane binding (47). We found that The N-terminal peptide of ANXA1 is cleaved by several pro- increased intracellular Ca2+ concentrations caused by treatment teases, including elastase, calpain, plasmin, cathepsin D, and pro- with DNP-BSA, A23187, and TG regulated not only the cleavage teinase 3 (11–14). ANXA1 cleavage promotes trans- of ANXA1, but also the phosphorylation of cPLA2 and release of endothelial migration (17) and regulates EGF-triggered signaling eicosanoids. In contrast, when intracellular Ca2+ levels were de- and AA production in normal and malignant squamous epithelial creased by pretreatment with BAPTA-AM and 2-APB, ANXA1 cells (14). Recently, an ANXA1 mutant (A11R/V22K/V36K) cleavage, cPLA2 phosphorylation, and eicosanoid production were was reported to be resistant to cleavage by all proteases present all inhibited (Fig. 3). We also observed that cPLA2 translocated in PMN cell extracts (18). In this study, we tested the effects of 5672 CLEAVAGE OF ANNEXIN A1 REGULATES PHOSPHORYLATION OF cPLA2

N-terminal cleavage on inflammatory reactions using RBL-2H3 16. Pepinsky, R. B., and L. K. Sinclair. 1986. Epidermal growth factor-dependent phosphorylation of lipocortin. Nature 321: 81–84. cells and BMMCs expressing mtANXA1. Expression of this 17. Williams, S. L., I. R. Milne, C. J. Bagley, J. R. Gamble, M. A. Vadas, mtANXA1 protein blocked the phosphorylation of cPLA2 in ac- S. M. Pitson, and Y. Khew-Goodall. 2010. A proinflammatory role for proteo- tivated RBL-2H3 cells and BMMCs, but ERK phosphoryla- lytically cleaved annexin A1 in neutrophil transendothelial migration. J. Immunol. 185: 3057–3063. tion was not affected (Fig. 6B, 6C, Supplemental Fig. 3B, 3C). 18. Pederzoli-Ribeil, M., F. Maione, D. Cooper, A. Al-Kashi, J. Dalli, M. Perretti, The release of cysLTs and PGE2 was inhibited in mtANXA1- and F. D’Acquisto. 2010. Design and characterization of a cleavage-resistant expressing RBL-2H3 cells and BMMCs; however, there were no Annexin A1 mutant to control inflammation in the microvasculature. Blood 116: 4288–4296. changes in degranulation activity or cytokine production (Fig. 7). 19. Chung, Y. W., H. Y. Oh, J. Y. Kim, J. H. Kim, and I. Y. Kim. 2004. Allergen- This could be attributed to the inhibition of cPLA2 phosphoryla- induced proteolytic cleavage of annexin-1 and activation of cytosolic phospho- tion by the mutant full-length ANXA1. lipase A2 in the lungs of a mouse model of asthma. Proteomics 4: 3328–3334. 20. Samuelsson, B. 1987. An elucidation of the arachidonic acid cascade. Discovery In summary, we suggest that ANXA1 cleavage is a necessary of , thromboxane and leukotrienes. Drugs 33(Suppl. 1): 2–9. process for inflammatory reactions in mast cells. cPLA2, which 21. Eum, S. Y., K. Maghni, Q. Hamid, H. Campbell, D. H. Eidelman, and J. G. Martin. 2003. Involvement of the cysteinyl-leukotrienes in allergen-induced constitutively interacts with ANXA1, is phosphorylated by airway eosinophilia and hyperresponsiveness in the mouse. Am. J. Respir. Cell cleavage of ANXA1 during mast-cell activation. Phosphorylated Mol. Biol. 28: 25–32. 22. Ghosh, M., D. E. Tucker, S. A. Burchett, and C. C. Leslie. 2006. Properties of the cPLA2 then produces AA, which is converted into various eico- Group IV phospholipase A2 family. Prog. Lipid Res. 45: 487–510. sanoids. Cleavage of ANXA1 may result in the loss of ANXA1’s 23. Glover, S., M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and inhibitory effect on cPLA2 phosphorylation and may promote M. H. Gelb. 1995. Translocation of the 85-kDa phospholipase A2 from cytosol to eicosanoid production during mast-cell activation. Although the the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen. J. Biol. Chem. 270: 15359–15367. mechanism of ANXA1 cleavage has not been fully elucidated, we 24. Evans, J. H., D. M. Spencer, A. Zweifach, and C. C. Leslie. 2001. Intracellular Downloaded from found that intracellular Ca2+ concentration is one of the most calcium signals regulating cytosolic phospholipase A2 translocation to internal important regulatory factors for ANXA1 cleavage in mast-cell membranes. J. Biol. Chem. 276: 30150–30160. 25. Lin, L. L., M. Wartmann, A. Y. Lin, J. L. Knopf, A. Seth, and R. J. Davis. 1993. activation. cPLA2 is phosphorylated and activated by MAP . Cell 72: 269–278. 26. Hefner, Y., A. G. Borsch-Haubold, M. Murakami, J. I. Wilde, S. Pasquet, D. Schieltz, F. Ghomashchi, J. R. Yates, III, C. G. Armstrong, A. Paterson, et al. Acknowledgments 2000. Serine 727 phosphorylation and activation of cytosolic phospholipase A2

We thank Drs. Jae-Hong Kim and Kyung-Jin Cho (College of Life Sciences by MNK1-related protein kinases. J. Biol. Chem. 275: 37542–37551. http://www.jimmunol.org/ and Biotechnology, Korea University) for BMMC culture technique. 27. Hirasawa, N., F. Santini, and M. A. Beaven. 1995. Activation of the - activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and se- Disclosures cretory granules. J. Immunol. 154: 5391–5402. 28. Kim, S., J. Ko, J. H. Kim, E. C. Choi, and D. S. Na. 2001. Differential effects of The authors have no financial conflicts of interest. I, II, III, and V on cytosolic phospholipase A2 activity: specific inter- action model. FEBS Lett. 489: 243–248. 29. Kim, K. M., D. K. Kim, Y. M. Park, C. K. Kim, and D. S. Na. 1994. Annexin-I References inhibits phospholipase A2 by specific interaction, not by substrate depletion. 1. Abraham, S. N., and A. L. St John. 2010. Mast cell-orchestrated immunity to FEBS Lett. 343: 251–255. pathogens. Nat. Rev. Immunol. 10: 440–452. 30. Cho, K. J., J. M. Seo, M. G. Lee, and J. H. Kim. 2010. BLT2 Is upregulated in by guest on September 28, 2021 2. Hofmann, A. M., and S. N. Abraham. 2009. New roles for mast cells in mod- allergen-stimulated mast cells and mediates the synthesis of Th2 cytokines. ulating allergic reactions and immunity against pathogens. Curr. Opin. Immunol. J. Immunol. 185: 6329–6337. 31. Cho, S. H., C. H. Woo, S. B. Yoon, and J. H. Kim. 2004. Protein kinase Cdelta 21: 679–686. 2+ 3. Brown, J. M., T. M. Wilson, and D. D. Metcalfe. 2008. The mast cell and allergic functions downstream of Ca mobilization in FcepsilonRI signaling to de- diseases: role in pathogenesis and implications for therapy. Clin. Exp. 38: granulation in mast cells. J. Allergy Clin. Immunol. 114: 1085–1092. 4–18. 32. Funk, C. D. 2001. Prostaglandins and leukotrienes: advances in eicosanoid bi- 4. Gilfillan, A. M., and C. Tkaczyk. 2006. Integrated signalling pathways for mast- ology. Science 294: 1871–1875. cell activation. Nat. Rev. Immunol. 6: 218–230. 33. Kamal, A. M., R. J. Flower, and M. Perretti. 2005. An overview of the effects of 5. Rivera, J. 2006. Adaptors discriminate mast-cell cytokine production from ei- annexin 1 on cells involved in the inflammatory process. Mem. Inst. Oswaldo cosanoid production and degranulation. Trends Immunol. 27: 251–253. Cruz 100(Suppl. 1): 39–47. 6. Mekori, Y. A., and D. D. Metcalfe. 2000. Mast cells in innate immunity. 34. Warne, J. P., C. D. John, H. C. Christian, J. F. Morris, R. J. Flower, D. Sugden, Immunol. Rev. 173: 131–140. E. Solito, G. E. Gillies, and J. C. Buckingham. 2006. deletion reveals roles 7. Bruhns, P., S. Fre´mont, and M. Dae¨ron. 2005. Regulation of allergy by Fc for annexin A1 in the regulation of lipolysis and IL-6 release in epididymal receptors. Curr. Opin. Immunol. 17: 662–669. adipose tissue. Am. J. Physiol. Endocrinol. Metab. 291: E1264–E1273. 8. Beaven, M. A., and R. A. Baumgartner. 1996. Downstream signals initiated in 35. Hannon, R., J. D. Croxtall, S. J. Getting, F. Roviezzo, S. Yona, M. J. Paul-Clark, mast cells by Fc epsilon RI and other receptors. Curr. Opin. Immunol. 8: 766– F. N. Gavins, M. Perretti, J. F. Morris, J. C. Buckingham, and R. J. Flower. 2003. 2/2 772. Aberrant inflammation and resistance to glucocorticoids in annexin 1 mouse. 9. Gerke, V., and S. E. Moss. 2002. Annexins: from structure to function. Physiol. FASEB J. 17: 253–255. Rev. 82: 331–371. 36. Yang, Y. H., E. F. Morand, S. J. Getting, M. Paul-Clark, D. L. Liu, S. Yona, 10. Lim, L. H., and S. Pervaiz. 2007. Annexin 1: the new face of an old molecule. R. Hannon, J. C. Buckingham, M. Perretti, and R. J. Flower. 2004. Modulation of FASEB J. 21: 968–975. inflammation and response to dexamethasone by Annexin 1 in antigen-induced 11. Wang, W., and C. E. Creutz. 1994. Role of the amino-terminal domain in reg- arthritis. Arthritis Rheum. 50: 976–984. ulating interactions of annexin I with membranes: effects of amino-terminal 37. Yazid, S., S. S. Ayoub, E. Solito, S. McArthur, P. Vo, N. Dufton, and R. J. Flower. truncation and mutagenesis of the phosphorylation sites. Biochemistry 33: 2010. Anti-allergic drugs and the Annexin-A1 system. Pharmacol. Rep. 62: 511– 275–282. 517. 12. Rescher, U., V. Goebeler, A. Wilbers, and V. Gerke. 2006. Proteolytic cleavage 38. Lloret, S., and J. J. Moreno. 1994. Effect of nonapeptide fragments of utero- of annexin 1 by human leukocyte elastase. Biochim. Biophys. Acta 1763: 1320– globin and lipocortin I on oedema and mast cell degranulation. Eur. J. Phar- 1324. macol. 264: 379–384. 13. Vong, L., F. D’Acquisto, M. Pederzoli-Ribeil, L. Lavagno, R. J. Flower, 39. Bandeira-Melo, C., A. G. Bonavita, B. L. Diaz, P. M. E Silva, V. F. Carvalho, V. Witko-Sarsat, and M. Perretti. 2007. Annexin 1 cleavage in activated neu- P. J. Jose, R. J. Flower, M. Perretti, and M. A. Martins. 2005. A novel effect for trophils: a pivotal role for proteinase 3. J. Biol. Chem. 282: 29998–30004. annexin 1-derived peptide ac2-26: reduction of allergic inflammation in the rat. 14. Sakaguchi, M., H. Murata, H. Sonegawa, Y. Sakaguchi, J. Futami, M. Kitazoe, J. Pharmacol. Exp. Ther. 313: 1416–1422. H. Yamada, and N. H. Huh. 2007. Truncation of annexin A1 is a regulatory lever 40. Kimata, M., N. Inagaki, T. Kato, T. Miura, I. Serizawa, and H. Nagai. 2000. for linking epidermal growth factor signaling with cytosolic phospholipase A2 in Roles of mitogen-activated protein kinase pathways for mediator release from normal and malignant squamous epithelial cells. J. Biol. Chem. 282: 35679– human cultured mast cells. Biochem. Pharmacol. 60: 589–594. 35686. 41.Kuehn,H.S.,E.J.Swindle,M.S.Kim,M.A.Beaven,D.D.Metcalfe,and 15. Solito, E., A. Mulla, J. F. Morris, H. C. Christian, R. J. Flower, and A. M. Gilfillan. 2008. The phosphoinositide 3-kinase-dependent activation of J. C. Buckingham. 2003. Dexamethasone induces rapid serine-phosphorylation Btk is required for optimal eicosanoid production and generation of reactive and membrane translocation of annexin 1 in a human folliculostellate cell line oxygen species in antigen-stimulated mast cells. J. Immunol. 181: 7706– via a novel nongenomic mechanism involving the receptor, pro- 7712. tein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein ki- 42. Qiu, Z. H., M. A. Gijo´n, M. S. de Carvalho, D. M. Spencer, and C. C. Leslie. nase. Endocrinology 144: 1164–1174. 1998. The role of calcium and phosphorylation of cytosolic phospholipase A2 in The Journal of Immunology 5673

regulating arachidonic acid release in macrophages. J. Biol. Chem. 273: 8203– 47. Tucker, D. E., M. Ghosh, F. Ghomashchi, R. Loper, S. Suram, B. S. John, 8211. M. Girotti, J. G. Bollinger, M. H. Gelb, and C. C. Leslie. 2009. Role of phos- 43. Schalkwijk, C. G., M. A. van der Heijden, G. Bunt, R. Maas, L. G. Tertoolen, phorylation and basic residues in the catalytic domain of cytosolic phospholipase P. M. van Bergen en Henegouwen, A. J. Verkleij, H. van den Bosch, and A2alpha in regulating interfacial kinetics and binding and cellular function. J. Boonstra. 1996. Maximal epidermal growth-factor-induced cytosolic phos- J. Biol. Chem. 284: 9596–9611. pholipase A2 activation in vivo requires phosphorylation followed by an in- 48. Oliani, S. M., H. C. Christian, J. Manston, R. J. Flower, and M. Perretti. 2000. creased intracellular calcium concentration. Biochem. J. 313: 91–96. An immunocytochemical and in situ hybridization analysis of annexin 1 ex- 44. Wong, A., M. N. Cook, S. M. Hwang, H. M. Sarau, J. J. Foley, and S. T. Crooke. pression in rat mast cells: modulation by inflammation and dexamethasone. Lab. 1992. Stimulation of leukotriene production and membrane translocation of 5- Invest. 80: 1429–1438. lipoxygenase by cross-linking of the IgE receptors in RBL-2H3 cells. Bio- 49. Kim, S. W., H. J. Rhee, J. Ko, Y. J. Kim, H. G. Kim, J. M. Yang, E. C. Choi, and chemistry 31: 4046–4053. D. S. Na. 2001. Inhibition of cytosolic phospholipase A2 by annexin I. Specific 45. Hundley, T. R., A. R. Prasad, and M. A. Beaven. 2001. Elevated levels of interaction model and mapping of the interaction site. J. Biol. Chem. 276: cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal 15712–15719. activation of p38 mitogen-activated protein kinase. J. Immunol. 167: 1629–1636. 50. Herbert, S. P., A. F. Odell, S. Ponnambalam, and J. H. Walker. 2007. The 46. Evans, J. H., D. J. Fergus, and C. C. Leslie. 2002. Inhibition of the MEK1/ERK confluence-dependent interaction of cytosolic phospholipase A2-alpha with pathway reduces arachidonic acid release independently of cPLA2 phosphory- annexin A1 regulates endothelial cell E2 generation. J. Biol. lation and translocation. BMC Biochem. 3: 30. Chem. 282: 34468–34478. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021