Group IID heparin-binding secretory phospholipase A 2 is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues Makoto Murakami 1 , Kumiko Yoshihara 1 , Satoko Shimbara 1 , Masatsugu Sawada 2 , Naoki Inagaki 2 , Hiroichi Nagai 2 , Mikihiko Naito 3 , Takashi Tsuruo 3 , Tae Churl Moon 4 , Hyeun Wook Chang 4 and Ichiro Kudo 1 1 Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo; 2 Pharmacological Department, Gifu College of Pharmacy, Gifu, Japan; 3 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan; 4 College of Pharmacy, Yeungnam University, Gyonsan, Korea Group IID secretory phospholipase A 2 (sPLA 2 -IID), a heparin-binding sPLA 2 that is closely related to sPLA 2 -IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA 2 -IIA. Here we identified the resi- dues of sPLA 2 -IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA 2 -IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus- induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell trans- fectants. As compared with several other group II subfamily sPLA 2 s, which were equally active on A23187- and IL-1- primed cellular membranes, sPLA 2 -IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA 2 -IID and sPLA 2 - X constitutively, the former of which was negatively regu- lated by IL-1. sPLA 2 -IID, but not other sPLA 2 isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA 2 -IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA 2 -IID may be involved in inflam- mation in cell- and tissue-specific manners under particular conditions. Keywords: phospholipase A 2 ; colon carcinoma; mast cell; inflammation. Phospholipase A 2 (PLA 2 ), which catalyzes the hydrolysis of the ester bond of the sn-2 position of glycerophospholipid to liberate free fatty acid and lysophospholipid, is structurally and functionally subdivided into four major classes: secre- tory PLA 2 (sPLA 2 ), cytosolic PLA 2 (cPLA 2 ), Ca 2+ -inde- pendent PLA 2 (iPLA 2 ) and platelet-activating factor acetylhydrolase [1]. The sPLA 2 family comprises Ca 2+ - dependent, disulfide-rich and low molecular mass (14–18 kDa) enzymes with histidine residue in the catalytic center. To date, 10 sPLA 2 isozymes (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals [1,2]. A subset of sPLA 2 s contributes to the release of arachidonic acid for eicosanoid generation and can also participate in a variety of physiological events. The regulatory functions of sPLA 2 -IIA, a prototypic proinflammatory sPLA 2 , have been investigated in a number of studies [3–18]. In general, this enzyme is exocytosed or newly synthesized and secreted by the cells after stimulation with proinflammatory agents [3–6] and plays an augmentative role in arachidonic acid release and prostaglandin generation [4–12], elimination of infectious bacteria [13–15], and other pathophysiological events [16–18]. Subsequently, several new group II subfamily sPLA 2 s (IIC, IID, IIE, IIF, and V), the genes for which are clustered in the same chromosome locus, have been identified [19–24]. Among them, sPLA 2 -V has the ability to augment cellular arachidonic acid release often more efficiently than does sPLA 2 -IIA [8–12,25,26], whereas the functions of the other novel group II sPLA 2 s are obscure. sPLA 2 -IB (pancreatic PLA 2 ) and -X, the genes for which each map to distinct chromosomes, have a unique N-terminal prepropeptide and proteolytic removal of this prepropeptide produces an active enzyme [27–29]. sPLA 2 - IB plays a role in the digestion of dietary phospholipids in the gastrointestinal tract and stimulates cellular responses by acting as a ligand for the sPLA 2 receptor [30,31]. sPLA 2 -X potently promotes arachidonic acid release through acting on the external plasma membrane of target cells, an event depending on its interfacial binding to zwitterionic phos- phatidylcholine [11,12,29,32,33]. Accumulating evidence has suggested that the cellular functions of the heparin-binding group II subfamily of sPLA 2 s (IIA and V) are influenced both positively [7–12] and negatively [34,35] by heparan sulfate proteoglycan Correspondence to M. Murakami, the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Fax: + 81 3 3784 8245, Tel.: + 81 3 3784 8197, E-mail: mako@pharm.showa-u.ac.jp Abbreviations:sPLA 2 , secretory phospholipase A 2 ; cPLA 2 , cytosolic phospholipase A 2 ; COX, cyclooxygemase; mPGES, microsomal PGE 2 synthase; cPGES, cytosolic PGE 2 synthase; HSPG, heparan sulfate proteoglycan; IL, interleukin; SCF, stem cell factor; LPS, lipopolysaccharide; DNFB, 2,4-dinitroflurobenzene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Enzyme: phospholipase A 2 (EC 3.1.1.4). (Received 28 January 2002, revised 15 April 2002, accepted 17 April 2002) Eur. J. Biochem. 269, 2698–2707 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02938.x (HSPG) on cell surfaces. In the former situation, the glycosylphosphatidylinositol-anchored HSPG glypican supports the arachidonic acid-releasing function of the HSPG-binding sPLA 2 s by sorting them into particular caveolin-rich punctate and perinuclear compartments [10,12]. Conversely, certain HSPG moieties facilitate inter- nalization and subsequent proteolytic degradation, thereby leading to inactivation of the HSPG-binding sPLA 2 s [34,35]. Thus, in addition to their enzymatic characteristics, the HSPG-binding properties of sPLA 2 s also dictate their cellular behaviors and functions. Cationic amino acid clusters in the N- and/or C-terminal domains of sPLA 2 - IIA [7,36] and sPLA 2 -V [8,34] are responsible for their functional association with HSPGs. sPLA 2 -IID, an isozyme most related to sPLA 2 -IIA, is reportedly expressed in immune and digestive organs and is proposed to replace sPLA 2 -IIA under certain conditions [21,22]. We have recently shown that sPLA 2 -IID, like sPLA 2 -IIA, binds to the HSPG glypican and augments the arachidonic acid-releasing response in HEK293 cells [12]. To better understand the regulatory functions of sPLA 2 - IID, we have determined its functional HSPG-binding site by site-directed mutagenesis. Furthermore, we show that this isozyme is expressed in human colon carcinoma cell lines and human mast cells as well as various mouse tissues. Importantly, the expression of sPLA 2 -IID is regulated both positively and negatively by proinflammatory stimuli. MATERIALS AND METHODS Materials HEK293 cells (Human Science Research Resources Bank, Osaka, Japan) and colon carcinoma cell lines (American Type Culture Collection) were cultured in RPMI 1640 medium(NissuiPharmaceuticalCo.,Tokyo,Japan)con- taining 10% fetal bovine serum [8–12]. cDNAs for human and mouse sPLA 2 s, human cyclooxygenase (COX)-2 and human microsomal prostaglandin E 2 (PGE 2 ) 1 synthase (mPGES) and their HEK293 cell transfectants were described previously [8–12,37]. To obtain human cord blood-derived mast cells [38], heparin-treated umbilical cord blood was obtained from normal full-term vaginal deliveries under auspices of the Kyungpook National University Hospital. Cord blood was diluted with the same volume of NaCl/P i and layered over Histopaque-1077 (Sigma) at room temperature within 4 h of delivery. The cord blood monoculcear cell fraction was obtained after centrifugation at 1000 g for 20 min at room temperature. The cells were washed twice with NaCl/P i and grown in tissue culture flasks in AIM-V medium (Life Technologies) in the presence of 100 ngÆmL )1 recombinant human stem cell factor (SCF) for 8 weeks. Non-adherent cells were then cultured for an additional 2 weeks with 100 ngÆmL )1 SCF and 50 ngÆmL )1 human interleukin (IL)- 6 in AIM-V medium. The mast cells thus obtained were > 97% tryptase- and  70% chymase-positive as demon- strated by immunocytostaining using specific antibodies, expressed functional c-kit and Fc e receptor I as assessed by flow cytometry, and responded to immunological and nonimmunological stimuli to secrete granule contents (T. C. Moon, M. Murakami, I. Kudo & H. W. Chang, unpub- lished data) 2 . The enzyme immunoassay kit for PGE 2 was from Cayman Chemicals (Ann Arbor, MI, USA). Rabbit antihuman COX-1 and antihuman cPLA 2 a antibodies were from Santa Cruz. Anti-human cytosolic PGE 2 synthase (cPGES) antibody was prepared as described previously [39]. Lipofectamine PLUS reagent, Opti-MEM medium, geneticin and TRIzol reagent were from Life Technologies. Horseradish peroxidase-conjugated antigoat and antirabbit IgGs were from Zymed. A23187 was from CalBiochem. Human IL-1b was obtained from Genzyme. Construction of sPLA 2 -IID mutants Mouse sPLA 2 -IID mutants were produced by PCR with the Advantage cDNA polymerase mix (Clontech). The condi- tion of PCR was 25 cycles at 94 °C, 55 °Cand72°Cfor 30 s each. The primers used were as follows: mIID-5¢,5¢-AT GAGACTCGCCCTGCTGTGTG-3¢;KE2,5¢-TTAGCA TGCTGGAGTCTCGCCTTCGCAAC-3¢; and KE2RS2, 5¢-GCATGCTGGAGTCTCGCCTTCGCAACAGGGCC ACCAGTA-3¢. PCR was preformed with mIID-5¢ and KE2 or KE2RS2 using mouse sPLA 2 -IID cDNA as a template. Each PCR product was ligated into pCR3.1 (Invitrogen) and was transfected into Top10F¢ supercom- petent cells (Invitrogen). The plasmids were isolated and sequenced using a Taq cycle sequencing kit (Takara, Ohtsu, Japan) and an autofluorometric DNA sequencer DSQ-1000 L (Shimadzu, Tokyo, Japan) to confirm the sequences. RT-PCR and Southern blotting Synthesis of cDNAs was performed using avian myeloblas- tosis virus reverse transcriptase and 0.5 lgtotalRNAfrom mouse tissues and human cell lines, according to the manufacturer’s instructions supplied with the RNA PCR kit (Takara). Subsequent amplification of the cDNA fragments was performed using 1 lL of the reverse- transcribed mixture as a template with specific oligonucle- otide primers (Greiner Japan) as follows: mIID-5¢ and mIID-3¢ (see above); human cPLA 2 a sense, 5¢-ATGTCATT TATAGATCCTTACC-3¢ and antisense, 5¢-TCAAAGTT CAAGAGACATTTCAG-3¢; human mPGES sense, 5¢-AT GCACTTCCTGGTCTTCCTCG-3¢ and antisense, 5¢-GC TTCCCCAGGAAGGCCACGG-3¢; human sPLA 2 -IB sense, 5¢-ATGAAACTCCTTGTGCTAGCTG-3¢ and anti- sense, 5¢-TCAACTCTGACAATACTTCTTGG-3¢; human sPLA 2 -hIIA sense, 5¢-CAGAATGATCAAGTTGACGAC AG-3¢ and antisense, 5¢-TCAGCAACGAGGGGTGCTC CTC-3¢; human sPLA 2 -hIID sense, 5¢-ATGGAACTTGCA CTGCTGTGTG-3¢ and antisense, 5¢-CAGTCGCTTCTG GTAGGTGTCC-3¢; human sPLA 2 -IIE sense, 5¢-ATGAA ATCTCCCCACGTGCTGG-3¢ and antisense, 5¢-TGTAG GTGCCCAGGTTGCGGCG-3¢; human sPLA 2 -IIF sense, 5¢-ATGAAGAAGTTCTTCACCGTG-3¢ and antisense, 5¢-CTAGCAGGTGACCTCCTCAGG-3¢; human sPLA 2 - Vsense,5¢-ATGAAAGGCCTCCTCCCACTGG-3¢ and antisense, 5¢-GGCCTAGGAGCAGAGGATGTTG-3¢; and human sPLA 2 -X sense, 5¢-ATGCTGCTCCTGCTAC TGCCG-3¢ and antisense, 5¢-TCAGTCACACTTGGGC GAGTC-3¢. PCR conditions were 94 °C for 30 s and then 30 cycles of amplification at 94 °Cfor5sand68°Cfor 4 min, using the Advantage cDNA polymerase mix. RT-PCR of glyceraldehyde-3-phosphate dehydrogenase Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2699 (GAPDH) was performed using specific primers (Clontech). The PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. The gels were further subjected to Southern blot hybridization using appropriate cDNAs as probes. Lipopolysaccharide treatment of mice Lipopolysaccharide (LPS) (5 mgÆkg )1 ) was administered intraperitoneally to 4-week-old male C57BL/6 mice (Nip- pon Bio-Supply Center, Tokyo, Japan). After 24 h, mice were sacrificed by bleeding, their organs were removed, and RNA was extracted by homogenization in TRIzol reagent using 10 strokes of a Potter homogenizer at 1000 r.p.m. 3 Mouse ear atopic dermatitis Five repeated topical applications of 2,4-dinitrofluoroben- zene(DNFB)totheearsofmiceresultincontact hypersensitivity of the ears as well as significant elevation of serum IgE levels, accompanied by the increased T H1 response for the onset of skin dermatitis and the T H2 response in the lymph node [40]. The ears of C57BL/6 mice (Nippon Bio-Supply Center) were painted with 25 lL 0.15% (w/v) DNFB or vehicle (acetone/olive oil 3 : 1) once a week. The ears were removed 24 h after the fifth painting and subjected to RNA extraction. Replicate ear sections were fixed by formalin, embedded in paraffin and stained with hematoxylin and eosin to verify the progress of inflammation. All procedures and analyses of other param- eters are detailed elsewhere [40]. Other procedures Northern and Western blottings, establishment and activa- tion of HEK293 transfectants, and measurement of in vitro sPLA 2 activity were performed as described in our previous reports [8–12]. RESULTS Determination of the heparin-binding site of mouse sPLA 2 -IID The amino-acid sequences of mouse and human sPLA 2 -IIDs reveal the presence of multiple cationic amino acid residues in their C-terminal regions [21,22]. Since the multiple cationic residues in the corresponding C-terminal portions of mouse and human sPLA 2 -IIAs and rat and human sPLA 2 -Vs serve as functional heparin-binding sites [7,8,34,36], we replaced some of these cationic residues in mouse sPLA 2 -IID with neutral and/or anionic amino acids by site-directed muta- genesis. The KE2 mutant, in which two lysine residues near the C-terminal end (Lys138 and Lys140) were replaced by glutamic acid, and the KE2RS2 mutant, in which two conserved arginine residues (Arg136 and Arg138) were additionally mutated to serine, were constructed (Fig. 1A). cDNAs for the native and mutant enzymes were transfected into HEK293 cells to establish drug-resistant stable clones. Comparable expression of the mutant and native enzymes was confirmed by Northern blotting (Fig. 1B). As the membrane distribution of sPLA 2 s expressed in HEK293 cells largely reflects their association with cell surface HSPG [7–12], we measured the enzyme activity in the supernatant and membrane-bound (i.e. 1 M NaCl- solubilized) fractions of the established transfectants (Fig. 1C). Consistent with our recent reports [7–12], the membrane-bound fraction contained more than 50% of the native enzyme (Fig. 1C). The distribution of the KE2 mutant between the two fractions was similar to that of the native enzyme (Fig. 1C). In contrast, the activity of the KE2RS2 mutant was detected mainly in the supernatants, with only a minor portion being recovered from the membrane-bound fraction (Fig. 1C). Thus, simultaneous mutation of the four cationic residues in the C-terminal domain of sPLA 2 -IID led to a marked reduction of its membrane-binding (and therefore HSPG-binding) capacity. Fig. 1. Mutation of basic amino acid residues near the C-terminus of sPLA 2 -IID affects its association with the cell surface. (A) Amino acid sequences of the C-terminal part of mouse sPLA 2 -IID (mIID) and its mutants, KE2 and KE2RS2. Two and four basic amino acids are replaced by glutamic acid or serine in KE2 and KE2RS2, respectively. (B) Expression of the wild-type (WT) and two mutants of mIID in HEK293 cells, as assessed by RNA blotting. (C) Membrane binding of the WT and two mutants of mIID. After collecting the culture sup- ernatants, the cells were incubated for 30 min with medium containing 1 M NaCl, which solubilizes the cell surface HSPG-bound form of sPLA 2 s. PLA 2 activities in the secreted (S) and cell membrane-bound (i.e. NaCl-solubilized) (C) fractions were measured. 2700 M. Murakami et al. (Eur. J. Biochem. 269) Ó FEBS 2002 This observation is in line with previous studies on the HSPG-binding of sPLA 2 -IIA, in which multiple cationic residues in the C-terminal domain are required for its proper association with heparanoids [7,8,34,36]. When the cells were prelabeled with [ 3 H]arachidonic acid and were then stimulated with A23187 for 30 min (Fig. 2A) or with IL-1 for 4 h (Figs 2,B,C) as models for the immediate and delayed responses, respectively [8–12], a marked elevation of [ 3 H]arachidonic acid release, which was accompanied by PGE 2 generation (Fig. 2C), was observed in cells transfected with the native enzyme or KE2 mutant, but not appreciably in those transfected with the KE2RS2 mutant. In the absence of stimulus, there were no increases in arachidonic acid release and PGE 2 generation even in cells transfected with the native enzyme (data not shown). Furthermore, IL-1-stimulated COX-2 expression was faci- litated in cells transfected with the native enzyme or KE2 mutant, whereas it occurred only minimally in cells trans- fected with KE2RS2 (Fig. 2D). These observations suggest that the cellular functions of sPLA 2 -IID are correlated with its membrane-binding property, and lend further support for the notion that this enzyme, as does sPLA 2 -IIA [7–12], acts on cells through an HSPG-dependent mechanism in this setting. sPLA 2 -IID prefers Ca 2+ ionophore-induced perturbed membrane While studying the arachidonic acid-releasing functions of the three heparin-binding group II subfamily enzymes (IIA, IID and V) in HEK293 transfectants, we noted that sPLA 2 - IID released arachidonic acid after A23187 stimulation more efficiently than it did after IL-1 stimulation under the condition where sPLA 2 -IIA and -V released equivalent levels of arachidonic acid in both responses (Fig. 3A). Thus, A23187-induced arachidonic acid release by these three sPLA 2 s reached comparable levels (net 4–6%), whereas IL-1-stimulated arachidonic acid release by sPLA 2 -IID (net 0.7%) was apparently lower than that by sPLA 2 -IIA and -V (net 4–5%) (Fig. 3A). When cells expressing sPLA 2 -IID were cocultured with those coexpressing COX-2 and mPGES and then stimulated (transcellular prostaglandin biosynthesis [9]), the increased production of PGE 2 in response to A23187 was higher than that in response to IL-1 (Fig. 3B, left). In comparison, coculture of cells expressing sPLA 2 -V with those coexpress- ing COX-2 and mPGES increased both the immediate and delayed PGE 2 -biosynthetic responses almost equally (Fig. 3B, right). These results indicate that sPLA 2 -IID secreted from the transfectants acts preferentially on the A23187-elicited membranes of neighboring cells, where the arachidonic acid released by the paracrine or juxtacrine action of sPLA 2 -IID is supplied to downstream COXs and mPGES. sPLA 2 -IID expression in human colon carcinoma cell lines Although sPLA 2 -IID has been reported to be expressed in tissues related to the immune response (spleen and thymus) and digestion (small and large intestines) of both human and mouse [21,22], which types of cell express this sPLA 2 isozyme remains obscure. We therefore surveyed the expression of sPLA 2 -IID in various human cell lines, and found that its transcript, as assessed by RT-PCR, was constitutively expressed in several human colon carcinoma cell lines, including HT29, KM12, KM20L2, Fig. 2. Mutation of basic amino acid residues near the C-terminus of sPLA 2 -IID affects its cellular arachidonic acid-releasing function. (A) Immediate arachidonic acid release. Control HEK293 cells and cells transfected with the WT or mutant mIID were prelabeled with [ 3 H]arachidonic acid and then stimulated for 30 min with 10 l M A23187 to assess [ 3 H]arachidonic acid release. (B–D) Delayed arachidonic acid release and PGE 2 generation. Control cells and cells transfected with the WT or mutant mIID were stimulated for 4 h with IL-1b to assess [ 3 H]arachidonic acid release (B), PGE 2 production (C) and COX-2 induction (D). In (D), COX-2 expression was assessed by RNA blotting. Equal loading of RNA on each lane was verified by ribosomal RNA staining with ethidium bromide (not shown). AA, arachidonic acid. Fig. 3. sPLA 2 -IID elicits the immediate response in preference to the delayed response. (A) [ 3 H]arachidonic acid release by control HEK293 cells and cells transfected with sPLA 2 -IIA, -IID or -V in response to A23187 (30 min) or IL-1b (4 h). (B) Transcellular PGE 2 production by sPLA 2 -IID (left) and sPLA 2 -V (right). Control, and COX-2/mPGES-coexpressing cells were cocultured for 4 days with control cells (–) or sPLA 2 -expressing cells (+), and were then stimulated for 4 h with IL-1b to assess PGE 2 generation. AA, arachidonic acid. Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2701 WiDr and HCT2998 cells (Fig. 4A). Unexpectedly, treat- ment of these cells with IL-1 consistently decreased the expression of sPLA 2 -IID in a time-dependent manner. sPLA 2 -X was also detected in these cell lines, in which its expression was unaffected by IL-1 except for HCT2998 cells, in which there was a slight increase in its expression (Fig. 4B). sPLA 2 -V was detected only in IL-1-stimulated HT29 cells, and sPLA 2 -IIA was weakly and constitutively expressed in HT29, KM12 and KM20L2 cells (Fig. 4B). The expression of other sPLA 2 s (IB, IIE and IIF) was undetectable. The expression of other enzymes involved in the PGE 2 - biosynthetic pathway in these colon carcinoma cell lines was also investigated (Fig. 4C). cPLA 2 a was detected in KM12, KM20L2 and WiDr cells. COX-1 was highly expressed in HT29 and WiDr cells and weakly expressed in KM20L2 cells. COX-2 was detected only in WiDr cells. The two terminal PGE 2 -biosynthetic enzymes, cPGES and mPGES, were expressed in all cell lines. Following IL-1 treatment, COX-2 expression was markedly induced in WiDr cells, whereas the expression levels of cPLA 2 a,COX-1,cPGES and mPGES in each cell line were unaltered. Among these cell lines, only WiDr cells produced a substantial amount of PGE 2 in response to IL-1 (Fig. 4D), most likely because COX-2 is a rate-limiting step for IL-1-dependent PGE 2 biosynthesis [6–12]. sPLA 2 -IID expression in human cultured mast cells We have previously reported that mouse bone marrow- derived cultured mast cells developed in the presence of IL-3 express all the group II subfamily sPLA 2 s[41].RT-PCR analyses revealed that, unlike mouse mast cells, human mast cells developed in the presence of SCF and IL-6 from cord blood cells [38] expressed only sPLA 2 -IID, but not the other sPLA 2 s including -IB, -IIA, -IIE, -IIF, -V and -X (Fig. 5). The expression of cPLA 2 a was readily detected under the same experimental conditions (Fig. 5). The expression of sPLA 2 -IID and cPLA 2 a in human mast cells was unchanged after treatment with various mast cell-poietic cytokines and immunological stimuli (T. C. Moon, M. Murakami, I. Kudo & H. W. Chang, unpublished data) 4 . sPLA 2 -IID expression in mouse tissues during inflammation The expression of sPLA 2 -IID in several tissues of mice before and 24 h after injection of LPS was examined by Fig. 4. Expression of sPLA 2 -IID and other PGE 2 -biosynthetic enzymes in human colon carcinoma cell lines. (A) Cells were stimulated for the indicated periods with 1 ngÆmL )1 IL-1b, and the expression of sPLA 2 -IID was assessed by 30 cycles of RT-PCR. After staining of the gel with ethidium bromide (top), samples were subjected to Southern blotting using 32 P-labeled human sPLA 2 -IID cDNA as a probe (middle). Equal loading of samples on each lane was verified by the expression of GAPDH, as assessed by RT-PCR (bottom). (B) The same samples [with (+) or without (–) 12-h stimulation with IL-1b] were subjected to RT-PCR (30 cycles) followed by Southern blotting to assess the expression of sPLA 2 -X, -V and -IIA. (C) Expression of cPLA 2 a, COX-1, COX-2, cPGES and mPGES with or without 12-h stimulation with IL-1b. The expression of cPLA 2 a, COX-1 and cPGES was assessed by immunoblotting, COX-2 by RNA blotting, and mPGES by RT-PCR (30 cycles) followed by Southern blotting. (D) Cells were stimulated for 12 h with IL-1b and PGE 2 released into the supernatants was quantified. 2702 M. Murakami et al. (Eur. J. Biochem. 269) Ó FEBS 2002 RT-PCR (Fig. 6A). After administration of LPS, sPLA 2 - IID expression was upregulated in the lung, thymus and heart in a dose-dependent manner. Conversely, sPLA 2 -IID expression was decreased in the kidney of LPS-treated mice. In the spleen, intestine and colon, in which the basal sPLA 2 - IID expression was high, as well as in the brain and liver, sPLA 2 -IID expression was largely unchanged after LPS challenge. In the ears of mice with DNFB-induced atopic dermatitis, there was a marked increase in sPLA 2 -IID expression (Fig. 6B). DISCUSSION sPLA 2 -IID, which was originally identified by searching nucleic acid data bases for expressed sequence tags repre- senting parts of genes for sPLA 2 homologs, displays all of the specific features of sPLA 2 -IIA: the homology between these two enzymes is  50% [21,22]. sPLA 2 -IID and -IIA also possess several common properties, one of which is their high affinity for heparanoids [7–12]. The major heparin-binding site of sPLA 2 -IIA is located near the C-terminus, where a highly localized site of basic residues affects its heparanoid affinity with diffuse basic residues throughout the molecule having a modifying role [7,36]. Similarly, the C-terminal basic amino acid cluster contri- butes to the binding of sPLA 2 -V to heparanoids [8,34]. In the present study, we have shown that a similar cluster of basic amino acids near the C-terminus of sPLA 2 -IID also crucially influences its binding to cellular HSPG (Fig. 1). Most importantly, as in the cases of sPLA 2 -IIA and -V, enzymes that act on ÔrearrangedÕ cellular membranes through the HSPG-dependent pathway [7,34,36], mutation of these basic residues of sPLA 2 -IIDledtoamarked reduction of its ability to release arachidonic acid, produce PGE 2 and induce COX-2 in HEK293 cells (Fig. 2), despite the fact that the mutation does not have a profound effect on enzyme activity (Fig. 1C). These results agree with our recent observation that sPLA 2 -IID augments arachidonic acid release from activated cells through the pathway dependent upon the HSPG glypican or other HSPG molecules [12]. The three-dimensional structure of sPLA 2 - IIA demonstrates that the C-terminal heparin-binding domain is located on the opposite side of a globular molecule to the interfacial binding surface [34], implying that this enzyme can interact simultaneously with substrates and heparanoids. Given the assumption that sPLA 2 -IID has a similar ternary structure, it is conceivable that its anchoring on the heparan sulfate chains of glypican (or other HSPG) through the C-terminal cationic surface allows sPLA 2 -IID to be locally concentrated and interact efficiently with phospholipids in adjacent cellular membranes. Fig. 6. Expression of sPLA 2 -IID in mouse during inflammation. RNAs obtained from various tissues of mice 24 h after administration of the indicated doses of LPS (A) and the ears of mice with or without five repeated treatments with DNFB (B) were subjected to RT-PCR (30 cycles) followed by Southern blotting to assess the expression of sPLA 2 -IID. To verify equal loading of RNA on each lane, RT-PCR (25 cycles) for GAPDH was also performed. R and L in (B) indicate right and left ears, respectively. Fig. 5. Expression of sPLA 2 -IID in human cord blood-derived mast cells. RNA obtained from human cord blood-derived mast cells was subjected to RT-PCR (30 cycles) using specific primers for human sPLA 2 -IB, IIA, IID, IIE, IIF, V and X (left) and for cPLA 2 a (right). After staining of the gel with ethidium bromide, samples were taken for Southern blotting using cDNA probes for the mixture of these sPLA 2 s. Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2703 Our transfection studies have revealed a subtle but substantial difference between sPLA 2 -IID and other group II subfamily enzymes (sPLA 2 -IIA and -V). These enzymes are in common active on ÔrearrangedÕ cellular membranes that have been primed by various cell activators [6–12], yet sPLA 2 -IID, relative to -IIA and -V, shows apparent preference for A23187-primed rather than IL-1-primed cellular membranes (Fig. 3). This is, in our hands, the first demonstration that a particular sPLA 2 isozyme exerts its arachidonic acid-releasing function more effectively in the Ca 2+ evoked immediate response than in the cytokine- induced delayed response. The membrane rearrangement that renders cells more susceptible to sPLA 2 s involves several processes, such as altered membrane phospholipid asymmetry (i.e. exposure of anionic phospholipids in the outer leaflet of the membrane), accelerated membrane oxidation and possibly sphingomyelin breakdown [1]. Although the precise mechanisms are still unclear, sPLA 2 - IID may be better suited to the particular perturbed membrane structures that are formed during prompt Ca 2+ signaling than to those formed during sustained cytokine signaling. In search of human cell lines that endogenously express sPLA 2 -IID, we found that several colon carcinoma cell lines constitutively express this particular sPLA 2 isozyme (Fig. 4). Most of these cell lines also express sPLA 2 -X, an observation reminiscent of the recent report by Morioka et al. [32] demonstrating the elevated expression of sPLA 2 - X in human colon adenocarcinoma neoplastic cells and tissues. A growing body of evidence has shown that nonsteroidal anti-inflammatory drugs that inhibit COX-2 can suppress colorectal tumorigenesis [42–45] and that PGE 2 , a major COX-2 product, is involved in this process [46–48]. Furthermore, targeted disruption of the cPLA 2 a gene has provided unequivocal evidence that this enzyme contributes significantly, if not solely, to the expansion of colorectal cancer, most probably by acting as a major supplier of arachidonic acid to COX-2 [49]. Our present results raise the intriguing possibility that, in addition to sPLA 2 -X [32,49], sPLA 2 -IID may also be able to promote certain phases of colorectal cancer development. Unfor- tunately, none of the cell lines used in this study (even WiDr cells, which express COX-2) turned out to depend on the COX products for their growth (data not shown), and the confirmation of this hypothesis awaits future study. Mast cells are highly specialized effector cells in the immune system, where they release a number of granule- associated preformed (e.g. histamine, serotonin, and pro- teases) and newly synthesized (e.g. PGD 2 , LTC 4 ,and cytokines) mediators following engagement of the Fc e receptor I on their surfaces by IgE and cognate antigen. Previous studies have established that mast cells represent a potent source of sPLA 2 s; mouse IL-3-dependent bone marrow-derived mast cells express all or some of the group II subfamily sPLA 2 s according to culture conditions [41,50], mouse mast cell line MMC-34 cells express sPLA 2 -V [51], and rat peritoneal mast cells express sPLA 2 -IIA [52]. These sPLA 2 s play augmentative roles in stimulus-coupled degranulation and lipid mediator generation in rodent mast cells [41,50–52]. Here we show that human cord blood- derived mast cells developed in SCF and IL-6 [38] express sPLA 2 -IID but not the other isozymes (Fig. 5). Given the experimental evidence that sPLA 2 -IID, as do the other group II subfamily sPLA 2 s, has the ability to augment IgE/ antigen-dependent exocytosis of granule-associated media- tors and generation of eicosanoids in rodent mast cells [12,41], it is tempting to speculate that sPLA 2 -IID may display similar functions in human mast cells. In this regard, sPLA 2 -IID may represent a novel therapeutic and prophy- lactic target for allergic diseases. It should be noted, however, that this finding does not necessarily mean that all mast cells distributed in human tissues express sPLA 2 - IID only, since mast cell phenotypes is crucially influenced by tissue microenvironments [53,54]. Indeed, a recent immunohistochemical analysis has demonstrated that human intestinal mast cells contain sPLA 2 -IIA [55]. We also recently found that sPLA 2 -V is located in mast cells in tissues from patients with allergic symptoms ( 5 M. Murakami & I. Kudo, unpublished data). Increased expression of sPLA 2 -IID was observed in some tissues (lung, thymus and heart) of mice with LPS- induced systemic inflammation and in the ears of mice with atopic dermatitis (Fig. 6), providing further support for the notion that the group II subfamily of sPLA 2 sare inducible enzymes. Consistent with our results, Ishizaki et al. [22] have shown that sPLA 2 -IID expression is increased in the thymus and lung of LPS-treated rats, and Shakhov et al.[56]haveshownthatsPLA 2 -IID expression is markedly reduced in lymphoid tissues of lymphotoxin a-deficient mice. However, this rather tissue-restricted induction of sPLA 2 -IID differs from the induction of sPLA 2 -IIA and -V [57,58], which is more widespread among tissues. Moreover, LPS treatment resulted in reduced expression of sPLA 2 -IID in the kidney (Fig. 6A), in which the expressions of sPLA 2 -IIA and -V [57,58] exhibit a reciprocal pattern. Decreased expression of sPLA 2 -IID, relative to increased expression of sPLA 2 -V, by proinflammatory stimulus was also observed in human colon carcinoma cell lines (Fig. 4A,B). These results argue that the regulatory mechanisms for gene expression, and perhaps functions, of sPLA 2 -IID and those of sPLA 2 -IIA and -V are not entirely identical and are even cell- and tissue-specific. Searching the nucleic acid database reveals the presence of the TATA box and the binding motifs for AP-1 and NFjB in the putative 5¢-flanking promoter region of the human sPLA 2 -IID gene, consistent with its proinflammatory signal-associated inducible nature. In comparison, the putative promoter region of the human sPLA 2 -V gene contains the C/EBP and CREB motifs as well as distal AP-1, NFjB and glucocorticoid-responsive elements. These motifs are also present in the promoter region of the sPLA 2 -IIA gene, albeit with a different alignment [59,60]. Such differences among the promoter regulatory regions of these sPLA 2 s may account for their distinct expression and induction. The present study implies that the structurally related group II subfamily sPLA 2 isozymes are not always functionally compensatory, even if they utilize common regulatory machinery under particular conditions. The expression and induction profiles of each sPLA 2 isozyme during inflammatory responses are tissue- and cell-specific. It is therefore likely that functional redundancy and segregation of sPLA 2 isozymes must occur in different physiological and pathological states and in different cells and tissues. 2704 M. Murakami et al. (Eur. J. 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Importantly, the expression of sPLA 2 -IID is regulated