Proinflammatory Secreted Phospholipase A2 Type IIA (sPLA-IIA) Induces Integrin Activation through Direct Binding to a Newly Identified Binding Site (Site 2) in Integrins αvβ3, α4β1, and α5β1*

Background: Besides inside-out signaling, integrins are activated by the binding of fractalkine to a newly identified binding site (site 2). Results: sPLA2-IIA induced integrin activation through site 2. A peptide from site 2 or a small compound that binds to sPLA2-IIA suppressed the activation. Conclusion: sPLA2-IIA activates integrins through direct binding to site 2. Significance: Integrin activation through site 2 may be a potential therapeutic target in inflammation. Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins ␣v␤3 and ␣4␤1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin ␣v␤3 and transmembrane ␣v␤3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of ␣v␤3. A peptide from site 2 of integrin ␤1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates ␣v␤3 through binding to site 2. sPLA2-IIA also activated integrins ␣4␤1 and ␣5␤1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)).
Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.
Secreted PLA2 type IIA (sPLA2-IIA) 2 was first isolated and purified from rheumatoid synovial fluid (1). sPLA2-IIA is an acute phase reactant, and its plasma concentration markedly increases in diseases that involve systemic inflammation such as sepsis, rheumatoid arthritis, and cardiovascular disease (up to 1000-fold and Ͼ1 g/ml). Inflammatory cytokines such as IL-6, TNF-␣, and IL-1␤ induce synthesis and release of sPLA2-IIA in arterial smooth muscle cells and hepatocytes, which are the major sources of the plasma sPLA2-IIA in these systemic inflammatory conditions (2,3). In addition to being a proinflammatory protein, sPLA2-IIA expression is elevated in neoplastic prostatic tissue (4), and dysregulation of sPLA2-IIA may play a role in prostatic carcinogenesis (5) and is a potential therapeutic target in prostate cancer (6).
Notably some biological effects associated with sPLA2-IIA are independent of its catalytic function (7). Catalytically inactive sPLA2-IIA mutants retain the ability to enhance cyclooxygenase-2 expression in connective tissue mast cells (7). Also, inactivation of sPLA2-IIA by bromophenacyl bromide does not affect the ability of sPLA2-IIA to induce secretion of ␤-glucuronidase, IL-6, and IL-8 from human eosinophils (8). It has thus been proposed that sPLA2-IIA action is mediated through interaction with specific receptors. Indeed the enzyme binds to a high affinity receptor of 180 kDa present on rabbit skeletal muscle (9). This so-called M (muscle)-type receptor belongs to the superfamily of C-type lectins and mediates some of the physiological effects of mammalian sPLA2-IIA, and binding of sPLA2-IIA to this receptor induces internalization of sPLA2-IIA (10). However, the interaction between sPLA2-IIA and the M-type receptor is species-specific, and human sPLA2-IIA binds to the human or mouse M-type receptor very weakly (11).
Integrins are a family of cell adhesion receptors that recognize ECM ligands and cell surface ligands (12). Integrins are transmembrane heterodimers, and at least 18 ␣ and 8 ␤ subunits are known (13). Integrins transduce signals to the cell upon ligand binding (12). We previously reported that sPLA2-IIA specifically binds to integrins ␣v␤3 and ␣4␤1 and induces proliferative signals in an integrin-dependent manner. sPLA2-IIA specifically binds to integrin v3 and 41 (14). The integrinbinding site does not include the catalytic center or the M-type receptor-binding site. WT and the catalytically inactive mutant (the H47Q mutant) of sPLA2-IIA induce intracellular signals in monocytic cells, but an integrin binding-defective mutant (the R74E/R100E mutant) does not (14). These results suggest that integrins may serve as receptors for sPLA2-IIA and mediate proinflammatory action of sPLA2-IIA in human. We screened small compounds that bind to sPLA-IIA and inhibit integrin binding. We obtained several compounds and compound 21 (Cmpd21) suppresses ␣v␤3-mediated cell adhesion and migration (15). These findings indicate direct binding of sPLA2-IIA to integrins is critical for proinflammatory actions of sPLA2-IIA.
It has been proposed that integrin activation is mediated by signaling from inside the cell (inside-out signaling) and that integrin activation is associated with global conformational changes of the integrin molecule (16,17). We recently discovered that the chemokine domain of fractalkine (FKN-CD) directly binds to several integrins, and this interaction is critical for fractalkine/CX3CR1 signaling (18). FKN-CD induces ternary complex formation (integrin-FKN-CD-CX3CR1) on the cell surface, suggesting that integrins act as the co-receptor for FKN-CD in FKN/CX3CR1 signaling (18). Notably we discovered that FKN-CD can activate integrins in the absence of CX3CR1 through direct binding to integrins probably in an allosteric mechanism (19). We identified a new FKN-CD-binding site in integrins (site 2) that is distinct from the classical RGD-binding site (site 1). The position of site 2 was predicted by docking simulation of interaction between FKN-CD and integrin ␣v␤3 that has a closed-headpiece conformation. This is based on the premise that site 2 is open in the closed-headpiece ␣v␤3. A peptide from site 2 (residues 267-286 of ␤3) directly binds to FKN-CD and suppresses FKN-CD-induced integrin activation (19). We thus propose a model in which FKN-CD binding to site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism).
The site 2-mediated activation of integrins may not be limited to FKN-CD. In the present paper we describe that sPLA2-IIA directly activates integrins (␣v␤3, ␣4␤1, and ␣5␤1) in cellfree conditions and/or on the cell surface. sPLA2-IIA mutants that are catalytically inactive or defective in binding to the M-type receptor still activate integrins, whereas the integrin binding-defective mutant did not. This suggests that direct integrin binding is required, but catalytic activity or M-type receptor is not. sPLA2-IIA is predicted to bind to site 2 in ␣v␤3 in a closed-headpiece conformation in docking simulation. Consistently, we obtained evidence that a peptide from site 2 effectively suppressed the sPLA-IIA-induced integrin activation, suggesting that this activation involves the binding of sPLA-IIA to site 2. Cmpd21 effectively suppressed sPLA2-IIAinduced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.
Synthesis of sPLA2-IIA-Recombinant sPLA2-IIA proteins (WT and mutants) were synthesized as described (14) using PET28a expression vector. The proteins were synthesized in Escherichia coli BL21 and induced by isopropyl ␤-D-thiogalactoside as insoluble proteins. The proteins were solubilized in 8 M urea, purified by nickel-nitrilotriacetic acid affinity chromatography under denatured conditions, and refolded as previously described (14). The refolded proteins were Ͼ90% homogeneous upon SDS-PAGE.
Remaining protein-binding sites were blocked by incubating with PBS, 0.1% BSA for 30 min at room temperature. After washing with PBS, soluble recombinant ␣v␤3 (5 g/ml) in the presence or absence of sPLA2-IIA (WT or mutants) was added to the wells and incubated in HEPES-Tyrodes buffer (10 mM HEPES, 150 mM NaCl, 12 mM NaHCO 3 , 0.4 mM NaH 2 PO 4 , 2.5 mM KCl, 0.1% glucose, 0.1% BSA) with 1 mM CaCl 2 for 2 h at room temperature. After unbound ␣v␤3 was removed by rinsing the wells with binding buffer, bound ␣v␤3 was measured using anti-integrin ␤3 mAb (AV-10) followed by HRP-conjugated goat anti-mouse IgG and peroxidase substrates.
Binding of Labeled Ligands to Integrins on the Cell Surface-The cells were cultured to nearly confluent in RPMI 1640, 10% FCS (K562 and U937) or DMEM, 10% FCS (CHO cells). The cells were resuspended with RPMI 1640, 0.02% BSA or DMEM, 0.02% BSA and incubated for 30 min at room temperature to block the remaining protein-binding sites. The cells were then incubated with WT sPLA2-IIA or mutants for 5 min at room temperature and then incubated with FITC-labeled integrin ligands (␥C399tr, FN-H120, FN8 -11, and ADAM15) for 15 min at room temperature. For blocking experiments, sPLA-IIA was preincubated with S2-␤1 peptide for 30 min at room temperature. The cells were washed with PBS, 0.02% BSA and analyzed by FACSCalibur (BD Biosciences). For inhibition studies using Cmpd21, sPLA2-IIA was preincubated with Cmpd21 for 30 min at room temperature.
Binding of S2 Peptide to Proteins-ELISA-type binding assays were performed as described previously (18). Briefly, wells of 96-well Immulon 2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 l of 0.1 M NaHCO 3 containing sPLA2-IIA, ␥C399tr, and FN-H120 for 2 h at 37°C. The remaining protein-binding sites were blocked by incubating with PBS, 0.1% BSA for 30 min at room temperature. After washing with PBS, S2 peptides were added to the wells and incubated in PBS for 2 h at room temperature. After unbound S2 peptides were removed by rinsing the wells with PBS, bound S2 peptides (GST-tagged) were measured using HRP-conjugated anti-GST antibody and peroxidase substrates.
Adhesion Assays-Adhesion assays were performed as described previously (18). Briefly, wells of a 96-well Immulon 2 microtiter plates were coated with 100 l of 0.1 M NaHCO 3 containing sPLA2-IIA (10 g/ml) and were incubated for 2 h at 37°C. The remaining protein-binding sites were blocked by incubating with PBS, 0.1% BSA for 30 min at room temperature. After washing with PBS, ␣4-K562 or K562 cells in 100 l of RPMI 1640 were added to the wells and incubated at 37°C for 1 h in the presence of Cmpd21 (0 -100 M). After unbound cells were removed by rinsing the wells with RPMI 1640, bound cells were quantified by measuring endogenous phosphatase activity.
Chemotaxis-Chemotaxis was measured in modified Boyden Chambers (Transwell). One g/ml sPLA2-IIA and 20 g/ml S2-␤1 peptide or control peptides in 600 l of RPMI 1640 medium were placed in the lower chamber, and U937 cells (2 ϫ 10 5 cells in 100 l of RPMI1640 medium) were placed in the upper chamber. After 5 h of incubation at 37°C, cells in the lower chamber were counted.
Other Methods-Treatment differences were tested using analysis of variance and a Tukey multiple comparison test to control the global type I error using Prism 5.0 (Graphpad Software). Surface plasmon resonance studies were performed as described (18).

RESULTS
sPLA2-IIA Activates Soluble Integrin ␣v␤3 in Cell-free Conditions-We recently reported that FKN-CD can activate integrins in the absence of CX3CR1 through direct binding to site 2 of integrins (19). A peptide from site 2 of integrin ␤3 (S2-␤3 peptide) directly binds to FKN-CD and suppresses FKN-CDinduced integrin activation (19). The newly identified site 2 is distinct from the classical RGD-binding site (site 1). We propose that FKN-CD binding to site 2 induces activation of site 1 through conformational changes (in an allosteric mechanism). The site 2-mediated activation of integrins may not be limited to FKN-CD, and we tested if other known integrin ligands activate ␣v␤3.
We previously reported that sPLA2-IIA binds to integrins ␣v␤3 and ␣4␤1 and induces signals through integrin pathways (14). We studied if sPLA2-IIA enhances the binding of recombinant soluble ␣v␤3 to ␥C399tr, an ␣v␤3-specific ligand (22,25) in cell-free conditions. We immobilized ␥C399tr to wells of microtiter plates and measured the binding of soluble ␣v␤3 to ␥C399tr in the presence of sPLA2-IIA. To keep soluble integrin inactive, we included 1 mM Ca 2ϩ in the assay. WT sPLA2-IIA enhanced the binding of ␥C399tr to ␣v␤3 in a concentrationdependent manner (Fig. 1, a and b). In contrast to WT sPLA2-IIA, the R74E/R100E mutant (integrin-binding-defective) (14) was defective in this function (Fig. 1b). H47Q (catalytically inactive) and G29S/D48K (M-type receptor-binding-defective) mutants behaved like WT sPLA2-IIA (Fig. 1c). These findings suggest that sPLA2-IIA activates ␣v␤3 in cell-free conditions, and this activation requires the integrin-binding site of sPLA2-IIA but does not require catalytic activity or receptor binding.
It is possible that the effect of sPLA2-IIA on ␣v␤3 may be specific to ␥C399tr. We thus used the disintegrin domain of human ADAM15, which has an RGD motif and specifically FIGURE 1. sPLA2-IIA activates ␣v␤3 integrin in cell-free conditions and on the cell surface (through direct integrin binding). a, activation of soluble ␣v␤3 by sPLA2-IIA as a function of ␥C399tr concentration. Binding of soluble ␣v␤3 (5 g/ml) to immobilized ␥C399tr in the presence or absence of WT sPLA2-IIA (50 g/ml) was performed as described under "Experimental Procedures." Data are shown as the means Ϯ S.E. of three independent experiments. b, activation of soluble ␣v␤3 by sPLA2-IIA as a function of sPLA2-IIA concentration. Wells of 96-well microtiter plates were coated with ␥C399tr (100 g/ml) and incubated with soluble ␣v␤3 (5 g/ml). Data are shown as the means Ϯ S.E. of three independent experiments. c, the effects of sPLA2-IIA mutations on integrin ␣v␤3 activation. Activation of soluble ␣v␤3 was measured as described above. SPLA2-IIA (50 g/ml) and ␥C399tr (100 g/ml for coating) were used. binds to ␣v␤3 (23). The binding of FITC-labeled ADAM15 disintegrin domain was markedly enhanced by WT sPLA2-IIA, but not by R74E/R100E, in ␣v␤3-K562 (Fig. 1g) and ␤3-CHO cells (Fig. 1h), suggesting that the effect of sPLA2-IIA on the ligand binding to ␣v␤3 is not unique to ␥C399tr. We confirmed that sPLA2-IIA does not directly interact with the integrin ligands used in this study (supplemental Fig. S1). sPLA2-IIA directly binds to integrins.
Docking Simulation Predicts That sPLA2-IIA Binds to Site 2 in an Inactive Form of ␣v␤3-We studied if sPLA2-IIA-induced activation of ␣v␤3 involves the binding of sPLA2-IIA to site 2 of ␣v␤3. Docking simulation of the interaction between sPLA2-IIA and the closed-headpiece form of ␣v␤3 (PDB code 1JV2) predicts that sPLA2-IIA binds to site 2 with high affinity (docking energy Ϫ22.1 kcal/mol) (Fig. 2a) as in the case of FKN-CD (19). Site 2 is located at the opposite side of site 1 (Fig. 2, b and  c). The RGD peptide binds to site 1 in the open-headpiece ␣v␤3 (Fig. 2d). Amino acid residues in sPLA2-IIA and integrin ␣v␤3 that are involved in sPLA2-IIA-␣v␤3 integrin are listed in Table  1. The docking model predicts that Arg-74 and Arg-100 are within the sPLA2-IIA/␣v␤3 interface at site 2 (Fig. 1a), suggesting that the integrin-binding interface in sPLA2-IIA at site 2 overlaps with that of site 1. This predicts that sPLA2-IIA may activate integrins through direct binding to site 2 and that the R74E/R100E mutant may be defective in this function.
sPLA2-IIA Enhances the Binding of the Fibronectin Fragment That Contains the RGD Motif to ␣5␤1-The interaction between the RGD-containing cell-binding fibronectin type III
sPLA2-IIA Suppresses H120 Binding to ␣4␤1 at High Concentrations-If sPLA2-IIA binds to site 1 (14) and site 2 (the present study), it is predicted that sPLA2-IIA competes with FIGURE 5. Cmpd21 blocks the binding of ␥C399tr to ␣v␤3 on the cell surface through binding to site 2. The effect of Cmpd21 on the binding of ␥C399tr to ␣v␤3 and on the sPLA2-IIA-induced integrin activation was studied. a, structure of Cmpd21. Amino acids with asterisks are D isomers. b, Cmpd21 suppresses the binding of sPLA2-IIA to ␣v␤3. Soluble ␣v␤3 was immobilized to a sensor chip, and Cmpd21 was added to the solution phase together with sPLA2-IIA in a surface plasmon resonance study. c-e, Cmpd21 suppressed the binding of FITC-labeled ␥C399tr enhanced by sPLA2-IIA (20 g/ml) to ␣v␤3 on U937 (c), ␣v␤3-K562 (d), and ␤3-CHO cells (e). The concentration of Cmpd21 in d and e is 50 M. Data are shown as the means Ϯ S.E. of median fluorescent intensity (MFI) of three independent experiments.

sPLA2-IIA Activates Integrins
ligands for binding to site 1. To address this question, we determined the effect of sPLA2-IIA as a function of sPLA2-IIA concentrations up to 500 g/ml. The binding of H120 to ␣4-CHO cells was maximum at 20 g/ml sPLA2-IIA and then reduced as sPLA2-IIA concentration increases (Fig. 8). This suggests that 1) sPLA2-IIA at low concentrations binds to site 2 of closed ␣4␤1 (site 1 closed, site 2 open) and activates ␣4␤1 (site 1 open), and 2) when site 2 is saturated with sPLA2-IIA, sPLA2-IIA competes with H120 for binding to site 1 (open) and reduces the binding of H120.

DISCUSSION
The present study establishes that sPLA2-IIA activates integrins ␣v␤3, ␣4␤1, and ␣5␤1 through direct binding to site 2. sPLA2-IIA activated recombinant soluble ␣v␤3 in cell-free conditions, suggesting that inside-out signals or other molecules are not involved. This process does not include catalytic activity or receptor binding of sPLA2-IIA as mutating the catalytic center or receptor-binding site of sPLA2-IIA did not affect sPLA2-IIA-mediated integrin activation. sPLA2-IIA induced integrin activation through binding to site 2 is a novel mechanism of integrin activation and proinflammatory action by sPLA2-IIA. sPLA2-IIA may activate other integrins through direct binding.
sPLA2-IIA-mediated integrin activation happens in biological fluids (at least in tears). In the present study Ͼ5 g/ml sPLA2-IIA were required to detect sPLA2-IIA-induced integrin activation. Notably, the concentration of sPLA2-IIA is exceptionally high in human tears (26 -28). In normal subjects the concentration of sPLA2-IIA in tears is 54.5 Ϯ 33.9 g/ml, one of the highest levels of sPLA2-IIA reported in any normal human secretions (29). Therefore, integrin activation by sPLA2-IIA happens at least in tears. sPLA2-IIA appears to be secreted by both the lacrimal glands and the goblet cells of conjunctival epithelia (26,30). Because sPLA2-IIA is bactericidal and kills Listeria at much lower concentrations (Ͻ0.1 nM), it is possible that the primary functions of sPLA2-IIA at such high concentrations in tears might be integrin activation. It is likely that sPLA2-IIA in tears may play a role in enhancing immune response to bacterial pathogens through local integrin activation in tears or perhaps in other tissues. Serum levels of sPLA2-IIA are increased only up to 1 g/ml during systemic inflammation (2,3). sPLA2-IIA may not effectively activate integrins at these concentrations. It is, however, possible that sPLA2-IIA may be highly concentrated in diseased tissues in chronic inflammation or on the cell surface through binding to proteoglycans.
The sPLA2-IIA-induced integrin activation is expected to enhance interaction between cells and extracellular matrix (e.g. fibrinogen and fibronectin) and thereby induce massive prolif-
We establish that site 2 is involved in integrin activation by sPLA2-IIA (the current study) and FKN-CD (19) (Fig. 9). This is a new mechanism of integrin activation. It has previously been reported that the binding of a RGD-mimetic peptide induces changes in the tertiary structure of ␣v␤3 (39) and ␣IIb␤3 (40) in the ␤3 I-like domain. RGD or ligand-mimetic peptides activate purified, non-activated ␣IIb␤3 (41) and ␣v␤3 (42). This process does not require inside-out signal transduction, and it appears that RGD or ligand-mimetic peptide triggers conformational changes that lead to full activation of integrins. These findings suggest that these peptides enhance integrin affinity by conformational changes in the headpiece possibly through additional ligand-binding sites in the integrin (41). A previous study suggests that there are two RGD-binding sites in integrin ␣IIb␤3 and that one binding site acts as an allosteric site based on binding kinetic studies (43). Also, another study suggests that two distinct cyclic RGD-mimetic peptides can simultaneously bind to distinct sites in ␣IIb␤3, and the estimated distance between two ligand-binding sites is about 6.1 Ϯ 0.5 nm (44). The possible allosteric ligand-binding site has not been pursued probably because the ␣v␤3 structure (ligand occupied, openheadpiece) contains only one RGD-binding site (39). In our docking model the distance between site 1 and site 2 is about 6 nm. Thus, the position of site 2 is consistent with the previous   We propose a model, in which sPLA2-IIA binds to site 2 of inactive/closedheadpiece integrins and induces conformational changes and enhance ligand binding to site 1 (the classical RGD-binding site). This activation is blocked by a peptide that is derived from site 2 or a small compound (Cmpd21) that binds to the integrin-binding site of sPLA2-IIA.

sPLA2-IIA Activates Integrins
report. Based on previous studies it is likely that the newly identified site 2 has ligand specificity that overlaps with that of site 1, interacts with integrin ligands other than FKN-CD (e.g. RGD), and is potentially involved in integrin regulation in an allosteric mechanism. It is reasonable to assume that FKN-CD or sPLA2-IIA binding to site 2 induces conformational changes in integrins. We suspect that other proteins also bind to site 2 and affect integrin functions. Also it is likely that integrins other than ␣v␤3, ␣4␤1, and ␣5␤1 are activated by site 2-mediated mechanism. It would be interesting to address this question in future studies.
It is unclear if sPLA2-IIA-induced integrin activation requires global conformational changes in integrins. In current models of integrin activation, activation of ␤1 integrins induces a swing-out movement of the hybrid domain and exposes epitopes recognized by activation-dependent antibodies (antihuman ␤1 HUTS4 and HUTS21) (45). The HUTS4 and HUTS21 epitopes are located in the hybrid domain of ␤1 (46,47). In our preliminary experiments sPLA-IIA did not change reactivity of ␤1 integrins to HUTS4 and HUTS21 in U937, K562, and ␣4-K562 cells in RPMI1640 medium under the conditions in which sPLA2-IIA enhanced the binding of ␤1 integrins to ligands. It is possible that the binding of sPLA2-IIA to site 2 induces only local conformational changes within the headpiece of integrins. Interestingly, the open-headpiece (PDB code 1L5G) and closed-headpiece (PDB code 1JV2) conformations of ␣v␤3 are very similar (39,48). Surprisingly, the docking simulation distinguished the two conformations and predicted the position of site 2 in the closed-headpiece form. We showed that sPLA2-IIA (the current study) and FKN-CD (19) actually bind to site 2 and the binding of sPLA2-IIA and FKN to site 2 is required for integrin activation using the peptides from site 2. It is likely that integrins that are activated by sPLA2-IIA and FKN-CD through site 2 have conformations similar to the open-headpiece ␣v␤3 that has no global conformational changes compared with the closed-headpiece form. The open-and closed-headpiece conformations of ␣v␤3 may really reflect the fact that integrins can be activated without global conformational changes through allosteric mechanism.
The site 2-derived integrin peptides were used for establishing that sPLA2-IIA really binds to site 2, and the interaction is involved in integrin activation. We studied if S2-␤1 peptide acts as an antagonist for sPLA2-IIA-mediated proinflammatory signals (supplemental Fig. S2). S2-␤1 peptide suppressed chemotaxis induced by sPLA2-IIA. Although it is unclear if S2-␤1 peptide suppressed the binding of sPLA2-IIA to site 1 or site 2 at this point, the results suggest that S2-␤1 peptide has potential as a therapeutic.
Specific inhibitors of sPLA2-IIA catalytic activity S-5920/ LY315920Na and S-3013/LY333013 failed to demonstrate a significant therapeutic effect in rheumatoid arthritis (49) and asthma (50). Our previous study suggests that sPLA2-IIA-integrin interaction may be a potential target for chronic inflammatory diseases (14). Cmpd21 was screened for its ability to bind to the integrin-binding site of sPLA2-IIA (15). Indeed Cmpd21 suppressed the binding of sPLA2-IIA to integrins, and in the present study we demonstrated that Cmpd21 suppressed the sPLA2-IIA-induced integrin activation via site 2 as well. It would be interesting to study if Cmpd21 or its variants suppress inflammation in vivo in future studies.