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. 2014 Nov 14;290(1):259–271. doi: 10.1074/jbc.M114.579946

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*

Masaaki Fujita ‡,§,, Kan Zhu ‡, Chitose K Fujita , Min Zhao ‡, Kit S Lam §, Mark J Kurth ‖, Yoko K Takada ‡,§, Yoshikazu Takada ‡,§,1
PMCID: PMC4281730  PMID: 25398877

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.

Keywords: Adhesion, Allosteric Regulation, Inflammation, Integrin, Peptides, Docking Simulation, Fractalkine, Integrin Activation, Secreted Phospholipase A2 Type IIA

Abstract

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.

Introduction

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 >1 μg/ml). Inflammatory cytokines such as IL-6, TNF-α, and IL-1β 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 β-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 α and 8 β subunits are known (13). Integrins transduce signals to the cell upon ligand binding (12). We previously reported that sPLA2-IIA specifically binds to integrins αvβ3 and α4β1 and induces proliferative signals in an integrin-dependent manner. sPLA2-IIA specifically binds to integrin v3 and 41 (14). The integrin-binding 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 αvβ3-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 αvβ3 that has a closed-headpiece conformation. This is based on the premise that site 2 is open in the closed-headpiece αvβ3. A peptide from site 2 (residues 267–286 of β3) 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 (αvβ3, α4β1, and α5β1) in cell-free 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 αvβ3 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-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.

EXPERIMENTAL PROCEDURES

Materials

U937 monocytic cells and Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection. K562 erythroleukemia cells that express human integrin αvβ3 (αvβ3-K562) (20) were provided by Eric Brown (University of California, San Francisco, CA). K562 cells that express human integrin α4 (α4-K562), CHO cells that express human integrin β3 (β3-CHO), or integrin α4 (α4-CHO) were described (14). Recombinant soluble αvβ3 was synthesized in CHO-K1 cells using the soluble αv and β3 expression constructs and purified by nickel-nitrilotriacetic acid affinity chromatography as described (21). Fibrinogen γ-chain C-terminal domain that lacks residues 400–411 (γC399tr) was synthesized as described (22). Fibronectin H120 fragment (FN-H120) (18), fibronectin type III domains 8–11 (FN8–11) (19), and ADAM15 (23) were synthesized as GST fusion proteins as described in the cited references. Anti-human β3 mAb AV10 was provided by B. Felding-Habermann (The Scripps Research Institute, La Jolla, CA). HRP-conjugated anti-His tag antibody was purchased from Qiagen (Valencia, CA). Cmpd21 was synthesized as described (15).

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 β-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 >90% homogeneous upon SDS-PAGE.

Synthesis of Site 2 Peptides

We introduced a His6 tag to the BamHI site of pGEX-2T using 5′-GATCTCATCATCACCATCACCATG-3′ and 5′-GATCCATGGTGATGGTGATGATGA-3′ (the resulting vector is designated pGEX-2THis6). We synthesized GST fusion protein of site 2 peptide (QPNDGQSHVGSDNHYSASTTM, residues 267–287 of β3, Cys-273 is changed to S) and a scrambled site 2 peptide (VHDSHYSGQGAMSDNTNSPQT) by subcloning oligonucleotides that encode these sequences into the BamHI/EcoRI site of pGEX-2THis6. We synthesized the proteins in E. coli BL21 and purified using glutathione-Sepharose affinity chromatography (18). The corresponding β1, β2, and β4 peptides were generated as described (18).

Binding of Soluble αvβ3 to γC399tr

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 μl of 0.1 m NaHCO3 containing γC399tr or ADAM15 for 2 h at 37 °C. 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 αvβ3 (5 μg/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 NaHCO3, 0.4 mm NaH2PO4, 2.5 mm KCl, 0.1% glucose, 0.1% BSA) with 1 mm CaCl2 for 2 h at room temperature. After unbound αvβ3 was removed by rinsing the wells with binding buffer, bound αvβ3 was measured using anti-integrin β3 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 (γC399tr, FN-H120, FN8–11, and ADAM15) for 15 min at room temperature. For blocking experiments, sPLA-IIA was preincubated with S2-β1 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 μl of 0.1 m NaHCO3 containing sPLA2-IIA, γC399tr, 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 μl of 0.1 m NaHCO3 containing sPLA2-IIA (10 μg/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, α4-K562 or K562 cells in 100 μl of RPMI 1640 were added to the wells and incubated at 37 °C for 1 h in the presence of Cmpd21 (0–100 μm). 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 μg/ml sPLA2-IIA and 20 μg/ml S2-β1 peptide or control peptides in 600 μl of RPMI 1640 medium were placed in the lower chamber, and U937 cells (2 × 105 cells in 100 μl of RPMI1640 medium) were placed in the upper chamber. After 5 h of incubation at 37 °C, cells in the lower chamber were counted.

Docking Simulation

Docking simulation of interaction between sPLA2-IIA (PDB code 1DCY), and integrin αvβ3 was performed using AutoDock3 as described (24). In the present study we used the headpiece (residues 1–438 of αv and residues 55–432 of β3) of αvβ3 (closed-headpiece form, PDB code 1JV2). Cations were not present in αvβ3 during docking simulation, as in the previous studies using αvβ3 (open-headpiece form, PDB code 1L5G) (14, 24).

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 αvβ3 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 β3 (S2-β3 peptide) directly binds to FKN-CD and suppresses FKN-CD-induced 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 αvβ3.

We previously reported that sPLA2-IIA binds to integrins αvβ3 and α4β1 and induces signals through integrin pathways (14). We studied if sPLA2-IIA enhances the binding of recombinant soluble αvβ3 to γC399tr, an αvβ3-specific ligand (22, 25) in cell-free conditions. We immobilized γC399tr to wells of microtiter plates and measured the binding of soluble αvβ3 to γC399tr in the presence of sPLA2-IIA. To keep soluble integrin inactive, we included 1 mm Ca2+ in the assay. WT sPLA2-IIA enhanced the binding of γC399tr to αvβ3 in a concentration-dependent 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 αvβ3 in cell-free conditions, and this activation requires the integrin-binding site of sPLA2-IIA but does not require catalytic activity or receptor binding.

FIGURE 1.

FIGURE 1.

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sPLA2-IIA activates αvβ3 integrin in cell-free conditions and on the cell surface (through direct integrin binding). a, activation of soluble αvβ3 by sPLA2-IIA as a function of γC399tr concentration. Binding of soluble αvβ3 (5 μg/ml) to immobilized γC399tr in the presence or absence of WT sPLA2-IIA (50 μg/ml) was performed as described under “Experimental Procedures.” Data are shown as the means ± S.E. of three independent experiments. b, activation of soluble αvβ3 by sPLA2-IIA as a function of sPLA2-IIA concentration. Wells of 96-well microtiter plates were coated with γC399tr (100 μg/ml) and incubated with soluble αvβ3 (5 μg/ml). Data are shown as the means ± S.E. of three independent experiments. c, the effects of sPLA2-IIA mutations on integrin αvβ3 activation. Activation of soluble αvβ3 was measured as described above. SPLA2-IIA (50 μg/ml) and γC399tr (100 μg/ml for coating) were used. R74E/R100E, defective in integrin binding; H74Q, catalytically inactive; G29S/D48K, defective in the binding to M-type receptor. d–f, the effects of sPLA2-IIA mutations on the binding of γC399tr to integrin αvβ3 on the cell surface. The binding of FITC-labeled γC399tr to the cells in the presence of sPLA2-IIA (up to 20 μg/ml in the case of U937 cells (d) and 20 μg/ml in αvβ3-K562 (e) and β3-CHO (f) cells) was measured using flow cytometry as described under “Experimental Procedures.” Data are shown as the means (median fluorescent intensity (MFI)) ± S.E. of three independent experiments. g and h, the effects of sPLA2-IIA mutations on the binding of ADAM15 to integrin αvβ3. The binding of FITC-labeled ADAM15 to αvβ3 in the presence of sPLA2-IIA (20 μg/ml) in αvβ3-K562 (g) and β3-CHO (h) cells was measured using flow cytometry. Data are shown as means (median fluorescent intensity) ± S.E. of three independent experiments.

We studied if sPLA2-IIA activates integrins on the cell surface by measuring the binding of FITC-labeled γC399tr to cells using flow cytometry. WT sPLA2-IIA activated αvβ3 on U937 (αvβ3+) (Fig. 1d), K562 cells that express recombinant αvβ3 (αvβ3-K562 cells) (Fig. 1e), and CHO cells that express hamster αv/human β3 hybrid (β3-CHO cells) (Fig. 1f). The effects of sPLA2-IIA mutations on sPLA2-IIA-induced αvβ3 activation were similar to those in soluble αvβ3. These findings suggest that sPLA2-IIA activates αvβ3 on the cell surface in a manner similar to that of soluble αvβ3 and that the sPLA2-IIA-induced αvβ3 activation is not cell type-specific.

It is possible that the effect of sPLA2-IIA on αvβ3 may be specific to γC399tr. We thus used the disintegrin domain of human ADAM15, which has an RGD motif and specifically binds to αvβ3 (23). The binding of FITC-labeled ADAM15 disintegrin domain was markedly enhanced by WT sPLA2-IIA, but not by R74E/R100E, in αvβ3-K562 (Fig. 1g) and β3-CHO cells (Fig. 1h), suggesting that the effect of sPLA2-IIA on the ligand binding to αvβ3 is not unique to γC399tr. 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 αvβ3

We studied if sPLA2-IIA-induced activation of αvβ3 involves the binding of sPLA2-IIA to site 2 of αvβ3. Docking simulation of the interaction between sPLA2-IIA and the closed-headpiece form of αvβ3 (PDB code 1JV2) predicts that sPLA2-IIA binds to site 2 with high affinity (docking energy −22.1 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 αvβ3 (Fig. 2d). Amino acid residues in sPLA2-IIA and integrin αvβ3 that are involved in sPLA2-IIA-αvβ3 integrin are listed in Table 1. The docking model predicts that Arg-74 and Arg-100 are within the sPLA2-IIA/αvβ3 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.

FIGURE 2.

FIGURE 2.

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Docking simulation predicts that sPLA2-IIA binds to a binding site that is distinct from the classical RGD-binding site in closed-headpiece αvβ3. a, a docking model of sPLA2-IIA-integrin αvβ3 (inactive) interaction. The headpiece of an inactive form of integrin αvβ3 (PDB code 1JV2) was used as a target. b, a docking model of sPLA2-IIA-integrin αvβ3 (active) interaction (14). The headpiece of the ligand-bound form of integrin αvβ3 (PDB code 1L5G) was used as a target. The model predicts that sPLA2-IIA (PDB code 1DCY) binds to the classical RGD-binding site of the integrin αvβ3 headpiece (Site 1). The model predicts the position of the second sPLA2-IIA-binding site (Site 2). c, superposition of two models shows that the positions of two predicted sPLA2-IIA-binding sites are distinct. d, open-headpiece αvβ3 structure (PDB code 1L5G) with an RGD-containing peptide (Site 1).

TABLE 1.

Amino acid residues involved in the interaction between sPLA2-IIA and integrin αvβ3

Amino acid residues within 6 Å between sPLA2-IIA and αvβ3 were selected using pdb viewer (Version 4.1). Amino acid residues in β3 site 2 peptide (S2-β3) are shown in bold.

αv β3 sPLA2-IIA
Glu-15, Lys-42, Asn-44, Gly-49, Ile-50, Val-51, Glu-52, Asn-77, Ser-90, His-91, Trp-93, Arg-122, Ala-397, Arg-398, Ser-399 Pro-160, Val-161, Ser-162, Met-165, Ser-168, Pro-169, Pro-170, Glu-171, Ala-172, Leu-173, Glu-174, Asn-175, Leu-185, Pro-186, Met-187, Phe188, His-192, Val-193, Leu-194, Glu-206, Ala-263, Gly-264, Gln-267, Gly-276, Ser-277, Asp-278, Asn-279, His-280, Ser-282, Ala-283, Thr-285, Thr-286 Leu-11, Thr-13, Gly-14, Lys-15
Ser-35, Pro-36, Lys-37, Asp-38, Ala-39, Arg-42, Val-45, Thr-46, His-47, Cy49, Cy50, Arg-53, Ser-71, Gly-72, Ser-73, Arg-74, Cys-97, Arg-100, Asn-101, Lys-102, Thr-103, Thr-104, Tyr-105, Asn-106, Lys-107, Lys-108, Tyr-109, Tyr-112, Arg-118, Ser-120, Pro-122, Arg-123, Cys-124
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sPLA2-IIA Directly Binds to a Peptide Derived from Site 2 of Integrin β1

We previously identified a peptide sequence (e.g. residues 256–288 of β3, S2-β3 peptide) from site 2 of αvβ3 that directly interacts with FKN-CD (Fig. 3a). The peptide suppresses FKN-CD-mediated integrin activation, but control scrambled peptide does not (19). We studied if site 2-derived peptides bind to sPLA2-IIA. It was expected that S2-β3 peptide binds to sPLA2-IIA because the amino acid residues in S2-β3 peptide are located within the integrin-binding interface of sPLA2-IIA in the docking model (Table 1). Interestingly, site 2 peptides from β1 (S2-β1 peptide) bound better to sPLA2-IIA in a concentration-dependent manner than S2-β3 peptide (Fig. 3, b and c). Control GST or scrambled S2-β3 peptide (S2-β3scr) did not bind to sPLA2-IIA. This suggests that site 2 has different binding specificity to FKN-CD and sPLA2-IIA. S2-β1 peptide suppressed sPLA2-IIA-mediated αvβ3 activation in U937 (Fig. 3d), αvβ3-K562 (Fig. 3e), and β3-CHO (Fig. 3f) cells, whereas control GST or S2-β3scr peptide did not. These findings suggest that sPLA2-IIA binds specifically to site 2 and that the binding of sPLA2-IIA to site 2 is critical for sPLA2-IIA-mediated αvβ3 activation.

FIGURE 3.

FIGURE 3.

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Site 2 peptide from β1 binds to sPLA2-IIA. a, alignment of peptides from site two from different integrin β subunits. b, binding of site 2 peptides from different integrin β subunits (S2-β1, β2, β3, and β4) to immobilized sPLA2-IIA (20 μg/ml). The binding of peptides to immobilized sPLA2-IIA was measured as described under “Experimental Procedures.” Data are shown as the means ± S.E. of three independent experiments. c, binding of S2-β1 peptide to sPLA2-IIA as a function of sPLA2-II concentrations. The binding of the peptide to immobilized sPLA2-IIA was measured as described in b. Data are shown as the means ± S.E. of three independent experiments. d, suppression of γC399tr binding to U937 cells by site 2 peptide. The binding of FITC-labeled γC399tr to αvβ3 on U937 was measured in flow cytometry as described under “Experimental Procedures.” Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments. e, suppression of γC399tr binding to αvβ3-K562 cells by site 2 peptide. The binding of FITC-labeled γC399tr to αvβ3 on αvβ3-K562 was measured in flow cytometry as described under “Experimental Procedures.” Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments. sPLA2-IIA (20 μg/ml) and site 2 peptides (200 μg/ml) were used. f, suppression of γC399tr binding to β3-CHO cells by site 2 peptide. The binding of FITC-labeled γC399tr to αvβ3 on β3-CHO was measured in flow cytometry as described under “Experimental Procedures.” Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments. sPLA2-IIA (20 μg/ml) and site 2 peptides (200 μg/ml) were used.

sPLA2-IIA Activates α4β1 in a Site 2-dependent Manner

We have reported that sPLA2-IIA directly binds to another integrin, α4β1, and induces signals in an α4β1-dependent manner (14). We found that sPLA2-IIA enhanced the binding of FITC-labeled fibronectin fragment specific to α4β1 (H120) to U937 cells (α4+) (Fig. 4a), K562 cells that express recombinant α4 (α4-K562) (Fig. 4c), and CHO cells that express recombinant α4 (α4-CHO) (Fig. 4e). This suggests that sPLA2-IIA activates α4β1. WT sPLA2-IIA markedly increased the binding of H120 to α4β1, whereas R74E/R100E did not (Fig. 4, a, c, and e). The H47Q or G29S/D48K mutants induced α4β1 activation, like WT sPLA2-IIA, suggesting that catalytic activity or receptor binding of sPLA2-IIA is not important. S2-β1 peptide suppressed the binding of H120 to α4β1 increased by sPLA2-IIA, whereas control GST or S2-β3scr peptide did not (Fig. 4, b, d, and e). These results suggest that sPLA2-IIA activates integrin α4β1 through direct binding to site 2 in a manner similar to that of αvβ3.

FIGURE 4.

FIGURE 4.

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sPLA2-IIA enhances the binding of the fibronectin fragment that contains CS-1 (H120) to α4β1 through binding to site 2. a, sPLA2-IIA activates α4β1. The binding of FITC-labeled FN H120 fragment (an α4β1-specific ligand) to α4β1 on U937 cells was measured by flow cytometry. WT sPLA2-IIA, catalytically inactive mutant (H47A), and the receptor-binding mutant (G29S/D48K) enhance the binding of H120 to α4β1, but the integrin-binding defective mutant (R74E/R100E) does not. Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments. b, site 2 peptide from β1 (S2-β1) suppressed sPLA2-IIA-induced α4β1 activation in U937 cells (20 μg/ml WT sPLA2-IIA was used). Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments. c, d, and e, sPLA2-IIA mutations and S2- β1 peptide affect α4β1 activation by sPLA2-IIA in α4-K562 (c and d) and α4-CHO (e) cells. The binding of FITC-labeled FN H120 fragment to α4β1+ cells was measured by flow cytometry. sPLA2-IIA (20 μg/ml) and site 2 peptides (200 μg/ml) were used. Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments.

Cmpd21 That Binds to sPLA2-IIA Inhibits sPLA2 IIA-mediated αvβ3 and α4β1 Activation

We recently identified small compounds that bind to sPLA2-IIA and suppress sPLA2-IIA binding to αvβ3 (15), including Cmpd21 (Fig. 5a). Cmpd21 was selected because of its ability to bind to WT sPLA2-IIA but not to R74E/R100E (15). Cmpd21 binds to the integrin-binding site of sPLA2-IIA and suppresses adhesion of αvβ3-K562 cells to γC399tr (15). Consistently, Cmpd21 suppressed the binding of sPLA2-IIA to αvβ3 in a concentration-dependent manner in surface plasmon resonance studies (Fig. 5b). We found that Cmpd21 suppressed the γC399tr binding to αvβ3-K562, U937, and β3-CHO cells in a concentration-dependent manner in three different cell types (Fig. 5, c, d, and e). Cmpd21 also suppressed the adhesion of α4-K562 cells to sPLA2-IIA in a concentration-dependent manner (Fig. 6a), suggesting that the effect of Cmpd21 is not limited to sPLA2-IIA-αvβ3 interaction. Cmpd21 suppressed sPLA2-IIA-induced binding of H120 to α4β1 in three different cell types (Fig. 6, b–d). These findings suggest that Cmpd21 suppresses sPLA2-IIA-mediated αvβ3 and α4β1 activation through site 2 by binding to the integrin-binding site of sPLA2-IIA.

FIGURE 5.

FIGURE 5.

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Cmpd21 blocks the binding of γC399tr to αvβ3 on the cell surface through binding to site 2. The effect of Cmpd21 on the binding of γC399tr to αvβ3 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 αvβ3. Soluble αvβ3 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 γC399tr enhanced by sPLA2-IIA (20 μg/ml) to αvβ3 on U937 (c), αvβ3-K562 (d), and β3-CHO cells (e). The concentration of Cmpd21 in d and e is 50 μm. Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments.

FIGURE 6.

FIGURE 6.

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Cmpd21 suppresses sPLA2-IIA-induced α4β1 activation in U937, α4-K562, and α4-CHO cells. a, Cmpd21 suppressed the binding of α4β1 to sPLA2-IIA. Adhesion of α4-K562 cells to sPLA2-IIA (at 10 μg/ml coating concentration) in RPMI1640 medium was measured. Data are shown as the means ± S.E. of three independent experiments. b–d, Cmpd21 suppressed the binding of FITC-labeled H120 enhanced by sPLA2-IIA (20 μg/ml) to α4β1 on U937 (b), α4-K562 (c), and α4-CHO cells (d). The concentration of Cmpd21 is 50 μm in d. Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments.

sPLA2-IIA Enhances the Binding of the Fibronectin Fragment That Contains the RGD Motif to α5β1

The interaction between the RGD-containing cell-binding fibronectin type III fragment and integrins has been extensively studied as a prototype cell-extracellular matrix interaction. We decided to study if sPLA2-IIA enhances the binding of this fragment to integrin α5β1 using rat FN8–11. We thus studied FN8–11 binding to α5β1. We found that sPLA2-IIA bound to integrin α5β1 in K562 cells, in which α5β1 is the only β1 integrin, and that mAb KH72 specific to α5 suppressed the binding, suggesting that sPLA2-IIA is a ligand of α5β1 (Fig. 7a). The binding of sPLA2-IIA to U937 cells was suppressed by KH73 (anti-α5), 7E3 (anti-αvβ3), and SG73 (anti-α4), suggesting αvβ3 and α4β1, in addition to α5β1, are involved in sPLA2-IIA binding to U937 cells (Fig. 7b). Cmpd21 effectively suppressed the adhesion of K562 cells to sPLA2-IIA (Fig. 7c). These findings suggest that sPLA2-IIA interacts with α5β1. We discovered that sPLA2-IIA markedly increased the binding of FITC-labeled FN8–11 to α5β1 on U937, K562, and CHO cells, whereas R74E/R100E did not (Fig. 7, d–f). The H47Q or G29S/D48K mutants induced α5β1 activation, like WT sPLA2-IIA. These findings suggest that sPLA2-IIA activates integrin α5β1 and that catalytic activity or receptor binding of sPLA2-IIA is not required for this process, as in the case of αvβ3 and α4β1. S2-β1 peptide suppressed the binding of FN8–11 to α5β1, increased by sPLA2-IIA, whereas control GST or scrambled β3 peptide did not (Fig. 7, g–i). Cmpd21 suppressed sPLA2-IIA-induced binding of FN8–11 to α5β1 in three different cell types (Fig. 7, j–l), suggesting Cmpd21 suppresses sPLA2-IIA-induced α5β1 activation through site 2 as well. Taken together, these results suggest that sPLA2-IIA enhances FN8–11 to integrin α5β1 through direct binding of sPLA2-IIA to site 2, as in the case of αvβ3 and α4β1.

FIGURE 7.

FIGURE 7.

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sPLA2-IIA enhances the binding of the RGD-containing fibronectin fragment (FN8–11) to α5β1 through binding to site 2. sPLA2-IIA at 20 μg/ml and FN8–11 at 20 μg/ml were used if not indicated otherwise. a and b, specific binding of sPLA2-IIA to integrin α5β1. FITC-labeled sPLA2-IIA (10 μg/ml) was incubated with K562 cells (a) or U937 cells (b) in the presence of mouse IgG, KH72 (anti-α5 mAb), SG73 (anti-α4 mAb), or 7E3 (anti-β3 mAb) (10 μg/ml). Bound FITC was measured using flow cytometry. Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments. c, Comp21 suppresses sPLA2 binding to α5β1. Adhesion of K562 cells to sPLA2-IIA (coating concentration 10 μg/ml) was measured in the presence of Cmpd21. Data are shown as the means ± S.E. of the % adhesion of three independent experiments. d–f, the effect of sPLA2-IIA mutants on the binding of FITC-FN8–11 was measured in U937 cells (d), K562 cells (e), and CHO cells (f). Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments. FITC-sPLA2-IIA was used at 20 μg/ml if not indicated otherwise. g–i, the binding of FITC-FN8–11 to U937 cells (g), K562 cells (h), or CHO cells (i) was measured in the presence of S2-β1 peptide. Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments. j–l, the binding of FITC-FN8–11 to U937 cells (j), K562 cells (k), or CHO cells (l) was measured in the presence of Cmpd21. Data are shown as the means ± S.E. of median fluorescent intensity of three independent experiments.

sPLA2-IIA Suppresses H120 Binding to α4β1 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 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 μg/ml. The binding of H120 to α4-CHO cells was maximum at 20 μg/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 α4β1 (site 1 closed, site 2 open) and activates α4β1 (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.

FIGURE 8.

FIGURE 8.

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sPLA2-IIA suppresses the binding of H120 to site 1 in α4β1. The binding of H120 to α4-CHO cells is shown as a function of sPLA2-IIA concentrations. The binding of FITC-H120 (10 μg/ml) was measured in flow cytometry (a). Data are shown as the means ± S.E. of median fluorescent intensity (MFI) of three independent experiments (b). *, p < 0.05 compared with the median fluorescent intensity at 20 μg/ml sPLA2-IIA (arrow).

DISCUSSION

The present study establishes that sPLA2-IIA activates integrins αvβ3, α4β1, and α5β1 through direct binding to site 2. sPLA2-IIA activated recombinant soluble αvβ3 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 >5 μg/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 ± 33.9 μg/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 (<0.1 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 μg/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 proliferative signals. Because integrins are involved in growth factor signaling through cross-talk with growth factor receptors, sPLA2-IIA-induced integrin activation is also expected to enhance cellular responsiveness to growth factors. We have reported that integrins cross-talk with several growth factor receptors through direct binding to growth factors (e.g. fibroblast growth factor-1 (24, 31–33), insulin-like growth factor-1 (34–37), neuregulin-1 (38), and fractalkine (18)). We propose that sPLA2-IIA-induced integrin activation indirectly affects intracellular signaling by these growth factors through enhancing integrin binding to growth factors.

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 αvβ3 (39) and αIIbβ3 (40) in the β3 I-like domain. RGD or ligand-mimetic peptides activate purified, non-activated αIIbβ3 (41) and αvβ3 (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 αIIbβ3 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 αIIbβ3, and the estimated distance between two ligand-binding sites is about 6.1 ± 0.5 nm (44). The possible allosteric ligand-binding site has not been pursued probably because the αvβ3 structure (ligand occupied, open-headpiece) 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 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 αvβ3, α4β1, and α5β1 are activated by site 2-mediated mechanism. It would be interesting to address this question in future studies.

FIGURE 9.

FIGURE 9.

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A model of sPLA2-IIA-induced integrin activation through site 2. We propose a model, in which sPLA2-IIA binds to site 2 of inactive/closed-headpiece 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.

It is unclear if sPLA2-IIA-induced integrin activation requires global conformational changes in integrins. In current models of integrin activation, activation of β1 integrins induces a swing-out movement of the hybrid domain and exposes epitopes recognized by activation-dependent antibodies (anti-human β1 HUTS4 and HUTS21) (45). The HUTS4 and HUTS21 epitopes are located in the hybrid domain of β1 (46, 47). In our preliminary experiments sPLA-IIA did not change reactivity of β1 integrins to HUTS4 and HUTS21 in U937, K562, and α4-K562 cells in RPMI1640 medium under the conditions in which sPLA2-IIA enhanced the binding of β1 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 αvβ3 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 αvβ3 that has no global conformational changes compared with the closed-headpiece form. The open- and closed-headpiece conformations of αvβ3 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-β1 peptide acts as an antagonist for sPLA2-IIA-mediated proinflammatory signals (supplemental Fig. S2). S2-β1 peptide suppressed chemotaxis induced by sPLA2-IIA. Although it is unclear if S2-β1 peptide suppressed the binding of sPLA2-IIA to site 1 or site 2 at this point, the results suggest that S2-β1 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.

*

This work was supported, in whole or in part, by National Institutes of Health Grant CA13015. This work was also supported by TRDRP (Tobacco-related Disease Research Program) Grant 18XT-0169 and Department of Defense Grant W81XWH-10-1-0312 (to Y. T.).

Inline graphic

This article contains supplemental Figs. 1 and 2.

2
The abbreviations used are:
sPLA2-IIA
secreted phospholipase 2 type IIA
FKN-CD
the chemokine domain of fractalkine
FN-H120
fibronectin H120 fragment
FN
fibronectin
γC399tr
fibrinogen γ-chain C-terminal domain that lacks residues 400–411
Cmpd21
compound 21.

REFERENCES

  • 1. Vadas P., Stefanski E., Pruzanski W. (1985) Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid. Life Sci. 36, 579–587 [DOI] [PubMed] [Google Scholar]
  • 2. Jaross W., Eckey R., Menschikowski M. (2002) Biological effects of secretory phospholipase A2 group IIA on lipoproteins and in atherogenesis. Eur J. Clin. Invest. 32, 383–393 [DOI] [PubMed] [Google Scholar]
  • 3. Niessen H. W., Krijnen P. A., Visser C. A., Meijer C. J., Erik Hack C. (2003) Type II secretory phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and ischemic damage to cardiomyocytes? Cardiovasc. Res. 60, 68–77 [DOI] [PubMed] [Google Scholar]
  • 4. Jiang J., Neubauer B. L., Graff J. R., Chedid M., Thomas J. E., Roehm N. W., Zhang S., Eckert G. J., Koch M. O., Eble J. N., Cheng L. (2002) Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma. Am. J. Pathol. 160, 667–671 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Dong Q., Patel M., Scott K. F., Graham G. G., Russell P. J., Sved P. (2006) Oncogenic action of phospholipase A2 in prostate cancer. Cancer Lett. 240, 9–16 [DOI] [PubMed] [Google Scholar]
  • 6. Sved P., Scott K. F., McLeod D., King N. J., Singh J., Tsatralis T., Nikolov B., Boulas J., Nallan L., Gelb M. H., Sajinovic M., Graham G. G., Russell P. J., Dong Q. (2004) Oncogenic action of secreted phospholipase A2 in prostate cancer. Cancer Res. 64, 6934–6940 [DOI] [PubMed] [Google Scholar]
  • 7. Tada K., Murakami M., Kambe T., Kudo I. (1998) Induction of cyclooxygenase-2 by secretory phospholipases A2 in nerve growth factor-stimulated rat serosal mast cells is facilitated by interaction with fibroblasts and mediated by a mechanism independent of their enzymatic functions. J. Immunol. 161, 5008–5015 [PubMed] [Google Scholar]
  • 8. Triggiani M., Granata F., Balestrieri B., Petraroli A., Scalia G., Del Vecchio L., Marone G. (2003) Secretory phospholipases A2 activate selective functions in human eosinophils. J. Immunol. 170, 3279–3288 [DOI] [PubMed] [Google Scholar]
  • 9. Lambeau G., Ancian P., Barhanin J., Lazdunski M. (1994) Cloning and expression of a membrane receptor for secretory phospholipases A2. J. Biol. Chem. 269, 1575–1578 [PubMed] [Google Scholar]
  • 10. Nicolas J. P., Lambeau G., Lazdunski M. (1995) Identification of the binding domain for secretory phospholipases A2 on their M-type 180-kDa membrane receptor. J. Biol. Chem. 270, 28869–28873 [DOI] [PubMed] [Google Scholar]
  • 11. Cupillard L., Mulherkar R., Gomez N., Kadam S., Valentin E., Lazdunski M., Lambeau G. (1999) Both group IB and group IIA secreted phospholipases A2 are natural ligands of the mouse 180-kDa M-type receptor. J. Biol. Chem. 274, 7043–7051 [DOI] [PubMed] [Google Scholar]
  • 12. Hynes R. O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 [DOI] [PubMed] [Google Scholar]
  • 13. Takada Y., Ye X., Simon S. (2007) The integrins. Genome Biol. 8, 215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Saegusa J., Akakura N., Wu C. Y., Hoogland C., Ma Z., Lam K. S., Liu F. T., Takada Y. K., Takada Y. (2008) Proinflammatory secretory phospholipase A2 type IIA binds to integrins αvβ3 and α4β1 and induces proliferation of monocytic cells in an integrin-dependent manner. J. Biol. Chem. 283, 26107–26115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Ye L., Dickerson T., Kaur H., Takada Y. K., Fujita M., Liu R., Knapp J. M., Lam K. S., Schore N. E., Kurth M. J., Takada Y. (2013) Identification of inhibitors against interaction between proinflammatory sPLA2-IIA protein and integrin αvβ3. Bioorg. Med. Chem. Lett. 23, 340–345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Zhu J., Zhu J., Springer T. A. (2013) Complete integrin headpiece opening in eight steps. J. Cell Biol. 201, 1053–1068 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Xiong J. P., Mahalingham B., Alonso J. L., Borrelli L. A., Rui X., Anand S., Hyman B. T., Rysiok T., Müller-Pompalla D., Goodman S. L., Arnaout M. A. (2009) Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment. J. Cell Biol. 186, 589–600 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Fujita M., Takada Y. K., Takada Y. (2012) Integrins αvβ3 and α4β1 act as coreceptors for fractalkine, and the integrin-binding defective mutant of fractalkine is an antagonist of CX3CR1. J. Immunol. 189, 5809–5819 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Fujita M., Takada Y. K., Takada Y. (2014) The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site. PLoS ONE 9, e96372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Blystone S. D., Graham I. L., Lindberg F. P., Brown E. J. (1994) Integrin αvβ3 differentially regulates adhesive and phagocytic functions of the fibronectin receptor α5β1. J. Cell Biol. 127, 1129–1137 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Takagi J., Erickson H. P., Springer T. A. (2001) C-terminal opening mimics “inside-out” activation of integrin α5β1. Nat. Struct. Biol. 8, 412–416 [DOI] [PubMed] [Google Scholar]
  • 22. Yokoyama K., Zhang X. P., Medved L., Takada Y. (1999) Specific binding of integrin αvβ3 to the fibrinogen γ and αE chain C-terminal domains. Biochemistry 38, 5872–5877 [DOI] [PubMed] [Google Scholar]
  • 23. Zhang X. P., Kamata T., Yokoyama K., Puzon-McLaughlin W., Takada Y. (1998) Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin αvβ3. J. Biol. Chem. 273, 7345–7350 [DOI] [PubMed] [Google Scholar]
  • 24. Mori S., Wu C. Y., Yamaji S., Saegusa J., Shi B., Ma Z., Kuwabara Y., Lam K. S., Isseroff R. R., Takada Y. K., Takada Y. (2008) Direct binding of integrin αvβ3 to FGF1 plays a role in FGF1 signaling. J. Biol. Chem. 283, 18066–18075 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Yokoyama K., Erickson H. P., Ikeda Y., Takada Y. (2000) Identification of amino acid sequences in fibrinogen γ-chain and tenascin C C-terminal domains critical for binding to integrin αvβ3. J. Biol. Chem. 275, 16891–16898 [DOI] [PubMed] [Google Scholar]
  • 26. Nevalainen T. J., Aho H. J., Peuravuori H. (1994) Secretion of group 2 phospholipase A2 by lacrimal glands. Invest. Ophthalmol. Vis. Sci. 35, 417–421 [PubMed] [Google Scholar]
  • 27. Qu X. D., Lehrer R. I. (1998) Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears. Infect. Immun. 66, 2791–2797 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Birts C. N., Barton C. H., Wilton D. C. (2010) Catalytic and non-catalytic functions of human IIA phospholipase A2. Trends Biochem. Sci 35, 28–35 [DOI] [PubMed] [Google Scholar]
  • 29. Saari K. M., Aho V., Paavilainen V., Nevalainen T. J. (2001) Group II PLA2 content of tears in normal subjects. Invest. Ophthalmol. Vis. Sci. 42, 318–320 [PubMed] [Google Scholar]
  • 30. Aho H. J., Saari K. M., Kallajoki M., Nevalainen T. J. (1996) Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands. Invest. Ophthalmol. Vis. Sci. 37, 1826–1832 [PubMed] [Google Scholar]
  • 31. Yamaji S., Saegusa J., Ieguchi K., Fujita M., Mori S., Takada Y. K., Takada Y. (2010) A novel fibroblast growth factor-1 (FGF1) mutant that acts as an FGF antagonist. PLoS ONE 5, e10273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Mori S., Takada Y. (2013) Crosstalk between fibroblast growth factor (FGF) receptor and integrin through direct integrin binding to FGF and resulting integrin-FGF-FGFR ternary complex formation. Med. Sci. 1, 20–36 [Google Scholar]
  • 33. Mori S., Tran V., Nishikawa K., Kaneda T., Hamada Y., Kawaguchi N., Fujita M., Saegusa J., Takada Y. K., Matsuura N., Zhao M., Takada Y. (2013) A dominant-negative FGF1 mutant (the R50E mutant) suppresses tumorigenesis and angiogenesis. PLoS ONE 8, e57927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Saegusa J., Yamaji S., Ieguchi K., Wu C. Y., Lam K. S., Liu F. T., Takada Y. K., Takada Y. (2009) The direct binding of insulin-like growth factor-1 (IGF-1) to integrin αvβ3 is involved in IGF-1 signaling. J. Biol. Chem. 284, 24106–24114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Fujita M., Ieguchi K., Davari P., Yamaji S., Taniguchi Y., Sekiguchi K., Takada Y. K., Takada Y. (2012) Cross-talk between integrin α6β4 and insulin-like growth factor-1 receptor (IGF1R) through direct α6β4 binding to IGF1 and subsequent α6β4-IGF1-IGF1R ternary complex formation in anchorage-independent conditions. J. Biol. Chem. 287, 12491–12500 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Fujita M., Ieguchi K., Cedano-Prieto D. M., Fong A., Wilkerson C., Chen J. Q., Wu M., Lo S. H., Cheung A. T., Wilson M. D., Cardiff R. D., Borowsky A. D., Takada Y. K., Takada Y. (2013) An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R. J. Biol. Chem. 288, 19593–19603 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Fujita M., Takada Y. K., Takada Y. (2013) Insulin-like growth factor (IGF) signaling requires αvβ3-IGF1-IGF type 1 receptor (IGF1R) ternary complex formation in anchorage independence, and the complex formation does not require IGF1R and Src activation. J. Biol. Chem. 288, 3059–3069 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Ieguchi K., Fujita M., Ma Z., Davari P., Taniguchi Y., Sekiguchi K., Wang B., Takada Y. K., Takada Y. (2010) Direct binding of the EGF-like domain of neuregulin-1 to integrins (αvβ3 and α6β4) is involved in neuregulin-1/ErbB signaling. J. Biol. Chem. 285, 31388–31398 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Xiong J. P., Stehle T., Zhang R., Joachimiak A., Frech M., Goodman S. L., Arnaout M. A. (2002) Crystal structure of the extracellular segment of integrin α Vβ3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 [DOI] [PubMed] [Google Scholar]
  • 40. Xiao T., Takagi J., Coller B. S., Wang J. H., Springer T. A. (2004) Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432, 59–67 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Du X. P., Plow E. F., Frelinger A. L., 3rd, O'Toole T. E., Loftus J. C., Ginsberg M. H. (1991) Ligands “activate” integrin αIIbβ3 (platelet GPIIb-IIIa). Cell 65, 409–416 [DOI] [PubMed] [Google Scholar]
  • 42. Legler D. F., Wiedle G., Ross F. P., Imhof B. A. (2001) Superactivation of integrin αvβ3 by low antagonist concentrations. J. Cell Sci. 114, 1545–1553 [DOI] [PubMed] [Google Scholar]
  • 43. Hu D. D., White C. A., Panzer-Knodle S., Page J. D., Nicholson N., Smith J. W. (1999) A new model of dual interacting ligand binding sites on integrin αIIbβ3. J. Biol. Chem. 274, 4633–4639 [DOI] [PubMed] [Google Scholar]
  • 44. Cierniewski C. S., Byzova T., Papierak M., Haas T. A., Niewiarowska J., Zhang L., Cieslak M., Plow E. F. (1999) Peptide ligands can bind to distinct sites in integrin αIIbβ3 and elicit different functional responses. J. Biol. Chem. 274, 16923–16932 [DOI] [PubMed] [Google Scholar]
  • 45. Luo B. H., Carman C. V., Springer T. A. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619–647 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Luque A., Gómez M., Puzon W., Takada Y., Sánchez-Madrid F., Cabañas C. (1996) Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β 1 chain. J. Biol. Chem. 271, 11067–11075 [DOI] [PubMed] [Google Scholar]
  • 47. Mould A. P., Barton S. J., Askari J. A., McEwan P. A., Buckley P. A., Craig S. E., Humphries M. J. (2003) Conformational changes in the integrin β A domain provide a mechanism for signal transduction via hybrid domain movement. J. Biol. Chem. 278, 17028–17035 [DOI] [PubMed] [Google Scholar]
  • 48. Xiong J. P., Stehle T., Diefenbach B., Zhang R., Dunker R., Scott D. L., Joachimiak A., Goodman S. L., Arnaout M. A. (2001) Crystal structure of the extracellular segment of integrin αVβ3. Science 294, 339–345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Bradley J. D., Dmitrienko A. A., Kivitz A. J., Gluck O. S., Weaver A. L., Wiesenhutter C., Myers S. L., Sides G. D. (2005) A randomized, double-blinded, placebo-controlled clinical trial of LY333013, a selective inhibitor of group II secretory phospholipase A2, in the treatment of rheumatoid arthritis. J. Rheumatol. 32, 417–423 [PubMed] [Google Scholar]
  • 50. Bowton D. L., Dmitrienko A. A., Israel E., Zeiher B. G., Sides G. D. (2005) Impact of a soluble phospholipase A2 inhibitor on inhaled allergen challenge in subjects with asthma. J Asthma 42, 65–71 [DOI] [PubMed] [Google Scholar]

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