Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin ␣v␤3 at a high affinity (K D ‫؍‬ 2 ؋ 10 ؊7 M). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to ␣4␤1. We showed that sPLA2-IIA competed with VCAM-1 for binding to ␣4␤1, and bound to a site close to those for VCAM-1 and CS-1 in the ␣4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins ␣v␤3 and ␣4␤1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrinsPLA2-IIA interaction is a novel therapeutic target. The phospholipase A2 (PLA2)2 family is a group of intracellular and secreted enzymes that hydrolyzes the sn-2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes to form nonesterified fatty acids and lysophospholipids. These products act as intracellular second messengers or are further metabolized into potent mediators of a broad range of cellular processes, including inflammation, apoptosis, and atherogenesis (1). The mammalian secretory PLA2 isoforms are comprised of the groups named IB, IIA, IIC, IID, IIE, IIF, V, X, and XII (2, 3). All secretory PLA2 isoforms have in common a Ca 2ϩ -dependent catalytic mechanism, a low molecular mass (13-16 kDa), several disulfide bridges, and a wellconserved overall three-dimensional structure (2, 4, 5). Secretory PLA2 type IIA (sPLA2-IIA) was first isolated and purified from rheumatoid synovial fluid. sPLA2-IIA is an acute phase reactant and is found in markedly increased plasma concentrations 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 (6, 7). In addition to being a pro-inflammatory ...

The incidence of distant metastases is higher in the tumours with low oxygen pressure than in those with high oxygen pressure. It is well known that hypoxia induces the transcription of various genes involved in angiogenesis and anaerobic metabolism necessary for the growth of tumour cells in vivo, suggesting that hypoxia may also induce the transcription of metastasis-associated genes. We sought to identify the metastasis-associated genes differentially expressed in tumour cells under hypoxic conditions with the use of a DNA microarray system. We found that hypoxia enhanced the expression of autocrine motility factor mRNA in various cancer cells and also enhanced the random motility of pancreatic cancer cells. Autocrine motility factor inhibitors abrogated the increase of motility under hypoxic conditions. In order to explore the roles of hypoxia-inducible factor-1a, we established hypoxia-inducible factor-1a-transfectants and dominant negative hypoxiainducible factor-1a-transfectants. Transfection with hypoxia-inducible factor-1a and dominant-negative hypoxia-inducible factor-1a enhanced and suppressed the expression of autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA and the random motility, respectively. These results suggest that hypoxia may promote the metastatic potential of cancer cells through the enhanced autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA expression and that the disruption of the hypoxia-inducible factor-1 pathway may be an effective treatment for metastasis. As metastasis is the major cause of death in cancer patients, control of metastasis is most important in the therapies for cancer patients. In order to control metastasis, we need to understand the details of metastatic process that is now thought to take multiple steps. Although a variety of factors have been documented to play important roles in the metastatic steps (Poste and Fidler, 1980;Liotta, 1986Liotta, , 1988Nicolson, 1988), many factors are yet to be elucidated. Several clinical investigations demonstrated that patients with hypoxic tumours had poor prognoses and that the incidence of distant metastases was higher in the tumours with low oxygen pressure than in those with high oxygen pressure (Gatenby et al, 1988;Brizel et al, 1996;Hockel et al, 1998;Rofstad, 2000). These studies suggest that the tumour cells exposed to hypoxia at the primary tumour sites acquire aggressive properties including metastatic potential more than the welloxygenated tumour cells do. In fact, recent reports have demonstrated that hypoxia enhances the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in tumour cells, resulting in an increase of metastatic potential (Claffey and Robinson, 1996;Shi et al, 1999;Biroccio et al, 2000). However, it remains poorly understood how hypoxia promotes tumour cells' metastatic potential.When tumour cells are exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1), which is a transcription factor composed of HIF-1a and HIF-1b subunits (Wang et al,...

Plasmin is a major extracellular protease that elicits intracellular signals to mediate platelet aggregation, chemotaxis of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types in a G protein-dependent manner. Angiostatin, a fragment of plasmin(ogen), is a ligand and an antagonist for integrin ␣ 9 ␤ 1 . Here we report that plasmin specifically interacts with ␣ 9 ␤ 1 and that plasmin induces migration of cells expressing recombinant ␣ 9 ␤ 1 (␣ 9 -Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with ␣ 9 ␤ 1 as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of ␣ 9 -CHO cells, but simultaneous activation of the G protein-coupled protease-activated receptor (PAR)-1 with an agonist peptide induced the migration on angiostatin, whereas PAR-2 or PAR-4 agonist peptides were without effect. Furthermore, a small chemical inhibitor of PAR-1 (RWJ 58259) and a palmitoylated PAR-1-blocking peptide inhibited plasmin-induced migration of ␣ 9 -CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to ␣ 9 ␤ 1 and simultaneous proteolytic activation of PAR-1.

It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling.

Fibrinogen is a major plasma protein (350 kDa) that induces proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing, angiogenesis, and tumor growth. Fibrin(ogen) degradation products generated during fibrinolysis are implicated in tissue injury. The fibrinogen ; chain has a COOH-terminal globular domain (;C, residues 151-411 of the ; chain, 30 kDa) to which several integrin cell adhesion receptors (e.g., platelet A IIb B 3 , endothelial A v B 3 , and leukocyte A M B 2 ) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that ;C and its truncation mutant (designated ;C399tr), with a deletion of the COOHterminal 12 residues, induced apoptosis of endothelial cells and blocked tube formation of endothelial cells. DLD-1 human colon cancer cells that secrete ;C or ;C399tr grew at similar levels in vitro but grew much slower in vivo than mocktransfected cells. The recombinant purified ;C399tr fragment markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis in vivo in the highly metastatic Met-1 breast cancer model. The determinant responsible for binding to endothelial cells is cryptic in native fibrinogen but is exposed in ;C and ;C399tr. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential. (Cancer Res 2006; 66(19): 9691-7)