Hitoshi Arita

Identification of the Product of Growth Arrest-specific Gene 6 as a Common Ligand for Axl, Sky, and Mer Receptor Tyrosine Kinases

Axl, Sky, and Mer, members of an Axl/Sky receptor tyrosine kinase subfamily, are typified by the cell adhesion molecule-related extracellular domain. The product of growth arrest-specific gene 6 (Gas6), structurally homologous to the anticoagulant protein S, was recently identified as the ligand for Axl and Sky, but the ligand for Mer remained unknown. We have now obtained evidence that Gas6 can also function as a ligand for Mer. Co-precipitation analysis, using soluble receptors of Axl, Sky, and Mer (Axl-Fc, Sky-Fc, and Mer-Fc) composed of the extracellular domain of receptors fused to the Fc domain of immunoglobulin G1, clearly showed that Gas6, but not protein S, specifically bound to Axl-Fc, Sky-Fc, and Mer-Fc fusion proteins. Quantitative kinetic analyses using a BIAcore biosensor instrument revealed dissociation constants (Kd) of the binding of rat Gas6 to Axl-Fc, Sky-Fc, and Mer-Fc are 0.4, 2.7, and 29 nM, respectively. We also found that Gas6 stimulated tyrosine phosphorylation of Axl, Sky, and Mer receptors ectopically expressed in Chinese hamster ovary cells. Taken together, these findings suggest that Gas6 is a common ligand for Axl, Sky, and Mer, all known members of an Axl/Sky receptor subfamily.

Cell Adhesion to Phosphatidylserine Mediated by a Product of Growth Arrest-specific Gene 6

Gas6, a product of a growth arrest-specific gene 6, potentiates proliferation of vascular smooth muscle cells and prevents cell death of vascular smooth muscle cells. It has been also demonstrated that Gas6 is a ligand of receptor tyrosine kinases Axl, Sky, and Mer. Gas6 contains γ-carboxyglutamic acid residues, which are found in some blood coagulation factors and mediate the interaction of the coagulation factors with negatively charged phospholipid. In this study, we clarified that Gas6 specifically bound to phosphatidylserine and the binding was dependent on Ca2+ and γ-carboxyglutamic acid residues. Furthermore, we found that U937 cells, which express Gas6 receptor on their surfaces, adhered to phosphatidylserine-coated enzyme-linked immunosorbent assay (ELISA) plate only in the presence of Gas6 and Ca2+. U937 cells also bound to ELISA plate coated with phosphatidylinositol, but the binding was independent of Gas6 and Ca2+. On the other hand, U937 cells did not adhere to phosphatidylcholine- or phosphatidylethanolamine-coated ELISA plate even in the presence of Gas6 and Ca2+. These findings suggest that Gas6 may play a role in recognition of cells exposing phosphatidylserine on their surfaces by phagocytic cells, which is supposed to be one of the mechanisms for clearing dying cells.

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL‐1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP‐elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL‐1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP‐elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL‐1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.

Potent Modification of Low Density Lipoprotein by Group X Secretory Phospholipase A2 Is Linked to Macrophage Foam Cell Formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.

Different Functional Aspects of the Group II Subfamily (Types IIA and V) and Type X Secretory Phospholipase A2s in Regulating Arachidonic Acid Release and Prostaglandin Generation IMPLICATIONS OF CYCLOOXYGENASE-2 INDUCTION AND PHOSPHOLIPID SCRAMBLASE-MEDIATED CELLULAR MEMBRANE PERTURBATION

We have recently reported that members of the heparin-binding group II subfamily of secretory PLA2s (sPLA2s) (types IIA and V), when transfected into 293 cells, released [3H]arachidonic acid (AA) preferentially in response to interleukin-1 (IL-1) and acted as “signaling” PLA2s that were functionally coupled with prostaglandin biosynthesis. Here we show that these group II subfamily sPLA2s and the type X sPLA2 behave in a different manner, the former being more efficiently coupled with the prostaglandin-biosynthetic pathway than the latter, in 293 transfectants. Type X sPLA2, which bound only minimally to cell surface proteoglycans, augmented the release of both [3H]AA and [3H]oleic acid in the presence of serum but not IL-1. Both types IIA and V sPLA2, the AA released by which was efficiently converted to prostaglandin E2, markedly augmented IL-1-induced expression of cyclooxygenase (COX)-2 in a heparin-sensitive fashion, whereas type X sPLA2 lacked the ability to augment COX-2 expression, thereby exhibiting the poor prostaglandin E2-biosynthetic response unless either of the COX isozymes was forcibly introduced into type X sPLA2-expressing cells. Implication of phospholipid scramblase, an enzyme responsible for the perturbation of plasma membrane asymmetry, revealed that the scramblase-transfected cells became more sensitive to types IIA and V, but not X, sPLA2, releasing both [3H]AA and [3H]oleic acid in an IL-1-independent manner. Thus, although phospholipid scramblase-mediated alteration in plasma membrane asymmetry actually led to the increased cellular susceptibility to the group II subfamily of sPLA2s, several lines of evidence suggest that it does not entirely mimic their actions on cells after IL-1 signaling. Interestingly, coexpression of type IIA or V, but not X, sPLA2 and phospholipid scramblase resulted in a marked reduction in cell growth, revealing an unexplored antiproliferative aspect of particular classes of sPLA2.

Purified Group X Secretory Phospholipase A2 Induced Prominent Release of Arachidonic Acid from Human Myeloid Leukemia Cells

Group X secretory phospholipase A2 (sPLA2-X) possesses several structural features characteristic of both group IB and IIA sPLA2s (sPLA2-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA2-X, the preparation of its antibody, and the purification of native sPLA2-X. The affinity-purified sPLA2-X protein migrated as various molecular species of 13–18 kDa on SDS-polyacrylamide gels, andN-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH2-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA2-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH2 termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA2-X has a relatively weak potency compared with the mature protein. The mature sPLA2-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA2groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA2-IB and -IIA with concomitant production of prostaglandin E2. A prominent release of arachidonic acid was also observed in sPLA2-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA2-X is a unique N-glycosylated sPLA2 that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA2-IB and -IIA.

Resistance to Endotoxic Shock in Phospholipase A2 Receptor-deficient Mice

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-α and interleukin-1β elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.

Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s

Mammalian secretory phospholipase A2s (sPLA2s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA2 has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA2 gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA2 expressed in the spleen of group IIA sPLA2-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA2 genes. The human mature sPLA2 protein consists of 125 amino acids (M r = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA2 with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA2 should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA2s. When the cDNA was expressed in COS-7 cells, PLA2 activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mm Ca2+. In assays with individual substrates,l-α-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA2 mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA2-deficient mice, suggesting its functional role in the progression of the inflammatory process.

Prevention of growth arrest-induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6

We have purified Gas6 as a growth‐potentiating factor for vascular smooth muscle cells (VSMCs) [Nakano, T. et al. (1995) J. Biol. Chem. 270, 5702‐57051. However, specific production of Gas6 in growth‐arrested cells raises an intriguing question as to the physiological function of Gas6. In this study, we found that serum‐starved VSMCs secreted some survival factors and depletion of the factors induced cell death of VSMCs. Finally, we demonstrated that cell death was prevented by the addition of Gas6, suggesting that one of the major biological activity of Gas6 is protection of growth‐arrested VSMCs from death.

Enhanced expression of group II phospholipase A2 gene in the tissues of endotoxin shock rats and its suppression by glucocorticoid.

We studied the regulation of group II phospholipase A2 (PLA2‐II) gene in vivo, using endotoxin shock rat as a model for systemic inflammation. Administration of endotoxin into rats increased PLA2 activity in the plasma, as described by Vadas and Hay, using endotoxin‐challenged rabbit. Specific absorption of this activity by anti‐PLA2‐II antibody indicated that the released PLA2 was PLA2‐II. The levels of PLA2‐II mRNA were elevated in the aorta, spleen, lung, and thymus but not in the liver and kidney. The tissues with high PLA2‐II mRNA contents released a greater amount of PLA2‐II than the tissues of control rats. These results suggest that in endotoxin shock rats, PLA2‐II is synthesized de novo in the above tissues and released into circulation. Furthermore, our present study demonstrates that glucocorticoid suppresses the enhanced expression of the PLA2‐II gene in the tissues of endotoxin shock rats.