Yasunori Yokota

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.

Suppression of murine endotoxic shock by sPLA2 inhibitor, indoxam, through group IIA sPLA2-independent mechanisms.

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A2 (sPLA2) have been implicated in this process. Group IB sPLA2 (PLA2-IB) binds to the PLA2 receptor (PLA2R), and PLA2R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha. Group IIA sPLA2 (PLA2-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA2 inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-alpha with a similar potency in PLA2-IIA-expressing and PLA2-IIA-deficient mice after LPS challenge. In PLA2-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1beta, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA2-IB binding to murine PLA2R with a high potency (Ki=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-alpha levels could not be observed in mice deficient in PLA2R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA2-IIA-independent mechanisms, possibly via blockade of the PLA2R function.

Identification of group X secretory phospholipase A2 as a natural ligand for mouse phospholipase A2 receptor

Phospholipase A2 receptor (PLA2R) mediates various biological responses elicited by group IB secretory phospholipase A2 (sPLA2‐IB). The recently cloned group X sPLA2 (sPLA2‐X) possesses several structural features characteristic of sPLA2‐IB. Here, we detected a specific binding site of sPLA2‐X in mouse osteoblastic MC3T3‐E1 cells. Cross‐linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA2R. In fact, sPLA2‐X was found to bind the recombinant PLA2R expressed in COS‐7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA2R. These findings demonstrate sPLA2‐X to be one of the high‐affinity ligands for mouse PLA2R.

Physiological Roles of Group X-secreted Phospholipase A2 in Reproduction, Gastrointestinal Phospholipid Digestion, and Neuronal Function

Although the secreted phospholipase A2 (sPLA2) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA2 (sPLA2-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA2-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA2-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA2-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.

Deficiency of Phospholipase A2 Receptor Exacerbates Ovalbumin-Induced Lung Inflammation

Secretory phospholipase A2 (sPLA2) plays a critical role in the genesis of lung inflammation through proinflammatory eicosanoids. A previous in vitro experiment showed a possible role of cell surface receptor for sPLA2 (PLA2R) in the clearance of extracellular sPLA2. PLA2R and groups IB and X sPLA2 are expressed in the lung. This study examined a pathogenic role of PLA2R in airway inflammation using PLA2R-deficient (PLA2R−/−) mice. Airway inflammation was induced by immunosensitization with OVA. Compared with wild-type (PLA2R+/+) mice, PLA2R−/− mice had a significantly greater infiltration of inflammatory cells around the airways, higher levels of groups IB and X sPLA2, eicosanoids, and Th2 cytokines, and higher numbers of eosinophils and neutrophils in bronchoalveolar lavage fluid after OVA treatment. In PLA2R−/− mice, intratracheally instilled [125I]-labeled sPLA2-IB was cleared much more slowly from bronchoalveolar lavage fluid compared with PLA2R+/+ mice. The degradation of the instilled [125I]-labeled sPLA2-IB, as assessed by trichloroacetic acid-soluble radioactivity in bronchoalveolar lavage fluid after instillation, was lower in PLA2R−/− mice than in PLA2R+/+ mice. In conclusion, PLA2R deficiency increased sPLA2-IB and -X levels in the lung through their impaired clearance from the lung, leading to exaggeration of lung inflammation induced by OVA treatment in a murine model.

Clearance of group X secretory phospholipase A2 via mouse phospholipase A2 receptor

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A2 (sPLA2‐X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA2‐X can also act as a ligand for mouse phospholipase A2 receptor (PLA2R). Here, we found that sPLA2‐X was internalized and degraded via binding to PLA2R associated with the diminished prostaglandin E2 (PGE2) formation in PLA2R‐expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA2‐X was co‐localized with PLA2R in the punctate structures in PLA2R‐expressing CHO cells. Moreover, in mouse osteoblastic MC3T3‐E1 cells that endogenously express the PLA2R, the internalized sPLA2‐X was localized in lysosomes. These findings demonstrate that PLA2R acts as a clearance receptor for sPLA2‐X to suppress its strong enzymatic activity.

Hair Follicular Expression and Function of Group X Secreted Phospholipase A2 in Mouse Skin

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A2 (PLA2) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA2 (sPLA2-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA2-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA2-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA2-X in hair follicles, the presence of skin-specific machinery leading to sPLA2-X activation, a functional link of sPLA2-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.

Lack of Phospholipase A2 Receptor Increases Susceptibility to Cardiac Rupture after Myocardial Infarction

Rationale: Recent evidence indicates that the biological effects of secretory phospholipase A2 (sPLA2) cannot be fully explained by its catalytic activity. A cell surface receptor for sPLA2 (PLA2 receptor 1 [PLA2R]) and its high-affinity ligands (including sPLA2-IB, sPLA2-IIE, and sPLA2-X) are expressed in the infarcted myocardium. Objective: This study asked whether PLA2R might play a pathogenic role in myocardial infarction (MI) using mice lacking PLA2R (PLA2R–/–). Methods and Results: MI was induced by permanent ligation of the left coronary artery. PLA2R–/– mice exhibited higher rates of cardiac rupture after MI compared with PLA2R wild-type (PLA2R+/+) mice (46% versus 21%, respectively; P=0.015). PLA2R–/– mice had a 31% decrease in collagen content and a 45% decrease in the number of &agr;-smooth muscle actin–positive fibroblasts in the infarcted region compared with PLA2R+/+ mice. PLA2R was primarily found in myofibroblasts in the infarcted region. PLA2R–/– myofibroblasts were impaired in collagen-dependent migration, proliferation, and activation of focal adhesion kinase in response to sPLA2-IB. Binding of sPLA2-IB to PLA2R promoted migration and proliferation of myofibroblasts through functional interaction with integrin &bgr;1, independent of the catalytic activity of sPLA2-IB. In rescue experiments, the injection of PLA2R+/+ myofibroblasts into the infarcted myocardium prevented post-MI cardiac rupture and reversed the decrease in collagen content in the infarcted region in PLA2R–/– mice. Conclusions: PLA2R deficiency increased the susceptibility to post-MI cardiac rupture through impaired healing of the infarcted region. This might be partly explained by a reduction in integrin &bgr;1–mediated migratory and proliferative responses of PLA2R–/– myofibroblasts.

Structural and functional roles of the amino‐terminal region and collagen‐like domain of human serum mannan‐binding protein

The serum mannan‐binding protein (S‐MBP) is a Ca2+‐dependent C‐type animal lectin specific for mannose and N‐acetylglucosamine, which plays an important role in first‐line host defense. To study the structure and function relationship of the lectin, a full‐length human S‐MBPcDNA was expressed in Sf9 insect cells using a baculovirus expression system, and a cDNA encoding the carbohydrate recognition domain (CRD) of human S‐MBP was expressed in E. coli. The properties of the recombinant S‐MBP and recombinant S‐MBP‐CRD were compared with those of the native human S‐MBP and the CRD of the native S‐MBP. The results indicated that functional human S‐MBP can be successfully expressed in Sf9 cells and functional S‐MBP‐CRD in E. coli. In addition, the amino‐terminal region and collagen‐like domain are required for higher oligomer formation and play important roles in complement activation.