Although group VIA Ca 2؉ -independent phospholipase A 2 ␤ (iPLA 2 ␤) has been implicated in various cellular events, the functions of other iPLA 2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA 2 ␥. Lentiviral transfection of iPLA 2 ␥ into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell deathassociated release of arachidonic acid (AA), which was converted to prostaglandin E 2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA 2 ␥ small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA 2 ␥. iPLA 2 ␥ protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA 2 ␥ and other intracellular PLA 2 enzymes acted on different phospholipid subclasses. Transfection of iPLA 2 ␥ into HCA-7 cells also led to increased AA release and prostaglandin E 2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA 2 ␥ in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA 2 ␤ and iPLA 2 ␥ in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA 2 ␥ to tumorigenesis.Phospholipase A 2 (PLA 2 ) 1 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to yield free fatty acids, including arachidonic acid (AA), and lysophospholipids. Mammalian PLA 2 enzymes have been classified into four major families, including secretory PLA 2 , cytosolic PLA 2 (cPLA 2 ), Ca 2ϩ -independent PLA 2 (iPLA 2 ), and platelet-activating factor acetyl hydrolases, each of which occurs as multiple isoforms (1, 2). Of these, the group IV cPLA 2 and group VI iPLA 2 families represent intracellular enzymes with a catalytic serine in their lipase consensus motif. Of the cPLA 2 family, cPLA 2 ␣, cPLA 2 ␤, and cPLA 2 ␦ possess a Ca 2ϩ -regulated C2 domain near the N terminus, and their function is Ca 2ϩ -dependent, whereas cPLA 2 ␥ is Ca 2ϩ -independent and membranebound (3-6). The regulatory roles for cPLA 2 ␣ in production of lipid mediators, including prostaglandins and leukotrienes, have been well documented in many studies, including gene targeting (7-10).Of the emerging iPLA 2 family, group VIA iPLA 2 ␤ has been implicated in various cellular events such as the basal fatty acid reacylation reaction (membrane remodeling) (11, 12), agonist-stimulated AA release and eicosanoid generation (13-16), cell proliferation (17), apoptosis (18 -21), exocytosis (22, 23), vascular relaxation (24), adipogenesis (25), and activation of store-operated channels and capacitative Ca 2ϩ influx (26). In iPLA 2 ␤ transgenic mice, the enzyme is activated during myocardial infarction and causes malignant ventr...

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Supplementary key words cardiolipin • mitochondria • muscle • phospholipidsPhospholipase A 2 (PLA 2 ) enzymes catalyze the cleavage of the sn -2 ester bond of glycerophospholipids to yield free fatty acids and lysophospholipids, thereby playing critical roles in cellular lipid metabolisms linked to energy storage, membrane remodeling, and lipid mediator signaling. In the membrane remodeling reaction, fatty acyl groups are fi rst removed (deacylated) by PLA 2 and then replaced (reacylated) with different fatty acyl groups by acyltransferases, which allow membrane phospholipids to acquire a variation of molecular species. In the signaling reaction, polyunsaturated fatty acids [typically arachidonic acid (AA)] and lysophospholipids released by the action of PLA 2 s are metabolized to various lipid mediators, such as prostaglandins (PG), leukotrienes, and platelet-activating factor, which exert a variety of biological actions through their cognate receptors. Abstract Group VIB Ca2+ -independent phospholipase A 2 ␥ (iPLA 2 ␥ ) is a membrane-bound iPLA 2 enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA 2 ␥ by disrupting its gene in mice. iPLA 2 ␥ -knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofi laments in iPLA 2 ␥ -KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA 2 ␥ -KO muscles. These results provide evidence that impairment of iPLA 2 ␥ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA 2 ␥ -KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA 2 ␥ may contribute to modulation of lipid mediator production in vivo. -Yoda, E., K. Hachisu, Y. Taketomi, K. Yoshida, M. Nakamura, K. Ikeda, R. Taguchi, Y. Nakatani, H. Kuwata, M. Murakami, I. Kudo, and S. Hara. Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca 2+ -independent phospholipase A2 ␥ -defi cient mice. J. Lipid Res.

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