Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE 2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE 2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE 2 involved in both tissue homeostasis and disease.Biosynthesis of prostaglandin (PG) 1 E 2 , which is produced by a variety of cells and tissues and exhibits diverse bioactivities, is mediated by three enzymatic reactions involving phospholipase A 2 (PLA 2 ), cyclooxygenase (COX), and PGE synthase (PGES). In this biosynthetic pathway, arachidonic acid (AA) released from membrane phospholipids by cytosolic or secretory PLA 2 s is converted to PGH 2 by COX-1 or COX-2 and is then isomerized to PGE 2 by terminal PGES enzymes.The constitutive COX-1 mainly promotes immediate PG production elicited by agonists promptly mobilizing intracellular Ca 2ϩ , a situation in which a burst release of AA occurs (1-5).The inducible COX-2 is essential for delayed PG generation induced by proinflammatory stimuli, during which AA is gradually supplied over long periods, and also promotes immediate PG production if it already exists in cells primed by particular stimuli (1-5). Current studies employing isozyme-specific inhibitors and knockout mice have revealed that the two COXs play distinct roles in vivo (6 -10), and segregated utilization of these enzymes at the cellular level has been explained not only by their distinct expression profiles but also by subtle differences in their AA requirement, hydroperoxide sensitivity, and subcellular localization (4, 11-13). In addition, selective coupling with various terminal PG synthases has also been shown to influence crucially the utilization of the two COX isoforms during the different phases of cell activation (14 -16). PGES enzymes, which lie downstream of COXs, occur in multiple forms in mammalian cells (1). Among them, a perinuclear membrane-bound form of PGES belonging to the MAPEG (for membrane-associated proteins involved in eicosanoid and glutathione metabolism) family, which we herein cal...

Among the many mammalian secreted phospholipase A 2 (sPLA 2 ) enzymes, PLA2G3 (group III secreted phospholipase A 2 ) is unique in that it possesses unusual N-and C-terminal domains and in that its central sPLA 2 domain is homologous to bee venom PLA 2 rather than to other mammalian sPLA 2 s. To elucidate the in vivo actions of this atypical sPLA 2 , we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA 2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA 2 s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5-or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A 2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.

Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions.

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A 2 (sPLA 2 -III), a member of the secreted phospholipase A 2 (sPLA 2 ) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3 -/-mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3 -/-mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3 +/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3 -/-mice. Moreover, the gonads of Pla2g3 -/-mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3 +/+ mice. Together, our results reveal a role for the atypical sPLA 2 family member sPLA 2 -III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A 2 (sPLA 2 ) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA 2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA 2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.