Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2 expression in cytokine-stimulated rat fibroblasts.

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A(2) (sPLA(2)-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA(2)-IIA by proinflammatory cytokines was under the control of both classical cPKCalpha and atypical aPKClambda/iota pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1beta/TNFalpha-dependent induction of sPLA(2)-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA(2)-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA(2)-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA(2)-IIA expression was markedly enhanced in cPKCalpha knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKClambda/iota isoform reduced the cytokine-induced expression of sPLA(2)-IIA. These results suggest that the aPKClambda/iota pathway is required for the induction of sPLA(2)-IIA expression and that the cPKCalpha pathway acts as a negative regulator of sPLA(2)-IIA expression in cytokine-stimulated rat fibroblasts.

Microsomal prostaglandin E synthase-1 in both cancer cells and hosts contributes to tumour growth, invasion and metastasis.

mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.

A negative regulator of delayed prostaglandin D2 production in mouse mast cells.

We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.

Establishment of the culture model system that reflects the process of terminal differentiation of connective tissue-type mast cells.

To understand physiological roles of tissue mast cells, we established a culture system where bone marrow-derived immature mast cells differentiate into the connective tissue-type mast cell (CTMC)-like cells through modifying the previous co-culture system with Swiss 3T3 fibroblasts. Our system was found to reproducibly mimic the differentiation of CTMCs on the basis of several criteria, such as granule maturation and sensitivity to cationic secretagogues. The gene expression profile obtained by the microarray analyses was found to reflect many aspects of the differentiation. Our system is thus helpful to gain deeper insights into terminal differentiation of CTMCs.

Association of microsomal prostaglandin E synthase 1 deficiency with impaired fracture healing, but not with bone loss or osteoarthritis, in mouse models of skeletal disorders.

OBJECTIVE: Prostaglandin E synthase (PGES) functions as the terminal enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)) and is a potent regulator of bone and cartilage metabolism. Among the 3 isozymes of PGES, microsomal PGES-1 (mPGES-1) is known to play the most critical role in the production of PGE(2) in pathophysiologic events. This study investigated the roles of mPGES-1 under normal physiologic and pathophysiologic conditions in the skeletons of mPGES-1-deficient (mPGES-1(-/-)) mice. METHODS: Skeletons of mPGES-1(-/-) mice and their wild-type littermates were compared by radiologic and histologic analyses. Four models of skeletal disorders were created: bone loss induced by ovariectomy, bone loss induced by hind limb unloading, osteoarthritis (OA) induced by instability in the knee joint, and bone fracture by osteotomy at the tibial midshaft. Expression of the PGES enzymes was examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction. The cellular mechanism of fracture healing was examined in ex vivo cultures of costal cartilage chondrocytes. RESULTS: Microsomal PGES-1(-/-) mice had unaffected skeletal phenotypes under normal physiologic conditions. In the bone fracture model, fracture healing was impaired by the mPGES-1 deficiency, with half of the mice remaining in a non-bone union state even after 21 days; normal fracture healing was restored by adenoviral reintroduction of mPGES-1. The other skeletal disorders were not affected by the mPGES-1 deficiency. In vivo and ex vivo analyses revealed an impaired proliferation of chondrocytes in cartilage with the mPGES-1 deficiency, at an early stage of fracture healing. CONCLUSION: In these mouse models of skeletal disorders, mPGES-1 was indispensable for bone repair through chondrocyte proliferation, but was not essential for the skeleton under normal physiologic conditions, nor did it play a role in the pathophysiologic conditions of bone loss due to ovariectomy, bone loss due to unloading, or stress-induced OA.

Human group III secreted phospholipase A2 promotes neuronal outgrowth and survival.

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.

Human group III phospholipase A2 suppresses adenovirus infection into host cells. Evidence that group III, V and X phospholipase A2s act on distinct cellular phospholipid molecular species.

Of 10 mammalian secreted phospholipase A(2) (sPLA(2)) enzymes identified to date, group V and X sPLA(2)s, which are two potent plasma membrane-acting sPLA(2)s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA(2), which is structurally more similar to bee venom PLA(2) than to other mammalian sPLA(2)s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA(2) hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA(2) domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA(2) depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA(2)s target distinct phospholipid molecular species in cellular membranes.

Impaired mast cell maturation and degranulation and attenuated allergic responses in Ndrg1-deficient mice.

We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.

Prostaglandin E synthase: a novel drug target for inflammation and cancer.

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin (PG) H(2) to PGE(2), occurs in multiple forms with distinct enzymatic properties, modes of expression, cellular and subcellular localizations and intracellular functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein belonging to the MAPEG (for membrane-associated proteins involved in eicosanoid and GSH metabolism) family. This enzyme is markedly induced by proinflammatory stimuli, is down-regulated by anti-inflammatory glucocorticoids, and is functionally coupled with cyclooxygenase (COX)-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE(2) production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes. In particular, recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs.

Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells.

sPLA2 (secretory phospholipase A2) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA2s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA2s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA2s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA2s. Thus hydrolysis of the plasma membrane by these sPLA2s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA2s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA2 is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA2s may play a role in innate immunity against adenoviral infection in the respiratory tract.

Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis.

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2 enzymes, cyclooxygenase (COX) enzymes, and various lineagespecific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived PGH2 specifically to PGE2, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by antiinflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE2 production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.

Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues.

The secretory phospholipase A2 (sPLA2) family in mammals contains more than 10 enzymes. In this study, we examined by immunohistochemistry the localization of six sPLA2s (IIA, IID, IIE, IIF, V and X) in human heart, kidney, liver and stomach. In normal hearts, sPLA2-IIA was detected in coronary vascular smooth muscle cells (VSMC) and sPLA2-V in cardiomyocytes beneath the endocardium. In infarcted hearts, expression of these two enzymes was markedly increased in damaged cardiomyocytes, and expression of sPLA2-IID and-IIE, which was undetectable in normal hearts, was elevated in damaged cardiomyocytes and VSMC, respectively. In infarcted kidneys, sPLA2-IIA and-V were markedly induced in the uriniferous tubular epithelium. In livers affected by viral hepatitis, sPLA2-IIA and-V were expressed in hepatocytes with fatty degeneration. In the gastric glands exhibiting intestinal metaplasia, sPLA2-IIA was localized in the glandular base, sPLA2-IID and-V in the glandular body epithelium, sPLA2-IIE and-IIF in goblet cells in the foveolar epithelium, and sPLA2-X in both glandular body epithelial cells and foveolar epithelial goblet cells. In the gastric submucosal tissues, sPLA2-IIA and-IIE were located in VSMC and sPLA2-V was in the interstitial fibroblasts. In addition, sPLA2-IIA,-IIE,-IIF and-X were highly expressed in gastric signet ring cell carcinoma. Thus, individual sPLA2s exhibit unique cellular localizations in each tissue, suggesting their distinct roles in pathophysiology.

Search of factors that intermediate cytokine-induced group IIA phospholipase A2 expression through the cytosolic phospholipase A2- and 12/15-lipoxygenase-dependent pathway.

Inducible expression of group IIA secretory phospholipase A2 (sPLA2-IIA) by interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) is under the control of group IVA cytosolic PLA2alpha and 12/15-lipoxygenase (12/15-LOX) in rat fibroblastic 3Y1 cells. We show here that this cytokine induction of sPLA2-IIA mRNA requires de novo protein synthesis. By means of cDNA array analysis, we found that the level of the CXC chemokine MIP-2 (macrophage inflammatory protein-2) was significantly elevated in 12/15-LOX-transfected cells compared with control cells. IL-1beta/TNFalpha-stimulated induction of endogenous MIP-2 preceded that of sPLA2-IIA, and exogenous MIP-2 induced sPLA2-IIA dose-dependently. Moreover, a MIP-2-specific antisense oligonucleotide and small interfering RNA attenuated the IL-1beta/TNFalpha-induced expression of sPLA2-IIA, suggesting that MIP-2 is an absolute intermediate requirement for optimal induction of sPLA2-IIA. In addition, the expression of c-jun and fra-1, which are components of the transcription factor AP-1, was elevated in 12/15-LOX-transfected cells, in which cytokine-dependent binding of AP-1 to the sPLA2-IIA promoter was increased significantly. Conversely, the receptors for transforming growth factor-beta and platelet-derived growth factor, which contributed to down-regulation of sPLA2-IIA expression, were decreased following 12/15-LOX overexpression. Taken together, 12/15-LOX-dependent up-regulation of sPLA2-IIA expression may result from the interplay between accelerated MIP-2 signaling, AP-1 activation, and attenuated transforming growth factor-beta and platelet-derived growth factor signaling.

Cellular distribution, post-translational modification, and tumorigenic potential of human group III secreted phospholipase A(2).

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.

Neuronal expression and neuritogenic action of group X secreted phospholipase A2.

Although individual mammalian secreted phospholipase A(2) (sPLA(2)) enzymes exhibit unique tissue and cellular distributions, the cell type-specific functions of each enzyme remain largely unknown. In this study, we found by immunohistochemistry that group X sPLA(2) (sPLA(2)-X) is uniquely located in the peripheral neuronal fibers, an observation that was supported by detection of its transcript and protein in the neuronal cell line PC12 and in primary dorsal root ganglia neurons. Adenoviral expression of sPLA(2)-X in PC12 cells facilitated neurite outgrowth, particularly when combined with a suboptimal concentration of nerve growth factor. In neuronally differentiated PC12 cells, sPLA(2)-X was preferentially localized in the Golgi apparatus and growth cones, and proteolytic conversion of the proenzyme to mature enzyme mainly occurred after the secretion process. The neurite-extending ability of sPLA(2)-X depended on the production of its catalytic product, lysophosphatidylcholine. Moreover, nerve growth factor-induced neurite extension of PC12 cells was modestly but significantly attenuated by an anti-sPLA(2)-X antibody or by a small interfering RNA for sPLA(2)-X. These observations suggest the potential contribution of sPLA(2)-X to neuronal differentiation, and possibly repair, under certain conditions.

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma 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 iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 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 iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.

Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells.

Although group IIA secretory phospholipase A2 (sPLA2-IIA) is known to be abundantly present in the joints of patients with rheumatoid arthritis (RA), expression of other sPLA2s in this disease has remained unknown. In this study, we examined the expression and localization of six sPLA2s (groups IIA, IID, IIE, IIF, V and X) in human RA. Immunohistochemistry of RA sections revealed that sPLA2-IIA was generally located in synovial lining and sublining cells and cartilage chondrocytes, sPLA2-IID in lymph follicles and capillary endothelium, sPLA2-IIE in vascular smooth muscle cells, and sPLA2-V in interstitial fibroblasts. Expression levels of these group II subfamily sPLA2s appeared to be higher in severe RA than in inactive RA. sPLA2-X was detected in synovial lining cells and interstitial fibers in both active and inactive RA sections. Expression of sPLA2-IIF was partially positive, yet its correlation with disease states was unclear. Expression of sPLA2 transcripts was also evident in cultured normal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively. Adenovirus-mediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ng x mL(-1) concentrations. Thus, multiple sPLA2s are expressed in human RA, in which they may play a role in the augmentation of arachidonate metabolism or exhibit other cell type-specific functions.

Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells.

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.

Group IIA secretory phospholipase A2 is a unique 12/15-lipoxygenase-regulated gene in cytokine-stimulated rat fibroblastic 3Y1 cells.

We have proposed previously that the expression of group IIA secretory phospholipase A(2) (sPLA(2)-IIA), an enzyme implicated in inflammation, is under the control of group IVA cytosolic phospholipase A(2) (cPLA(2)) and 12/15-lipoxygense (12/15-LOX) in cytokine-stimulated rat fibroblastic 3Y1 cells. Here, we show that the reduction of cytokine-stimulated sPLA(2)-IIA induction by the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)) is partially overcome by the addition of various lysophospholipids, such as lysophosphatidylcholine (LysoPC). Furthermore, this lysophospholipid effect was enhanced by further addition of 12/15-LOX products, such as 12(S)- or 15(S)-hydroxyeicosatetraenoic acid (HETE) and 13(S)-hydroxyoctadecadienoic acid (HODE), thus substantiating the hypothesis that the expression of sPLA(2)-IIA is selectively regulated by lipid products of the cPLA(2)-12/15-LOX pathway. In an attempt to identify a set of 12/15-LOX-regulated genes, the cDNA subtraction technique, followed by Northern blotting, was performed to screen particular clones, the expression of which was suppressed by the LOX inhibitor nordihydroguaiaretic acid (NDGA). NDGA-sensitive clones identified thus far included sPLA(2)-IIA, cytoplasmic signaling intermediates, several oxygenases, extracellular matrices, secretory proteins, and other cellular proteins. Of these genes, however, only the expression of sPLA(2)-IIA and 14-3-3eta was enhanced by 12/15-LOX expression. Taken together, our data suggest that sPLA(2)-IIA represents a particular group of genes, the transcription of which is up-regulated by 12/15-LOX metabolites.

Localization of various secretory phospholipase A2 enzymes in male reproductive organs.

Current evidence suggests the presence of transcripts for several secretory phospholipase A(2) (sPLA(2)) enzymes in male genital organs. In this study, we examined by immunohistochemistry the localization of group IIA, IIC, IID, IIE, IIF, V and X sPLA(2)s in male genital organs. In sPLA(2)-IIA-deficient C57BL/6 mouse testis, sPLA(2)-IIC, -IID, -IIE, -IIF, -V and -X were diversely expressed in spermatogenic cells within the seminiferous tubules. Immunoblotting revealed the presence of these sPLA(2)s in mouse spermatozoa. In addition, sPLA(2)-IIF, -V and -X were localized in the interstitial Leydig cells. The same set of sPLA(2)s was detected in a mouse cultured Leydig cell line, and adenovirus-mediated transfer of these sPLA(2)s into Leydig cells resulted in increased prostaglandin production. sPLA(2)-IIC, -IID, -IIE, -IIF, -V and -X were also detected diversely in the epithelium of the epididymis, vas deferens, seminal vesicles, and prostate. In a sPLA(2)-IIA-positive FVB strain, weak expression of sPLA(2)-IIA was detected in Leydig cells. Notable differences in the sPLA(2) expression profiles were found in the seminal vesicles and prostate between mice and humans. Taken together, individual sPLA(2)s exhibit distinct or partially overlapping localizations in male reproductive organs, suggesting both specific and redundant functions.