Background: Arachidonic acid and its metabolites regulate pancreatic glucose-stimulated insulin secretion (GSIS) through multiple mechanisms. Results: Group X secretory phospholipase A(2) (GX sPLA(2)) suppresses GSIS; suppression was abolished when COX-2 activity or PGE2-EP3 receptor signaling were inhibited. Conclusion: GX sPLA(2) inhibits GSIS by augmenting PGE2 production. Significance: GX sPLA(2) may be targeted for ameliorating beta cell dysfunction in type 2 diabetes.Group X secretory phospholipase A(2) (GX sPLA(2)) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA(2) is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA(2)-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA(2) overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA(2) (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA(2) on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA(2) plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.

Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes phospholipids within membrane bilayers, liberating free fatty acids and lysophospholipids. We reported previously that C57BL/6 mice deficient in GX sPLA2 (GX KO) have increased plasma corticosterone levels under both basal and adrenocorticotropic hormone (ACTH) stimulated conditions (Shridas et al. 2010, J. Biol. Chem. 285:20031-9). Conversely, overexpression of GX sPLA2 but not a catalytically inactive mutant, suppresses steroid production in mouse Y1 adrenal cells. GX sPLA2 is produced as a proenzyme that contains an N-terminal 11 amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase. While propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for GX sPLA2 activation have not been identified. In this study we report that ACTH treatment increases the phospholipase activity secreted by Y1 adrenal cells stably expressing GX sPLA2 (Y1-GX cells). Western blot analysis showed an increase in the ratio of mature/total GX sPLA2 in the media of ACTH-treated Y1-GX cells. Use of broad specificity proprotein convertase inhibitors confirmed the involvement of furin-like proteases in the processing and subsequent activation of GX sPLA2. Results from qRT-PCR analysis indicated that furin and PACE4, but not other members of the convertase family, are upregulated by ACTH in Y1 cells. siRNA-mediated gene targeting indicated that both furin and PACE4 mediate GX sPLA2 activation under both basal and ACTH-induced conditions. These results clearly demonstrate a role for furin and PACE4 in the proteolytic activation of GX sPLA2 in Y1 adrenal cells.

Objective: Abdominal aortic aneurysm (AAA) is a complex vascular disease characterized by matrix degradation and inflammation and is a major cause of mortality in older men. Specific interventions that prevent AAA progression remain to be identified. In this study, we tested the hypothesis that Group X secretory phospholipase A(2) (GX sPLA(2)), an enzyme implicated in inflammatory processes, mediates AAA.Methods and results: GX sPLA(2) was detected by immunostaining in human aneurysmal tissue and in angiotensin II (Ang II)-induced AAAs in apolipoprotein E-deficient (apoE(-/-)) mice. GX sPLA(2) mRNA was increased significantly (11-fold) in abdominal aortas of apoE(-/-) mice in response to Ang II infusion. To define the role of GX sPLA(2) in experimental AAAs, apoE(-/-) and apoE(-/-) x GX sPLA(2)(-/-) (GX DKO) mice were infused with Ang II for either 10 (n = 7) or 28 (n = 24-26) days. Deficiency of GX sPLA(2) significantly reduced the incidence and severity of AAAs, as assessed by ultrasound measurements in vivo of aortic lumens and by computer-assisted morphometric analyses ex vivo of external diameter. Results from gene expression profiling indicated that the expression of specific matrix metalloproteinases and inflammatory mediators was blunted in aortas from GX DKO mice compared to apoE(-/-) mice after 10-day Ang II infusion. Ang II induction of cyclooxygenase-2, interleukin-6, matrix metalloproteinase (MMP)-2, MMP-13 and MMP-14 was reduced significantly in GX DKO mice compared to apoE(-/-) mice.Conclusion: GX sPLA(2) promotes Ang II-induced pathological responses leading to AAA formation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Studies in vitro indicate that group X secretory phospholipase A(2) (GX sPLA(2)) potently releases arachidonic acid (AA) and lysophosphatidylcholine from mammalian cell membranes. To define the function of GX sPLA(2) in vivo, our laboratory recently generated C57BL/6 mice with targeted deletion of GX sPLA(2) (GX(-/-) mice). When fed a normal rodent diet, GX(-/-) mice gained significantly more weight and had increased adiposity compared to GX(+/+) mice, which was not attributable to alterations in food consumption or energy expenditure. When treated with adipogenic stimuli ex vivo, stromal vascular cells isolated from adipose tissue of GX(-/-) mice accumulated significantly more (20%) triglyceride compared to cells from GX(+/+) mice. Conversely, overexpression of GX sPLA(2), but not catalytically inactive GX sPLA(2), resulted in a significant 50% reduction in triglyceride accumulation in OP9 adipocytes. The induction of genes encoding adipogenic proteins (PPAR gamma, SREBP-1c, SCD1, and FAS) was also significantly blunted by 50-80% in OP9 cells overexpressing GX sPLA(2). Activation of the liver X receptor (LXR), a nuclear receptor known to up-regulate adipogenic gene expression, was suppressed in 3T3-L1 and OP9 cells when GX sPLA(2) was overexpressed. Thus, hydrolytic products generated by GX sPLA(2) negatively regulate adipogenesis, possibly by suppressing LXR activation.-Li, X., Shridas, P., Forrest, K., Bailey, W., Webb, N. R. Group X secretory phospholipase A(2) negatively regulates adipogenesis in murine models. FASEB J. 24, 4313-4324 (2010). www.fasebj.org

Objective— In vitro data indicate that human LDL modified by Group V secretory phospholipase A 2 (GV sPLA 2 ) is proatherogenic. Consistent with this, gain and loss of function studies demonstrated that GV sPLA 2 promotes atherosclerosis in LDLR −/− mice. The current study investigates whether GV sPLA 2 promotes atherosclerotic processes in apoE −/− mice. Methods and Results— LDL ( d =1.019 to 1.063) from apoE −/− and LDLR −/− mice fed chow or Western diet were hydrolyzed by GV sPLA 2 . Phosphatidylcholine on LDL from LDLR −/− mice fed either a chow or Western diet was hydrolyzed to a greater extent (61.1±0.4% and 45.3±4.6%) than the corresponding fractions from apoE −/− mice (41.7±3.6% and 39.4±1.2%). ApoE −/− LDL induced macrophage foam cell formation in vitro without modification by GV sPLA 2 , whereas hydrolysis of LDLR −/− LDL was a prerequisite for foam cell formation. In contrast to findings in LDLR −/− mice, GV sPLA 2 deficiency did not significantly reduce atherosclerosis in apoE −/− mice, although collagen content was significantly reduced in lesions of apoE −/− mice lacking GV sPLA 2 . Conclusions— The ability of GV sPLA 2 to promote atherosclerotic lipid deposition in apoE −/− and LDLR −/− mice may be related to its ability to increase the atherogenic potential of LDL from these mice as assessed in vitro.

Introduction: Of the 10 secretory phospholipase A 2 (sPLA 2 ) enzymes expressed in humans, Group X (GX) sPLA 2 is the most potent in hydrolyzing phospholipids on cell membranes. In addition to its lipolytic activity, GX sPLA 2 is also a high affinity ligand for the M-type sPLA 2 receptor. Although numerous studies have implicated GX sPLA 2 in important biological processes in vitro , data from studies in vivo are lacking. To elucidate the physiological functions of this enzyme we recently developed C57BL/6 mice with targeted deletion of the GX sPLA 2 gene. Results : Analysis by real time RT-PCR showed that GX sPLA 2 mRNA is widely distributed in mouse tissues, with highest expression in intestine, testes, brain, thymus, spleen, fat, lung, and heart. Unlike some of the other members of the sPLA 2 family, tissue expression of GX sPLA 2 was not highly upregulated in mice injected with lipopolysaccharide. However, GX sPLA 2 was induced almost 5-fold in retroperitoneal fat of mice fed a high-fat (60 kcal%) diet for 17 weeks.Targeted disruption of GX sPLA 2 resulted in increased body weight in 1.5 year-old mice fed a normal laboratory diet (male GX sPLA 2 +/+ mice: 35.8±0.4 g; male GX sPLA 2 −/− mice: 45.033.2 g; p<0.05). The increase in body weight was associated with significantly increased percent body fat (GX sPLA 2 +/+ mice: 16.0±1.0%; GX sPLA 2 −/− mice: 19.6±1%; p<0.05), increased adipocyte size (GX sPLA 2 +/+ mice: 1300±26 um 2 GX sPLA 2 −/− mice: 2700±78 um 2 ; p<0.001), and decreased fasting plasma triglyceride levels (GX sPLA 2 +/+ mice: 46.3±4.9 mg/dl; GX sPLA 2 −/− mice: 33.8±2.9 mg/dl; p<0.05) at 3 months of age. Food consumption, fasting blood glucose, and plasma total cholesterol levels were not significantly different between GX sPLA 2 −/− and GX sPLA 2 +/+ mice. Compared to 3 month old GX sPLA 2 +/+ mice, the relative expression of IL-6 and F4/80 mRNAs in adipose tissue of age-matched GX sPLA 2 −/− mice was increased 9- and 4-fold, respectively, suggesting that the increased adiposity in GX sPLA 2 −/− mice was associated with an increase in the inflammatory profile of adipose tissue Conclusions: Our data points to a previously unrecognized role for Group X sPLA 2 in the development of obesity and associated adipose tissue inflammation.

Few data exist on the modulation of cytokine receptor signaling by the actin or tubulin cytoskeleton. Therefore, we studied interleukin-2 receptor (IL-2R) signaling in phytohemagglutinine (PHA)-pretreated human T cells in the context of alterations in the cytoskeletal system induced by cytochalasin D (CyD), jasplaklinolide (Jas), taxol (Tax), or colchicine (Col). We found that changes in cytoskeletal tubulin polymerization altered the strength of several IL-2-triggered signals. Moreover, Tax-induced tubulin hyperpolymerization augmented the surface expression of the IL-2R β -chain and enhanced the association of the IL-2R γ -chain with cytoskeletal tubulin. The IL-2R β -chain, in turn, was constitutively associated with tubulin and, more weakly, actin. To exclude the possibility that these associations are artifacts caused by PHA, we confirmed them in T cells from TCR-transgenic DO11.10 mice stimulated with their nominal antigen. We conclude that altered polymerization of cytoskeletal components, especially tubulin, is accompanied by modulation of IL-2 signaling at the receptor level.

Serum amyloid A (SAA) is a major acute phase reactant whose expression can increase 1000-fold in response to inflammation. Clinical studies have suggested that serum levels of SAA may be associated with processes involved in the early phases of abdominal aortic aneurysm (AAA) formation. The most prominent characteristics of developing AAA are localized inflammation and enzymatic degradation of elastic lamellae and extracellular matrix proteins. Mounting evidence suggests that matrix metalloproteinases (MMPs) are the predominant proteinases in AAA. Given previous findings that SAA induces MMP secretion by a variety of cell types, we postulated that SAA plays a direct role in MMP activation and consequently, AAA formation. To test this hypothesis, we analyzed AAA induced in apoE -/mice by 2 week infusion of angiotensin II (1000 ng/kg/min). Immunohistochemical analysis using anti-SAA antibody revealed intense SAA staining in aneurismal tissue. We have investigated the effects of SAA on the production of MMP-13 and MMP-9 in the murine macrophage-like cell line J774 by real-time RT-PCR, Western Blot and zymography. MMP activity was stimulated by SAA in a dose-dependent manner. Lipoxin A4, a known endogenous ligand for the formyl peptide receptor-like 1/lipoxin A4 receptor (FPRL1/LXA4R), blocked SAA induction of MMPs. Pertussis toxin also significantly reduced SAA induction of MMPs in J774 cells. These results implicate SAA in the early phases of AAA formation through transcriptional upregulation of MMPs via a signaling pathway involving the G-protein-coupled FPRL1/LXA4R receptor.

Stimulated human T cells from healthy volunteers demonstrate attenuated early interleukin (IL)-2 receptor (R) signaling in the presence of daclizumab (Dac). Aiming to confirm that this ex-vivo effect of Dac is also observed in-vivo, we studied T cells from 3 kidney transplant recipients before and 2-3 weeks and 4-6 months after transplantation. We found by flow cytometry that T cells obtained pre-transplant and stimulated ex-vivo with phytohemeagglutinine upregulated the IL-2R alpha-(CD25) and beta-(CD122) chains as expected. Moreover, exogenous IL-2 induced characteristic tyrosine phosphorylation events detectable by immunoblotting in these cells. However, T cells studied post-transplant neither exhibited CD25 or -122 upregulation nor IL-2-induced tyrosine phosphorylation events, indicating broad, persistent suppression of the IL-2R signaling machinery which thus appears largely inaccessible for Dac in actual transplant recipients. We therefore conclude that the clinical efficacy of this agent may depend on additional mechanisms in-vivo other than those identified ex-vivo.

Although the immunomodulatory properties of statins are in part independent of their lipid-lowering effects, cholesterol is a major component of lipid rafts. We therefore studied the effects of atorvastatin (AS) on the raft enrichment of the interleukin-2 receptor (IL-2R) beta chain previously described by us and on early IL-2R signaling events in activated human T cells. We found that concomitant AS exposure during a 3-day stimulation with phytohemagglutinin (PHA) attenuates activation-associated events, such as the enhanced surface expression of the raft marker GM-1 and the induced expression of the activation marker CD25 (the IL-2R alpha chain). In contrast, brief AS treatment after PHA stimulation increased GM-1 surface expression and virtually abolished the selective raft enrichment of the IL-2R beta chain. Although this AS-associated increase in GM-1 expression resembled that seen in the presence of the raft-disrupting cholesterol chelator methyl-beta-cyclodextrin (MBCD), the two agents had contrasting effects on the tyrosine phosphorylation of the IL-2R beta chain by exogenous IL-2: MBCD essentially abolished this event, whereas AS tended to enhance it and shifted its occurrence out of rafts. We conclude that AS affects IL-2R signaling by altering the raft enrichment of the IL-2R beta chain and propose that this effect is one mechanism underlying the immunomodulatory properties of statins.

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.