Group IVA Cytosolic Phospholipase A2 Research Articles

Identification of MAP3K4 as a novel regulation factor of hepatic lipid metabolism in non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a metabolic disorder with abnormal lipid metabolism. The present study was to identify regulatory genes related to lipid droplets (LDs) abnormal accumulation in NAFLD.Methodstranscriptomic analysis and bioinformatics analysis (GEO database) were used to identify potential genes in abnormal lipid metabolism of NAFLD. A candidate gene MAP3K4 expression were detected by immunohistochemistry staining in NAFLD and controls. RNA interference and immunoblotting were used to verify the roles of MAP3K4 in the formation of hepatic LDs.ResultsA total of 134 candidate genes were screened, including 44 up-regulated genes and 90 down-regulated genes. 29 genes in the protein–protein interaction (PPI) were selected as hub genes, including MAP3K4. The expression levels of MAP3K4 were positively correlated with NAFLD activity score (r = 0.702, p = 0.002). Furthermore, we found a positive correlation of MAP3K4 expression with serum total cholesterol (r = 0.564, p = 0.023), uric acid levels (r = 0.520, p = 0.039), and body mass index (r = 0.574, p = 0.020). Downregulation of MAP3K4 decreased LDs accumulation in HepG2 cells and reduced the expression of CGI-58 and Plin-2 by imbibition of JNK and group IVA cytosolic phospholipase A2 (cPLA2) activation.ConclusionThe study revealed a number of regulatory genes related to hepatic lipid metabolism of NAFLD, and demonstrated that MAP3K4 played a pivotal role in the hepatic lipogenesis of NAFLD.

Segregated functions of two cytosolic phospholipase A2 isoforms (cPLA2α and cPLA2ε) in lipid mediator generation

Among the phospholipase A2 (PLA2) superfamily, group IVA cytosolic PLA2 (cPLA2α) is currently attracting much attention as a central regulator of arachidonic acid (AA) metabolism linked to eicosanoid biosynthesis. Following cell activation, cPLA2α selectively releases AA, a precursor of a variety of eicosanoids, from phospholipids in perinuclear membrane compartments. cPLA2α-null mice display various phenotypes that could be largely explained by reduced eicosanoid signaling. In contrast, group IVE cPLA2ε, another member of the cPLA2 family, acts as a Ca2+-dependent N-acyltransferase rather than a PLA2, thereby regulating the biosynthesis of N-acylethanolamines (NAEs), a unique class of lipid mediators with an anti-inflammatory effect. In response to Ca2+ signaling, cPLA2ε translocates to phosphatidylserine-rich organelle membranes in the endocytic/recycling pathway. In vivo, cPLA2ε is induced in keratinocytes of psoriatic skin, and its genetic deletion exacerbates psoriatic inflammation due to a marked reduction of NAE-related lipids. cPLA2ε also contributes to NAE generation in several if not all mouse tissues. Thus, the two members of the cPLA2 family, cPLA2α and cPLA2ε, catalyze distinct enzymatic reactions to mobilize distinct sets of lipid mediators, thereby differently regulating pathophysiological events in health and disease. Such segregation of the cPLA2α-eicosanoid and cPLA2ε-NAE pathways represents a new paradigm of research on PLA2s and lipid mediators.

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.

Choline Glycerophospholipid-Derived Prostaglandins Attenuate TNFα Gene Expression in Macrophages via a cPLA2α/COX-1 Pathway.

Macrophages are professional antigen presenting cells with intense phagocytic activity, strategically distributed in tissues and cavities. These cells are capable of responding to a wide variety of innate inflammatory stimuli, many of which are signaled by lipid mediators. The distribution of arachidonic acid (AA) among glycerophospholipids and its subsequent release and conversion into eicosanoids in response to inflammatory stimuli such as zymosan, constitutes one of the most studied models. In this work, we used liquid and/or gas chromatography coupled to mass spectrometry to study the changes in the levels of membrane glycerophospholipids of mouse peritoneal macrophages and the implication of group IVA cytosolic phospholipase A2 (cPLA2α) in the process. In the experimental model used, we observed that the acute response of macrophages to zymosan stimulation involves solely the cyclooxygenase-1 (COX-1), which mediates the rapid synthesis of prostaglandins E2 and I2. Using pharmacological inhibition and antisense inhibition approaches, we established that cPLA2α is the enzyme responsible for AA mobilization. Zymosan stimulation strongly induced the hydrolysis of AA-containing choline glycerophospholipids (PC) and a unique phosphatidylinositol (PI) species, while the ethanolamine-containing glycerophospholipids remained constant or slightly increased. Double-labeling experiments with 3H- and 14C-labeled arachidonate unambiguously demonstrated that PC is the major, if not the exclusive source, of AA for prostaglandin E2 production, while both PC and PI appeared to contribute to prostaglandin I2 synthesis. Importantly, in this work we also show that the COX-1-derived prostaglandins produced during the early steps of macrophage activation restrict tumor necrosis factor-α production. Collectively, these findings suggest new approaches and targets to the selective inhibition of lipid mediator production in response to fungal infection.

Lipidomics-based assays coupled with computational approaches can identify novel phospholipase A2 inhibitors.

Phospholipase A2 (PLA2) enzymes play a major role in many diseases including the inflammatory cascade and specific potent small molecule inhibitors could be useful in studying their physiological role as well as for the development of drugs. In order to discover novel small molecule inhibitor platforms for members of the PLA2 superfamily of enzymes, we have applied computational approaches to determine the binding mode of potent inhibitors specific for particular PLA2s to the screening of chemical libraries. This has including the U.S. National Institutes of Health (NIH) National Cancer Institute (NCI) Diversity Set V and the ChemBridge commercial compound libraries. We have then subjected identified inhibitor structures to recently developed lipidomics based screening assays to determine the XI(50) and specificity of the identified compounds for specific PLA2s. Herein we review this approach and report the identity of initial hits for both the Group IVA cytosolic PLA2 and the Group VIA calcium-independent PLA2 that are worthy of further structural modification to develop novel platforms for inhibitor development.

CPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

DMSO-induced drastic changes in cellular processes and epigenetic landscape in vitro

Classical FcεRI-induced mast cell (MC) activation causes synthesis of arachidonic acid (AA)–derived eicosanoids (leukotriene [LT] C4, prostaglandin [PG] D2, and thromboxane A2), which mediate vascular leak, bronchoconstriction, and effector cell chemotaxis. Little is known about the significance and regulation of eicosanoid generation in response to nonclassical MC activation mechanisms.We sought to determine the regulation and significance of MC-derived eicosanoids synthesized in response to IL-33, a cytokine critical to innate type 2 immunity.We used an ex vivo model of mouse bone marrow–derived mast cells and an IL-33–dependent in vivo model of aspirin-exacerbated respiratory disease (AERD).IL-33 potently liberates AA and elicits LTC4, PGD2, and thromboxane A2 production by bone marrow–derived mast cells. Unexpectedly, the constitutive function of COX-1 is required for IL-33 to activate group IVa cytosolic phospholipase A2 with consequent AA release for synthesis of all eicosanoids, including CysLTs. In contrast, COX-1 was dispensable for FcεRI-driven CysLT production. Inhibition of COX-1 prevented IL-33–induced phosphorylation of extracellular signal-related kinase, an upstream effector of cytosolic phospholipase A2, which was restored by exogenous PGH2, implying that the effects of COX-1 required its catalytic function. Administration of a COX-1–selective antagonist to mice completely prevented the generation of both PGD2 and LTC4 in a model of AERD in which MC activation is IL-33 driven.MC-intrinsic COX-1 amplifies IL-33–induced activation in the setting of innate type 2 immunity and might help explain the phenomenon of therapeutic desensitization to aspirin by nonselective COX inhibitors in patients with AERD.

Cytosolic phospholipase A2 contributes to innate immune defense against Candida albicans lung infection.

BackgroundThe lung is exposed to airborne fungal spores, and fungi that colonize the oral cavity such as Candida albicans, but does not develop disease to opportunistic fungal pathogens unless the immune system is compromised. The Group IVA cytosolic phospholipase A2 (cPLA2α) is activated in response to Candida albicans infection resulting in the release of arachidonic acid for eicosanoid production. Although eicosanoids such as prostaglandins and leukotrienes modulate inflammation and immune responses, the role of cPLA2α and eicosanoids in regulating C. albicans lung infection is not understood.MethodsThe responses of cPLA2α+/+ and cPLA2α−/− Balb/c mice to intratracheal instillation of C. albicans were compared. After challenge, we evaluated weight loss, organ fungal burden, and the recruitment of cells and the levels of cytokines and eicosanoids in bronchoalveolar lavage fluid. The ability of macrophages and neutrophils from cPLA2α+/+ and cPLA2α−/− mice to recognize and kill C. albicans was also compared.ResultsAfter C. albicans instillation, cPLA2α+/+ mice recovered a modest weight loss by 48 h and completely cleared fungi from the lung by 12 h with no dissemination to the kidneys. In cPLA2α−/− mice, weight loss continued for 72 h, C. albicans was not completely cleared from the lung and disseminated to the kidneys. cPLA2α−/− mice exhibited greater signs of inflammation including higher neutrophil influx, and elevated levels of albumin and pro-inflammatory cytokines/chemokines (IL1α, IL1β, TNFα, IL6, CSF2, CXCL1, CCL20) in bronchoalveolar lavage fluid. The amounts of cysteinyl leukotrienes, thromboxane B2 and prostaglandin E2 were significantly lower in bronchoalveolar lavage fluid from C. albicans-infected cPLA2α−/− mice compared to cPLA2α+/+ mice. Alveolar macrophages and neutrophils from uninfected cPLA2α−/− mice exhibited less killing of C. albicans in vitro than cells from cPLA2α+/+ mice. In addition alveolar macrophages from cPLA2α−/− mice isolated 6 h after instillation of GFP-C. albicans contained fewer internalized fungi than cPLA2α+/+ macrophages.ConclusionsThe results demonstrate that cPLA2α contributes to immune surveillance and host defense in the lung to prevent infection by the commensal fungus C. albicans and to dampen inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0165-9) contains supplementary material, which is available to authorized users.

Anti-vascular effects of the cytosolic phospholipase A2 inhibitor AVX235 in a patient-derived basal-like breast cancer model.

BackgroundGroup IVA cytosolic phospholipase A2 (cPLA2α) plays an important role in tumorigenesis and angiogenesis. It is overexpressed in basal-like breast cancer (BLBC), which is aggressive and usually triple-negative, making it unresponsive to current targeted therapies. Here, we evaluated the anti-angiogenic effects of a specific cPLA2α inhibitor, AVX235, in a patient-derived triple-negative BLBC model.MethodsMice bearing orthotopic xenografts received i.p. injections of AVX235 or DMSO vehicle daily for 1 week and then every other day for up to 19 days. Six treated and six control mice were terminated after 2 days of treatment, and the tumors excised for high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and prostaglandin E2 (PGE2) enzyme immunoassay (EIA) analysis. A 1-week imaging study was performed on a separate cohort of mice. Longitudinal dynamic contrast enhanced (DCE)-MRI was performed before, after 4 days, and after 1 week of treatment. The mice were then perfused with a radiopaque vascular casting agent, and the tumors excised for micro-CT angiography. Subsequently, tumors were sectioned and stained with lectin and for Ki67 or α-smooth muscle actin to quantify endothelial cell proliferation and vessel maturity, respectively. Partial least squares discriminant analysis was performed on the multivariate HR MAS MRS data, and non-parametric univariate analyses using Mann–Whitney U tests (α = 0.05) were performed on all other data.ResultsGlycerophosphocholine and PGE2 levels, measured by HR MAS MRS and EIA, respectively, were lower in treated tumors after 2 days of treatment. These molecular changes are expected downstream effects of cPLA2α inhibition and were followed by significant tumor growth inhibition after 8 days of treatment. DCE-MRI revealed that AVX235 treatment caused a decrease in tumor perfusion. Concordantly, micro-CT angiography showed that vessel volume fraction, density, and caliber were reduced in treated tumors. Moreover, histology showed decreased endothelial cell proliferation and fewer immature vessels in treated tumors.ConclusionsThese results demonstrate that cPLA2α inhibition with AVX235 resulted in decreased vascularization and perfusion and subsequent inhibition of tumor growth. Thus, cPLA2α inhibition may be a potential new therapeutic option for triple-negative basal-like breast cancer.

Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages

In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

Group IVA Cytosolic Phospholipase A2 Regulates the G2-to-M Transition by Modulating the Activity of Tumor Suppressor SIRT2.

The G2-to-M transition (or prophase) checkpoint of the cell cycle is a critical regulator of mitotic entry. SIRT2, a tumor suppressor gene, contributes to the control of this checkpoint by blocking mitotic entry under cellular stress. However, the mechanism underlying both SIRT2 activation and regulation of the G2-to-M transition remains largely unknown. Here, we report the formation of a multiprotein complex at the G2-to-M transition in vitro and in vivo. Group IVA cytosolic phospholipase A2 (cPLA2α) acts as a bridge in this complex to promote binding of SIRT2 to cyclin A-Cdk2. Cyclin A-Cdk2 then phosphorylates SIRT2 at Ser331. This phosphorylation reduces SIRT2 catalytic activity and its binding affinity to centrosomes and mitotic spindles, promoting G2-to-M transition. We show that the inhibitory effect of cPLA2α on SIRT2 activity impacts various cellular processes, including cellular levels of histone H4 acetylated at K16 (Ac-H4K16) and Ac-α-tubulin. This regulatory effect of cPLA2α on SIRT2 defines a novel function of cPLA2α independent of its phospholipase activity and may have implications for the impact of SIRT2-related effects on tumorigenesis and age-related diseases.

Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells.

Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation.

Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Inhibition of group IVA cytosolic phospholipase A2 by thiazolyl ketones in vitro, ex vivo, and in vivo.

Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA2, exhibiting an XI(50) value of 0.011 mole fraction in a mixed micelle assay and an IC50 of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC50 value of 0.6 μM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE2 levels.

Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s Act on Distinct Phospholipid Pools in Zymosan-Stimulated Mouse Peritoneal Macrophages

Phospholipase A2s generate lipid mediators that constitute an important component of the integrated response of macrophages to stimuli of the innate immune response. Because these cells contain multiple phospholipase A2 forms, the challenge is to elucidate the roles that each of these forms plays in regulating normal cellular processes and in disease pathogenesis. A major issue is to precisely determine the phospholipid substrates that these enzymes use for generating lipid mediators. There is compelling evidence that group IVA cytosolic phospholipase A2 (cPLA2α) targets arachidonic acid-containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca(2+)-independent group VIA phospholipase A2 (iPLA2β) has not been clearly defined. We applied mass spectrometry-based lipid profiling to study the substrate specificities of these two enzymes during inflammatory activation of macrophages with zymosan. Using selective inhibitors, we find that, contrary to cPLA2α, iPLA2β spares arachidonate-containing phospholipids and hydrolyzes only those that do not contain arachidonate. Analyses of the lysophospholipids generated during activation reveal that one of the major species produced, palmitoyl-glycerophosphocholine, is generated by iPLA2β, with minimal or no involvement of cPLA2α. The other major species produced, stearoyl-glycerophosphocholine, is generated primarily by cPLA2α. Collectively, these findings suggest that cPLA2α and iPLA2β act on different phospholipids during zymosan stimulation of macrophages and that iPLA2β shows a hitherto unrecognized preference for choline phospholipids containing palmitic acid at the sn-1 position that could be exploited for the design of selective inhibitors of this enzyme with therapeutic potential.

Cytosolic Phospholipase A2 Regulates TNF-Induced Production of Joint Destructive Effectors in Synoviocytes

IntroductionRheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target.MethodsThe involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes (synovial fibroblast-like cells) was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively. ResultsInhibitors of cPLA2α enzyme activity (AVX002, ATK) significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes. ConclusionscPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.

Cytosolic Phospholipase A2α and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α+/+ macrophages. Representative genes expressed lower in cPLA2α+/+ macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5' distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1β. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPβ with the distal novel enhancer. The association of C/EBPβ with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.

The molecular basis of ceramide-1-phosphate recognition by C2 domains.

Group IVA cytosolic phospholipase A₂ (cPLA₂α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain β-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA₂α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg⁵⁹, Arg⁶¹, and His⁶² (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition.