Arachidonic Acid Release Research Articles

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils

Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. Arabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. Weused confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils. These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulatedby proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications forthe treatment of eosinophilic disorders such as asthma.

Cytosolic Phospholipase A2α Is Essential for Renal Dysfunction and End-Organ Damage Associated With Angiotensin II-Induced Hypertension.

The kidney plays an important role in regulating blood pressure (BP). cPLA2α in the kidney is activated by various agents including angiotensin II (Ang II) and selectively releases arachidonic acid (AA) from tissue lipids, generating pro- and antihypertensive eicosanoids. Since activation of cPLA2α is the rate-limiting step in AA release, this study was conducted to determine its contribution to renal dysfunction and end-organ damage associated with Ang II-induced hypertension. cPLA2α(+/+) and cPLA2α(-/-) mice were infused with Ang II (700 ng/ kg/min) or its vehicle for 13 days. Mice were placed in metabolic cages to monitor their food and water intake, and urine was collected and its volume was measured. Doppler imaging was performed to assess renal hemodynamics. On the 13th day of Ang II infusion, mice were sacrificed and their tissues and blood collected for further analysis. Ang II increased renal vascular resistance, water intake, and urine output and Na(+) excretion, decreased urine osmolality, and produced proteinuria in cPLA2α(+/+) mice. Ang II also caused accumulation of F4/80(+) macrophages and CD3(+) T cells and renal fibrosis, and increased oxidative stress in the kidneys of cPLA2α(+/+) mice. All these effects of Ang II were minimized in cPLA2α(-/-) mice. cPLA2α contributes to renal dysfunction, inflammation, and end-organ damage, most likely via the action of pro-hypertensive eicosanoids and increased oxidative stress associated with Ang II-induced hypertension. Thus, cPLA2α could serve as a potential therapeutic target for treating renal dysfunction and end-organ damage in hypertension.

Abstract 483: Non-redundant and Opposing Roles of Group X and Group V Secretory Phospholipase A2s on Pancreatic Beta-cell Function

Background: Group X and group V secretory phospholipase A2s (GX and GV sPLA2s) potently release arachidonic acid (AA) from the plasma membrane of intact cells. AA is an activator of glucose-stimulated insulin secretion (GSIS) by β-islet cells. However, the AA metabolite prostaglandin E2 (PGE2) is a known inhibitor of GSIS. Both GX and GV sPLA2s are expressed in mouse pancreatic islet cells. We previously demonstrated that GX sPLA2 negatively regulates GSIS by a PGE2-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. Methods and Results: GSIS was measured in pancreatic islet cells isolated from WT and GV sPLA2-deficient (GV KO) mice. To complement these studies, GSIS was also assessed in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. In marked contrast to our findings in GX KO mice, GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice. Similarly, there was a significant decrease in GSIS in MIN6 cells with siRNA-mediated GV sPLA2 suppression. Consistent with these findings, MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. As expected, the amount of AA released into the media by MIN6-GV cells was significantly increased compared to control MIN6 cells. However, unlike MIN6 cells overexpressing GX sPLA2, MIN6-GV cells did not exhibit enhanced PGE2 production or decreased cAMP content compared to control MIN6 cells, despite similar amounts of sPLA2 activity produced by the two cell lines. Conclusions: We conclude that GX and GV sPLA2s play opposing and non-redundant roles in pancreatic β-cell function. Whereas GV sPLA2 activates GSIS, GX sPLA2 suppresses this process. This functional difference appears to be due to the extent to which AA generated by the respective sPLA2’s is coupled to PGE2 production.

Sphingomyelinase D/ceramide 1-phosphate in cell survival and inflammation.

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.

Inhibitory effect of acteoside on melittin-induced catecholamine exocytosis through inhibition of Ca(2+)-dependent phospholipase A2 and extracellular Ca(2+) influx in PC12 cells.

To investigate the inhibitory effect of acteoside on the process of exocytosis induced by melittin, we measured Ca(2+) mobilization, arachidonic acid (AA) release and catecholamine exocytosis in PC12 chromaffin cells. Melittin significantly increased the intracellular Ca(2+) mobilization via receptor-operated calcium channel but not the intracellular Ca(2+) release. It caused AA release via activation of Ca(2+)-dependent phospholipase A2 (PLA2) and catecholamine secretion in a dose-dependent manner. Acteoside dose-dependently inhibited the release of AA and intracellular Ca(2+) mobilization induced by melittin. Acteoside reduced the catecholamine release and raised the amount of intracellular chromogranin A which is co-released with catecholamine from melittin-stimulated PC12 cells. Taken together, our results suggest that acteoside could suppress the exocytosis via inhibition of Ca(2+)-dependent PLA2 and extracellular Ca(2+) influx in PC12 cells stimulated by melittin.

Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs) as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

Metabolic plasticity in resting and thrombin activated platelets.

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.

A new era of secreted phospholipase A2

A new era of secreted phospholipase A₂

Anti-inflammatory Activity of Magnesium Isoglycyrrhizinate Through Inhibition of Phospholipase A2/Arachidonic Acid Pathway.

Glycyrrhiza glabra (licorice) has been known to possess various pharmacological properties including anti-inflammatory, antioxidants, antiviral, and hepatoprotective activities. Magnesium isoglycyrrhizinate (MgIG), a magnesium salt of 18-α glycyrrhizic acid stereoisomer, is clinically used for the treatment of inflammatory liver diseases. However, the mechanism by which MgIG exerts its anti-inflammatory effects remains unknown. In the present study, we investigated the inhibitory potential of MgIG in phospholipase A2 (PLA2)/arachidonic acid (AA) pathway and release of the pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Results revealed that MgIG suppressed LPS-induced activation of PLA2 and production of AA metabolites such as prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane 2 (TXB2), and leukotrienes (LTB4) in macrophages. Furthermore, LPS-induced AA-metabolizing enzymes including COX-2, COX-1, 5-LOX, TXB synthase, and PGI2 synthase were significantly inhibited by MgIG. Taken together, our data suggest that modulation of cyclooxygenase (COXs) and 5-lipoxygenase (LOX) pathways in AA metabolism could be a novel mechanism for the anti-inflammatory effects of MgIG.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca2+-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10–100nM in cells from females and at 1μM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca2+ was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

Cytosolic phospholipase A2α is critical for angiotensin II-induced hypertension and associated cardiovascular pathophysiology.

Angiotensin II activates cytosolic phospholipase A(2)α (cPLA2α) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α(+/+)) and cPLA2α(-/-) mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α(+/+) mice was abolished in cPLA2α(-/-) mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α(+/+) mice. Angiotensin II in cPLA2α(+/+) mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2α(+/+) mice; these changes were diminished in cPLA2α(-/-) mice. Angiotensin II also increased cardiac infiltration of F4/80(+) macrophages and CD3(+) T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58(IPK), and CHOP in cPLA2α(+/+) but not cPLA2α(-/-) mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α(+/+) but not cPLA2α(-/-) mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc.

BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx mori.

PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemocytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa protein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24h followed by post-48 and post-72h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva implied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.

Mechanisms for proteinase-activated receptor 1-triggered prostaglandin E2 generation in mouse osteoblastic MC3T3-E1 cells.

We analyzed signaling mechanisms for prostaglandin E2 (PGE2) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE2 release, an effect suppressed by inhibitors of COX-1, COX-2, iPLA2, cPLA2, MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-κB. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1-triggered PGE2 release. Exogenously applied PGE2 facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE2 production in MC3T3-E1 cells appears to involve iPLA2 and cPLA2 for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE2 formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE2 system in the early stage of the bone healing.

The Site of Arachidonic Acid Release Drives Calcium Dynamics in β-Cells

Insulin is the blood glucose-lowering hormone essential for glucose homeostasis. β-Cells secrete insulin in pulses in response to glucose. Loss of the oscillatory nature of insulin secretion is associated with the development of type II diabetes. Calcium is the most important trigger of insulin release even though the exact mechanism of insulin secretion regulation by calcium is elusive. Arachidonic acid (AA) is an essential signalling molecule involved in regulation of physiological functions of many cell types. In insulin secreting pancreatic β-cells AA was shown to modulate calcium levels and as result to trigger insulin secretion. However, the exact interplay between AA signalling and insulin secretion is still a matter of speculation. In the present work we investigated the difference of AA action at the internal membranes (IMs) and the plasma membrane (PM) of β-cells. To perform this study, we developed a caging group which permits localization, visualization and quantitative photorelease of AA exclusively on the PM of living cells. We applied it in combination with a previously reported caging group used to release AA on the IMs. We found that the release of AA on the PM and the IMs leads to a significantly different modulation of intracellular calcium dynamics. Uncaging of AA on the PM induces calcium oscillations in non-oscillating cells and increases the duration of calcium transients in oscillating cells, leading to overall higher calcium levels. Release of AA on the IM results in transiently or permanently diminished calcium oscillations in β-cells and lower average calcium levels. We attribute the observed effects to direct action of AA on Arachidonic acid Regulated Calcium (ARC) channels localized on the PM and potentially a negative feedback mechanism triggered by higher levels of AA at the internal membranes.

Molecular characterisation of group IVA (cytosolic) phospholipase A2 in murine osteoblastic MC3T3-E1 cells.

Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.

PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation.

Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.

The role of PGE2 in intestinal inflammation and tumorigenesis

Release of the free fatty acid arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) and its subsequent metabolism by the cyclooxygenase and lipoxygenase enzymes produces a broad panel of eicosanoids including prostaglandins (PGs). This study sought to investigate the roles of these mediators in experimental models of inflammation and inflammation-associated intestinal tumorigenesis. Using the dextran sodium sulfate (DSS) model of experimental colitis, we first investigated how a global reduction in eicosanoid production would impact intestinal injury by utilizing cPLA2 knockout mice. cPLA2 deletion enhanced colonic injury, reflected by increased mucosal ulceration and pro-inflammatory cytokine expression. Increased disease severity was associated with a significant reduction in the levels of several eicosanoid metabolites, including PGE2. We further assessed the precise role of PGE2 synthesis on mucosal injury and repair by utilizing mice with a genetic deletion of microsomal PGE synthase-1 (mPGES-1), the terminal synthase in the formation of inducible PGE2. DSS exposure caused more extensive acute injury as well as impaired recovery in knockout mice compared to wild-type littermates. Increased intestinal damage was associated with both reduced PGE2 levels as well as altered levels of other eicosanoids including PGD2. To determine whether this metabolic redirection impacted inflammation-associated intestinal tumorigenesis, ApcMin/+ and ApcMin/+:mPGES-1−/− mice were exposed to DSS. DSS administration caused a reduction in the number of intestinal polyps only in ApcMin/+:mPGES-1−/− mice. These results demonstrate the importance of the balance of prostaglandins produced in the intestinal tract for maintaining intestinal homeostasis and impacting tumor development.

Calcium/calmodulin-dependent protein kinase II regulates cyclooxygenase-2 expression and prostaglandin E2 production by activating cAMP-response element-binding protein in rat peritoneal macrophages.

Prostaglandin E2 (PGE2 ) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE2 production is regulated by arachidonic acid release and cyclooxygenase-2 (COX-2) expression. In the present study, we found that COX-2 expression can be achieved by activating Ca(2+) /Calmodulin (CaM)-dependent protein kinase II (CaMKII) and cAMP-response element-binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca(2+) ]i ), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX-2 expression and PGE2 production in a chronological order. These results suggested that Ca(2+) /CaM-dependent CaMKII plays an important role in mediating COX-2 expression and PGE2 production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE2 production, which plays an essential role in inflammation and cancers.