5-Lipoxygenase-activating protein homodimer in human neutrophils: evidence for a role in leukotriene biosynthesis.
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ABSTRACT:
SUBMITTER: Plante H
PROVIDER: S-EPMC1383679 | biostudies-other | 2006 Jan
REPOSITORIES: biostudies-other
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Project description:The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.
| S-EPMC4099325 | biostudies-literature
Project description:5-Lipoxygenase-activating protein (FLAP) is a regulator of cellular leukotriene biosynthesis, which governs the transfer of arachidonic acid (AA) to 5-lipoxygenase for efficient metabolism. Here, the synthesis and FLAP-antagonistic potential of fast synthetically accessible 1,2,4-triazole derivatives based on a previously discovered virtual screening hit compound is described. Our findings reveal that simple structural variations on 4,5-diaryl moieties and the 3-thioether side chain of the 1,2,4-triazole scaffold markedly influence the inhibitory potential, highlighting the significant chemical features necessary for FLAP antagonism. Comprehensive metabololipidomics analysis in activated FLAP-expressing human innate immune cells and human whole blood showed that the most potent analogue 6x selectively suppressed leukotriene B4 formation evoked by bacterial exotoxins without affecting other branches of the AA pathway. Taken together, the 1,2,4-triazole scaffold is a novel chemical platform for the development of more potent FLAP antagonists, which warrants further exploration for their potential as a new class of anti-inflammatory agents.
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Project description:Background and purposeLeukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis.Experimental approachWe analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis.Key resultsBRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels.Conclusions and implicationsBRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development.
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Project description:5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.
| S-EPMC4128154 | biostudies-other
Project description:Individuals with sickle cell disease (SCD) have increased inflammation, a high incidence of airway hyperreactivity (AH), and increased circulating leukotrienes (LT). We show that expression of 5-lipoxygenase and 5-lipoxygenase activating protein (FLAP), key catalytic molecules in the LT pathway, were significantly increased in peripheral blood mononuclear cells (MNCs) in patients with SCD, compared with healthy controls. Placenta growth factor (PlGF), elaborated from erythroid cells, activated MNC and THP-1 monocytic cells to induce LT production. PlGF-mediated increased FLAP mRNA expression occurred via activation of phosphoinositide-3 (PI-3) kinase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and hypoxia inducible factor-1alpha (HIF-1alpha). HIF-1alpha small interfering RNA (siRNA) reduced PlGF-induced FLAP expression. FLAP promoter-driven luciferase constructs demonstrated that PlGF-mediated luciferase induction was abrogated upon mutation of HIF-1alpha response element (HRE), but not the nuclear factor-kappaB (NF-kappaB) site in the FLAP promoter; a finding confirmed by chromatin immunoprecipitation (ChIP) analysis. PlGF also increased HIF-1alpha binding to the HRE in the FLAP promoter. Therefore, it is likely that the intrinsically elevated levels of PlGF in SCD subjects contribute to increased LT, which in turn, mediate both inflammation and AH. Herein, we identify a mechanism of increased LT in SCD and show HIF-1alpha as a hypoxia-independent target of PlGF. These studies provide new avenues to ameliorate these complications.
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Project description:Arachidonic acid (AA) can be converted into prostaglandins (PGs) or leukotrienes (LTs) by the enzymatic actions of cyclooxygenases (COX-1 and COX-2) or 5-lipoxygenase (5-LO), respectively. PGs and LTs are lipid signaling molecules that have been implicated in various diseases, including multiple cancers. 5-LO and its activating protein (FLAP) work together in the first two conversion steps of LT production. Previous work has suggested a role for LTs in cancer development and progression. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression post-transcriptionally, and have previously been shown to be involved in cancer. Here, we show that high FLAP expression is associated with lower overall survival in lung adenocarcinoma patients, and FLAP protein is overexpressed in lung cancer cells compared to normal lung cells. Our lab has previously shown that miR-146a regulates COX-2 in lung cancer cells, and this miRNA is also predicted to target FLAP. Transient and stable transfections of miR-146a repress endogenous FLAP expression in lung cancer cells, and reporter assays show this regulation occurs through a direct interaction between the FLAP 3' untranslated region (UTR) and miR-146a. Restoration of miR-146a also results in decreased cancer cell Leukotriene B4 (LTB4) production. Additionally, methylation analysis indicates the miR-146a promoter is hypermethylated in lung cancer cell lines. Taken together, this study and previous work from our lab suggest miR-146a is an endogenous dual inhibitor of AA metabolism in lung cancer cells by regulating both PG and LT production through direct targeting of the COX-2 and FLAP 3' UTRs.
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Project description:Leukotrienes are inflammatory mediators that actively participate in the inflammatory response and host defense against pathogens. However, leukotrienes also participate in chronic inflammatory diseases. 5-lipoxygenase is a key enzyme in the biosynthesis of leukotrienes and is thus a validated therapeutic target. As of today, zileuton remains the only clinically approved 5-lipoxygenase inhibitor; however, its use has been limited due to severe side effects in some patients. Hence, the search for a better 5-lipoxygenase inhibitor continues. In this study, we investigated structural analogues of caffeic acid phenethyl ester, a naturally-occurring 5-lipoxygenase inhibitor, in an attempt to enhance the inhibitory activity against 5-lipoxygenase and determine structure-activity relationships. These compounds were investigated for their ability to attenuate the biosynthesis of leukotrienes. Compounds 13 and 19, phenpropyl and diphenylethyl esters, exhibited significantly enhanced inhibitory activity when compared to the reference molecules caffeic acid phenethyl ester and zileuton.
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Project description:Leukotrienes are among the most potent mediators of inflammation, and inhibition of their biosynthesis, is becoming increasingly important in the treatment of many pathologies. In this work, we demonstrated that preincubation of human neutrophils with the mitochondria targeted antioxidant SkQ1 (100 nM) strongly inhibits leukotriene synthesis induced by three different stimuli: the Ca2+ ionophore A23187, the chemotactic formyl-peptide fMLP in combination with cytocholasin B, and opsonized zymosan. The SkQ1 analogue lacking the antioxidant quinone moiety (C12TPP) was ineffective, suggesting that mitochondrial production of reactive oxygen species (ROS) is critical for activating of leukotriene synthesis in human neutrophils. The uncoupler of oxidative phosphorylation FCCP also inhibits leukotriene synthesis, indicating that a high membrane potential is a prerequisite for stimulating leukotriene synthesis in neutrophils. Our data show that activation of mitogen-activated protein kinases p38 and ERK1/2, which is important for leukotriene synthesis in neutrophils is a target for SkQ1: 1) the selective p38 inhibitor SB203580 inhibited fMLP-induced leukotriene synthesis, while the ERK1/2 activation inhibitor U0126 suppressed leukotriene synthesis induced by any of the three stimuli; 2) SkQ1 effectively prevents p38 and ERK1/2 activation (accumulation of phosphorylated forms) induced by all three stimuli. This is the first study pointing to the involvement of mitochondrial reactive oxygen species in the activation of leukotriene synthesis in human neutrophils. The use of mitochondria-targeted antioxidants can be considered as a promising strategy for inhibiting leukotriene synthesis and treating various inflammatory pathologies.
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Project description:Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.
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Project description:Lipoxygenases (LOX) transform arachidonic acid (AA, C20:4) and docosahexaenoic acid (DHA, C22:6) into bioactive lipid mediators (LMs) that comprise not only pro-inflammatory leukotrienes (LTs) but also the specialized pro-resolving mediators (SPMs) that promote inflammation resolution and tissue regeneration. The 5-LOX-activating protein (FLAP) is known to provide AA as a substrate to 5-LOX for generating LTs, such as LTB4, a potent chemoattractant and activator of phagocytes. Notably, 5-LOX is also involved in the biosynthesis of certain SPMs, namely, lipoxins and D-resolvins, implying a role of FLAP in SPM formation. FLAP antagonists have been intensively developed as LT biosynthesis inhibitors, but how they impact SPM formation is a matter of debate. Here, we show that FLAP antagonism suppresses the conversion of AA by 5-LOX to LT and lipoxins, while the conversion of DHA to SPM is unaffected. Screening of multiple prominent FLAP antagonists for their effects on LM formation in human M1- and M2-monocyte-derived macrophages by comprehensive LM profiling showed that all nine compounds reduced the production of 5-LOX-derived LTs but increased the formation of SPMs from DHA, e.g., resolvin D5. Some FLAP antagonists, especially those that contain an indole or benzimidazole moiety, even elicited SPM formation in resting M2-monocyte-derived macrophages. Intriguingly, in coincubations of human neutrophils and platelets that produce substantial AA-derived lipoxin and DHA-derived RvD5, FLAP antagonism abolished lipoxin formation, but resolvin D5 levels remained unaffected. Conclusively, antagonism of FLAP suppresses the conversion of AA by 5-LOX to LTs and lipoxins but not the conversion of DHA by 5-LOX to SPM, which should be taken into account for the development of such compounds as anti-inflammatory drugs.
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