Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogen-activated protein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3.
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Interleukin-13 (IL-13) is a cytokine secreted by Th2 lymphocytes that is capable of inducing expression of 15-lipoxygenase (15-LO) in primary human monocytes. We recently demonstrated that induction of 15-LO requires the activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6. Since IL-13-induced 15-LO expression was inhibited by H7 (a serine-threonine kinase inhibitor), we predicted that Stat serine phosphorylation may also be crucial for 15-LO expression. In this study, we present evidence indicating that IL-13-induced 15-LO mRNA expression was detectable as early as 1 h by real-time reverse transcription-PCR. We found that IL-13 induced a time-dependent serine phosphorylation of both Stat1 and Stat3, detectable at 15 min after IL-13 treatment. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) was detected in a time-dependent fashion, with peak phosphorylation at 15 min after IL-13 treatment. SB202190, a p38 MAPK-specific inhibitor, markedly inhibited IL-13-induced Stat1 and Stat3 serine phosphorylation as well as DNA binding. Furthermore, treatment of cells with Stat1 or Stat3 decoys significantly impaired IL-13-induced 15-LO expression. Taken together, our results provide the first evidence that IL-13 induces p38 MAPK phosphorylation/activation, which regulates Stat1 and Stat3 serine 727 phosphorylation. Both of these events are important steps in IL-13-induced 15-LO expression in human monocytes. (+info)
The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells.
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Diminished apoptosis, a critical event in tumorigenesis, is linked to down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer cells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primary product of 15-LOX-1 metabolism of linoleic acid, restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for linoleic and arachidonic acid metabolites. PPAR-delta promotes colonic tumorigenesis. NSAIDs suppress PPAR-delta activity in colon cancer cells. The mechanistic relationship between 15-LOX-1 and PPAR-delta was previously unknown. Our current study shows that (i) 13-S-HODE binds to PPAR-delta, decreases PPAR-delta activation, and down-regulates PPAR-delta expression in colorectal cancer cells; (ii) the induction of 15-LOX-1 expression is a critical step in NSAID down-regulation of PPAR-delta and the resultant induction of apoptosis; and (iii) PPAR-delta is an important signaling receptor for 13-S-HODE-induced apoptosis. The in vivo relevance of these mechanistic findings was demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our findings indicate that the down-regulation of PPAR-delta by 15-LOX-1 through 13-S-HODE is an apoptotic signaling pathway that is activated by NSAIDs. (+info)
Interleukin-13 upregulates vasodilatory 15-lipoxygenase eicosanoids in rabbit aorta.
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OBJECTIVE: Vasorelaxation of rabbit aorta is mediated by factors released from the vascular endothelium. In the aortic endothelium, arachidonic acid (AA) is metabolized via the 15-lipoxygenase pathway to the vasodilatory compounds 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA). Interleukin-13 (IL-13) increases 15-lipoxygenase expression and activity in several types of cells. We tested the hypothesis that IL-13 upregulates the 15-lipoxygenase pathway in rabbit aorta by inducing 15-lipoxygenase expression, thus increasing vascular relaxation mediated by THETA and HEETA. METHODS AND RESULTS: Aorta rings and cultured endothelial cells were treated with IL-13, and 15-lipoxygenase expression was analyzed by reverse transcription-polymerase chain reaction and immunoblotting. 15-Lipoxygenase expression was increased by IL-13 in a concentration- and time-dependent manner. Aortic rings were incubated with [14C]AA, and the metabolites were extracted and resolved by high-performance liquid chromatography. IL-13 treatment increased the production of 15-hydroxyeicosatetraenoic acid, HEETA, and THETA. Indomethacin-resistant vasorelaxation to AA was significantly greater in IL-13-treated vessels than in controls. The relaxation responses to sodium nitroprusside were not altered by IL-13 treatment. CONCLUSIONS: These data indicate that in the vascular endothelium, IL-13 induces the expression of 15-lipoxygenase and increases the production of the vasodilatory eicosanoids HEETA and THETA. (+info)
Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase.
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Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages. (+info)
Investigations into calcium-dependent membrane association of 15-lipoxygenase-1. Mechanistic roles of surface-exposed hydrophobic amino acids and calcium.
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Among mammalian lipoxygenases the 15-lipoxygenase-1 is somewhat special because of its capability of oxygenating complex lipid-protein assemblies (biomembranes, lipoproteins) and previous investigations have implicated calcium in enzyme/membrane interaction. We investigated the mechanism of calcium-dependent membrane association and obtained the following results. (i) Membrane binding of 15-lipoxygenase-1 involves electrostatic forces as well as hydrophobic interactions of solvent-exposed apolar amino acids (Tyr(15), Phe(70), Leu(71), Trp(181), and Leu(195)) with the hydrophobic core of membrane phospholipids. These sequence determinants of membrane association are clustered at the membrane contact plane of the enzyme that also involves the entrance to the substrate binding pocket. Site-directed mutagenesis of these determinants to negatively charged residues strongly impaired membrane binding. (ii) Calcium at micromolar concentrations (5-50 microM) is required for efficient membrane binding. For direct 15-lipoxygenase/calcium interaction a dissociation constant of 2-5 x 10(-4) m was determined (low affinity binding) and we failed to detect high affinity calcium-binding sites at the enzyme. Reversible low affinity calcium binding induces subtle structural alterations of the enzyme, which did not impact catalytic activity. (iii) Increasing calcium concentrations failed to reverse impairment of membrane binding induced by mutagenesis of the sequence determinants indicating the priority of hydrophobic interactions. Taken together these data suggest that 15-lipoxygenase-1 associates to biomembranes primarily via hydrophobic interactions between surface-exposed apolar amino acid side chains and membrane lipids. Calcium supports membrane binding probably by forming salt bridges between the negatively charged head groups of membrane phospholipids and acidic surface amino acids of the membrane contact plane and this interaction might contribute to overcome repulsive forces. (+info)
Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators.
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PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features. (+info)
A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice.
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The 12/15-lipoxygenases (12/15-LOX) catalyze the stereo-specific oxygenation of arachidonic and linoleic acids into a complex series of signaling molecules, including the hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs). Our previous studies, using high density oligonucleotide microarrays, suggested a novel link between progesterone receptor (PR) signaling and 12/15-LOX-mediated fatty acid metabolism in preimplantation mouse uterus. In this paper, using PR knockout mice, we established that the transcripts encoding leukocyte-12/15-LOX (L-12/15-LOX) and epidermal-12/15-LOX (E-12/15-LOX) are indeed downstream targets of regulation by PR in the uterine surface epithelium. Maximal induction of both L- and E-12/15-LOX on the day of implantation resulted in a marked increase in the uterine levels of the eicosanoids, 12-HETE, 15-HETE, and 13-HODE. Mice with null mutation in L-12/15-LOX had significantly reduced uterine levels of arachidonic acid metabolites and exhibited a partial impairment in implantation. Complete blockade of uterine 12/15-LOX activity by a specific inhibitor led to greater than 80% reduction in a number of implantation sites relative to untreated controls. Cell-based assays indicated that 12-HETE, 15-HETE, and 13-HODE function as activating ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), suggesting that this nuclear receptor could be a downstream target of 12/15-LOX-derived metabolites in the preimplantation uterus. Consistent with this hypothesis, administration of rosiglitazone, a potent PPARgamma-selective agonist, efficiently reversed inhibition of implantation by the 12/15-LOX-specific inhibitor. Rosiglitazone also induced a number of potential target genes of 12/15-LOX-derived metabolites in the pregnant uterus, indicating their regulation by PPARgamma. Collectively, our results uncovered a novel signaling pathway in which progesterone-induced synthesis of the 12/15-LOX-derived lipid mediators activates PPARgamma and its downstream gene networks, which in turn function as critical regulators of implantation in the mouse. (+info)
Regulation of bone mass in mice by the lipoxygenase gene Alox15.
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The development of osteoporosis involves the interaction of multiple environmental and genetic factors. Through combined genetic and genomic approaches, we identified the lipoxygenase gene Alox15 as a negative regulator of peak bone mineral density in mice. Crossbreeding experiments with Alox15 knockout mice confirmed that 12/15-lipoxygenase plays a role in skeletal development. Pharmacologic inhibitors of this enzyme improved bone density and strength in two rodent models of osteoporosis. These results suggest that drugs targeting the 12/15-lipoxygenase pathway merit investigation as a therapy for osteoporosis. (+info)