Systemic deficiency of mouse arachidonate 15-lipoxygenase induces defective erythropoiesis and transgenic expression of the human enzyme rescues this phenotype.

Arachidonic acid 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes, which has previously been implicated in the maturational breakdown of intracellular organelles and plasma membrane remodeling during reticulocyte-erythrocyte transition. Conventional Alox15-/- mice are viable, develop normally but do not exhibit a major defective erythropoietic phenotype. To characterize the putative in vivo relevance of Alox15 for red blood cell development, we explored the impact of systemic inactivation of the Alox15 gene on mouse erythropoiesis. We found that Alox15-/- mice exhibited reduced erythrocyte counts, elevated reticulocyte counts and red cell hyperchromia. The structure of the plasma membrane of Alox15-/- erythrocytes is altered and a significant share of the red cells was present as echinocytes and/or acanthocytes. An increased share of the Alox15-/- erythrocytes cells were annexin V positive, which indicates a loss of plasma membrane asymmetry. Erythrocytes of Alox15-/- mice were more susceptible to osmotic hemolysis and exhibited a reduced ex vivo life span. When we transgenically expressed human ALOX15 in Alox15-/- mice under the control of the aP2 promoter the defective erythropoietic system was rescued and the impaired osmotic resistance was normalized. Together these data suggest the involvement Alox15 in the maturational remodeling of the plasma membrane during red cell development.

Eosinophils promote corneal wound healing via the 12/15-lipoxygenase pathway.

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.

12-Lipoxygenase Regulates Cold Adaptation and Glucose Metabolism by Producing the Omega-3 Lipid 12-HEPE from Brown Fat.

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and ß3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway.

12(S)-hydroxyeicosatetraenoic acid impairs vascular endothelial permeability by altering adherens junction phosphorylation levels and affecting the binding and dissociation of its components in high glucose-induced vascular injury.

AIMS/INTRODUCTION: Diabetes is an important risk factor for atherosclerotic disease. The initiating factor of atherosclerosis is local endothelial cell injury. The arachidonic acid metabolite, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), might be involved in this process. In recent years, some studies have discussed the effect of 12(S)-HETE on vascular endothelial cell function. In the present study, we investigated the effect of 12(S)-HETE on vascular endothelial cell function in high-glucose conditions and the mechanisms involved. MATERIALS AND METHODS: Human umbilical vein endothelial cells were cultured in conventional M199 medium and high-glucose M199 medium. Human umbilical vein endothelial cells were stimulated with 12(S)-HETE and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (a 12/15-lipoxygenases inhibitor). A type 1 diabetes mellitus model was established in C57BL/6 or 12/15-lipoxygenases knockout mice with streptozotocin. Aortic tissue was harvested for subsequent testing. The transmembrane transport of dextran and human acute monocytic leukaemia cell line (THP-1) cells was measured. The adherens junction protein, IkBα, nuclear factor kappa Bp65 (P65), intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression and phosphorylation, and the binding/dissociation of endothelial cell components were observed. RESULTS: Transendothelial migration of dextran and THP-1 cells was significantly increased by stimulation of human umbilical vein endothelial cell monolayers with high glucose and 12(S)-HETE (P < 0.05). High glucose and 12(S)-HETE altered the vascular endothelial cadherin and ß-catenin phosphorylation level, and promoted the dissociation of ß-catenin and vascular endothelial cadherin. Expression levels of P-Ikbα, P-P65, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 were elevated in high glucose and 12(S)-HETE treated cells and diabetic mice compared with controls (P < 0.05). CONCLUSIONS: The lipoxygenases metabolite, 12(S)-HETE, can impair vascular endothelial permeability by altering adherens junction phosphorylation levels, and affecting the binding and dissociation of its components in high-glucose conditions.

Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice.

15-lipoxygenase is involved in the generation of specialized pro-resolving lipid mediators that play essential roles in resolution and inflammatory responses. Here, we investigated anti-inflammatory role of Alox15 in skin homeostasis. We demonstrated that knockout (KO) of Alox15 led to hair loss and disrupted the structural integrity of the dorsal skin. Alox15 KO resulted in loss of hair follicle stem cells and abnormal transition of dermal adipocytes into fibroblasts. Alox15 deficiency increased infiltration of proinflammatory macrophages and upregulated proinflammatory and necroptotic signaling in dermal adipose tissue in the dorsal skin. Lipidomic analysis revealed severe loss of resolvin D2 in the dorsal skin of Alox15 KO mice compared to wild type controls. Treatment with resolvin D2 reduced skin inflammation in Alox15 KO mice. Collectively, these results indicate that Alox15-mediated production of resolvin D2 is required to maintain skin integrity by suppressing dermal inflammation.

Deletion of 12/15-lipoxygenase accelerates the development of aging-associated and instability-induced osteoarthritis.

OBJECTIVE: 12/15-Lipoxygenase (12/15-LOX) catalyzes the generation of various anti-inflammatory lipid mediators, and has been implicated in several inflammatory and degenerative diseases. However, there is currently no evidence that 12/15-LOX has a role in osteoarthritis (OA). The aim of this study was to investigate the role of 12/15-LOX in the pathogenesis of OA. METHODS: The development of aging-associated and destabilization of the medial meniscus (DMM)-induced OA were compared in 12/15-LOX-deficient (12/15-LOX-/-) and wild-type (WT) mice. The extent of cartilage damage was evaluated by histology. The expression of OA markers was evaluated by immunohistochemistry and RT-PCR. Cartilage explants were stimulated with IL-1α in the absence or presence of the 12/15-LOX metabolites, 15-hydroxyeicosatetraenoic acids (15-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) or lipoxin A4 (LXA4), and the levels of matrix metalloproteinases-13 (MMP-13), Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. The effect of LXA4 on the progression of OA was evaluated in wild type (WT) mice. RESULTS: The expression of 12/15-LOX in cartilage increased during the progression of DMM-induced OA and with aging in WT mice. Cartilage degeneration was more severe in 12/15-LOX-/- mice compared to WT mice in both models of OA, and this was associated with increased expression of MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs, aggrecanases (ADAMTS5), inducible NO synthases (iNOS), and mPGES-1. Treatment of cartilage explants with 12/15-LOX metabolites, suppressed IL-1α-induced production of MMP-13, NO and PGE2, with LXA4 being the most potent. Intra-peritoneal injection of LXA4 reduced the severity of DMM-induced cartilage degradation. CONCLUSIONS: These data suggest an important role of 12/15-LOX in the pathogenesis of OA. They also suggest that activation of this pathway may provide a novel strategy for prevention and treatment of OA.

Activation of peroxisome proliferator-activated receptor-γ by a 12/15-lipoxygenase product of arachidonic acid: a possible neuroprotective effect in the brain after experimental intracerebral hemorrhage.

OBJECTIVE In this study, the authors investigated the involvement of 15( S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in the regulation of peroxisome proliferator-activated receptor-γ (PPARγ) after intracerebral hemorrhage (ICH) and its effects on hemorrhage-induced inflammatory response and oxidative stress in an experimental rodent model. METHODS To simulate ICH in a rat model, the authors injected autologous whole blood into the right striatum of male Sprague-Dawley rats. The distribution and expression of 12/15-lipoxygenase (12/15-LOX) were determined by immunohistochemistry and Western blot analysis, respectively. Immunofluorescent double labeling was used to study the cellular localization of 12/15-LOX, and 15(S)-HETE was measured with a 15(S)-HETE enzyme immunoassay kit. Neurological deficits in the animals were assessed through behavioral testing, and apoptotic cell death was determined with terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling. RESULTS Rats with ICH had increased expression of 12/15-LOX predominantly in neurons and also in oligodendrocytes, astrocytes, and microglia. Moreover, ICH elevated production of 15(S)-HETE in the brain area ipsilateral to the blood injection. The PPARγ agonist, exogenous 15(S)-HETE, significantly increased PPARγ protein levels and increased PPARγ-regulated gene (i.e., catalase) expression in the ICH rats. Reduced expression of the gene for the proinflammatory protein nuclear factor κB coincided with decreased neuron damage and improved functional recovery from ICH. A PPARγ antagonist, GW9662, reversed the effects of exogenous 15(S)-HETE on the PPARγ-regulated genes. CONCLUSIONS The induction of 15(S)-HETE during simulated ICH suggests generation of endogenous signals of neuroprotection. The effects of exogenous 15(S)-HETE on brain hemorrhage-induced inflammatory responses and oxidative stress might be mediated via PPARγ.

12/15-Lipoxygenase Inhibition Reverses Cognitive Impairment, Brain Amyloidosis, and Tau Pathology by Stimulating Autophagy in Aged Triple Transgenic Mice.

BACKGROUND: The 12/15-lipoxygenase (12/15-LO) enzyme is upregulated in the brains of patients with Alzheimer's disease (AD), and its expression levels influence the onset of the AD-like phenotype in mouse models. However, whether targeting this pathway after the neuropathology and behavioral impairments have been established remains to be investigated. METHODS: Triple transgenic (3xTg) mice received either PD146176-a selective and specific pharmacological inhibitor of 12/15-LO-or placebo starting at 12 months of age for 12 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. RESULTS: At the end of the study, mice in the control group showed a worsening of memory and learning abilities, whereas mice receiving PD146176 were undistinguishable from wild-type mice. The same group also had significantly lower amyloid beta levels and deposition, less tau neuropathology, increased synaptic integrity, and autophagy activation. Ex vivo and in vitro genetic and pharmacological studies found that the mechanism involved in these effects was the activation of neuronal autophagy. CONCLUSIONS: Our findings provide new insights into the disease-modifying action of 12/15-LO pharmacological inhibition and establish it as a viable therapeutic approach for patients with AD.

A role for 12/15-lipoxygenase-derived proresolving mediators in postoperative ileus: protectin DX-regulated neutrophil extravasation.

Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1(+)/Ly6C(+) infiltrating monocytes rather than Ly6G(+) neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G(+)/Ly6C(+) neutrophil extravasation after protectin DX treatment, whereas Ly6G(-)/Ly6C(+) monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.

12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

The 12/15-lipoxygenase as an emerging therapeutic target for Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative condition characterized by progressive memory loss. Mutations in genes involved in the production of amyloid-ß (Aß) are linked to the early-onset variant of AD. However, the most common form, sporadic AD, is considered to be the result of an interaction between environmental risk factors and various genes. Among them, recent work has highlighted the potential role that the 12/15-lipoxygenase (12/15LO) pathway may play in AD pathogenesis. 12/15LO is widely distributed in the central nervous system, and its levels are upregulated in patients with AD or mild cognitive impairments. Studies using animal models have implicated 12/15LO in the molecular pathology of AD, including the metabolism of Aß and tau, synaptic integrity, and cognitive functions. We provide an overview of this pathway and its relevance to AD pathogenesis, discuss the mechanism(s) involved, and provide an assessment of how targeting 12/15LO could lead to novel AD therapeutics.

ROS-dependent Syk and Pyk2-mediated STAT1 activation is required for 15(S)-hydroxyeicosatetraenoic acid-induced CD36 expression and foam cell formation.

15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1/2 (15-LO1/2) metabolite of arachidonic acid (AA), induces CD36 expression through xanthine oxidase and NADPH oxidase-dependent ROS production and Syk and Pyk2-dependent STAT1 activation. In line with these observations, 15(S)-HETE also induced foam cell formation involving ROS, Syk, Pyk2, and STAT1-mediated CD36 expression. In addition, peritoneal macrophages from Western diet-fed ApoE(-/-) mice exhibited elevated levels of xanthine oxidase and NADPH oxidase activities, ROS production, Syk, Pyk2, and STAT1 phosphorylation, and CD36 expression compared to those from ApoE(-/-):12/15-LO(-/-) mice and these events correlated with increased lipid deposits, macrophage content, and lesion progression in the aortic roots. Human atherosclerotic arteries also showed increased 15-LO1 expression, STAT1 phosphorylation, and CD36 levels as compared to normal arteries. Together, these findings suggest that 12/15-LO metabolites of AA, particularly 12/15(S)-HETE, might play a crucial role in atherogenesis by enhancing foam cell formation.

Interaction between cytokines and inflammatory cells in islet dysfunction, insulin resistance and vascular disease.

Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in sustained inflammation in diabetes and atherosclerosis. 12- and 15-lipoxygenase (LO), such as 12/15-LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12- and 15-LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue-specific knockout of 12/15-LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12- and 15-LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12- and 15-LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)-12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1) in islets and IL-17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.

Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size.

Glutathione depletion and 12-lipoxygenase-dependent metabolism of arachidonic acid are known to be implicated in neurodegeneration associated with acute ischemic stroke. The objective of this study was to investigate the significance of miR-29 in neurodegeneration associated with acute ischemic stroke. Neural cell death caused by arachidonic acid insult of glutathione-deficient cells was preceded by a 12-lipoxygenase-dependent loss of miR-29b. Delivery of miR-29b mimic to blunt such loss was neuroprotective. miR-29b inhibition potentiated such neural cell death. 12-Lipoxygenase knockdown and inhibitors attenuated the loss of miR-29b in challenged cells. In vivo, stroke caused by middle-cerebral artery occlusion was followed by higher 12-lipoxygenase activity and loss of miR-29b as detected in laser-captured infarct site tissue. 12-Lipoxygenase knockout mice demonstrated protection against such miR loss. miR-29b gene delivery markedly attenuated stroke-induced brain lesion. Oral supplementation of α-tocotrienol, a vitamin E 12-lipoxygenase inhibitor, rescued stroke-induced loss of miR-29b and minimized lesion size. This work provides the first evidence demonstrating that loss of miR-29b at the infarct site is a key contributor to stroke lesion. Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke.

Aloxe3 knockout mice reveal a function of epidermal lipoxygenase-3 as hepoxilin synthase and its pivotal role in barrier formation.

Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway.

12/15-lipoxygenase during the regulation of inflammation, immunity, and self-tolerance.

12/15-Lipoxygenase (12/15-LO) catalyzes the oxidation of free and esterified fatty acids thereby generating a whole spectrum of bioactive lipid mediators. This enzyme is involved in the regulation of various homeostatic processes as well as in the pathogenesis of multiple diseases. During the innate and adaptive immune response, 12/15-LO and its products exert both pro- and anti-inflammatory effects. Likewise, this enzyme has been implicated in the pathogenesis of autoimmune disease as well as in the maintenance of self-tolerance. This review will summarize our current knowledge about the role of 12/15-LO and will try to examine the two faces of this enzyme within the context of inflammation and immunity.

DNA hypomethylation at ALOX12 is associated with persistent wheezing in childhood.

RATIONALE: Epigenetic changes may play a role in the occurrence of asthma-related phenotypes. OBJECTIVES: To identify epigenetic marks in terms of DNA methylation of asthma-related phenotypes in childhood, and to assess the effect of prenatal exposures and genetic variation on these epigenetic marks. METHODS: Data came from two cohorts embedded in the Infancia y Medio Ambiente (INMA) PROJECT: Menorca (n = 122) and Sabadell (n = 236). Wheezing phenotypes were defined at age 4-6 years. Cytosine-guanine (CpG) dinucleotide site DNA methylation differences associated with wheezing phenotypes were screened in children of the Menorca study using the Illumina GoldenGate Panel I. Findings were validated and replicated using pyrosequencing. Information on maternal smoking and folate supplement use was obtained through questionnaires. Dichlorodiphenyldichloroethylene was measured in cord blood or maternal serum. Genotypes were extracted from genome-wide data. MEASUREMENT AND MAIN RESULTS: Screening identified lower DNA methylation at a CpG site in the arachidonate 12-lipoxygenase (ALOX12) gene in children having persistent wheezing compared with those never wheezed (P = 0.003). DNA hypomethylation at ALOX12 loci was associated with higher risk of persistent wheezing in the Menorca study (odds ratio per 1% methylation decrease, 1.13; 95% confidence interval, 0.99-1.29; P = 0.077) and in the Sabadell study (odds ratio, 1.16; 95% confidence interval, 1.03-1.37; P = 0.017). Higher levels of prenatal dichlorodiphenyldichloroethylene were associated with DNA hypomethylation of ALOX12 in the Menorca study (P = 0.033), but not in the Sabadell study (P = 0.377). ALOX12 DNA methylation was strongly determined by underlying genetic polymorphisms. CONCLUSIONS: DNA methylation of ALOX12 may be an epigenetic biomarker for the risk of asthma-related phenotypes.

Transcriptional regulation of ß-secretase-1 by 12/15-lipoxygenase results in enhanced amyloidogenesis and cognitive impairments.

OBJECTIVE: 12/15-Lipoxygenase (12/15-LO) is an enzyme widely distributed in the central nervous system, and it has been involved in the neurobiology of Alzheimer disease (AD). However, the mechanism involved remains elusive. METHODS: We investigated the molecular mechanism by which 12/15-LO regulates amyloid ß (Aß)/Aß precursor protein (APP) metabolism in vivo and in vitro by genetic and pharmacologic approaches. RESULTS: Here we show that overexpression of 12/15-LO leads to increased levels of ß-secretase-1 (BACE1) mRNA and protein, a significant elevation in Aß levels and deposition, and a worsening of memory deficits in AD transgenic mice. In vitro and in vivo studies demonstrate that 12/15-LO regulates BACE1 mRNA expression levels via the activation of the transcription factor Sp1. Thus, 12/15-LO-overexpressing mice had elevated levels of Sp1 and BACE1, whereas 12/15-LO-deficient mice had reduced levels of both. Preventing Sp1 activation by pharmacologic inhibition or dominant-negative mutant blocks the 12/15-LO-dependent elevation of Aß and BACE1 levels. INTERPRETATION: Our findings demonstrate a novel pathway by which 12/15-LO increases the amyloidogenic processing of APP through a Sp1-mediated transcriptional control of BACE1 levels that could have implications for AD pathogenesis and therapy.

12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response.

Central obesity is associated with chronic inflammation, insulin resistance, ß-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.