Lipoxygenase mRNA Research Articles

The potential of acetylsalicylic acid and vitamin E in modulating inflammatory cascades in chickens under lipopolysaccharide-induced inflammation

Distinct enzymes, including cyclooxygenase 1 and 2 (COX-1 and COX-2), lipoxygenase (LOXs), and cytochrome P450 monooxygenase (CYP450), produce different stress mediators and mediate inflammation in birds. Bioactive agents such as acetylsalicylic acid (ASA) and vitamin E (vE) may affect enzyme activities and could be used in poultry production to control the magnitude of acute phase inflammation. Here, we characterized COX, LOX, and CYP450 mRNA expression levels in chicken immune tissues in response to Escherichia coli lipopolysaccharide (LPS) challenge and investigated whether ASA and vE could alter gene expression. Additionally, for the first time in chickens, we evaluated oxygen consumption by platelet mitochondria as a biomarker of mitochondria function in response to ASA- and vE. LPS challenge compromised bird growth rates, but neither dietary ASA nor vE significantly ameliorated this effect; however, gradually increasing dietary vE levels were more effective than basal levels. ASA regulated arachidonic acid metabolism, providing an eicosanoid synthesis substrate, whereas gradually increasing vE levels evoked aspirin resistance during challenge. Gene expression in immune tissues was highly variable, indicating a complex regulatory network controlling inflammatory pathways. However, unlike COX-1, COX-2 and CYP450 exhibited increased mRNA expression in some cases, suggesting an initiation of novel anti-inflammatory and pro-resolving signals during challenge. Measuring oxygen consumption rate, we revealed that neither the ASA nor vE levels applied here exerted toxic effects on platelet mitochondria.

Parainfluenza virus infection enhances NSAIDs–induced inhibition of PGE2 generation and COX-2 expression in human airway epithelial cells

PurposeRespiratory viral infection and nonsteroidal anti-inflammatory drugs (NSAIDs) may affect arachidonic acid (AA) metabolism in the airway epithelium, however their joint effect has not been studied. We hypothesized, that alternations of AA metabolism in human airway epithelial cells (ECs) – induced by Parainfluenza virus type 3 (PIV3) – may be modified by concomitant treatment with NSAIDs. Materials and methodsNasal (RPMI 2650) and bronchial (BEAS-2B) epithelial cells were cultured into confluence and then infected with PIV3. Prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) levels in cell supernatants were measured by ELISA and expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) mRNA in cells was evaluated after reverse transcription with real-time polymerase chain reactions. ResultsPGE2 generation was decreased by PIV3 infection in the upper airway epithelial cells, and increased in the lower airway epithelial cells. Both naproxen and celecoxib induced significant reduction in PGE2 release in both infected and non-infected upper and lower airway epithelial cells. However, in PIV3-infected epithelial cells celecoxib inhibited PGE2 release and COX-2 expression to significantly higher degree as compared to non-infected cells. 15-HETE generation or COX-1, 5-LO and 15-LO expression were not affected by the virus infection or by NSAIDs. ConclusionVirus infection in airway epithelial cells enhances inhibitory effect of NSAIDs on prostaglandin E2 generation.

Evolution and Expansion of the Prokaryote-Like Lipoxygenase Family in the Brown Alga Saccharina japonica.

Lipoxygenase (LOX) plays important roles in fatty acid oxidation and lipid mediator biosynthesis. In this study, we give first insights into brown algal LOX evolution. Whole genome searches revealed four, three, and eleven LOXs in Ectocarpus siliculosus, Cladosiphon okamuranus, and Saccharina japonica, respectively. In phylogenetic analyses, LOXs from brown algae form a robust clade with those from prokaryotes, suggesting an ancestral origin and slow evolution. Brown algal LOXs were divided into two clades, C1 and C2 in a phylogenetic tree. Compared to the two species of Ectocarpales, LOX gene expansion occurred in the kelp S. japonica through tandem duplication and segmental duplication. Selection pressure analysis showed that LOX genes in brown algae have undergone strong purifying selection, while the selective constraint in the C2 clade was more relaxed than that in the C1 clade. Furthermore, within each clade, LOXs of S. japonica evolved under more relaxed selection constraints than E. siliculosus and C. okamuranus. Structural modeling showed that unlike LOXs of plants and animals, which contain a β barrel in the N-terminal part of the protein, LOXs in brown algae fold into a single domain. Analysis of previously published transcriptomic data showed that LOXs in E. siliculosus are responsive to hyposaline, hypersaline, oxidative, and copper stresses. Moreover, clear divergence of expression patterns was observed among different life stages, as well as between duplicate gene pairs. In E. siliculosus, all four LOXs are male-biased in immature gametophytes, and mature gametophytes showed significantly higher LOX mRNA levels than immature gametophytes and sporophytes. In S. japonica, however, our RNA-Seq data showed that most LOXs are highly expressed in sporophytes. Even the most recently duplicated gene pairs showed divergent expression patterns, suggesting that functional divergence has likely occurred since LOX genes duplicated, which potentially contributes to the production of various oxylipins in brown algae.

Inhibitory effects of aspirin-triggered resolvin D1 on spinal nociceptive processing in rat pain models.

BackgroundHarnessing the actions of the resolvin pathways has the potential for the treatment of a wide range of conditions associated with overt inflammatory signalling. Aspirin-triggered resolvin D1 (AT-RvD1) has robust analgesic effects in behavioural models of pain; however, the potential underlying spinal neurophysiological mechanisms contributing to these inhibitory effects in vivo are yet to be determined. This study investigated the acute effects of spinal AT-RvD1 on evoked responses of spinal neurones in vivo in a model of acute inflammatory pain and chronic osteoarthritic (OA) pain and the relevance of alterations in spinal gene expression to these neurophysiological effects.MethodsPain behaviour was assessed in rats with established carrageenan-induced inflammatory or monosodium iodoacetate (MIA)-induced OA pain, and changes in spinal gene expression of resolvin receptors and relevant enzymatic pathways were examined. At timepoints of established pain behaviour, responses of deep dorsal horn wide dynamic range (WDR) neurones to transcutaneous electrical stimulation of the hind paw were recorded pre- and post direct spinal administration of AT-RvD1 (15 and 150 ng/50 μl).ResultsAT-RvD1 (15 ng/50 μl) significantly inhibited WDR neurone responses to electrical stimuli at C- (29 % inhibition) and Aδ-fibre (27 % inhibition) intensities. Both wind-up (53 %) and post-discharge (46 %) responses of WDR neurones in carrageenan-treated animals were significantly inhibited by AT-RvD1, compared to pre-drug response (p < 0.05). These effects were abolished by spinal pre-administration of a formyl peptide receptor 2 (FPR2/ALX) antagonist, butoxy carbonyl-Phe-Leu-Phe-Leu-Phe (BOC-2) (50 μg/50 μl). AT-RvD1 did not alter evoked WDR neurone responses in non-inflamed or MIA-treated rats. Electrophysiological effects in carrageenan-inflamed rats were accompanied by a significant increase in messenger RNA (mRNA) for chemerin (ChemR23) receptor and 5-lipoxygenase-activating protein (FLAP) and a decrease in 15-lipoxygenase (15-LOX) mRNA in the ipsilateral spinal cord of the carrageenan group, compared to controls.ConclusionsOur data suggest that peripheral inflammation-mediated changes in spinal FLAP expression may contribute to the novel inhibitory effects of spinal AT-RvD1 on WDR neuronal excitability, which are mediated by FPR2/ALX receptors. Inflammatory-driven changes in this pathway may offer novel targets for inflammatory pain treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0676-6) contains supplementary material, which is available to authorized users.

Lipoxin Inhibits Fungal Uptake by Macrophages and Reduces the Severity of Acute Pulmonary Infection Caused by Paracoccidioides brasiliensis.

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages.

Acute arsenic treatment alters arachidonic acid and its associated metabolite levels in the brain of C57Bl/6 mice.

The toxic effects of arsenic on the whole brain, as well as the discrete regions, has been previously reported for mice. We investigated the effects of acute arsenite (As(III)) on brain levels of arachidonic acid (AA) and its associated metabolites generated through cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) pathways. Our results demonstrated that acute As(III) treatment (12.5 mg·(kg body mass)(-1)) decreases cytosolic phospholipase A2 (cPLA2) with a subsequent decrease in its catalytic activity and brain AA levels. In addition, As(III) differentially altered CYP epoxygenases and CYP ω-hydroxylases, but it did not affect brain Ephx2 mRNA or sEH catalytic activity levels. As(III)-mediated effects on Cyps caused an increase in brain 5,6-epoxyeicosatrienoic acid (5,6-EET) and 16/17-hydroxyeicosatetreinoic acid (16/17-HETE) levels, and a decrease in 18- and 20-HETE levels. Furthermore, As(III) increased cyclooxygenase-2 (COX-2) mRNA while decreasing prostaglandins F2α (PGF2α) and PGJ2. As(III) also increased brain 5-lipoxygenase (5-LOX) and 15-LOX mRNA, but decreased 12-LOX mRNA. These changes in LOX mRNA were associated with a decrease in 8/12-HETE levels only. In conclusion, this is the first demonstration that As(III) decreases AA levels coinciding with alterations to EET, HETE, and PG levels, which affects brain development and neurochemistry.

Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (kcat/KM) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2).

Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids.

Abstract Analysis of arginine methylation, which affects specific protein interactions in eukaryotic cells, requires access to methylated protein for biophysical and biochemical studies. Methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon co-expression with protein arginine methyltransferase 1 in E. coli was monitored by mass spectrometry and found to be identical to the modification of hnRNP K purified from mammalian cells. Recombinant non-methylated and arginine-methylated hnRNP K (MethnRNP K) were used to characterize self-aggregation and nucleic acid binding. Analytical ultracentrifugation and static light scattering experiments revealed that hnRNP K methylation does not impact reversible self-aggregation, which can be prevented by high ionic strength and organic additives. Filter binding assays were used to compare the binding of non-methylated and MethnRNP K to the pyrimidine repeat-containing differentiation control element (DICE) of reticulocyte 15-lipoxygenase mRNA 3' UTR. No affinity differences were detected for both hnRNP K variants. A series of oligonucleotides carrying various numbers of C4 motifs at different positions was used in steady state competition assays with fluorescently-labeled functional differentiation control element (2R). Quantitative evaluation indicated that all hnRNP K homology domains of hnRNP K contribute differentially to RNA binding, with KH1-KH2 acting as a tandem domain and KH3 as an individual binding domain.

Insulin‐like growth factor I (IGF‐1) suppressed foam cells via downregulation of 12/15‐lipoxygenase: potential mechanism responsible for IGF‐1‐induced atheroprotective effect (832.14)

We have shown that IGF‐1 reduced foam cells and suppressed atherosclerosis, however mechanism mediating these effects was not identified. Macrophage (MΦ) 12/15‐lipoxygenase (LOX) known to mediate oxidation of low density lipoprotein (LDL) and promote formation of foam cells contributing to atherogenesis. We hypothesized that IGF‐1 downregulates LOX and via this mechanism reduces foam cells and plaque size.IGF‐1 infusion into Apoe‐null mice (1.5 mg/kg/d, 12 weeks on a Western diet) reduced LOX plaque immunopositivity (74±9% decrease), suppressed plaque oxidative stress (64±7% decrease, immunohistochemistry with 8‐oxodG a/b), reduced plaque lipids (35%±6 decrease, Oil Red staining) and decreased atherosclerotic lesion area (IGF‐1: 0.259 ± 0.018 mm2, saline: 0.356 ± 0.031 mm2).To further define mechanisms THP‐1‐derived MΦ were exposed to IGF‐1. IGF‐1 (50 ng/ml, 16h) reduced LOX mRNA (49±6% decrease), decreased LOX protein (42±6% decrease, immunoblotting) and also IGF‐1 suppressed STAT6 transcription factor binding to LOX gene promoter region (CHIP assay with STAT6 a/b) suggesting that IGF‐1 downregulates LOX via STAT6‐dependent transcriptional mechanism. IGF‐1 reduced oxidation of LDL mediated by THP‐1 or mouse peritoneal MΦ (85±6% and 67±5% decrease, respectively; MDA assay) and also IGF‐1 decreased cellular LDL uptake (i.e. formation of foam cells) by THP‐1 or WT peritoneal MΦ (45±8% and 51±3% decrease, respectively), however both lipid uptake and IGF‐1 effect were blocked in LOX‐deficient peritoneal MΦ. These data indicate that LOX downregulation mediates IGF‐1‐induced suppression of LDL oxidation and foam cell formation.In summary, IGF‐1 reduced LOX expression in atherosclerotic plaque and in vitro in cultured MΦ. We demonstrated that LOX downregulation mediates IGF‐1‐induced reduction in foam cells representing potential mechanism mediating IGF‐1‐induced atheroprotective effect.Grant Funding Source: Supported by NIH/NHLBI

A Cell Wall Extract from Piriformospora indica Promotes Tuberization in Potato (Solanum tuberosum L.) Via Enhanced Expression of Ca+2 Signaling Pathway and Lipoxygenase Gene

Piriformospora indica is an axenically cultivable phytopromotional endosymbiont that mimics capabilities of arbuscular mycorrhizal fungi. This is a basidiomycete of the Sebacinaceae family, which promotes growth, development, and seed production in a variety of plant species. We report that the cell wall extract (CWE) from P. indica induces tuberization in vitro and promotes tuber growth and yield in potato. The CWE altered the calcium signaling pathway that regulates tuberization process. An increase in tuber number and size was correlated with increased transcript expression of the two Ca(2+)-dependant proteins (CaM1 and St-CDPK1) and the lipoxygenase (LOX) mRNA, which are known to play distinct roles in potato tuberization. External supplementation of Ca(2+) ions induced a similar set of tuberization pathway genes, indicating presence of an active Ca(2+) in the CWE of P. indica. Since potato tuberization is directly influenced by the presence of microflora in nature, the present study provides an insight into the novel mechanism of potato tuberization in relation to plant-microbe association. Ours is the first report on an in vitro tuber-inducing beneficial fungus.

Age-dependent responsiveness of human female bone cells to vitamin D analog and PTH

Vitamin D less-calcemic analog JKF 1624 F2-2 (JKF) and PTH 1-34 stimulate in human female cultured osteoblasts (Ob) DNA synthesis (DNA), creatine kinase specific activity (CK), 1α, 25 vitamin D hydroxylase mRNA (1OHase) expression and 1,25(OH)2D3 (1,25) production, estrogen receptors (ER) mRNA expression and intracellular and membranal estrogen binding. In the present study, cultured Ob from different ages were subjected to hormonal stimulations and analyzed for different parameters. We found: 1) ERα expression is higher and ERβ expression is lower in pre-meno - pausal Ob (prOb), with similar intracellular and membranal binding. 2) JKF and PTH up-regulated ERα and JKF downregulated ERβ in both Ob, while PTH stimulated it in post- (poOb) and inhibited it in prOb. 3) There is higher expression of 1OHase mRNA in prOb, but 1,25 production is similar. Both parameters were hormonally stimulated to higher extent in prOb. 4) Ob express 12 and 15 lipoxygenase (LO) mRNA and produce 12- and 15-hydroxyeicosatetraenoic acid (H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. JKF inhibited 12LO expression in prOb and stimulated in poOb, whereas PTH stimulated it to higher extent in prOb. JKF stimulated and PTH inhibited 15LO expression in both; 12 and 15H were stimulated by both hormones in both Ob. 5. PTH and JKF stimulated DNA and CK in both Ob. In conclusion Ob demonstrate some age-dependent response to calciotrophic hormones, but the mechanism and beneficial outcome for human is unclear.

Upregulated expression of brain enzymatic markers of arachidonic and docosahexaenoic acid metabolism in a rat model of the metabolic syndrome

BackgroundIn animal models, the metabolic syndrome elicits a cerebral response characterized by altered phospholipid and unesterified fatty acid concentrations and increases in pro-apoptotic inflammatory mediators that may cause synaptic loss and cognitive impairment. We hypothesized that these changes are associated with phospholipase (PLA2) enzymes that regulate arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-6) acid metabolism, major polyunsaturated fatty acids in brain. Male Wistar rats were fed a control or high-sucrose diet for 8 weeks. Brains were assayed for markers of AA metabolism (calcium-dependent cytosolic cPLA2 IVA and cyclooxygenases), DHA metabolism (calcium-independent iPLA2 VIA and lipoxygenases), brain-derived neurotrophic factor (BDNF), and synaptic integrity (drebrin and synaptophysin). Lipid concentrations were measured in brains subjected to high-energy microwave fixation.ResultsThe high-sucrose compared with control diet induced insulin resistance, and increased phosphorylated-cPLA2 protein, cPLA2 and iPLA2 activity and 12-lipoxygenase mRNA, but decreased BDNF mRNA and protein, and drebrin mRNA. The concentration of several n-6 fatty acids in ethanolamine glycerophospholipids and lysophosphatidylcholine was increased, as was unesterified AA concentration. Eicosanoid concentrations (prostaglandin E2, thromboxane B2 and leukotriene B4) did not change.ConclusionThese findings show upregulated brain AA and DHA metabolism and reduced BDNF and drebrin, but no changes in eicosanoids, in an animal model of the metabolic syndrome. These changes might contribute to altered synaptic plasticity and cognitive impairment in rats and humans with the metabolic syndrome.

Thiamine seed treatment enhances LOX expression, promotes growth and induces downy mildew disease resistance in pearl millet

Seeds of pearl millet [Pennisetum glaucum (L). R.Br.] susceptible cv. 7042S were treated with thiamine at 5, 10, 15, 20 and 25 mM concentrations and growth promotion and downy mildew resistance were tested. Seed treatment with 20 mM thiamine resulted in 72 and 70 % disease protection under greenhouse and field conditions, respectively, and enhanced vegetative and reproductive growth parameters. Analysis of lipoxygenase (LOX) activity in inoculated pearl millet seedlings at different time intervals indicated that increased LOX activity was initiated at 3 h after inoculation (hai) and maximum activity was observed at 24 hai. Northern analysis showed that LOX mRNA transcript accumulation was higher in the resistant seedlings (cv. IP18292) than in susceptible seedlings. Thiamine seed treatment induces rapid LOX gene expression and results in significant disease protection against downy mildew disease.

Thiamine seed treatment enhances LOX expression, promotes growth and induces downy mildew disease resistance in pearl millet

Seeds of pearl millet [Pennisetum glaucum (L). R.Br.] susceptible cv. 7042S were treated with thiamine at 5, 10, 15, 20 and 25 mM concentrations and growth promotion and downy mildew resistance were tested. Seed treatment with 20 mM thiamine resulted in 72 and 70 % disease protection under greenhouse and field conditions, respectively, and enhanced vegetative and reproductive growth parameters. Analysis of lipoxygenase (LOX) activity in inoculated pearl millet seedlings at different time intervals indicated that increased LOX activity was initiated at 3 h after inoculation (hai) and maximum activity was observed at 24 hai. Northern analysis showed that LOX mRNA transcript accumulation was higher in the resistant seedlings (cv. IP18292) than in susceptible seedlings. Thiamine seed treatment induces rapid LOX gene expression and results in significant disease protection against downy mildew disease.

The effects of estrogen receptors α‐ and β‐specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations with cerebrovascular symptoms

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors.The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77).Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11–1.56], n=102; and 0.79 [0.55–1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue.In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.

Age-Dependent Responsiveness of Human Female Cultured Bone Cells to Estrogenic Compounds

Human cultured female osteoblasts (Obs) respond age-dependently to estradiol-17β (E2) and to phytoestrogens by increased DNA synthesis (DNA) and creatine kinase specific activity (CK). ERα mRNA expression is higher than ERβ mRNA in Obs. Pre-menopausal Ob (prOb) reveals higher expression of ERα than post-menopausal Ob (poOb), but similar intracellular and membranal E2 bindings. ERα mRNA is stimulated in prOb and inhibited in poOb by different estrogens. ERβ mRNA in prOb is stimulated by 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4ʹ,4ʺ-[4-Propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) and raloxifene (Ral) and inhibited by genistein (G) and daidzein (D) while in poOb only E2 and DPN inhibited it. All phytoestrogenic carboxy- derivatives stimulated ERα mRNA in both Ob, while the protein bound- carboxy were ineffective. All compounds except carboxybiochainin A (cBA) had no effect on ERβ. There is higher expression of 1α 25 hydroxy- vitamin D (1OHase) mRNA in poOb, whereas 1,25(OH)2D3 (1,25) production is similar, but all compounds increased 1OHase mRNA and 1,25 to higher extent in prOb. Obs express 15 and 12 lipoxygenase (12LO and 15LO) mRNA and produce 12 and 15 HETE (12H and 15H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. Both LOs are increased to higher extent in poOb by all estrogens except Ral and PPT. 12H is increased by DPN, PPT and carboxy- derivatives similarly in both Obs, while 15H is increased by biochainin A (BA), Ral and carboxyderivatives. DNA synthesis and CK are stimulated by all compounds in both Obs, but to higher extent in prOb. The 1,25 less-calcemic analog JK 1624F2-2 (JKF) up-regulated DNA and CK response to all estrogens except BA and the carboxy- derivatives in both Obs. JKF stimulated intracellular binding of E2 similarly by all compounds except BA, but inhibited its membranal binding in both Obs. In conclusion Obs respond age-dependently to estrogens in a yet unknown mechanism and the beneficial outcome for human female bones is not yet clear.

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D 3 (25D), 1,25(OH) 2D 3 (1,25D), 24,25(OH) 2D 3 (24,25D), JKF1624F 2-2 (JKF) or QW1624F 2-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.

DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules.

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.

Vitamin D analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cells

Vitamin D metabolites or its less-calcemic analogs (JKF or QW) are beneficial for bone biology. We analyzed whether or not 25(OH)D 3 (25), 1,25(OH) 2D 3 (1,25), JKF or QW regulate lipooxygenase (LO) enzymes expression and their products hydroxyeicosatetraenoic acid (12 and 15 HETE) formation as well as reactive oxygen species (ROS) production in human bone cell lines (SaOS2 and hFOB) and primary cultured human bone cells (Obs) from males or females. All compounds except 25 increased LOs mRNA expression and HETE production in female or male Obs. ROS formation was induced by JKF and QW in both cell lines, and was inhibited by different inhibitors. Baicalein (baic) an inhibitor of 12 and 15 LO activity, inhibited partially ROS formation by JKF or QW in SaSO2 and hFOB. JKF-stimulated DNA synthesis in female Obs was inhibited by baic but unchanged by addition of HETE or HETE with baic. These results indicate that vitamin D increased oxidative stress in bone cells is in part via induction of LO expression and activity. This new feature of vitamin D is probably mediated by intracellular and/or membranal receptors and its potential hazard could lead to potential damage due to increased lipid oxidation.