Publications by authors named "Toshiaki Okuno"

57 Publications

Eicosanoid profiling in patients with complete form of pachydermoperiostosis carrying SLCO2A1 mutations.

J Dermatol 2021 Jun 11. Epub 2021 Jun 11.

Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Pachydermoperiostosis (PDP) is a genetic disease characterized by digital clubbing, periostosis, and pachydermia caused by mutated HPGD or SLCO2A1. Plasma prostaglandin (PG)E levels are increased in these patients. However, other eicosanoids have not been quantitated. We aimed to quantitate plasma eicosanoid levels in four patients carrying SLCO2A1 mutations by high-performance liquid chromatography-tandem mass spectrometry. PGE level was elevated in all patients; PGD and 11β-PGF α levels were also increased in some patients, whereas eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid levels were decreased in all patients. Our data indicate a dysfunctional eicosanoid homeostasis and varied levels of PG in patients with a complete form of PDP carrying SLCO2A1 mutations. PGE levels seem to mostly affect the symptoms, with other eicosanoids possibly having a minor effect.
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http://dx.doi.org/10.1111/1346-8138.16012DOI Listing
June 2021

Up-regulation of cytosolic prostaglandin E synthase in fetal-membrane and amniotic prostaglandin E2 accumulation in labor.

PLoS One 2021 23;16(4):e0250638. Epub 2021 Apr 23.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Prostaglandin E2 (PGE2) is known to have important roles in labor, but the detailed mechanism underlying the spontaneous human labor remains unknown. Here, we examined the involvement of prostaglandin biosynthetic enzymes and transporter in the accumulation of PGE2 in amniotic fluid in human labor. PGE2 and its metabolites were abundant in amniotic fluid in deliveries at term in labor (TLB), but not at term not in labor (TNL). In fetal-membrane Transwell assays, levels of PGE2 production in both maternal and fetal compartments were significantly higher in the TLB group than the TNL group. In fetal-membrane, the mRNA level of PTGES3, which encodes cytosolic prostaglandin E synthase (cPGES), was significantly higher in TLB than in TNL, but the mRNA levels of the other PGE2-synthase genes were not affected by labor. Moreover, the mRNA level of PTGS2, which encodes cyclooxygenase-2 (COX-2) in the amnion was significantly higher in TLB than in TNL. Western blot analyses revealed that the levels of COX-1 and COX-2 were comparable between the two groups, however, the level of cPGES was relatively higher in TLB than in TNL. COXs, cPGES, and prostaglandin transporter (SLCO2A1) proteins were all expressed in both chorionic trophoblasts and amniotic epithelium. These findings suggest that COXs, cPGES and SLCO2A1 contribute to PGE2 production from fetal-membrane in labor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250638PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064594PMC
April 2021

Eicosanoids in Skin Wound Healing.

Int J Mol Sci 2020 Nov 10;21(22). Epub 2020 Nov 10.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.

Wound healing is an important process in the human body to protect against external threats. A dysregulation at any stage of the wound healing process may result in the development of various intractable ulcers or excessive scar formation. Numerous factors such as growth factors, cytokines, and chemokines are involved in this process and play vital roles in tissue repair. Moreover, recent studies have demonstrated that lipid mediators derived from membrane fatty acids are also involved in the process of wound healing. Among these lipid mediators, we focus on eicosanoids such as prostaglandins, thromboxane, leukotrienes, and specialized pro-resolving mediators, which are produced during wound healing processes and play versatile roles in the process. This review article highlights the roles of eicosanoids on skin wound healing, especially focusing on the biosynthetic pathways and biological functions, i.e., inflammation, proliferation, migration, angiogenesis, remodeling, and scarring.
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http://dx.doi.org/10.3390/ijms21228435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698125PMC
November 2020

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy.

PLoS One 2020 9;15(11):e0241869. Epub 2020 Nov 9.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241869PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652309PMC
January 2021

Metabolism and biological functions of 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid.

Prostaglandins Other Lipid Mediat 2021 02 16;152:106502. Epub 2020 Oct 16.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, Japan.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is a 17-carbon hydroxy fatty acid that is biosynthesized either by enzymatic pathways, like thromboxane synthase (TXAS) and cytochrome P450 or a non-enzymatic pathway. TXAS catalyzes the isomerization reaction from PGH to 12-HHT, malondialdehyde, and TXA at a ratio of 1:1:1. Furthermore, 12-HHT has been considered as a mere byproduct of TXA biosynthesis, and its biological function has long been uncertain. BLT2 was initially identified as a low-affinity leukotriene B (LTB) receptor, which is also activated by various hydroxy-eicosatetraenoic acids (HETEs), suggesting that BLT2 may be activated by other endogenous ligands apart from LTB and HETEs. By unbiased ligand screening using crude lipids from rat organs, 12-HHT has been identified as an endogenous agonist for BLT2. The 12-HHT-BLT2 axis induces mast cell migration and contributes to allergic inflammation. BLT2 is also expressed in epithelial cells of the small intestine and skin in mice and contributes to in vivo epithelial barrier functions.
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http://dx.doi.org/10.1016/j.prostaglandins.2020.106502DOI Listing
February 2021

Expression of leukotriene B receptor 1 defines functionally distinct DCs that control allergic skin inflammation.

Cell Mol Immunol 2021 Jun 9;18(6):1437-1449. Epub 2020 Oct 9.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, 113-8421, Japan.

Leukotriene B (LTB) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1 and BLT1 DCs. We also found that BLT1 and BLT1 DCs differentially migrated toward LTB and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB-producing enzyme LTAH knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1 DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1 DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1 DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.
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http://dx.doi.org/10.1038/s41423-020-00559-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167169PMC
June 2021

Leukotriene A hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

FASEB J 2020 10 26;34(10):13949-13958. Epub 2020 Aug 26.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A hydrolase (LTA H), a key enzyme in the synthesis of the lipid mediator leukotriene B (LTB ), in diet-induced obesity. LTA H-deficient (LTA H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA H-KO mice. In contrast, LTB receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA H-KO mice is independent of the LTB /BLT1 axis. These results indicate that LTA H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.
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http://dx.doi.org/10.1096/fj.202001148RDOI Listing
October 2020

Leukotriene B receptor 1 exacerbates inflammation following myocardial infarction.

FASEB J 2020 06 8;34(6):8749-8763. Epub 2020 May 8.

Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

Leukotriene B receptor 1 (BLT1), a high-affinity G-protein-coupled receptor for leukotriene B4 (LTB ), is expressed on various inflammatory cells and plays critical roles in several inflammatory diseases. In myocardial infarction (MI), various inflammatory cells are known to be recruited to the infarcted area, but the function of BLT1 in MI is poorly understood. Here, we investigated the role of BLT1 in MI and the therapeutic effect of a BLT1 antagonist, ONO-4057, on MI. Mice with infarcted hearts showed increased BLT1 expression and LTB levels. BLT1-knockout mice with infarcted hearts exhibited attenuated leukocyte infiltration, proinflammatory cytokine production, and cell death, which led to reduced mortality and improved cardiac function after MI. Bone-marrow transplantation studies showed that BLT1 expressed on bone marrow-derived cells was responsible for the exacerbation of inflammation in infarcted hearts. Furthermore, ONO-4057 administration attenuated the inflammatory responses in hearts surgically treated for MI, which resulted in reduced mortality and improved cardiac function after MI. Our study demonstrated that BLT1 contributes to excessive inflammation after MI and could represent a new therapeutic target for MI.
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http://dx.doi.org/10.1096/fj.202000041RDOI Listing
June 2020

The role of propofol hydroxyl group in 5-lipoxygenase recognition.

Biochem Biophys Res Commun 2020 05 11;525(4):909-914. Epub 2020 Mar 11.

Department of Biochemistry, Juntendo University Faculty of Medicine, Bunkyo-Ku, Tokyo, 113-8421, Japan. Electronic address:

Propofol is a clinically important intravenous anesthetic. We previously reported that it directly inhibited 5-lipoxygenase (5-LOX), a key enzyme for leukotriene biosynthesis. Because the hydroxyl group in propofol (propofol 1-hydroxyl) is critical for its anesthetic effect, we examined if its presence would be inevitable for 5-lipoxygenase recognition. Fropofol is developed by substituting the hydroxy group in propofol with fluorine. We found that propofol 1-hydroxyl was important for 5-lipoxygenase recognition, but it was not absolutely necessary. Azi-fropofol bound to 5-LOX at one of the two propofol binding sites of 5-LOX (pocket around Phe-187), suggesting that propofol 1-hydroxyl is important for 5-LOX inhibition at the other propofol binding site (pocket around Val-431). Interestingly, 5-hydroperoxyeicosatetraenoic acid (5-HpETE) production was significantly increased by stimulation with calcium ionophore A23187 in HEK293 cells expressing 5-LOX, suggesting that the fropofol binding site is important for the conversion from 5-HpETE to leukotriene A. We also indicated that propofol 1-hydroxyl might have contributed to interaction with wider targets among our body.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167355PMC
May 2020

n-3 Fatty Acid and Its Metabolite 18-HEPE Ameliorate Retinal Neuronal Cell Dysfunction by Enhancing Müller BDNF in Diabetic Retinopathy.

Diabetes 2020 04 5;69(4):724-735. Epub 2020 Feb 5.

Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Diabetic retinopathy (DR) is a widespread vision-threatening disease, and neuroretinal abnormality should be considered as an important problem. Brain-derived neurotrophic factor (BDNF) has recently been considered as a possible treatment to prevent DR-induced neuroretinal damage, but how BDNF is upregulated in DR remains unclear. We found an increase in hydrogen peroxide (HO) in the vitreous of patients with DR. We confirmed that human retinal endothelial cells secreted HO by high glucose, and HO reduced cell viability of MIO-M1, Müller glia cell line, PC12D, and the neuronal cell line and lowered BDNF expression in MIO-M1, whereas BDNF administration recovered PC12D cell viability. Streptozocin-induced diabetic rats showed reduced BDNF, which is mainly expressed in the Müller glia cell. Oral intake of eicosapentaenoic acid ethyl ester (EPA-E) ameliorated BDNF reduction and oscillatory potentials (OPs) in electroretinography (ERG) in DR. Mass spectrometry revealed an increase in several EPA metabolites in the eyes of EPA-E-fed rats. In particular, an EPA metabolite, 18-hydroxyeicosapentaenoic acid (18-HEPE), induced BDNF upregulation in Müller glia cells and recovery of OPs in ERG. Our results indicated diabetes-induced oxidative stress attenuates neuroretinal function, but oral EPA-E intake prevents retinal neurodegeneration via BDNF in Müller glia cells by increasing 18-HEPE in the early stages of DR.
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http://dx.doi.org/10.2337/db19-0550DOI Listing
April 2020

Prostaglandin E and its receptor EP2 trigger signaling that contributes to YAP-mediated cell competition.

Genes Cells 2020 Mar 7;25(3):197-214. Epub 2020 Feb 7.

Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.

Cell competition is a biological process by which unfit cells are eliminated from "cell society." We previously showed that cultured mammalian epithelial Madin-Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by "normal" MDCK cells. However, the molecular mechanism underlying the elimination of active YAP-expressing cells was unknown. Here, we used high-throughput chemical compound screening to identify cyclooxygenase-2 (COX-2) as a key molecule triggering cell competition. Our work shows that COX-2-mediated PGE secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase-cyclic AMP-PKA pathway that, in the presence of active YAP, induces E-cadherin internalization leading to apical extrusion. Thus, COX-2-induced PGE appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.
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http://dx.doi.org/10.1111/gtc.12750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078805PMC
March 2020

Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.

FASEB J 2019 12 2;33(12):14528-14541. Epub 2019 Nov 2.

Department of Anesthesiology, Critical Care, and Pain Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

General anesthesia has been the requisite component of surgical procedures for over 150 yr. Although immunomodulatory effects of volatile anesthetics have been growingly appreciated, the molecular mechanism has not been understood. In septic mice, the commonly used volatile anesthetic isoflurane attenuated the production of 5-lipoxygenase products and IL-10 and reduced CD11b and intercellular adhesion molecule-1 expression on neutrophils, suggesting the attenuation of TLR4 signaling. We confirmed the attenuation of TLR4 signaling and their direct binding to TLR4-myeloid differentiation-2 (MD-2) complex by photolabeling experiments. The binding sites of volatile anesthetics isoflurane and sevoflurane were located near critical residues for TLR4-MD-2 complex formation and TLR4-MD-2-LPS dimerization. Additionally, TLR4 activation was not attenuated by intravenous anesthetics, except for a high concentration of propofol. Considering the important role of TLR4 system in the perioperative settings, these findings suggest the possibility that anesthetic choice may modulate the outcome in patients or surgical cases in which TLR4 activation is expected.-Okuno, T., Koutsogiannaki, S., Hou, L., Bu, W., Ohto, U., Eckenhoff, R. G., Yokomizo, T., Yuki, K. Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.
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http://dx.doi.org/10.1096/fj.201901570RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894077PMC
December 2019

The volatile anesthetic sevoflurane reduces neutrophil apoptosis Fas death domain-Fas-associated death domain interaction.

FASEB J 2019 11 30;33(11):12668-12679. Epub 2019 Aug 30.

Cardiac Anesthesia Division, Department of Anesthesiology, Critical Care, and Pain Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

Sepsis remains a significant health care burden, with high morbidities and mortalities. Patients with sepsis often require general anesthesia for procedures and imaging studies. Knowing that anesthetic drugs can pose immunomodulatory effects, it would be critical to understand the impact of anesthetics on sepsis pathophysiology. The volatile anesthetic sevoflurane is a common general anesthetic derived from ether as a prototype. Using a murine sepsis model induced by cecal ligation and puncture surgery, we examined the impact of sevoflurane on sepsis outcome. Different from volatile anesthetic isoflurane, sevoflurane exposure significantly improved the outcome of septic mice. This was associated with less apoptosis in the spleen. Because splenic apoptosis was largely attributed to the apoptosis of neutrophils, we examined the effect of sevoflurane on FasL-induced neutrophil apoptosis. Sevoflurane exposure significantly attenuated apoptosis. Sevoflurane did not affect the binding of FasL to the extracellular domain of Fas receptor. Instead, analysis suggested that sevoflurane would bind to the interphase between Fas death domain (DD) and Fas-associated DD (FADD). The effect of sevoflurane on Fas DD-FADD interaction was examined using fluorescence resonance energy transfer (FRET). Sevoflurane attenuated FRET efficiency, indicating that sevoflurane hindered the interaction between Fas DD and FADD. The predicted sevoflurane binding site is known to play a significant role in Fas DD-FADD interaction, supporting our and apoptosis results.-Koutsogiannaki, S., Hou, L., Babazada, H., Okuno, T., Blazon-Brown, N., Soriano, S. G., Yokomizo, T., Yuki, K. The volatile anesthetic sevoflurane reduces neutrophil apoptosis Fas death domain-Fas-associated death domain interaction.
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http://dx.doi.org/10.1096/fj.201901360RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988854PMC
November 2019

Dietary supplementation of omega-3 fatty acid eicosapentaenoic acid does not ameliorate pruritus in murine models of atopic dermatitis and psoriasis.

J Dermatol Sci 2019 09 27;95(3):130-133. Epub 2019 Jul 27.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8421, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jdermsci.2019.07.010DOI Listing
September 2019

The Role of Leukotrienes as Potential Therapeutic Targets in Allergic Disorders.

Int J Mol Sci 2019 Jul 22;20(14). Epub 2019 Jul 22.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.

Leukotrienes (LTs) are lipid mediators that play pivotal roles in acute and chronic inflammation and allergic diseases. They exert their biological effects by binding to specific G-protein-coupled receptors. Each LT receptor subtype exhibits unique functions and expression patterns. LTs play roles in various allergic diseases, including asthma (neutrophilic asthma and aspirin-sensitive asthma), allergic rhinitis, atopic dermatitis, allergic conjunctivitis, and anaphylaxis. This review summarizes the biology of LTs and their receptors, recent developments in the area of anti-LT strategies (in settings such as ongoing clinical studies), and prospects for future therapeutic applications.
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http://dx.doi.org/10.3390/ijms20143580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679143PMC
July 2019

An Alternative Pathway to Leukotriene B Enantiomers Involving a 1,8-Diol-Forming Reaction of an Algal Oxylipin.

Org Lett 2019 06 13;21(12):4667-4670. Epub 2019 Jun 13.

Institute for Inorganic and Analytical Chemistry, Department of Instrumental Analytics/Bioorganic Analytics , Friedrich-Schiller-University Jena , 07743 Jena , Germany.

Oxidized lipids function as tissue hormones in mammals. An important class of these oxylipins are the immunomodulatory leukotrienes (LTs). Besides mammals, marine algae produce bioactive oxylipins, including LTs. The novel acid-labile oxylipin, (5 R,8 S)-dihydroxy eicosatetraenoic acid, from the edible alga Gracilaria vermiculophylla rearranges via a 1,8-diol-forming mechanism to inflammatory LTB enantiomers that exhibit an enantio-specific potency toward LTB receptor 1. This alternative pathway to a well-known leukotriene may explain food poisoning after Gracilaria consumption.
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http://dx.doi.org/10.1021/acs.orglett.9b01554DOI Listing
June 2019

Biological functions of 12()-hydroxyheptadecatrienoic acid as a ligand of leukotriene B receptor 2.

Inflamm Regen 2018 29;38:29. Epub 2018 Oct 29.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Although 12()-hydroxyheptadecatrienoic acid (12-HHT) is an abundant fatty acid, it is long considered a byproduct of thromboxane A production. We identified a leukotriene B receptor 2 (BLT2)-specific agonistic activity in lipid extracts from rat small intestine, and mass spectrometric analysis of partially purified lipids containing BLT2 agonistic activity revealed that 12-HHT is an endogenous ligand of BLT2. In a dextran sulfate sodium (DSS)-induced inflammatory colitis model, BLT2-deficient mice exhibited enhanced intestinal inflammation, possibly due to impaired epithelial barrier function. In a skin wound healing model, BLT2-deficient mice exhibited delayed wound healing via dampened keratinocyte migration. BLT2 also accelerates corneal wound healing, and eye drops containing a non-steroidal anti-inflammatory drug (NSAID) inhibit the production of 12-HHT, resulting in delayed corneal wound healing. Furthermore, BLT2 is expressed in pulmonary epithelial type II cells and vascular endothelial cells in the mouse lung, and BLT2-deficient mice are more susceptible to lung damage by pneumolysin. In this review, we summarize the identification and characterization of 12-HHT as a ligand for BLT2 and discuss recent research on the physiological and pathophysiological roles of the 12-HHT-BLT2 axis. Some side effects of NSAIDs such as delayed wound healing may be caused by reduced 12-HHT production rather than diminished production of prostaglandins.
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http://dx.doi.org/10.1186/s41232-018-0087-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205785PMC
October 2018

Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating T2 immune responses.

FASEB J 2019 03 1;33(3):3392-3403. Epub 2018 Nov 1.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Allergic conjunctivitis (AC) is one of the most common ocular surface diseases in the world. In AC, T helper type 2 (T2) immune responses play central roles in orchestrating inflammatory responses. However, the roles of lipid mediators in the onset and progression of AC remain to be fully explored. Although previous reports have shown the beneficial effects of supplementation of ω-3 fatty acids in asthma or atopic dermatitis, the underlying molecular mechanisms are poorly understood. In this study, a diet rich in ω-3 fatty acids alleviated AC symptoms in both early and late phases without affecting T2 immune responses, but rather by altering the lipid mediator profiles. The ω-3 fatty acids completely suppressed scratching behavior toward the eyes, an allergic reaction provoked by itch. Although total serum IgE levels and the expression levels of T2 cytokines and chemokines in the conjunctiva were not altered by ω-3 fatty acids, eosinophil infiltration into the conjunctiva was dramatically suppressed. The levels of ω-6-derived proinflammatory lipid mediators, including those with chemoattractant properties for eosinophils, were markedly reduced in the conjunctivae of ω-3 diet-fed mice. Dietary ω-3 fatty acids can alleviate a variety of symptoms of AC by altering the lipid mediator profile.-Hirakata, T., Lee, H.-C., Ohba, M., Saeki, K., Okuno, T., Murakami, A., Matsuda, A., Yokomizo, T. Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating T2 immune responses.
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http://dx.doi.org/10.1096/fj.201801805RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404575PMC
March 2019

Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models.

JCI Insight 2018 09 20;3(18). Epub 2018 Sep 20.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.
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http://dx.doi.org/10.1172/jci.insight.96902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237215PMC
September 2018

Profiling of bioactive lipids in different dendritic cell subsets using an improved multiplex quantitative LC-MS/MS method.

Biochem Biophys Res Commun 2018 10 12;504(3):562-568. Epub 2018 Jun 12.

Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, Japan.

Lipids are an energy source and key components of the cell membrane; however, they are also bioactive mediators of physiological and pathophysiological phenomena. Quantification of bioactive lipids is not easy because they have diverse chemical properties and are present in trace amounts. Here, we improved a multiplex method of quantifying bioactive lipids, thereby enabling measurement of 90 compounds simultaneously. We then used this system to quantify bioactive lipids produced by two subsets of dendritic cells (DCs): all-trans retinoic acid-treated DCs (RA-DCs) (a type of tolerogenic DC (tDC)) and conventional DCs (cDCs). We found that cDCs produced inflammatory lipid mediators such as leukotrienes, whereas RA-DCs produced anti-inflammatory lipid mediators such as prostaglandin I. Consistent with this, cDCs expressed larger amounts of mRNA encoding 5-lipoxygenase and LTA hydrolase (both responsible for leukotriene biosynthesis) and RA-DCs produced larger amounts of mRNA encoding prostaglandin I synthase. Taken together, the results suggest that the method is useful for clarifying the roles of bioactive lipids during immune responses.
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http://dx.doi.org/10.1016/j.bbrc.2018.06.026DOI Listing
October 2018

Endurance exercise training and high-fat diet differentially affect composition of diacylglycerol molecular species in rat skeletal muscle.

Am J Physiol Regul Integr Comp Physiol 2018 06 14;314(6):R892-R901. Epub 2018 Feb 14.

Graduate School of Health and Sports Science, Juntendo University, Inzai, Chiba, Japan.

Insulin resistance of peripheral muscle is implicated in the etiology of metabolic syndrome in obesity. Although accumulation of glycerolipids, such as triacylglycerol and diacylglycerol (DAG), in muscle contributes to insulin resistance in obese individuals, endurance-trained athletes also have higher glycerolipid levels but normal insulin sensitivity. We hypothesized that the difference in insulin sensitivity of skeletal muscle between athletes and obese individuals stems from changes in fatty acid composition of accumulated lipids. Here, we evaluated the effects of intense endurance exercise and high-fat diet (HFD) on the accumulation and composition of lipid molecular species in rat skeletal muscle using a lipidomic approach. Sprague-Dawley female rats were randomly assigned to three groups and received either normal diet (ND) in sedentary conditions, ND plus endurance exercise training, or HFD in sedentary conditions. Rats were fed ND or HFD between 4 and 12 wk of age. Rats in the exercise group ran on a treadmill for 120 min/day, 5 days/wk, for 8 wk. Soleus muscle lipidomic profiles were obtained using liquid chromatography/tandem mass spectrometry. Total DAG levels, particularly those of palmitoleate-containing species, were increased in muscle by exercise training. However, whereas the total DAG level in the muscle was also increased by HFD, the levels of DAG molecular species containing palmitoleate were decreased by HFD. The concentration of phosphatidylethanolamine molecular species containing palmitoleate was increased by exercise but decreased by HFD. Our results indicate that although DAG accumulation was similar levels in trained and sedentary obese rats, specific changes in molecular species containing palmitoleate were opposite.
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http://dx.doi.org/10.1152/ajpregu.00371.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032301PMC
June 2018

Altered eicosanoid production and phospholipid remodeling during cell culture.

J Lipid Res 2018 03 20;59(3):542-549. Epub 2018 Jan 20.

Department of Pharmacology, Anschutz Medical Campus, University of Colorado Denver, Aurora, CO 80045

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B, PGE, and PGD, and the 5-lipoxygenase pathway, leukotriene (LT)B, LTC, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB, all -LTB, LTC, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.
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http://dx.doi.org/10.1194/jlr.M083030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832926PMC
March 2018

Na-mimicking ligands stabilize the inactive state of leukotriene B receptor BLT1.

Nat Chem Biol 2018 03 8;14(3):262-269. Epub 2018 Jan 8.

RIKEN Structural Biology Laboratory, Tsurumi-ku, Yokohama, Kanagawa, Japan.

Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B (LTB) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D66, and mimics the entire sodium ion-centered water cluster. Thus, BLT1 is fixed in the inactive state, and the transmembrane helices cannot change their conformations to form the active state. Moreover, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. As the residues involved in the benzamidine binding are widely conserved among GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs. Consequently, the present structure will enable the rational development of inverse agonists specific for each GPCR.
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http://dx.doi.org/10.1038/nchembio.2547DOI Listing
March 2018

Non-steroidal anti-inflammatory drug delays corneal wound healing by reducing production of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B receptor 2.

Sci Rep 2017 10 16;7(1):13267. Epub 2017 Oct 16.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce inflammation by suppressing cyclooxygenases (COXs). NSAID eye drops are frequently prescribed after ocular surgery to reduce inflammation and pain, but this treatment has clinically significant side effects, including corneal ulcer and perforation. The molecular mechanisms underlying these side effects remain unknown. Recently, the COX product 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as an endogenous ligand for leukotriene B receptor 2 (BLT2), which is important in maintenance of epithelial homeostasis. We hypothesized that NSAID-dependent corneal damage is caused by reduced production of 12-HHT. Diclofenac eye drops decreased the abundance of downstream products of COX and delayed corneal wound healing in BALB/c mice. Expression of BLT2 was observed in murine ocular tissues including cornea, and in human corneal epithelial cell line and human primary corneal epithelial cells. In BLT2-knockout mice, corneal wound healing was delayed, but the diclofenac-dependent delay in corneal wound healing disappeared. 12-HHT accelerated wound closure both in BLT2-transfected corneal cell line and human primary corneal epithelial cells. Thus, our results reveal that NSAIDs delay corneal wound healing by inhibiting 12-HHT production, and suggest that stimulation of the 12-HHT/BLT2 axis represents a novel therapeutic approach to corneal wound healing.
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http://dx.doi.org/10.1038/s41598-017-13122-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643301PMC
October 2017

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1.

PLoS One 2017 18;12(9):e0185133. Epub 2017 Sep 18.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185133PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602668PMC
October 2017

Urinary prostaglandin D metabolite excretion during the first six months of life was significantly lower in breast-fed than formula-fed infants.

Acta Paediatr 2018 Jan 27;107(1):95-100. Epub 2017 Sep 27.

Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Aim: The metabolic changes that occur during the postnatal weaning period appear to be particularly important for future health, and breast milk is considered to provide the optimal source of infant nutrition. This pilot study from September 2013 to May 2015 examined the effect of breastfeeding on prostaglandin metabolism in healthy term infants.

Methods: Urine samples were collected from 19 infants at one month of age in the Juntendo University Hospital, Tokyo, Japan. The 13 infants in the breast-fed group received less than 540 mL/week of their intake from formula, and the other six were exclusively fed on formula. At six months, we sampled 14 infants: nine breast-fed and five receiving formula. The infants were from normal single pregnancies and free from perinatal complications. We analysed urinary prostaglandin metabolites-tetranor prostaglandin E metabolite (t-PGEM) and tetranor prostaglandin D metabolite (t-PGDM)-using liquid chromatography tandem-mass spectrometry.

Results: Urinary t-PGDM excretion at one and six months was significantly lower in breast-fed infants than formula-fed infants. However, urinary t-PGEM excretion at one and six months was not significantly different between the groups.

Conclusion: Our study showed that the type of feeding in early infancy affected prostaglandin metabolism in healthy term infants.
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http://dx.doi.org/10.1111/apa.14068DOI Listing
January 2018

Determination of Double Bond Positions in Polyunsaturated Fatty Acids Using the Photochemical Paternò-Büchi Reaction with Acetone and Tandem Mass Spectrometry.

Anal Chem 2017 08 2;89(16):8545-8553. Epub 2017 Aug 2.

Department of Pharmacology, University of Colorado Denver , Anschutz Medical Campus, 12801 E. 17th Ave., Aurora, Colorado 80045, United States.

The positions of double bonds along the carbon chain of methylene interrupted polyunsaturated fatty acids are unique identifiers of specific fatty acids derived from biochemical reactions that occur in cells. It is possible to obtain direct structural information as to these double bond positions using tandem mass spectrometry after collisional activation of the carboxylate anions of an acetone adduct at each of the double bond positions formed by the photochemical Paternò-Büchi reaction with acetone. This reaction can be carried out by exposing a small portion of an inline fused silica capillary to UV photons from a mercury vapor lamp as the sample is infused into the electrospray ion source of a mass spectrometer. Collisional activation of [M - H] yields a series of reverse Paternò-Büchi reaction product ions that essentially are derived from cleavage of the original carbon-carbon double bonds that yield an isopropenyl carboxylate anion corresponding to each double bond location. Aldehydic reverse Paternò-Büchi product ions are much less abundant as the carbon chain length and number of double bonds increase. The use of a mixture of D/D-acetone facilitates identification of these double bonds indicating product ions as shown for arachidonic acid. If oxygen is present in the solvent stream undergoing UV photoactivation, ozone cleavage ions are also observed without prior collisional activation. This reaction was used to determine the double bond positions in a 20:3 fatty acid that accumulated in phospholipids of RAW 264.7 cells cultured for 3 days.
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http://dx.doi.org/10.1021/acs.analchem.7b02375DOI Listing
August 2017

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

Biochem Biophys Res Commun 2017 May 1;486(4):1077-1082. Epub 2017 Apr 1.

Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan. Electronic address:

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.
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http://dx.doi.org/10.1016/j.bbrc.2017.03.165DOI Listing
May 2017
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