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Linköping University Medical Dissertations No. 1287 The enzymatic machinery of leukotriene biosynthesis: Studies on ontogenic expression, interactions and function Tobias Strid Division of Cell Biology Department of Clinical and Experimental Medicine Faculty of Health Sciences, Linköping University SE-‐581 85 Linköping, Sweden www.liu.se © Tobias Strid 2012 Cover: Pixel-‐inverted scan of LTA4H in situ hybridized mouse e18.5 fetus. Printed by LiU-‐Tryck, Linköping, Sweden 2012 ISBN: 978-‐91-‐7519-‐987-‐0 ISSN 0345-‐0082 Published articles were reprinted with permission from the copyright holder Elsevier Ltd. According to Elsevier’s policy on author postings Research is the act of going up alleys to see if they are blind // Plutarch Supervisor: Faculty opponent: Professor Sven Hammarström Professor Ralf Morgenstern Department of Clinical and Experimental Institute of Environmental Medicine Medicine, Faculty of Health Sciences, Karolinska Institute, Stockholm Board committee: Dr. Mats Söderström Dr. David Engblom Department of Clinical and Experimental Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Medicine, Faculty of Health Sciences, Linköping University Linköping University Linköping University Co-‐supervisor: Professor Jan Ernerudh Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University Professor Ernst Oliw Department of Pharmaceutical Biosciences Uppsala University Professor Christer Tagesson Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University List of Papers LIST OF PAPERS This thesis is based upon the following papers referred to in the text by roman numerals: I: Strid T., Söderström M., and Hammarström S. (2008) Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity. Biochem Biophys Res Commun. 366: 80-‐85. http://www.journals.elsevier.com/biochemical-‐and-‐biophysical-‐research-‐ communications/ II: Strid T., Svartz J., Franck N., Hallin E., Ingelsson B., Söderström M., and Hammarström S. (2009) Distinct parts of leukotriene C4 synthase interact with 5-‐lipoxygenase and 5-‐lipoxygenase activating protein, Biochem Biophys Res Commun. 381: 518-‐522. III: Strid T., Karlsson C., Söderström M., Zhang J., Qian H., Sigvardsson M., and Hammarström S. (2009) Fetal hepatic expression of 5-‐lipoxygenase activating protein is confined to colonizing hematopoietic cells, Biochem Biophys Res Commun. 383: 336-‐339. IV: Strid T., Sigvardsson M., Karlsson C., Söderström M., Qiang H., and Hammarström S. (2012) Expression of leukotriene biosynthesis proteins in fetal and adult hematopoietic cells and its functional effects on hematopoiesis. Manuscript. I Populärvetenskaplig Sammanfattning POPULÄRVETENSKAPLIG SAMMANFATTNING Leukotriener tillhör gruppen eikosanoider som är biologiskt aktiva substanser bildade från fleromättade fettsyror. De verkar proinflammatoriskt och deltar i utvecklandet av inflammatoriska sjukdomar såsom astma, allergi, hjärtinfarkt och stroke. De bildas genom att enzymet 5-‐lipoxygenas (5-‐LO) tillsammans med aktiveringsproteinet FLAP, omvandlar arakidonsyra, till leukotrien A4 (LTA4). FLAP saknar egen enzymatisk aktivitet, och dess roll anses vara att överföra arakidonsyra från fosfolipas A2 till 5-‐LO. LTA4 är en instabil molekyl som spontant bryts ned om den inte omvandlas till LTB4 av enzymet LTA4 hydrolas eller till LTC4 av LTC4 syntas. Leukotriener bildas runt cellens kärnmembran där FLAP och LTC4S är belägna och dit 5-‐LO förflyttas i samband med inflammatorisk aktivering av cellen. Vi har studerat proteinerna som möjliggör syntes av LTC4 och visat att FLAP och LTC4S kan binda till varandra genom sina membrangenomträngande delar. Proteinerna binder till 5-‐LO genom sina vattenlösliga delar som sticker upp ur membranet. De proteiner som behövs för att bilda leukotriener finns i vissa typer av vita blodkroppar. LTA4H finns i de flesta celler medan övriga proteiner finns i betydligt färre celltyper. Vi undersökte LTC4S genens uttryck genom att konstruera en vektor som uttrycker det lätt påvisbara proteinet GFP under reglering av promotorn för LTC4S. Denna vektor gav ett cellspecifikt uttryck liknande det naturliga för LTC4S när vektorn fördes in i olika celltyper. GFP uttrycket ökade av ämnen som tidigare visats stimulera LTC4S. Vektorn är alltså lämplig som rapportör för LTC4S uttryck. II Populärvetenskaplig Sammanfattning DNA från vektorn användes för att ta fram genmodifierade möss som uttrycker GFP som LTC4S markör. Leukotrienbiosyntesproteiners uttryck under fosterutveckling undersöktes med två andra tekniker som specifikt påvisar enskilda proteiners mRNA respektive proteinet självt. Resultaten visade att ett komplett maskineri för leukotriensyntes uttrycks i levern under fosterutvecklingen. Under denna tid sker här blodbildning från celler som koloniserar levern. Vi särskilde olika celltyper i fetal lever genom cellsortering och undersökte vilka som uttryckte mRNA kodande för proteiner viktiga för leukotriensyntes. Störst mängd fanns i mogna myeloida celler, men även i omogna blodceller fann vi FLAP. Detta fick oss att spekulera kring leukotrieners roll i att reglera själva blodbildningen. Vi undersökte detta genom att analysera cellsammansättningen i blod och benmärg från möss som saknar FLAP och leukotrien produktion. Det var tidigare känt att sådana möss har dämpade inflammatoriska svar. Våra resultat visade att förhållandet av B-‐ till T-‐lymfocyter var lägre hos dessa möss jämfört med kontrolldjur. Resultaten tyder på att leukotriener deltar i reglering av blodcellers differentiering och därmed är möjliga mål vid behandling av sjukdomar som drabbar blodbildningen. II Abstract ABSTRACT Leukotrienes (LTs) are biologically active arachidonic acid (AA) derivatives generated by the 5-‐lipoxygenase (5-‐LO) pathway. They are produced by myeloid cells. 5-‐LO converts AA to LTA4 in cooperation with 5-‐LO activating protein (FLAP). LTA4 is converted to LTB4, by LTA4-‐hydrolase (LTA4H) or to LTC4 by LTC4-‐synthase (LTC4S). LTs act on cells through plasma membrane bound G-‐protein coupled receptors found on leukocytes, smooth muscle and endothelial cells. We report here protein-‐protein interactions of proteins involved in LTC4 synthesis. 5-‐LO interacts with cytosolic domains of the integral membrane proteins FLAP and LTC4S at the nuclear envelope, in addition LTC4S interacts with FLAP through its hydrophobic membrane spanning regions. We constructed an LTC4S promoter controlled GFP reporter vector, displaying cell specific expression and sensitivity to agents known to affect LTC4S expression. The vector was used to create transgenic mice expressing GFP as a reporter for LTC4S. Ontogenic mouse expression studies revealed that the complete LT biosynthesis machinery was present at e11.5 primarily in the hematopoietic cells colonizing the liver. Although mature myeloid cells were the main contributors, a substantial amount of FLAP message was also detected in hematopoietic stem and progenitor cells, indicating possible functions for FLAP in hematopoietic regulation. Functional analyses using FLAP knockout mice suggested fine-‐tuning roles for LTs during differentiation, primarily along the B-‐lymphocyte differentiation path. III LSC, leukemic stem cell LSK, lineage-‐ Sca1+ cKit+ cells LT, leukotriene LTA4H, leukotriene A4 hydrolase LTC4S, leukotriene C4 synthase LT-‐HSC/LSC: long term HSC/LSC MAPEG, membrane-‐associated proteins in eicosanoid and glutathione metabolism MAPK, mitogen activated protein kinase MEP, megakaryocyte and erythroid progenitor mGST, microsomal glutathione S-‐ transferase MPP, multipotential progenitor MRP1, multidrug resistance associated protein 1 MS, multiple sclerosis NE, nuclear envelope NK-‐cell, natural killer cell OAG, 1-‐oleoyl-‐2-‐acteyl-‐sn-‐glycerol PAF, platelet activating factor PG, prostaglandin PI3K, phosphoinositide 3-‐kinase PIP2, phosphatidylinositol-‐2-‐phosphate PKC, protein kinase C PPARα, peroxisome proliferator-‐ activated receptor-‐α PTX, pertussis toxin PUFA, polyunsaturated fatty acid RA, retinoic acid ROS, reactive oxygen spices SNP, single nucleotide polymorphism SP1, specificity protein 1 SP3, specificity protein 3 SRS-‐A, slow reacting substance of anaphylaxis STAT3, signal transducer and activator of transcription 3 ST-‐HSC/LSC, short term HSC/LSC TGF-‐ β, transforming growth factor β TPA, 12-‐O-‐tetradecanoylphorbol-‐13-‐ acetate TX, thromboxane UCB, umbilical cord blood 12-‐HHT, 12(S)-‐hydroxyheptadeca-‐5(Z), 8(E), 10(E)-‐trienoic acid ABBREVIATIONS AA, arachidonic acid AGM, aorta-‐gonad-‐mesonephros AP1, activating protein 1 AP2, activating protein 2 BLT1R, leukotriene B4 receptor 1 BLT2R, leukotriene B4 receptor 2 cAMP, cyclic AMP CLP, common lymphoid progenitors CMP, common myeloid progenitors COX, cyclooxygenase cPLA2, cytosolic phospholipase A2 CysLT, cysteinyl leukotriene CysLT1R, CysLT receptor 1 CysLT2R, CysLT receptor 2 DAG, diacyl glycerol DHGLA, dihomo-‐γ-‐linolenic acid eGFP, enhanced green fluorescent protein EPA, eicosapentaenoic acid ER, endoplasmatic reticulum ERK, extracellular regulated kinase EX, eoxin FACS, fluorescence-‐activated cell sorting FLAP, 5-‐lipoxygenase activating protein FLIM, fluorescence lifetime imaging microscopy GMLP, granulocyte, macrophage, lymphoid progenitor GMP, granulocyte and macrophage progenitor GPCR, G-‐protein coupled receptor GSH, reduced glutathione GST, glutathione S-‐transferase HETE, hydroxyeicosatetraenoic acid HPC, hematopoietic progenitor cell HpETE, hydroperoxyeicosatetraenoic acid HSC, hematopoietic stem cell IP3, Inositol tris-‐phosphate ISH, in situ hybridization LMPP, lymphoid primed multipotential progenitor LO, lipoxygenase LPS, lipopolysaccharide Abbreviations IV Table of contents TABLE OF CONTENTS LIST OF PAPERS ...................................................................................................... I POPULÄRVETENSKAPLIG SAMMANFATTNING .................................... II ABSTRACT ............................................................................................................. III ABBREVIATIONS ................................................................................................. IV 1. INTRODUCTION ............................................................................................. 13 1.1 Lipids and inflammation .......................................................................... 13 1.2 Biosynthesis of eicosanoids ................................................................... 15 1.2.1 Cyclooxygenases .................................................................................................................. 17 1.2.2 Lipoxygenases ....................................................................................................................... 18 2. LEUKOTRIENES AND THE 5-‐LO PATHWAY ...................................... 21 2.1 Biosynthesis of leukotrienes ................................................................. 21 2.1.1 Biochemistry of Leukotrienes ........................................................................................ 21 2.1.2 5-‐LO ..................................................................................................................................... 23 2.1.3 FLAP ..................................................................................................................................... 27 2.1.4 LTC4S ..................................................................................................................................... 29 2.1.5 LTA4H ..................................................................................................................................... 32 2.2 Leukotriene receptors .............................................................................. 35 2.2.1 LTB4-‐receptors ..................................................................................................................... 36 2.2.2 CysLT receptors. ................................................................................................................... 38 2.3 Leukotriene actions ................................................................................... 41 2.3.1 Modulators of inflammatory responses .................................................................... 41 2.3.2 Regulators of hematopoiesis .......................................................................................... 44 3. AIMS .................................................................................................................... 49 Table of contents 4. RESULTS AND DISCUSSION ...................................................................... 51 4.1 Paper I ............................................................................................................. 51 4.2 Generation of GFP mice (unpublished results) .............................. 53 4.3 Paper II ............................................................................................................ 55 4.4 Paper III .......................................................................................................... 56 4.5 Paper IV ........................................................................................................... 57 5. CONCLUSIONS ............................................................................................... 61 6. GENERAL DISCUSSION AND FUTURE PERSPECTIVES ................. 63 7. ACKNOWLEDGEMENTS .............................................................................. 67 7.1 Financial support ........................................................................................ 67 7.2 Personal thank you / Personligt tack ................................................. 67 8. REFERENCES ................................................................................................... 73 9. REPRINTS OF PUBLISHED ARTICLES AND MANUSCRIPTS .... 105 Introduction 1. INTRODUCTION 1.1 Lipids and inflammation An inflammatory process is initiated as an important and immediate response conducted by our bodies to protect us against infections or injuries. This process typically leads to redness, swelling, heat and pain. These hallmarks of inflammation are caused by increases in blood flow, vascular permeability and leukocyte migration as well as stimulation of pain receptors. The purpose of an inflammatory response is to initiate host defense reactions to eliminate intruders, such as bacteria, viruses and parasites and to initiate repair of injured tissues. Inflammation can be viewed upon as a double-‐edged sword that needs a strict and precise control to be neither too weak nor too strong, either of which may be detrimental. The responses of cells involved in the inflammatory reaction are directed by a complicated array of signaling molecules (the topic of inflammation has been reviewed in [1-‐5]). Proinflammatory mediators may be quite diverse at the molecular level. Some of them are bioactive lipids generated from essential fatty acids, and one such family of very potent lipid mediators are the eicosanoids (reviewed in [6, 7]). Essential fatty acids are necessary for survival of mammals and they are found in the phospholipids of most cell membranes. The term “essential” indicates that they cannot be synthesized by the human body and therefore must be obtained by dietary intake. The essential fatty acids are divided into two series; ω-‐6 fatty acids derived from linoleic acid and ω-‐3 fatty acids derived from α-‐linolenic acid (reviewed in [8]). 13 Introduction Essential fatty acids COOH COOH α-Linolenic acid Linoleic acid COOH COOH Dihomo-γ-linolenic acid Eicosapentaenoic acid COOH Arachidonic acid Eicosanoids Fig. 1 Essential fatty acids. The precursors of eicosanoids (dihomo-‐γ-‐linolenic acid, arachidonic acid and eicosapentaenoic acid) are 20-‐carbon fatty acids formed from two 18-‐carbon essential fatty acids: linoleic acid and α-‐linolenic acid. 14 Introduction 1.2 Biosynthesis of eicosanoids Eicosanoids are oxygenated derivatives of 20-‐carbon polyunsaturated fatty acids (PUFAs), which in turn are formed from 18-‐carbon essential fatty acids (Fig. 1). Both the 20-‐ and the 18-‐carbon fatty acids are normal components of phospholipids in the cell membranes. Dihomo-‐γ-‐linolenic acid (DHGLA), AA and eicosapentaenoic acid (EPA) give rise to different series of eicosanoids [9, 10]. The conversion of linoleic acid to AA occurs mainly in the liver. From there AA is distributed to cells throughout the body for incorporation into cell membrane phospholipids [11]. AA is formed by desaturation and elongation of linoleic acid, an essential ω-‐6 fatty acid. These conversions are catalyzed by Δ6-‐desaturase and fatty acid elongase respectively, giving rise to DHGLA which is further converted by Δ5-‐desaturase to AA [12]. AA is incorporated into membrane phospholipids by acylation of 2-‐lysophospholipids with arachidonyl-‐CoA or by transacylation of existing phospholipids [13]. During evolution, human diet presumably contained an equal ratio οf ω-‐6 to ω-‐3 fatty acids. In modern western diets however, this ratio is approximately 16:1[14-‐16]. This has lead to increased formation of eicosanoids derived from AA and less from EPA [15]. AA constitutes around 20% of membrane phospholipid fatty acids in immune cells from people consuming a western diet [17-‐23], whereas EPA occupies < 1% [20, 22]. Increased intake of long chain ω-‐3 PUFAs increases the proportion of these fatty acids in membrane phospholipids [17-‐23] and leads to lower production of prostaglandin E2 (PGE2), thromboxane A2 (TXA2), leukotriene B4 (LTB4), 5-‐hydroxyeicosatetraenoic acid (5-‐HETE) and leukotriene C4 (LTC4) [17-‐19, 21, 24]. 15 Introduction Instead it increases the production of the 3 series prostaglandins (PGs), thromboxane (TXA3) and the 5 series leukotrienes (LTs) [17, 19, 24, 25] some of which are less potent than the corresponding eicosanoids formed from AA [26, 27] (reviewed in [7, 28]). COOH Arachidonic acid (20:4) COX 5-LO O OOH COOH COOH O O PGG2 5-HpETE HO COX 5-LO O O COOH O COOH OH LTA4 PGH2 Fig. 2 Eicosanoid formation. Eicosanoids are formed from arachidonic acid (AA) primarily by two distinct enzymatic pathways: the cyclooxygenase (COX) pathway by which AA is oxygenated in two-‐steps to an unstable prostaglandin endoperoxide intermediate (PGH2) and the 5-‐lipoxygenase (5-‐LO) pathway, which in two-‐steps converts AA to the unstable epoxide intermediate LTA4. Enzymes involved in eicosanoid formation use free AA as substrate and the hydrolytic release of AA is a rate limiting step in eicosanoid biosynthesis [29]. The availability of free AA in cells is strictly regulated [30] and the cellular level of free AA is low [31]. AA and other PUFAs are released by phospholipase A2 enzymes [30], most commonly cytosolic phospholipase A2 (cPLA2) [32, 33]. 16 Introduction cPLA2 is activated by an increase in intracellular Ca2+ concentration [34, 35] and by phosphorylation [36-‐39]. Ca2+ activates cPLA2 directly by interaction with a Ca2+-‐binding C2 domain in the N-‐terminal part of the protein and indirectly through protein kinases [40, 41]. Activation results in translocation of the protein from cytosol to the nuclear envelope (NE), endoplasmic reticulum (ER) and Golgi [42-‐47] which have higher AA content than other cell membranes [48, 49] (reviewed in [30]). Conversion of free AA to eicosanoids takes place via two main enzymatic pathways: the cyclooxygenase (COX) and lipoxygenase (LO) pathways (Fig. 2). A third pathway (the cytochrome P450 pathway) catalyzes conversion of AA to vasoactive hydroxyeicosatetraenoic and epoxyeicosatrienoic acids (reviewed in [50]). 1.2.1 Cyclooxygenases The COX isoenzymes, COX-‐1[51] and COX-‐2 [52, 53], metabolize AA to the prostaglandin endoperoxide PGH2. Both isoenzymes are localized to the NE and ER. COX-‐1 is constitutively expressed while COX-‐2 expression is inducible [54, 55]. The first steps catalyzed by COX involve dual oxygenation of AA to PGG2. This is followed by reduction of a 15-‐hydroperoxy group in PGG2 to a 15-‐hydroxyl in PGH2. PGH2 is the immediate precursor of the primary PGs, prostacyclin (PGI2) and TXA2 and the subsequent products are formed by specific synthase enzymes (Fig. 3). A more detailed description of the COX pathway is beyond the scoop of this thesis. This topic has been reviewed in [56-‐58]. 17 Introduction OH COOH O COOH O OH O TXA2 OH PGD2 O O COOH COOH O OH OH PGH2 OH PGE2 OH CO OH O COOH OH OH OH PGF2α PGI2 Fig. 3 The COX pathway. The precursor prostaglandin endoperoxide PGH2 is generated from AA by cyclooxygenases. Specific synthases convert PGH2 to primary prostaglandins (PGD2, PGE2 and PGF2 ), prostacyclin (PGI2) and thromboxane A2 (TXA2). α 1.2.2 Lipoxygenases Lipoxygenases are a group of enzymes, which oxygenate PUFAs containing methylene interrupted cis-‐double bonds, notably the ω-‐3 and the ω-‐6 series essential fatty acids. They are named after the carbon atom where the O2 is introduced. The products from AA are hydroperoxy-‐eicosatetraenoic-‐acids (HpETE). One 1,4-‐cis-‐cis-‐diene is transformed into a 1-‐hydroperoxy-‐2-‐ trans-‐4-‐cis-‐diene group [59]. AA contains three 1,4-‐cis-‐cis-‐diene groups and O2 can be introduced at 6 different positions to give 5-‐, 8-‐, 9-‐, 11-‐, 12-‐, or 15-‐HpETE (Fig. 4). HpETEs are reduced to HETEs by peroxidases. 18 Introduction Lipoxygenases occur in fungi, plants, and animals [60]. In addition to the 5-‐LO pathway, described in detail below, human cells may express 12-‐ and/or 15-‐lipoxygenases. Platelet and leukocyte forms of 12-‐LO and reticulocyte/leukocyte and epidermis types of 15-‐LO are expressed in humans. Leukocyte 12-‐LO and reticulocyte 15-‐LO form the same products and are also referred to as 12/15-‐LO. Expression of 12-‐ and 15-‐LO differs between species; mice do not express 15-‐LO, but express leukocyte type 12-‐LO with both 12-‐LO and 15-‐LO activity (reviewed in [61]). 12-‐LO and 15-‐LO are involved in lipoxin formation (reviewed in [62, 63]). A role in cancer metastasis has been proposed for 12-‐LO and epidermal 15-‐LO has been associated with suppression of carcinogenesis (reviewed in [64]). COOH Arachidonic acid (20:4) 5-LO 15-LO 12-LO OOH COOH COOH OOH 5-HpETE COOH 15-HpETE OOH 12-HpETE Fig. 4 The LO pathway. Lipoxygenases convert AA to HpETEs. The reaction is stereospecific and the lipoxygenases are named after the carbon atom where molecular oxygen is introduced. Human 5-‐, 12-‐ and 15-‐lipoxygenases have been identified. 19 20 Leukotrienes and the 5-‐LO pathway 2. LEUKOTRIENES AND THE 5-‐LO PATHWAY 2.1 Biosynthesis of leukotrienes The 5-‐LO pathway generates the proinflammatory lipid mediators leukotrienes. The main protein components of this pathway are 5-‐LO, FLAP, LTA4H and LTC4S. 5-‐LO, assisted by FLAP, transforms AA to LTA4, which is metabolized either by LTA4H to LTB4, or by LTC4S to LTC4. 2.1.1 Biochemistry of Leukotrienes Leukotrienes of the 4 series are formed from AA following its release from membrane phospholipids by cPLA2 (see above on pages 16-‐17). Major determinants for the magnitude of LT biosynthesis are the concentration of free AA [65] and its accessibility [66]. Most stimuli of LT synthesis activate both 5-‐LO and cPLA2 [67]. 5-‐LO catalyzes a two-‐step conversion of AA via 5-‐HpETE to LTA4 (reviewed in [68, 69]). These steps are stereospecific and initiated by abstraction of a hydrogen atom from carbon C-‐7 of AA and insertion of molecular oxygen at C-‐5, giving 5-‐HpETE [70] (Fig. 4). In the second reaction the 10D (pro-‐R) hydrogen is removed followed by radical migration and formation of the allylic epoxide LTA4 [71-‐73]1 (Fig. 5). LTA4 is unstable and rapidly metabolized to either LTB4 or LTC4. LTA4H catalyzes a hydrolytic reaction to the dihydroxy acid LTB4 [68, 69] (Fig. 5), which is exported out of the cell by a carrier-‐mediated, temperature sensitive and energy-‐dependent mechanism [74, 75]. LTC4S catalyzes a glutathione-‐S-‐ transferase reaction yielding the peptidolipid LTC4. 1 In [73] EPA was used instead of AA and the absolute configuration of the 10D-‐hydrogen in EPA is pro(S) and 5-‐LO conversion yields LTA5. 21 Leukotrienes and the 5-‐LO pathway The discovery of LTC4 in 1979 [76, 77] was a result of investigations to find the chemical structure of a factor termed slow reacting substance of anaphylaxis (SRS-‐A) [78]. LTC4 is formed by conjugation of LTA4 with reduced glutathione (GSH) catalyzed by LTC4S [76, 77]. Shortly after that LTD4 [79-‐81] and LTE4 [81, 82] were found to be formed by metabolism of the peptide part of LTC4. OH O OH COOH COOH LTA4H LTB4 LTA4 LTC4S + GSH OH COOH Gly C5H11 γGluCys LTC4 γ-Glutamyl transpeptidase / γ-Glutamyl leukotrienase OH COOH C5H11 CysGly LTD4 Dipeptidase OH COOH C5H11 Cys LTE4 22 Fig. 5 Leukotriene biochemistry. LTA4, formed from AA by the action of 5-‐LO, is an unstable epoxide which is converted to either LTB4 or LTC4. LTB4 is formed by hydrolysis catalyzed by LTA4H which introduces an OH group at carbon 12 and converts the epoxide group in LTA4 to an OH group at C-‐5. LTC4 is formed by the action of LTC4S which conjugates LTA4 with GSH at carbon 6 and converts the epoxide to a C-‐5 OH group. LTC4 is actively transported out of the cell and converted extracellularly to LTD4 by γ -‐glutamyl-‐ transpeptidase or γ-‐ glutamyl-‐leukotrienase. LTD4 is metabolized to LTE4 by a dipeptidase. Leukotrienes and the 5-‐LO pathway LTD4 is generated from LTC4 by cleavage of the isopeptide bond of the GSH moiety by γ-‐glutamyl transpeptidase [79, 83] or γ-‐glutamyl leukotrienase [84] and LTE4 is formed from LTD4 by elimination of the glycine residue by a dipeptidase [82, 85] (Fig. 5). LTC4 is actively transported out of the cells by multidrug resistance-‐associated protein 1 (MRP1) [86-‐88] and the conversions to LTD4 and LTE4 take place extracellularly. LT production may also occur in cells not expressing all the necessary enzymes. LTB4 was formed when red blood cells, which lack 5-‐LO, were incubated with LTA4 [89]. Conversion of exogenous LTA4 to LTC4 was demonstrated in bone marrow-‐derived mast cells [90] and co-‐incubations of red blood cells with neutrophils provided the first evidence for cooperation between different types of cells in LT synthesis [91]. Transcellular formation of LTC4 was reported soon thereafter [92]. This phenomenon has later been reported for a number of other cell types [93-‐ 100]. Another example of transcellular biosynthesis is the generation of the antiinflammatory lipoxins which requires cooperation between 5-‐LO and 12/15-‐LO expressing cells (reviewed in [62, 63]). Resolvins and protectins are generated by cooperation between COX-‐2 and lipoxygenase enzymes (reviewed in [101]). 2.1.2 5-‐LO Human 5-‐LO, is a 674 amino acid protein with a molecular weight of 78 kDa [102, 103] which requires non-‐heme iron (III) for enzymatic activity [104]. cDNA encoding human 5-‐LO was cloned in 1988 [102, 103]. 23 Leukotrienes and the 5-‐LO pathway Sequence comparisons with other lipoxygenases and mutagenesis studies indicated that iron is bound to His367, His372 and His550, Asn554 and Ile673 of the active site in 5-‐LO [105]. The crystal structure confirmed this binding pattern except that Asn554 is not involved [106]. Resting inflammatory cells harbor 5-‐LO in the cytosol or in the nucleus, depending on cell type [107-‐113]. Upon cell activation, cPLA2 and 5-‐LO translocate to the NE and/or ER, where FLAP resides [114-‐116]. Most stimuli simultaneously activate 5-‐LO and cPLA2 (Fig. 6, reviewed in [67]). In the absence of functional FLAP, 5-‐LO is unable to convert endogenous AA to LTs in cells (reviewed in [117]). However, exogenous AA has been suggested to be converted also by non-‐translocated cytosolic 5-‐LO [118]. The crystal structure of 5-‐LO revealed that the catalytic site is located in the C-‐terminal part of the protein, while the N-‐terminal part contains the C2-‐ like calcium-‐binding domain [106]. Binding of Ca2+ is reversible [119] and serves as an activator of 5-‐LO [120]. Asn43, Asp44 and Glu46 located in loop 2 of the C2-‐like domain, are important for this interaction and for the stimulatory effects of Ca2+ [121]. ATP has been reported as another activator of 5-‐LO [122] and additional activating factors e.g. microsomal membranes [120, 123] were identified during purification. In addition, glycerides such as 1-‐oleoyl-‐2-‐acteyl-‐sn-‐glycerol (OAG) activated 5-‐LO [124]. 5-‐LO binding to coactosin-‐like protein is essential for activation of the enzyme by Ca2+ [125, 126]. The interaction, but not enzyme activity occurs in the absence of Ca2+ [126]. Nitric oxide [127, 128] and certain peroxidases [129, 130] suppress 5-‐LO activity. 24 Leukotrienes and the 5-‐LO pathway Another control mechanism is regulation of subcellular distribution: e.g. neutrophils, monocytes and peritoneal macrophages harbor inactive 5-‐LO in cytosol while alveolar macrophages and Langerhans cells harbor it in the nuclear matrix (reviewed in [67]). These localizations of the protein in resting cells affect their ability to produce LTs. Nuclear localization has been associated with increased LT synthesis, except in eosinophils where the opposite effect was observed (reviewed in [131]). 5-‐LO has both nuclear import [131, 132] and nuclear export sequences [133]. Nuclear import is induced by glycogen, cytokines and adhesion to cell surfaces (reviewed in [131]). Nuclear import and export are both regulated by phosphorylation [66, 134, 135] of Ser271 by MAPkinase-‐activated protein kinase 2 [136, 137], Ser663 by extracellular-‐signal regulated kinases (ERK) [138] and Ser523 by PKA [139]. Ser523 phosphorylation prevented nuclear localization and suppressed 5-‐LO activity [139, 140]. Control also takes place at the transcriptional level. The human 5-‐LO gene [141] is located at chromosome 10q11.2. Expression of 5-‐LO is mainly restricted to granulocytes, monocytes/macrophages, mast cells, dendritic cells, Langerhans cells of the skin and B-‐lymphocytes (reviewed in [142, 143]). Age-‐dependent increase of 5-‐LO expression has also been detected in mouse hippocampus [144]. 5-‐LO expression was upregulated in differentiated compared to undifferentiated myeloid cells [143], e.g. differentiated tissue macrophages compared to blood monocytes [145, 146]. Elevated levels of 5-‐LO mRNA were detected in leukocytes from asthmatic patients [147]. 5-‐LO expression appears to require growth factors since human monocytes lost both 5-‐LO and FLAP expression when kept in culture for 7 days [148]. 25 Leukotrienes and the 5-‐LO pathway 5-‐LO has also been detected in several types of cancer cells (reviewed in [149, 150]), where its products have been suggested to serve as autocrine growth factors [151]. 5-‐LO products have further been proposed to induce DNA damage, and increase formation of HεdGuo-‐adducts (a lipid peroxide derived DNA adduct) in Ca2+ ionophore A23187 stimulated cells, whereas treatment with a FLAP inhibitor returned HεdGuo-‐adducts to basal levels [152]. The human 5-‐LO gene promoter contains GC rich regions but lacks both TATA and CCAAT boxes [141]. An important factor for silencing 5-‐LO gene expression appears to be DNA methylation [153]. Transcription factors Sp1 and Egr1 interacted with five of eight tandem arranged GC boxes [154-‐156]. Naturally occurring mutations in the human 5-‐LO promoter are either deletions or addition of extra Sp1 or Egr1 sites [155]. Interestingly, the mouse 5-‐LO promoter lacks tandem GC boxes and contains only one Sp1 or Sp3 site [157]. Promoter analyses have revealed two positive and two negative regulatory regions [154]. A slight increase in promoter activity was observed after treatment with phorbol ester [154]. A positive regulatory region containing several vitamin D3 response elements was detected between positions -‐779 and -‐229 [158]. Additional vitamin D3 response elements as well as response elements for the TGF-‐β effectors, Smad3 and 4 appear further upstream in the promoter as well as in intron 4 and exons 10-‐14, respectively [159, 160]. Recent findings suggest involvement of male sex hormones in the regulation of 5-‐LO [161, 162]. Decreased LT formation was detected in male compared to female blood cells due to variations in ERK activation as a result of higher androgen levels. 26 Leukotrienes and the 5-‐LO pathway Part of the 5-‐LO pool in male cells reside in the perinuclear region prior to activation, a distribution previously associated with impaired enzyme activity upon Ca2+ ionophore A23187 activation [163] (for reviews on the regulation of 5-‐LO, see [151, 164]). 2.1.3 FLAP The integral membrane protein, FLAP, localized to NE and ER [109, 112, 165-‐167] is structurally related to LTC4S (described below). The two proteins belong to the same gene superfamily, referred to as Membrane-‐ Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) [168]. FLAP was discovered in studies using the FLAP inhibitor MK886, at the time known as an inhibitor of 5-‐LO in intact cells but not in broken cell preparations [169, 170]. Transfection experiments showed that both 5-‐LO and FLAP were required for Ca2+-‐ionophore induced LT formation from endogenous AA [171]. FLAP also stimulates conversion of exogenous AA to LTs [172] but in this case the requirement for FLAP is not absolute. Several functions of FLAP have been suggested: 1) It may serve as a scaffold protein assembling 5-‐LO and cPLA2 at the NE and ER involving important protein-‐ protein interactions [169, 173, 174], e.g. between FLAP and 5-‐LO [116]. However, others report that MK886 inhibited LT biosynthesis without affecting translocation and membrane association of 5-‐LO [112, 163, 175, 176]. 2) FLAP is an AA binding protein, which donates AA to 5-‐LO [172, 177]. AA and other PUFAs compete with FLAP inhibitors for binding to FLAP (Fig. 6) [178, 179]. Inhibitors such as MK886 bind to the first hydrophilic loop of FLAP as judged by immuneprecipitation of FLAP peptide fragments and mutagenesis studies [180, 181]. 27 Leukotrienes and the 5-‐LO pathway The crystal structure of inhibitor bound FLAP [182] showed four trans-‐ membrane α-‐helices connected by two long cytosolic loops and one short luminal loop. Amino acid residues 38-‐47 and 102-‐115 make up cytosolic loops 1 and 2, respectively. The crystal structure also revealed that FLAP is a homotrimer [182] and suggested that its cytosolic loops interact with the C-‐terminal catalytic domain of 5-‐LO. The calcium-‐binding domain of 5-‐LO anchors it to the nuclear membrane [182]. One FLAP trimer binds one 5-‐LO monomer [182]. The essential role of FLAP in LT biosynthesis was established using FLAP null mice. Like 5-‐LO-‐/-‐ mice, FLAP-‐/-‐ mice lack detectable LT production [183]. It is not known whether FLAP influences AA metabolism by 12-‐ and 15-‐LO. FLAP, however, stimulated 5-‐LO oxygenation of both 12(S)-‐ and 15(S)-‐HETE and this effect was abolished by the FLAP inhibitor MK886 as well as when certain FLAP mutant variants were used [184]. A significant reduction of 12-‐HETE formation was described in FLAP knockout mice [185]. The human FLAP gene [186] is composed of 5 small exons, four large introns, and a promoter region containing a potential TATA box, as well as an AP-‐2 and several glucocorticoid receptor binding sites. FLAP expression, which occurs mainly in myeloid, 5-‐LO expressing cells [187], is altered by treatment of cells with TGF-‐β1, 1,25-‐dihydroxy-‐vitamin D3, and phorbol ester [188, 189]. Transcription factors of the C/EBP family were identified as important for FLAP expression modulated by TNF-‐α [190]. Prolonged exposure to lipopolysaccharide (LPS) induced FLAP mRNA and protein. The effect was mediated by NF-‐κB and a C/EBP element located within the first 134 bp of the FLAP promoter [191]. 28 Leukotrienes and the 5-‐LO pathway Increased FLAP expression has been observed in several pathological conditions. Single nucleotide polymorphisms (SNP) identified in the FLAP gene have been correlated with increased risk of developing myocardial infarction and stroke [192]. FLAP was highly upregulated in aorta and adipose tissue in a mouse model of atherosclerosis and the increased FLAP expression was accompanied by elevated LTB4 production [193]. FLAP protein in aorta and adipose tissue was found in infiltrating macrophages, but not in T-‐cells. Treatment with FLAP inhibitor, however, reduced not only atherosclerotic lesion size but also T-‐cell content, a finding suggesting FLAP as a ”potential link between innate and adaptive immunity” [193]. 2.1.4 LTC4S Leukotriene A4 is enzymatically converted to LTC4 [194, 195] by a glutathione S transferase (GST) with narrow substrate specificity [196]. LTC4S is a membrane-‐bound protein [196, 197] located in the microsomal cell fraction [198-‐201]. LTC4S was purified by two groups in 1992 and 1993 [202, 203] and its molecular weight, 18 kDa, was determined. The amino-‐ terminal sequence was also reported [203]. The cloning of LTC4S cDNA was reported by two groups in 1994 [204, 205], revealing a 150 amino acid protein. Evidence for “homo-‐oligomerization of LTC4S in living cells” was published in 2003 [206]. The two-‐dimensional crystal structure of recombinant LTC4S indicated that it forms homotrimers [207]. This observation was confirmed by the 3D crystal structure reported in 2007 [208, 209]. The enzyme contains 5 α-‐helices, of which four are membrane spanning, with the N-‐ and C-‐termini at the same side of the membrane and an overall structure that resembles that of FLAP. 29 Leukotrienes and the 5-‐LO pathway Human LTC4S shares 31% amino acid identity with FLAP, and both belong to the MAPEG gene-‐superfamily [168]. Other closely related MAPEG proteins are the microsomal Glutathione S-‐transferases (mGSTs) [210]. Helices 1 and 2 in LTC4S are connected by a long cytosolic loop [208, 209] as in FLAP [182]. Helices 3 and 4 are connected by just a short turn in LTC4S compared to a long second cytosolic loop in FLAP. The GSH binding site of LTC4S is formed at the interface between two neighboring monomers close to the protein surface and the top of the binding site is covered by the long cytosolic loop between helices 1 and 2 [208, 209]. Site directed mutagenesis showed that residues important for catalytic activity are located in hydrophilic segments. Arg51 and Tyr93 were suggested as critical residues at the active site [211]. The crystal structure confirmed that Arg30, Arg51, Asn55, Glu58, Tyr59, Tyr93, Tyr97 and Arg104 are important for GSH binding to the protein [208, 209]. LTC4S is distributed in the outer and excluded from the inner part of the nuclear membrane. FLAP, on the other hand, occurs in both the outer and inner segments of the NE [212]. The third hydrophobic region of LTC4S is important for membrane localization [213] and amino acids 114-‐150 are sufficient for homo-‐ oligomerization to occur [206]. The human [214, 215] and mouse [216] LTC4S genes have five exons and identical intron /exon boundaries. Murine intron 1 is 1/3 (ca. 500 bp) shorter than its human counterpart. The mouse gene is localized to chromosome 11B1.1-‐1.2. The promoter region of the mouse gene has consensus sites for AP-‐2, CCAAT (enhancer binding protein) and polyoma virus enhancer-‐3 [216]. The human gene is localized to chromosome 5q35 [214, 215] adjacent to a gene cluster implicated in asthma (5q31-‐33) (reviewed in [217]). 30 Leukotrienes and the 5-‐LO pathway The human LTC4S and FLAP genes have almost identical intron / exon organization but LTC4S has much shorter introns [186]. The human LTC4S promoter contains a binding motif for the ubiquitously expressed Sp1 and Sp3 transcription factors [218, 219]. Other regulatory elements would therefore be required to explain the limited expression of LTC4S. 5-‐LO and LTC4S co-‐express in cells of myeloid origin (basophils, eosinophils, mast cells and monocytes / macrophages) [220]. Platelets contain LTC4S but lack 5-‐LO [221]. LTC4S expression also occurs in the plexus choroideus and other parts of the brain [222-‐228]. Treatment of human erythroleukemia cells with the PKC activator TPA induced LTC4S [229] in agreement with the presence of PKC-‐responsive elements (AP-‐1 and AP-‐2) in the promoter [214-‐216]. Other reports have indicated inhibition of LTC4 formation in cells pre-‐treated with TPA [230, 231]. LPS [232, 233] and TNF-‐α [234] down-‐regulate expression of LTC4S whereas TGF-‐β [235] upregulates it in mononuclear-‐like cell lines. Retinoic acid upregulated LTC4S promoter and enzyme activity in rat basophilic leukemia cells [236, 237]. LTC4S expression is thus differentially regulated depending on cell type. This is in agreement with a report suggesting that cell-‐specific transcription involves Sp1 and a Kruppel-‐like transcription factor [219]. Several LTC4S promoter polymorphisms have been associated with inflammatory disorders. A Polish study identified a -‐444 A to C polymorphism with the C allele being more frequent in patients with aspirin-‐induced asthma [238] but this association was not found in Australia, Japan or the United States [239-‐241]. In addition, a G/A polymorphism located at -‐1072 in the LTC4S promoter was associated with an increased risk of developing ischemic brain injury [242]. 31 Leukotrienes and the 5-‐LO pathway LTC4S knockout mice [243] develop and breed normally but have a reduced inflammatory response in some models. Data obtained from these mice showed that LTC4S accounted for the major part of their LTC4 production, with only a small contribution from other GST enzymes. A few human case reports have indicated individuals lacking LTC4S. These children had no CysLTs in their cerebrospinal fluid and showed impaired CNS development and mental retardation [244] suggesting developmental roles for CysLTs. Eosinophils produce a different spectrum of eicosanoids compared to other granulocytes. When incubated with AA human eosinophils produce a positional isomer of LTC4 with an absorbance maximum at 282 rather than 280 nm and a shorter retention time than LTC4 when analyzed by RP-‐HPLC. The product was identified as 14,15-‐LTC4 and named eoxin (EX) C4 [245]. Like LTC4, EXC4 was metabolized to EXD4 and EXE4 by elimination of its γ-‐ glutamyl followed by its glycine residue, respectively. Incubation of human eosinophils with AA alone favored EX production whereas incubation with AA plus a Ca2+ ionophore resulted in mainly LT products [245]. It is not known whether LTC4S is involved in the biosynthesis of eoxins or other 15-‐ LO or 12-‐LO products. 2.1.5 LTA4H LTA4H is a cytosolic 70 kDa zinc-‐containing metalloprotease [246]. The protein has dual enzyme activities and is responsible for the conversion of LTA4 to LTB4. In contrast to its usual cytosolic localization one report has described a nuclear localization in alveolar macrophages [247]. 32 Leukotrienes and the 5-‐LO pathway The deduced amino acid sequence [248] revealed similarities to zinc-‐ containing aminopeptidases [249] an observation, which has been experimentally verified [250]. Inhibition of the peptidase activity also inhibited the conversion of LTA4 to LTB4 [251]. A specific role for the aminopeptidase activity could not be established using LTA4H knockout mice [252]. Cloning of the human cDNA encoding LTA4H [248, 253] identified a 610 amino acid protein. The crystal structure of the protein, bound to a competitive inhibitor, revealed a protein folded into three domains, together creating a deep cleft harboring the catalytic Zn2+ site [254]. LTA4H is self-‐inactivated during catalysis due to covalent modification of the enzyme by its substrate LTA4 [255]. Phosphorylation of Ser415 has been reported, but present evidence does not support regulation of LTA4H by phosphorylation [256, 257]. The human LTA4H gene is 35 kbp and localized to chromosome position 12q22. It has 19 exons and its 5´-‐flanking region has several putative transcription factor binding sites including an AP-‐2 site and two xenobiotic response elements [258]. Promoter studies identified a negative control element between -‐1702 and -‐1196, one or more positive elements between -‐1196 and -‐123 and a cis-‐acting cell specific control element between -‐123 and -‐40 [259]. Transcriptional regulation of LTA4H is distinct from that of 5-‐ LO and the other proteins involved in LT synthesis since this enzyme is expressed much more widely [260]. 33 Leukotrienes and the 5-‐LO pathway LTA4H LTB4 LTA4 AA 5LO LTC4 LTC4S PM FLAP cPLA2 5LO 5LO cPLA2 NM Fig. 6 Cellular leukotriene synthesis. Activation of cells by inflammatory stimuli results in translocation of cPLA2 and 5-‐LO to the nuclear membrane. cPLA2 liberates AA from membrane phospholipids. AA is transferred to 5-‐LO by FLAP and converted to LTA4. LTA4 is either hydrolyzed by LTA4H to LTB4 or conjugated with glutathione by LTC4S to LTC4 depending on the expression of these enzymes. NM: nuclear membrane, PM: plasma membrane 34 Leukotrienes and the 5-‐LO pathway 2.2 Leukotriene receptors Leukotrienes exert their actions through specific cell surface receptors (reviewed in [261, 262]). Two receptors (BLT1R and BLT2R) mediate intracellular signals in response to LTB4. Cys-‐LTs mediate their effects through the two cell surface receptors CysLT1R and CysLT2R. Additional membrane proteins may also serve as receptors for CysLTs. The leukotriene receptors are seven-‐transmembrane G-‐protein coupled receptors (GPCRs). To date more than 30 GPCRs are known to respond to lipids [261]. The G-‐proteins involved are heterotrimeric GTP-‐binding proteins consisting of α-‐, β-‐ and γ-‐subunits. Upon receptor activation the α-‐ subunit is released and stimulates or inhibits multiple intracellular signaling pathways. The β,γ-‐subunits are also involved in signaling. GPCR activation may lead to increased concentrations of intracellular calcium ions, increased or decreased cAMP production, Rho activation and modification of ion channels. The heterotrimeric G-‐proteins are grouped based on their α-‐subunits (Gαs, Gαi, Gαq and Gα12 reviewed in [263]). Gαs stimulates and Gαi inhibits adenylate cyclase. Gαq stimulates phospholipase C which cleaves phosphatidylinositol-‐2-‐phosphate (PIP2) to inositol tris-‐ phosphate (IP3) and diacylglycerol (DAG) resulting in increased Ca2+ concentrations and PKC activation. Gα12 activates Rho GDP / GTP exchange factor resulting in actin rearrangements (reviewed in [261]). 35 Leukotrienes and the 5-‐LO pathway 2.2.1 LTB4-‐receptors BLT1R, was discovered in 1997 [264] and BLT2R, in 2000 [265]. Expression of BLT1R occurs mainly in inflammatory cells e.g. neutrophils, eosinophils, dendritic cells, macrophages, B-‐lymphocytes and certain T-‐lymphocytes [266-‐272] but low expression has also been detected in spleen and thymus [264]. The human BLT1R gene is located on chromosome 14, (14q11.2-‐q12) [273]. The precise mechanism behind the leukocyte-‐restricted expression is not clear. Sp1 binds to the promoter but, due to a GC rich sequence, the gene is heavily methylated in non-‐expressing cells and this may explain its restricted expression [274]. BLT2R is a low affinity receptor for LTB4 [275, 276]. In man it is expressed mainly in skeletal muscle, heart, lung, spleen, liver, ovaries, and leukocytes, but low expression levels are detected in most tissues [261, 265]. The coding region for BLT2R was identified as a putative intronless open reading frame in close proximity to the BLT1R gene [275, 276]. Intracellular signaling evoked by LTB4 receptor activation is cell type specific depending on the type of G-‐protein involved. BLT1R signaling in granulocytes is commonly mediated by Gαi2, while Gαi1 and Gα0 has been found to mediate BLT1R signaling in the nervous system. Signaling through Gαq has also been reported there (reviewed in [261, 262]). Downstream effects of BLT1R signaling involve activation of kinases [277], MAPKs [278, 279], phosphatidylinositol 3-‐kinase (PI3-‐K) [280, 281] and tyrosine kinases [281, 282]. LTB4 signaling also increases expression of inflammatory genes e.g. IL-‐6 and monocyte chemoattractant protein-‐1 (MCP-‐1, see Fig. 7) [283-‐285] (reviewed in [261, 262, 286]). BLT2R activation inhibits adenylate-‐cyclase and increases intracellular Ca2+, but less potently than BLT1R [265]. Gαi-‐mediated chemotaxis has been described [265, 275]. 36 Leukotrienes and the 5-‐LO pathway Generation of reactive oxygen spices (ROS) mediated by activation of Rac-‐ ERK has also been proposed to occur through BLT2R [287]. As mentioned above, BLT2R is a low affinity LTB4 receptor, consequently higher LTB4 concentrations are needed to inhibit adenylate-‐cyclase and to induce chemotaxis than when BLT1R is activated [265, 275]. BLT1R G αi1 G αq G α16 Gαi2 Gα0 BLT2R Gα0 cAMP 2+ Ca PKC cAMP 2+ Ca Gαi Rac-ERK MAPKs PI3-K Tyrosine kinases IL-5 IL-6 IL-10 Fig. 7 LTB4 receptors. LTB4 interacts with two GPCRs; BLT1R and BLT2R. The receptors couple to different G-‐ proteins in different cell types. LTB4 signaling activates several kinases and induces expression of inflammatory genes e.g. the IL-‐5, IL-‐6 and IL-‐10 genes (revived in [261, 262, 286]). The ligand potency ranking for BLT1R is LTB4 > 20-‐OH-‐LTB4 = 12-‐oxo-‐LTB4 > 12(R)-‐HETE > 20-‐COOH-‐LTB4 [264] while that of BLT2R is LTB4 > 12-‐epi-‐ LTB4 > 12(S)HETE > 12(S)HpETE > 12(R)HETE > 20-‐OH-‐LTB4 [288]. 12(S)-‐ hydroxyheptadeca-‐5(Z), 8(E), 10(E)-‐trienoic acid (12-‐HHT) generated from PGH2 during its conversion by thromboxane A2 synthase [289] was recently identified as a high affinity ligand for BLT2R having higher affinity than LTB4 [290]. In addition to BLT1R and BLT2R, LTB4 also binds to and activates the nuclear orphan receptor peroxisome proliferator-‐activated receptor-‐α (PPARα) in vitro [291] and in cells [292]. 37 Leukotrienes and the 5-‐LO pathway 2.2.2 CysLT receptors. Pharmacological evidence for the existence of CysLT (SRS-‐A) receptors was reported 6 years before the structure of LTC4 was reported [293]. Subsequent studies on venous preparations where LTC4 and LTD4 antagonists failed to block the response indicated the presence of a second CysLT receptor [294]. Cysteinyl leukotriene receptors 1(CysLT1R) and 2 (CysLT2R) have been cloned and characterized [295-‐298]. Human CysLT1R is a 337 amino acid protein [295, 297] expressed in adipose tissue, brain, bronchus, colon, heart, kidney, liver, lung, pancreas, peripheral blood leukocytes, placenta, prostate gland, skeletal muscle, small intestine and spleen [295, 297, 299]. At the cell level CysLT1R occurs in B-‐lymphocytes, endothelial cells, eosinophils, fibroblasts, fibrocytes, mast cells, monocytes/macrophages, neutrophils, pregranulocytic CD34+ cells and smooth muscle cells [300-‐305]. CysLT2R, a 346 amino acid protein, is expressed in adrenal glands, brain, heart, leukocytes, lymph nodes, peripheral blood, placenta and spleen [296, 298, 306]. At the cell level expression occurs in macrophages and in smooth muscle cells of the human lung [296] (reviewed in [261]). It also occurs in neurons of the myenteric plexus where lack of expression in knockout mice dampened experimental colitis progression [307]. CysLT2R dominates over CysLT1R in heart and eosinophils [296, 298, 308] while only CysLT1R is expressed in trachea [298]. Similar CysLT2R patterns occur in mice but the pharmacological characteristics of the mouse receptor differ from those of the human receptor [309, 310]. The CysLT1R gene is located on the X chromosome both in man (Xq13.3-‐ q21.1, [295]) and mouse [311]. The human gene has 5 variably spliced exons and a promoter region with multiple transcription start sites [299]. 38 Leukotrienes and the 5-‐LO pathway The human CysLT2R gene is located on chromosome 13 (13q14.12-‐q21.1) in a region with several markers of asthma or atopi [312, 313]. The gene has 6 variably spliced exons and the whole coding sequence is located in exon 6 [314]. CysLT1R signaling employs multiple second messengers (e.g. DAG, inositol phosphates and Ca2+) and PKC and involves differentiated G-‐protein coupling, including pertussis toxin (PTX)-‐sensitive (Gαi/o) and insensitive (Gαq/ii) ones [315, 316] (Reviewed in [261]). MAPK pathways are activated in several cell types (Fig. 8) [317-‐324]. CysLT effects on mouse embryonic stem cells involve STAT3, PI3-‐K/Akt, glycogen synthase kinase-‐3β/β-‐ catenin and Ca2+-‐calcineurin pathways [325]. Like LTB4, CysLTs, induce expression of inflammation-‐associated genes [326, 327] (reviewed in [261, 262, 286]). GPCRs, including CysLT1R, have recently been localized in the cell nucleus in contrast to the usual plasma membrane localization and a nuclear localization signal was recognized in CysLT1R [328]. Evidence for intracrine signaling activating ERK 1/2 and vesicular transport mediated IL-‐4 secretion has been reported [328]. Detailed information on CysLT2R signal transduction is not available due to a lack of selective antagonists: coupling to both PTX sensitive and insensitive G proteins has been suggested (Fig. 8, [296, 329] reviewed in [261, 262]). 39 Leukotrienes and the 5-‐LO pathway CysLT1R CysLT2R Gα11 Gα0 G αq Gαi Gαi DAG 2+ Ca cAMP PKC Rho GEF 2+ Ca cAMP MAPK PI3-K STAT3 AA-release Actin reorganization Gα11 Gαq Gαo NO IL-8 IL-4 VEGF IL-13 Integrins Fig. 8 CysLT receptors. CysLTs interact with two GPCRs; CysLT1R and CysLT2R. Additional receptors have been reported to respond to CysLTs. G-‐protein coupling of the receptor depends on the expressing cell type. Signaling primarily involves kinases including MAPK. Signaling also induces expression of inflammatory genes (revived in [261, 262, 286]). The ligand affinity order for CysLT1R is LTD4 > LTC4 > LTE4 [297] and for CysLT2R LTD4 = LTC4 > LTE4 [296]. Short-‐hairpin (sh)RNA-‐mediated knockdown of CysLT2R enhanced the surface expression of CysLT1R, suggesting a coherent regulation of the two receptors [330]. Additional CysLT receptors with specificity for LTC4 [331-‐333] and LTE4 [334] have been postulated (reviewed in [261]) and recently an orphan GPCR (GPR17 [335]) was recognized as a phylogenetic intermediate between purinergic P2Y and CysLT receptors [336]. GPR17 is a dual receptor for uracil nucleotides and CysLTs [336, 337] and negatively regulates CysLT1R signaling in a ligand-‐independent manner [338, 339]. 40 Leukotrienes and the 5-‐LO pathway 2.3 Leukotriene actions 2.3.1 Modulators of inflammatory responses At the site of inflammation, recruited leukocytes produce LTs in response to microbes interacting via cell surface receptors e.g. Fc-‐γ receptors [340, 341], Toll-‐like receptors [342-‐344] and β-‐glycan receptors [345] (reviewed in [346]). LTs facilitate recruitment of additional leukocytes in several ways: LTB4 is a potent chemoattractant for neutrophils and monocytes [347-‐349] and upregulates the expression of cell surface adhesion glycoproteins. Interactions with blood vessel endothelial cells slow down and stop the circulating leukocytes so they can migrate into surrounding tissues [350]. LTB4 also aids leukocyte accumulation by attenuation of leukocytes apoptosis [351, 352]. CysLTs contribute to leukocyte accumulation by increasing vascular permeability and by attracting subsets of T-‐cells [353-‐355]. CysLT receptor expression is upregulated by activation of CD4+ T-‐cells and is responsible for their chemotaxis to LTD4 [355, 356]. CysLT signaling on eosinophils induces adhesion, IL-‐4 release, migration, recruitment, and increases eosinophil survival [352, 357-‐359]. It activates dendritic cells and their cytokine release [360, 361], mast-‐cell cytokine production [362] and activation of basophils [363]. CysLT receptor signaling also enhances IgG and IgE production in IL-‐4 / CD40-‐activated B-‐ cells [364]. These proinflammatory actions of LTs on the immune system are a major contributor to the pathogenesis of several diseases. 41 Leukotrienes and the 5-‐LO pathway Elevated levels of LTB4 (LTB4-‐like immunereactivity) has been reported in ulcerative colitis and other types of inflammatory bowel disease [365-‐367], psoriasis [368], cystic fibrosis [369], rheumatoid arthritis [370, 371] and in neutrophils from patients treated surgically for aortic abdominal aneurysm [372, 373]. The main symptoms of asthma come from CysLT formation. These compounds are potent stimulators of bronchial smooth muscle [374] and induce airway mucus secretion [375]. CysLTs also constrict coronary (but not pulmonary) artery and pulmonary vein [376, 377]. They potently increase vascular permeability throughout the body [353, 354, 378]. Th2-‐ biased allergic responses due to CysLT signaling affect both acute and chronic phases of asthmatic inflammation. The effects are primarily mediated by CysLT1R signaling but CysLT2R may be involved in certain cases [379, 380] (reviewed in [381]). CysLTs have also been implicated in the pathogenesis of allergic rhinitis [382] and atopic dermatitis [383, 384]. Clinical studies using inhibitors of LT biosynthesis or antagonists of LT receptors in asthmatic patients blocked both the early and the late phase of an allergic response [385, 386] (reviewed in [261]). The CyLT1R antagonists montelukast2, pranlukast and safirlukast and the 5-‐LO inhibitor zileuton are approved drugs for the treatment of asthma (reviewed in [387]). Some features of asthma may also be mediated by LTB4 [388], e.g. proliferative effects on airway smooth muscle cells has been described [279]. Leukotrienes are also important players of the inflammatory component of cardiovascular diseases (reviewed in [389-‐391]). 2 Montelukast under the trade name Singulair is one of Merk Sharp & Dohme´s best selling drugs. 42 Leukotrienes and the 5-‐LO pathway Effects mediated by CysLT1R [392-‐396] and CysLT2R [397] were shown to be involved in ischemic brain injury. The proposed CysLT receptor GPR17 was upregulated in damaged tissues, and knockdown reduced neuronal injury after ischemia [337, 398]. Elevated LT levels were detected in cerebrospinal fluid of MS patients [399]. In spite of detrimental roles in inflammatory diseases, LTs are also important players in host defense reactions against microbial infections (reviewed in [286]). LTs stimulate phagocytosis [341, 400-‐402] and microbe killing [346, 403, 404] the latter by induction of NO synthesis and production of ROS [404-‐408]. LTs induce release of α-‐defensin [409] and antimicrobial peptide LL-‐37 [410, 411]. In malnutrition [412-‐414], vitamin D deficiency [415], HIV infection [416, 417], liver cirrhosis [418], diabetes [419] and among cigarette smokers [420, 421] there is correlation between the susceptibility to infectious diseases and attenuated LT synthesis. Several studies have postulated LTs as mediators in different types of cancer. For example: LTB4 increases proliferation and survival of a colon cancer cell line and proliferation of pancreatic cancer cells. CysLT signaling increased proliferation of colorectal cancer cell lines and CysLT1R antagonists induced apoptosis in a pancreatic cancer cell line. Effects have also been seen on metastasis: BLTR antagonists reduced metastasis in a mouse model of pancreatic cancer (reviewed in [422]). 43 Leukotrienes and the 5-‐LO pathway 2.3.2 Regulators of hematopoiesis The hematopoietic system is one of the largest and most proliferative cell systems in the body. Studies in mice revealed that the first hematopoietic cells develop in the yolk sac at around day 8 of gestation. Around day 9 hematopoietic stem cells (HSC) appear in the aorta-‐gonad-‐mesonephros (AGM) region [423] and somewhat later in the placenta and liver [424]. HSC from yolk sac, AGM region and placenta all appear to contribute to the colonization of the liver, the main hematopoietic organ during fetal life [423, 425] (reviewed in [426]). Shortly before birth, bone marrow starts to take over this role and becomes the main hematopoietic organ for the remaining life [427, 428]. All types of blood cells derive from a common HSC (reviewed in [429]). This cell can a), self-‐renew and b), differentiate into the different blood cell lineages (Fig. 9). Flow cytometry is widely used to fractionate hematopoietic cells into different cell types. Stem cell activity is found in the so-‐called LSK population, i.e. cells lacking lineage specific surface markers (B220, Mac1, Gr1, IL7Rα, Ter119, CD3, CD4, and CD8) and amply expressing the markers Sca1 and cKit [430, 431] (reviewed in [432]). LSK cells include both long term (LT)-‐HSC, short term (ST)-‐HSC, multipotent progenitors (MPPs), lymphoid primed multipotent progenitors (LMPPs) and granulocyte-‐macrophage-‐lymphocyte progenitors (GMLPs). Further compartmentalization of the LSK population is achieved using antibodies against CD34 [433] and Flt3 [434, 435]. LSK cells lacking expression of CD34 and Flt3 are LT-‐HSC. ST-‐HSC and MPPs express CD34 while LMPPs and GMLPs express both CD34 and Flt3 [436, 437] (reviewed in [438]). Lymphoid, natural killer (NK) and dendritic cell potential lies within the Lin-‐/Flt3+/IL7R+/cKitlow/Sca1+ common lymphoid progenitor (CLP) population [438-‐440]. 44 Leukotrienes and the 5-‐LO pathway The cKithigh/Sca1-‐ myeloid, megakaryocyte and erythroid progenitor population contains three subpopulations: megakaryocyte and erythroid progenitors (MEP; CD34low/CD16/CD32low), common myeloid progenitors (CMP; CD34low/CD16/CD32+), and granulocyte and macrophage progenitors (GMP; CD34high/CD16/CD32high) [441]. Only MEP has megakaryocyte potential while erythroid potential is found in both MEP and CMP cells [438] (Fig. 9). The CLP population has B-‐ and T-‐lymphoid potential [439], but is considered to develop mainly into B-‐lymphocytes in vivo. T-‐lymphocytes primarily mature from early thymic progenitors seeding the thymus [442-‐444] (Fig. 9, reviewed in [445]). 45 Leukotrienes and the 5-‐LO pathway LSK LT-HSC MPP ST-HSC LMPP/ GMLP ELP CMP MEP CLP ETP MkP PreCFU-E Pre-GMP CFU-E GMP Granulocytes Erythrocytes Megakaryocytes T-lymphocytes Dendritic cells/ NK-cells Monocytes/ Macrophages B-lymphocytes Fig. 9 Hematopoietic development. All hematopoietic cells are derived from a single ancestor stem cell with potential of regeneration as well as differentiation. During proliferation, hematopoietic cells gradually become specialized and concomitantly lose Zandi eal PMID: 20969584 potential to differentiate into other cell types (reviewed in [438, 445]). LT-‐HSC, long term hematopoietic stem cell; ST-‐HSC, short term HSC; MPP, multipotent progenitor cell; LMPP, lymphoid primed MPP; GMLP, granulocyte, macrophage and lymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte and erythroid progenitor; MkP, megakaryocyte progenitor; GMP, granulocyte and macrophage progenitor; CFU-‐E, colony forming unit erythrocyte; ELP, early lymphoid progenitor; CLP, common lymphoid progenitor; ETP, early thymic progenitor. 46 Leukotrienes and the 5-‐LO pathway The mechanisms regulating the hematopoietic system are complex and both intrinsic (involving transcription factor modulation) and extrinsic (cytokine-‐mediated) pathways are employed [446]. It is not yet understood to which extent eicosanoids are involved in this system or their precise roles. There is an excellent review on the topic; “Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking” [447]. PGE2 inhibits in vitro proliferation of mouse and human cells of the myeloid lineage and an abnormal inhibitory response to PGE2 has been reported in cells derived from leukemia patients [448-‐455]. There is a prognostic connection between disordered CFU-‐GM inhibition by PGE2 and myelodysplastic syndromes [456]. However other groups did not observe inhibition by PGE2 on hematopoiesis; PGE2 treatment of murine bone marrow increased proliferation of hematopoietic cells and increased production of HSC [457-‐459]. LT effects on hematopoietic regulation have also been reported. The expression of 5-‐LO and FLAP in human CD34+ cells is low, but increases when the cells are differentiated in vitro to dendritic cells [460]. In vitro differentiation of CD34+ cells also leads to increased expression of BLT1R [269]. Other in vitro studies suggest that LTs promote hematopoietic progenitor cell (HPC) proliferation [461-‐463]. These proliferative effects were reduced by 5-‐LO inhibitors [461, 464]. LTB4 and LTC4 decreased erythroid and granulocyte-‐macrophage colony formation [465] whereas a 5-‐LO inhibitor had the opposite effect. LTB4 increased HPC proliferation in umbilical cord blood (UCB) and decreased the number of CD34+ cells whereas a BLTR antagonist had opposite effects (increased the number of CD34+ cells and blocked HPC proliferation) [466]. LTD4 induced chemotaxis and transendothelial migration of CD34+ progenitor cells and affected their trafficking [467]. 47 Leukotrienes and the 5-‐LO pathway LTD4 promoted hematopoietic progenitor cell expansion and stimulated integrin-‐dependent adhesion by upregulating α4β1 and α5β1 integrins [468]. LTD4 also increased growth of eosinophil progenitor cells from atopic patients [462]. The 5-‐LO gene has been identified as a critical regulator of leukemia stem cells (LSC) in a mouse model of chronic myeloid leukemia, since induction of the disease failed in 5-‐LO-‐/-‐ mice [469]. Lack of 5-‐LO impaired differentiation, cell division and survival of long term LSC, but not of normal HSC. BM cell compositions were not altered in 5-‐LO knockout mice compared to wild type animals [469] but reduced numbers of memory B-‐cells and mature germinal centers in spleen after immunization were reported in knockout compared to wild type mice [470]. A reduction was also seen in spleen follicular B-‐cells and the ability to generate a sub-‐ population of helper T-‐cells (Tfh) in the spleen was nearly lost in 5-‐LO deficient mice [470]. 5-‐LO deficient mouse spleen dendritic cells have altered cytokine production [471]. Other LO products have also been proposed to influence hematopoietic regulation. 12-‐HETE and 15-‐HETE stimulated proliferation and accelerated differentiation of CD34+ cells toward the erythroid lineage [472]. 48 Aims 3. AIMS The aims of this thesis were to investigate subcellular localization and protein-‐protein interactions taking place within the LT machinery, to investigate the expression pattern of the LT machinery during mouse fetal development compared to the expression pattern in adult animals and to identify functional roles of LTs in the hematopoietic system. 49 50 Results and Discussion 4. RESULTS AND DISCUSSION 4.1 Paper I To investigate the mechanisms behind LTC4S gene expression we constructed a vector expressing enhanced green fluorescent protein (eGFP) under the control of the mouse leukotriene C4 synthase promoter (Fig. 1A, Paper I). This vector was used to study promoter activity in various cell lines. GFP expression was observed in human monocytic leukemia (THP-‐1) and rat basophilic leukemia (RBL-‐1) myeloid cells which both normally express leukotriene C4 synthase, but not in human embryonic kidney (HEK-‐293T) epithelial cells which do not express this enzyme (Fig. 1, Paper I). In the myeloid cells, but not in the epithelial cells, we observed that LTC4S promoter activity was stimulated by 12-‐O-‐tetradecanoylphorbol-‐13-‐ acetate (TPA) in both RBL-‐1 and THP-‐1 cells and by all-‐trans-‐retinoic acid (RA) in RBL-‐1 cells. In contrast dimethyl sulfoxide did not affect promoter activity (Figs. 2 and 3, Paper I). In a previous study TPA stimulated leukotriene C4 synthase activity in human erythroleukemia cells whereas dimethyl sulfoxide inhibited the activity and retinoic acid had no effect [229]. The stimulatory effect of TPA was confirmed in this study suggesting that it occurs at the promoter level. The lack of effect of the various treatments in HEK-‐293T control cells and in cells transfected with control plasmid further supported this notion. The 5´-‐flanking region of the mouse leukotriene C4 synthase gene contains consensus sequences for AP-‐2, CCAAT (enhancer binding protein) and polyoma virus enhancer-‐3 [216]. 51 Results and Discussion AP-‐1 and AP-‐2 sites, known to be affected by TPA, are found in the human LTC4S promoter [214, 215]. Sp1 and a Kruppel-‐like transcription factor have been suggested to determine cell-‐specific LTC4S transcription [219]. TPA induced leukotriene C4 synthesis in certain cell lines [229], while it decreased leukotriene C4 production in HL-‐60 cells, an effect that was blocked by PKC specific inhibitors [230]. Furthermore, retinoic acid upregulated leukotriene C4 synthase in rat basophilic leukemia cells [236, 237]. Thus it appears that leukotriene C4 synthase gene expression is differentially regulated depending on the cellular environment, in agreement with the results presented in Paper I. Our recombinant vector is regulated in an LTC4S specific manner suggesting that the promoter sequence and intron 2 are sufficient for LTC4S regulation and furthermore that the vector should be useful as a reporter of LTC4S expression. 52 Results and Discussion 4.2 Generation of GFP mice (unpublished results) The vector expressing GFP under control of the regulatory regions of the mouse LTC4S promoter and intron 2 (described in Paper I) and an analogous vector where intron 2 was replaced by LTC4S intron 1, were used to produce transgenic mice. The intron 1 vector was constructed by PCR amplification of a sequence containing mouse LTC4S intron 1 (primer 1: 5´-‐ CACCCTCGTGGGAGTTCTGTTGC-‐3´, primer 2: 5´-‐CAGAGATCACCTGTCTCGA-‐ GAAGTAGGC-‐3´) and subsequently subcloned into the vector described in Paper I after excision of its intron 2 sequence by Nhe I / Xho I digestion. Transgenic mice were created by pronuclear injection of cassettes containing LTC4S regulatory regions, eGFP and a poly A tail, excised from the recombinant vectors. Injected mouse oocytes were implanted in the uteri of pseudopregnant mice and transgenic litters were identified by PCR screening using one primer matching a sequence in the LTC4S promoter and a second primer matching the eGFP region (primer 1: 5´-‐AAGTGGCTCTTCT-‐ GGCTACCG-‐3´, primer 2: 5´-‐GCTTCTCGTTGGGGTCTTTGC-‐3´). To establish the new strains, positive offspring was mated with C57BL/6 mice. eGFP expression was analyzed by fluorescence microscopy using 478-‐495 nm blue light. Analysis of blood from transgenic litter revealed eGFP expression in nucleated white blood cells. Prior to analysis the cells were counterstained with DAPI to visualize the cell nuclei (Fig. 10). Tissues and embryos from sacrificed transgenic animals were analyzed for the expression of GFP by in situ hybridization using a short GFP anti sense probe created by primer 1: 5´-‐CTCGTGAGGATCCTGACCTACGGCG-‐3´, primer 2: 5´-‐GATGTTGTGGCGGATCTAGAAGTTCACCTT-‐3 ´or a long probe using same primers as in Paper I to amplify eGFP. 53 Results and Discussion GFP DAPI pLTC4S-intron2-GFP pLTC4S-intron1-GFP Fig. 10 eGFP expression in leukocytes. Blood from transgenic mice carrying a cassette expressing eGFP regulated by the LTC4S promoter plus intron 2 or intron 1 were examined under 478-‐495 nm blue light in a fluorescence microscope. Prior to mounting, cells were treated with DAPI nuclear stain. DAPI was visualized in UV-‐light Analysis of tissues or embryos from transgenic mice by in situ hybridization revealed expression of eGFP message in the fetal and neonatal liver (Fig. 11). Tissues from non-‐transgenic littermates were used as negative controls. The results from fluorescence microscopy and in situ hybridization confirmed that GFP was expressed at the mRNA and protein levels under control of the LTC4S promoter combined with either intron 1 or 2, with a localization resembling that of wild type LTC4S expression. + GFP P0 tissue GFP P0 tissue Fig. 11 In situ hybridization using eGFP antisense probes. Tissues from mice carrying a transgenic cassette expressing eGFP under control of pLTC4S + intron 1 or tissues from non-‐transgenic mice were examined using in situ hybridization with antisense probes for eGFP. Expression was detected in liver. AS, anti-‐sense. long AS GFP probe short AS GFP probe 54 Results and Discussion 4.3 Paper II In this paper we report interactions between proteins involved in LTC4 biosynthesis. Fluorescent FLAP-‐ and LTC4S-‐fusion proteins co-‐localized at the ER and nuclear envelope (NE) in transfected cells (Fig. 2, Paper II). Upon activation with Ca2+ ionophore A23187, 5-‐LO translocated from cytosol/nucleus to ER and NE (Fig. 3, Paper II). Both FLAP and LTC4S interacted with 5-‐LO in vitro (Fig. 1, Paper II). 5-‐LO and FLAP have previously been shown to interact by fluorescence lifetime imaging microscopy (FLIM) and immunoprecipitation [116] but no interaction between 5-‐LO and LTC4S was reported. Using GST-‐pulldown technique, we showed preferential interaction between 5-‐LO and the second hydrophilic segment of LTC4S in vitro (Fig. 1, Paper II). In contrast, both hydrophilic loops of FLAP bound 5-‐LO (Fig. 1, Paper II). Thus the binding of 5-‐LO to FLAP and LTC4S, respectively, appears to be different, even though FLAP and LTC4S have a very similar 3-‐D structure. FLAP and LTC4S interacted via hydrophobic regions of LTC4S (Fig. 1, Paper II), suggesting that membrane-‐ spanning regions of one LTC4S trimer contacts membrane-‐spanning regions of a neighboring FLAP trimer. Alternatively, heterotrimers or higher multimers might form (see below). The interaction between LTC4S and FLAP was more efficient with an N-‐terminal part of LTC4S than with the full length protein and interestingly this N-‐terminal part did not bind 5-‐LO (Fig. 1, Paper II). It is not known if formation of LTC4S and FLAP heterooligomers competes with formation of homotrimers of each protein or if larger protein complexes are formed. The in vitro finding of 5-‐LO/ LTC4S interaction was supported by BRET experiments, which showed a similar interaction in transfected cells following activation with calcium ionophore A23187 (Fig. 4, Paper II). 55 Results and Discussion 4.4 Paper III The expression of FLAP message was investigated in mouse fetuses at different stages of development. High levels of expression was detected in liver by in situ hybridization (ISH) and fluorescence-‐activated cell sorting (FACS) plus qRT-‐PCR analyses. FLAP expression in liver was detected as early as e11.5 (Fig. 1, Paper III). The ISH-‐images obtained revealed a non-‐ homogenous patchy expression pattern. Sorting of e15.5 fetal liver hematopoietic cells and CD45-‐ stromal cells (Fig. 2, Paper III) followed by qPCR analyses, showed that hematopoietic cells colonizing the liver during embryogenesis express FLAP (Fig. 4, Paper III). The liver is formed as a diverticulum from foregut epithelium between e8.5 and e9.5 in the mouse [473]. Hematopoietic cells are not generated in the liver but migrate there from other tissues notably the yolk sac and AGM region [426, 474]. By e10.5 the colonized liver is the major hematopoietic organ of the developing mouse embryo. The hematopoietic cells from embryonic liver were sorted as mature (CD45+/Lin+) and immature (CD45+/Lin-‐) cells (Fig. 2, Paper III). The highest FLAP expression was found in mature hematopoietic cells, but also immature cells expressed FLAP (Fig. 4, Paper III). These results demonstrated that hematopoietic but not stromal cells expressed the FLAP gene. Of the adult hematopoietic cells that are known to express FLAP [117], neutrophils and lymphocytes are present in fetal mouse liver from e12.5 [475]. These cell types may thus be the source of, or part of the FLAP expression in the mature (CD45+, Lin+) cell population. Interestingly, significant FLAP expression was also observed in the immature hematopoietic cell (CD45+, Lin-‐) population suggesting that FLAP may have roles prior to or during the maturation of hematopoietic cells. 56 Results and Discussion 4.5 Paper IV Additional studies of LT biosynthesis protein expression in fetal tissues revealed that in addition to FLAP (Paper III), 5-‐LO, LTC4S and LTA4H were also expressed during fetal development (Figs. 1 & 2, Paper IV). Co-‐ expression of the four proteins was seen in the hematopoietic cells of fetal liver (Fig. 2, Paper IV). Expression was also analyzed in adult bone marrow. LTC4S expression was very low, while higher expression was observed for 5-‐LO, FLAP, and LTA4H. The relative expression pattern of these genes was similar in fetal liver and adult bone marrow. However LTA4H mRNA, relative to the other messages examined, was higher in fetal liver than in adult bone marrow, probably since also liver stromal cells express LTA4H. 5-‐LO expression has been reported in granulocytes, monocytes / macrophages, mast cells, dendritic cells, Langerhans cells of the skin and B-‐ lymphocytes while platelets, endothelial cells, T-‐lymphocytes and erythrocytes lack expression of 5-‐LO message [142, 143]. Expression of FLAP was previously found only in 5-‐LO expressing cells but not in cells lacking 5-‐LO [187]. Co-‐expression of LTC4S and 5-‐LO has been found in cells of myeloid lineage e.g. basophils, eosinophils, mast cells and monocytes / macrophages [220]. LTC4S has also been found in platelets, which lack 5-‐LO, but express 12-‐LO [221]. Platelets can form LTC4 by transcellular synthesis using LTA4 provided by other cells [95]. In mouse embryos LTA4H expression was detected in several tissues compared to the other LT biosynthetic enzymes. Also FLAP was detected at sites lacking detectable 5-‐ LO expression. For example, thymus and spleen expressed FLAP and LTA4H but not 5-‐LO (Fig. S1, Paper IV). A more refined sorting of hematopoietic cells revealed a similar expression pattern for cells from fetal liver and adult bone marrow. 57 Results and Discussion The highest expression was found in mature myeloid cells (Figs. 3 & 4, Paper IV). 5-‐LO expression was low in most of the isolated cell populations whereas FLAP expression was high in the LSK cell compartment (Fig. 4, Paper IV). Interestingly, FLAP expression in LSK cells was the second highest, after myeloid cells. This population contains the hematopoietic stem cells. Although 5-‐LO expression in this compartment was very low (Fig 4, Paper IV) increased expression of both 5-‐LO and FLAP has been reported after differentiation of human CD34+ cells into dendritic cells in vitro [460]. Our results suggested a role for leukotrienes in hematopoietic regulation. To probe the functional importance of LTs, or LT-‐independent FLAP effects in the hematopoietic system, we used FLAP-‐/-‐ mice. HPLC analysis confirmed that these mice did not form LTs from AA. Interestingly, a significant reduction of 5(S)-‐HETE formation after incubations of bone marrow cells with AA + Ca2+ ionophore accompanied the FLAP deficiency (Figs. 5 & S2, Paper IV). We analyzed peripheral blood and bone marrow from FLAP deficient mice and control animals using flow cytometry and detected an increased frequency of T-‐lymphocytes and a decreased frequency of B-‐lymphocytes in peripheral blood (Fig. 6, Paper IV). A slight, but not statistically significant, reduction of total white blood cells was also observed. When bone marrow was investigated, no difference was seen between FLAP knockouts and controls for B-‐cells or B-‐cell progenitors (Fig. 8 and table III, Paper IV), but a statistically significant increase of common lymphoid progenitors (CLP) was found in the knockout animals (Fig. 7 and table II, Paper IV). 58 Results and Discussion Interestingly, our experiments with FLAP knockout mice showed significant differences between these animals and control mice in bone marrow and peripheral blood cell compositions. No such effects were observed in another study using 5-‐LO knockout mice [469]. However, effects on spleen B-‐ and helper T-‐cell composition have been reported in 5-‐LO-‐/-‐ mice [470]. In summary our results suggest a mild block of B-‐cell differentiation at an early stage of lineage commitment in FLAP knockout mice. The CLP population of bone marrow is considered to develop in vivo into mainly B-‐ lymphocytes (reviewed in [445]). Interestingly, 5-‐LO knockout mice had a tendency to reduce the CLP compartment in BM [469]. The distinctly different results obtained with FLAP-‐/-‐ compared to 5-‐LO-‐/-‐ mice may suggest important LT-‐independent functions of FLAP in hematopoiesis. 59 60 Conclusions 5. CONCLUSIONS The key enzyme in CysLT biosynthesis, LTC4S has a restricted expression. This may be regulated differently depending on cell type. A transfected sequence containing 1230 bp of the upstream promoter region and LTC4S intron 2 regulated GFP expression in a LTC4S-‐specific manner. This included upregulated expression in THP-‐1 and RBL-‐1 cells after treatment with TPA and in RBL-‐1 after RA treatment. The LTC4S promoter plus intron 2 or intron 1 also drove expression of GFP in a LTC4S specific manner in transgenic mice as judged by GFP expression in white blood cells and fetal liver. The leukotriene synthesis protein machinery is arranged around the nuclear envelope and ER where FLAP and LTC4S reside as integral membrane proteins. LTC4S interacted with FLAP in vitro through hydrophobic, membrane spanning regions. FLAP and LTC4S also interacted with 5-‐LO, which translocates from cytosol and nuclear matrix to the ER and nuclear envelope upon cell activation. The complete protein machinery for production of proinflammatory leukotrienes was expressed already at stage e11.5 during mouse embryogenesis and is restricted to the hematopoietic cells of the liver. 61 Conclusions Expression of LT synthesis proteins was similar in various hematopoietic populations of fetal liver and adult bone marrow. FLAP, but not 5-‐LO mRNA was upregulated in hematopoietic stem and progenitor cells and there was a decreased ratio of B to T-‐lymphocytes in peripheral blood and an increased ratio of common lymphoid progenitor cells in FLAP-‐/-‐ animals, suggesting a block in lymphocyte maturation in the absence of LT-‐ biosynthesis. 62 General discussion and future perspectives 6. GENERAL DISCUSSION AND FUTURE PERSPECTIVES Following the discovery of leukotrienes they were considered as mediators of allergic and “classical” inflammatory disorders. More recently however, it has been realized that LTs are important for a much wider range of diseases such as cardiovascular disease [389-‐391] including ischemic brain injury [242, 337, 392-‐398] and in cancer (reviewed in [149, 150, 422]). A few case studies have suggested CysLT roles during fetal development of the central nervous system, where congenital lack of LTC4S was associated with developmental CNS abnormalities [244]. Several reports, including our work in this thesis, suggest a possible role for LTs in hematopoietic regulation [269, 460-‐470] (Papers III & IV). Other reports suggest LTs as important factors for cancer cells, possibly implying direct effects on regulation of cell differentiation and proliferation. Further insights to roles of leukotrienes have been provided by studies of gene knockout animals. However, generation of these mice gave somewhat surprising results. Animals lacking functional genes for 5-‐LO, FLAP or LTC4S did not give profound phenotypes [183, 243, 476]. This could mean that LTs are not essential regulators of embryonic development and cell differentiation, or that redundant regulating systems exist. The results are however, compatible with LT roles as fine-‐tuning regulators of cell differentiation, as suggested in Paper IV, as well as with roles in induction or maintenance of malignant cell states. 63 General discussion and future perspectives The impaired LT synthesis in knockout mice is associated with a reduction of certain inflammatory reactions [183, 243, 476], which in many cases may be favorable. However, recent reports have shown also negative effects of impaired LT synthesis (reviewed in [286]). Thus, production of LTs needs to be well balanced and a shift from normal LT synthesis, in either direction, may have adverse effects. Too much LTs will lead to hyperreactive inflammatory disorders like asthma and allergy. Todays diet consumed in the western world is rich in ω-‐6 PUFAs and leads to an increased cell content of AA [14-‐23]. Hence, this could lead to an increased potential of leukotriene production that may in part explain the increased frequency of asthma and other allergic disorders observed in several parts of primarily the industrialized world [477]. Too little LTs on the other hand might lead to impaired inflammatory responses and a reduced defense capacity to microbe infections. Changes in LT production in either direction may also have mild effects on differentiation of e.g. hematopoietic cells, and possibly also other tissues where the outcome still needs to be explored. The effects observed in FLAP null mice on the lymphocyte lineage differentiation are interesting and should be investigated further. FLAP is essential for LT production from endogenous AA. In addition, FLAP affected the yield of metabolites formed by 12-‐lipoxygenase [185]. This suggests that studies of 5-‐LO-‐ and LT-‐ independent effects of FLAP may be another interesting field for investigations. Pharmaceutical research has been directed to the development of LT synthesis inhibitors and antagonists of LT receptors. The medical indications to use such drugs are primarily asthma and allergic rhinitis, but the involvement of LTs in other inflammatory conditions suggests that LT modulating drugs could be beneficial also in other diseases. 64 General discussion and future perspectives The ability to pharmacologically increase LT effects with receptor agonists might also be favorable in certain cases, such as malnutrition, vitamin D deficiency, HIV infection, liver cirrhosis or diabetes [412-‐419], where endogenous production is low. To allow a broader use of drugs affecting the LT system drugs of greater specificity will probably be needed. Highly specific agonists or receptor-‐blockers may be of great clinical importance since the effects mediated by the different subclasses of receptors in certain cells have been reported to oppose each other. Detailed information regarding protein-‐protein interactions taking place between the proteins of the LT machinery (cf. Paper II) may provide information which will aid the development of new more specific modulators of the LT synthesis machinery, such as peptides designed to compete at interaction sites between LT synthesis proteins. Studies regarding expression and function of LTs (Papers III and IV), contribute to better understanding of where beneficial or detrimental LT synthesis can be expected. Hence, indicate when and where there may be a gain to pharmacologically modulate production or actions of LTs. In the future, pharmacological modulation of the LT system will probably be used for an increasing number of disorders. The data presented in this thesis may indicate possibilities for new therapeutic approaches to diseases such as malignancies of the blood forming system using agents directed against the leukotriene system. 65 66 Acknowledgements 7. ACKNOWLEDGEMENTS 7.1 Financial support The work of this thesis was financially supported by: The Swedish Research Council, projects 31X-‐5914 & 31BI-‐14751 Östergötlands County Council Research Fund Märtha Lundqvists stiftelse Stiftelsen Lars Hiertas minne Fredrik och Ingrid Thurings stiftelse Stiftelsen för Gamla Tjänarinnor The Swedish Foundation for Strategic Research Lions forskningsfond mot folksjukdomar The Swedish Cancer Society (to Mikael Sigvardsson) 7.2 Personal thank you/Personligt tack The work behind a thesis like this is not solely the work of a single person. In addition to myself, several others have, directly or indirectly, contributed so that this book was ever written. Some of you were directly involved in the research work. Others were there as colleagues and friends and as parts of our nice milieu in the Division of cell biology in general and at floor 12 in particular. I would also like to acknowledge friends and family outside the university. All of you deserve a thank you… First of all, my supervisors Sven Hammarström; Thank you for giving me the opportunity to do the work, which has now resulted in this thesis and for sharing your great knowledge and amazing proof reading skills. 67 Acknowledgements Mats Söderström; Thank you for sharing your great methodological knowledge in the lab. Thanks to you a lot of trial and error experiments did not need to be done. Thank you also for being, not only a supervisor, but also a friend at work and outside of work and for sharing the great interest in Ice hockey. Former members of the Sven and Mats lab, either as undergraduate students or PhD students: Jesper, my predecessor as a PhD student working for Sven and Mats. I started as an undergraduate student when you were still there but we did not get to work that much together and you left before I started my PhD studies. However you still made a lasting impression on me and in the lab and I guess on everybody working on floor 12 (not until recently people dare to eat directly out of their lunch boxes again J). Elisabeth, when I started you were already there as a student from “forskarskolan”. During our time together in the lab we worked a lot together on similar projects and had a lot of fun (at least I did, I´m not sure about you since you first moved to the next room and later across the Atlantic ocean). Kristina, you were also a part of the lab when I started. Even though you did not work in the LT field you did not hesitate to help me with my questions. That you have left the lab is very obvious; I have not heard E-‐type on the stereo since. Björn and Cissi started off as undergraduate students in our lab when I had started my run towards the PhD degree. We had a lot of fun working together, it is always more fun than working alone. But after a while Björn climbed to floor 13 to work with trees and Cissi moved to “Strålforskningsgruppen” and left me doing the DIPPING alone J. 68 Acknowledgements Mikael Sigvardsson and Hong Qiang for a lot of help with Papers III and IV and the hematopoietic connection in this work. A lot of the work behind these papers could not have been made without your help. Micke; thanks for letting me work with this in your lab and thanks also for taking the time to talk science, always in a very enthusiastic way. Hong thanks for taking the time to help me perform a lot of work using FACS and also for introducing me to the field of hematopoiesis. Thanks also for being a patient teacher regarding questions of hematopoiesis and cell sorting. Michael G Rosenfeld and Christopher K Glass at UCSD for giving me the opportunity to learn important molecular techniques and Jie Zhang in the Rosenfeld lab for being an excellent teacher of in situ hybridization. Johan Ruud in the Blomqvist lab for pointers on DAB immuno, even though I abandoned your floating method. Thanks also for many hours of teaching histology together, to more or less motivated students. Åsa and Anette in ”cellkärnan” for help with numerous sequences and questions over the years and for always being friendly and nice. All other PhD student colleagues and friends at floor 12: Sebastian “Tysken” Schultz, my current room-‐mate. Thanks for all our good talks in the office. Those could deal with scientific questions as well as completely trivial things, sometimes just moaning about stuff together. It has been great to share office with a friend who understands the situation you are in. In many things we are very alike in others we are not that alike. In this part of YOUR thesis you suggested that I might join you on a camping trip. We will see, but may you first explain to me what that is?? J. 69 Acknowledgements My former roommates; Johan, Sofia and Veronica. It was great fun sharing office with all of you. Since you left the room it is bit quieter and (for some reason) seems to have a less active phone. All other present or former PhD student colleagues at floor 12; Åsa, Siri, Ia: it has been nice to always be able to stop by your office for a chat. I have missed you all during your times away from work. Marie, too bad that you left for Uppsala, you were great fun to have around, always one comment away from a laugh. Niclas thanks for nice discussions both on science and other stuff and for great “kräftskivor” at your “cottage”. Thanks also to everybody who has worked or works at “tolvan” for being a part of the nice atmosphere on our floor over the last 5-‐6 years. Other group leaders at floor 12 during my time here. It would have been a very different place without you: Gunilla Westermark: Thanks for always being helpful and for the spirit you created here and also for providing me with a lot of teaching jobs. Peter Strålfors: Thanks for many interesting and fun talks in the Lunch room and for being a big part of the nice working atmosphere at our floor. PhD student from other parts of HU whom I have got to know during my time here: Anders C, Jonas U, Johan J, Jocke H, and many more. Thanks for great fun and all great parties together. The rest of the staff in the cell biology building and in entire IKE for making it such a nice place to work in. 70 Acknowledgements Tack också till släkt och vänner utanför jobbet som också varit ett viktigt stöd för att kunna koppla bort jobbet ibland och tänka på andra saker. Mamma, Pappa, Syrran Sofia och hennes familj Pontus och Theo. Tack för att ni alltid är uppmuntrande även om ni inte har en aning om vad det är jag sysslat med på jobbet. And last but not least: Victoria, ÄNTLIGEN tror jag är ett ord som kan beskriva dina känslor kring att den här boken blivit klar. 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