The enzymatic machinery of leukotriene biosynthesis:

(1)

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

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© Tobias Strid 2012

Cover: Pixel-‐inverted scan of LTA

4

H 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

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Research is the act of going up alleys to see if they are blind

// Plutarch

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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 Linköping University

Co-‐supervisor:

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

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

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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 C

4

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 C

4

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

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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 A

4

(LTA

4

). FLAP saknar egen

enzymatisk aktivitet, och dess roll anses vara att överföra arakidonsyra från

fosfolipas A

2

till 5-‐LO. LTA

4

är en instabil molekyl som spontant bryts ned

om den inte omvandlas till LTB

4

av enzymet LTA

4

hydrolas eller till LTC

4

av

LTC

4

syntas. Leukotriener bildas runt cellens kärnmembran där FLAP och

LTC

4

S är belägna och dit 5-‐LO förflyttas i samband med inflammatorisk

aktivering av cellen. Vi har studerat proteinerna som möjliggör syntes av

LTC

4

och visat att FLAP och LTC

4

S 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. LTA

4

H finns i de flesta celler medan övriga proteiner finns i

betydligt färre celltyper. Vi undersökte LTC

4

S genens uttryck genom att

konstruera en vektor som uttrycker det lätt påvisbara proteinet GFP under

reglering av promotorn för LTC

4

S. Denna vektor gav ett cellspecifikt uttryck

liknande det naturliga för LTC

4

S när vektorn fördes in i olika celltyper. GFP

uttrycket ökade av ämnen som tidigare visats stimulera LTC

4

S. Vektorn är

alltså lämplig som rapportör för LTC

4

S uttryck.

(8)

DNA från vektorn användes för att ta fram genmodifierade möss som

uttrycker GFP som LTC

4

S 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.

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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 LTA

4

in cooperation with 5-‐LO activating

protein (FLAP). LTA

4

is converted to LTB

4

, by LTA

4

-‐hydrolase (LTA

4

H) or to

LTC

4

by LTC

4

-‐synthase (LTC

4

S). 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 LTC

4

synthesis. 5-‐LO interacts with cytosolic domains

of the integral membrane proteins FLAP and LTC

4

S at the nuclear envelope,

in addition LTC

4

S interacts with FLAP through its hydrophobic membrane

spanning regions. We constructed an LTC

4

S promoter controlled GFP

reporter vector, displaying cell specific expression and sensitivity to agents

known to affect LTC

4

S expression. The vector was used to create transgenic

mice expressing GFP as a reporter for LTC

4

S. 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

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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

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

(11)

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

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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

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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]).

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COOH Arachidonic acid COOH Eicosapentaenoic acid COOH Dihomo-γ-linolenic acid

Eicosanoids

Essential fatty acids

Linoleic acid α-Linolenic acid

COOH COOH

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.

(15)

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 Δ

6

_{-‐desaturase
and
fatty
acid}

elongase respectively, giving rise to DHGLA which is further converted by

Δ

5

_{-‐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 E

2

(PGE

2

)

,

thromboxane A

2

(TXA

2

), leukotriene

B

4

(LTB

4

), 5-‐hydroxyeicosatetraenoic acid (5-‐HETE) and leukotriene C

4

(16)

Instead it increases the production of the 3 series prostaglandins (PGs),

thromboxane (TXA

3

) 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]).

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 A

2

enzymes [30], most commonly cytosolic phospholipase A

2

(cPLA

2

) [32, 33].

Arachidonic acid (20:4) HO COOH OOH 5-HpETE COOH O LTA4 O O COOH PGH2 OH O O COOH O 5-LO COX PGG2 5-LO COX COOH

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.

(17)

cPLA

2

is activated by an increase in intracellular Ca

2+

concentration [34, 35]

and by phosphorylation [36-‐39]. Ca

2+

_{activates
cPLA}

₂

_{directly
by
interaction}

with a Ca

2+

_{-‐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 P

450

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 PGH

2

. 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 PGG

2

. This is followed by reduction of a

15-‐hydroperoxy group in PGG

2

to a 15-‐hydroxyl in PGH

2

. PGH

2

is the

immediate precursor of the primary PGs, prostacyclin (PGI

2

) and TXA

2

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].

(18)

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 O

2

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

O

2

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.

COOH PGD2 OH O O COOH PGH2 OH COOH TXA2 OH COOH PGE2 OH COOH PGI2 OH COOH PGF2 OH O O OH O O OH OH OH O α

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 PGF2α), prostacyclin (PGI2) and

(19)

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 OOH 5-HpETE Arachidonic acid (20:4) COOH 12-HpETE OOH 15-HpETE COOH OOH COOH 5-LO 12-LO 15-LO

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.

(20)

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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,

LTA

4

H and LTC

4

S. 5-‐LO, assisted by FLAP, transforms AA to LTA

4

, which is

metabolized either by LTA

4

H to LTB

4

, or by LTC

4

S to LTC

4

.

2.1.1 Biochemistry of Leukotrienes

Leukotrienes of the 4 series are formed from AA following its release from

membrane phospholipids by cPLA

2

(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 cPLA

2

[67]. 5-‐LO catalyzes a two-‐step conversion of AA via

5-‐HpETE to LTA

4

(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 LTA

4

[71-‐73]

1

(Fig. 5). LTA

4

is

unstable and rapidly metabolized to either LTB

4

or LTC

4

. LTA

4

H catalyzes a

hydrolytic reaction to the dihydroxy acid LTB

4

[68, 69] (Fig. 5), which is

exported out of the cell by a carrier-‐mediated, temperature sensitive and

energy-‐dependent mechanism [74, 75]. LTC

4

S catalyzes a glutathione-‐S-‐

transferase reaction yielding the peptidolipid LTC

4

.

(22)

The discovery of LTC

4

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]. LTC

4

is formed by conjugation of LTA

4

with

reduced glutathione (GSH) catalyzed by LTC

4

S [76, 77]. Shortly after that

LTD

4

[79-‐81] and LTE

4

[81, 82] were found to be formed by metabolism of

the peptide part of LTC

4

.

COOH O LTA4 COOH OH OH LTB4 COOH OH γGluCysGly C5H11 LTC4 COOH OH CysGly C5H11 LTD4 COOH OH Cys C5H11 LTE4 LTA4H LTC4S + GSH γ-Glutamyl transpeptidase / γ-Glutamyl leukotrienase Dipeptidase 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.

(23)

LTD

4

is generated from LTC

4

by cleavage of the isopeptide bond of the GSH

moiety by γ-‐glutamyl transpeptidase [79, 83] or γ-‐glutamyl leukotrienase

[84] and LTE

4

is formed from LTD

4

by elimination of the glycine residue by

a dipeptidase [82, 85] (Fig. 5). LTC

4

is actively transported out of the cells

by multidrug resistance-‐associated protein 1 (MRP1) [86-‐88] and the

conversions to LTD

4

and LTE

4

take place extracellularly.

LT production may also occur in cells not expressing all the necessary

enzymes. LTB

4

was formed when red blood cells, which lack 5-‐LO, were

incubated with LTA

4

[89]. Conversion of exogenous LTA

4

to LTC

4

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 LTC

4

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].

(24)

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, cPLA

2

and 5-‐LO

translocate to the NE and/or ER, where FLAP resides [114-‐116]. Most

stimuli simultaneously activate 5-‐LO and cPLA

2

(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 Ca

2+

_{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 Ca

2+

_{[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 Ca

2+

_{[125,
126].
The
interaction,
but
not
enzyme
activity
occurs}

in the absence of Ca

2+

_{[126].
Nitric
oxide
[127,
128]
and
certain
peroxidases}

[129, 130] suppress 5-‐LO activity.

(25)

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].

(26)

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 Ca

2+

_{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 D

3

response elements was

detected between positions -‐779 and -‐229 [158]. Additional vitamin D

3

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.

(27)

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 Ca

2+

_{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 LTC

4

S (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 Ca

2+

_{-‐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 cPLA

2

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].

(28)

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 D

3

, 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].

(29)

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 LTB

4

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 LTC

4

S

Leukotriene A

4

is enzymatically converted to LTC

4

[194, 195] by a

glutathione S transferase (GST) with narrow substrate specificity [196].

LTC

4

S is a membrane-‐bound protein [196, 197] located in the microsomal

cell fraction [198-‐201]. LTC

4

S 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 LTC

4

S cDNA was

reported by two groups in 1994 [204, 205], revealing a 150 amino acid

protein. Evidence for “homo-‐oligomerization of LTC

4

S in living cells” was

published in 2003 [206]. The two-‐dimensional crystal structure of

recombinant LTC

4

S 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.

(30)

Human LTC

4

S 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 LTC

4

S 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 LTC

4

S

compared to a long second cytosolic loop in FLAP. The GSH binding site of

LTC

4

S 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]. LTC

4

S 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 LTC

4

S 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]

LTC

4

S 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]).

(31)

The human LTC

4

S and FLAP genes have almost identical intron / exon

organization but LTC

4

S has much shorter introns [186]. The human LTC

4

S

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 LTC

4

S. 5-‐LO and

LTC

4

S co-‐express in cells of myeloid origin (basophils, eosinophils, mast

cells and monocytes / macrophages) [220]. Platelets contain LTC

4

S but lack

5-‐LO [221]. LTC

4

S 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 LTC

4

S [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 LTC

4

formation in

cells pre-‐treated with TPA [230, 231]. LPS [232, 233] and TNF-‐α [234]

down-‐regulate expression of LTC

4

S whereas TGF-‐β [235] upregulates it in

mononuclear-‐like cell lines. Retinoic acid upregulated LTC

4

S promoter and

enzyme activity in rat basophilic leukemia cells [236, 237]. LTC

4

S

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 LTC

4

S 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 LTC

4

S promoter was associated with

(32)

LTC

4

S knockout mice [243] develop and breed normally but have a reduced

inflammatory response in some models. Data obtained from these mice

showed that LTC

4

S accounted for the major part of their LTC

4

production,

with only a small contribution from other GST enzymes. A few human case

reports have indicated individuals lacking LTC

4

S. 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 LTC

4

with an absorbance maximum at 282 rather than

280 nm and a shorter retention time than LTC

4

when analyzed by RP-‐HPLC.

The product was identified as 14,15-‐LTC

4

and named eoxin (EX) C

4

[245].

Like LTC

4,

EXC

4

was metabolized to EXD

4

and EXE

4

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 Ca

2+

_{ionophore
resulted
in
mainly
LT
products
[245].
It
is
not}

known whether LTC

4

S is involved in the biosynthesis of eoxins or other 15-‐

LO or 12-‐LO products.

2.1.5 LTA

4

H

LTA

4

H is a cytosolic 70 kDa zinc-‐containing metalloprotease [246]. The

protein has dual enzyme activities and is responsible for the conversion of

LTA

4

to LTB

4

The enzymatic machinery of leukotriene biosynthesis: