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Pharmacological actions and targets of boswellic acids in human leukocytes and platelets [Elektronische Ressource] / von Daniel Pöckel

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Pharmacological actions and targets of boswellic acids in human leukocytes and platelets Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe–Universität Frankfurt am Main von Daniel Pöckel aus Frankfurt am Main Frankfurt am Main (2006) (D 30) vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Dr. Harald Schwalbe Gutachter: Prof. Dr. Oliver Werz Prof. Dr. Theo Dingermann Datum der Disputation: 16.11.2006 Meinen Eltern Table of Contents 1 Table of Contents 1 ABBREVIATIONS ____________________________________________3 2 INTRODUCTION_____________________________________________6 2.1. Inflammation 6 2.1.1. Inducers/Mediators of inflammation 7 2.2. Physiology of haematopoietic cells 8 2.2.1. Polymorphonuclear leukocytes (PMNL) 8 2.2.2. Monocytes and macrophages 9 2.2.3. Platelets 10 2.3. Typical signalling pathways 12 2.3.1. Cell surface receptors 12 2+2.3.2. The role of Ca 13 2.3.3. MAP kinase cascades 14 2.4. The arachidonic acid cascade 17 2.4.1. Cytosolic phospholipase A 17 22.4.2. Lipoxygenases 19 2.4.2.1. 5-Lipoxygenase 20 2.4.2.2. 12-Lipoxygenase 22 2.4.2.3. 15-Lipoxygenase 24 2.4.3. LTA hydrolase and LTC synthase 25 4 42.4.4.

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Published 01 January 2006
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Pharmacological actions and targets
of boswellic acids in human leukocytes and platelets



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe–Universität
Frankfurt am Main




von
Daniel Pöckel
aus Frankfurt am Main



Frankfurt am Main (2006)
(D 30)





vom Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
als Dissertation angenommen.












Dekan: Prof. Dr. Harald Schwalbe
Gutachter: Prof. Dr. Oliver Werz
Prof. Dr. Theo Dingermann

Datum der
Disputation: 16.11.2006









Meinen Eltern Table of Contents 1

Table of Contents


1 ABBREVIATIONS ____________________________________________3
2 INTRODUCTION_____________________________________________6
2.1. Inflammation 6
2.1.1. Inducers/Mediators of inflammation 7
2.2. Physiology of haematopoietic cells 8
2.2.1. Polymorphonuclear leukocytes (PMNL) 8
2.2.2. Monocytes and macrophages 9
2.2.3. Platelets 10
2.3. Typical signalling pathways 12
2.3.1. Cell surface receptors 12
2+2.3.2. The role of Ca 13
2.3.3. MAP kinase cascades 14
2.4. The arachidonic acid cascade 17
2.4.1. Cytosolic phospholipase A 17 2
2.4.2. Lipoxygenases 19
2.4.2.1. 5-Lipoxygenase 20
2.4.2.2. 12-Lipoxygenase 22
2.4.2.3. 15-Lipoxygenase 24
2.4.3. LTA hydrolase and LTC synthase 25 4 4
2.4.4. Cyclooxygenases 25
2.5. Biological functions of eicosanoids 27
2.5.1. Leukotrienes 27
2.5.2. 5- and 12-H(P)ETE 28
2.5.3. 15-HETE, oxo-ETEs, lipoxins, hepoxilins, and EETs 28
2.6. Boswellic acids 30
2.6.1. Historical overview and sources 30
2.6.2. BAs in disease treatment 32
2.6.3. BAs in immunity and inflammation 33
2.6.3.1. Modulation of the immune response 33
2.6.3.2. Anti-inflammatory effects 33 Table of Contents 2

2.6.4. Molecular targets 34
2.6.4.1. 5-Lipoxygenase 34
2.6.4.2. Other molecular targets 35
2.6.5. Modulation of intracellular signalling networks 36
2.6.6. Effects of BAs on cell proliferation, differentiation, and death 37
2.7. Aim of this work 39
3 METHODS ________________________________________________41
4 RESULTS AND DISCUSSION ___________________________________42
2+4.1. Paper I: Coupling of boswellic acid-induced Ca mobilisation and MAPK
activation to lipid metabolism and peroxide formation in human leucocytes 43
4.2. Paper II: Induction of central signalling pathways and select functional effects
in human platelets by β-boswellic acid 47
4.3. Paper III: Boswellic acids stimulate arachidonic acid release and 12-
2+lipoxygenase activity in human platelets independent of Ca and differentially interact
with platelet-type 12-lipoxygenase 51
TM4.4. Paper IV: Immobilisation of Boswellic acids at EAH Sepharose for “target
fishing” 54
2+4.5. Paper V: 3-O-Acetyl-11-keto-boswellic acid decreases basal intracellular Ca
2+levels and inhibits agonist-induced Ca mobilisation and MAP kinase activation in
human monocytic cells 57
5 SUMMARY ________________________________________________62
6 ZUSAMMENFASSUNG _______________________________________68
7 REFERENCES______________________________________________75
8 APPENDIX (PAPER I-V) _____________________________________92
9 PUBLICATION LIST ________________________________________146
10 ACKNOWLEDGMENTS______________________________________149
11 CURRICULUM VITAE151
Abbreviations 3
1 Abbreviations

AA arachidonic acid
AC adenylyl cyclase
A( β)BA 3-O-acetyl-( β-)BA
(A)KBA (3-O-acetyl-)11-keto-BA
A(D/T)P adenosine (bis/tris)phosphate
ATL aspirin-triggered lipoxin
( β-)BA ( β-)boswellic acid
BC-4 mixture of α-BA and β-ABA (1:1) extracted from B. carterii
B. spec Boswellia species
2+CaLB Ca /lipid binding domain
2+CaMK Ca /calmodulin-dependent kinase
2+(c/s/i)PLA (cytosolic/secretory/Ca -independent) phospholipase A2 2
cGMP cyclic guanosine monophosphate
CNS central nervous system
CSF colony-stimulating factor
COX cyclooxygenase
CYP cytochrome P450
cys-LT cysteinyl leukotriene
DAG diacylglycerol
DCF-DA dichlorofluorescein diacetate
EET epoxyeicosatrienoic acid
ER/SR endoplasmatic/sarcoplasmatic reticulum
ERK extracellular signal-regulated kinase
ETP endogenous thrombin potential
FLAP 5-lipoxygenase-activating protein
fMLP N-formyl-methionyl-leucyl-phenylalanine
G(D/T)P guanosine (bis/tris)phosphate
GEF guanine exchange factor
GM-CSF granulocyte/macrophage colony stimulating factor
GP glycoprotein
GPx glutathione peroxidase
GPCR G protein-coupled receptor
Grb-2 growth factor receptor-bound protein 2
GSH glutathione (reduced state)
(12-)HHT (12-)hydroxyheptadecatrienoic acid
HL-60 human leukaemia cell line
HLE human leukocyte elastase
H(P)ETE hydro(pero)xy-eicosatetraenoic acid
(13-)HPODE (13-)hydroperoxyoctadecadienoic acid
IFN interferone
IKK inhibitor of NF- κB (I κB)-kinase Abbreviations 4
IL interleukin
IP inositol trisphosphate 3
JNK c-Jun N-terminal kinases
LDL low-density lipoprotein
LPS lipopolysaccharide
LO lipoxygenase
LT leukotriene
LTAH LTA hydrolase 4 4
LTC S LTC synthase 4 4
LX lipoxin
Mac-1 macrophage antigen-1
MAPEG membrane associated proteins in eicosanoid and GSH metabolism
MAPK(KK) mitogen-activated protein kinase (kinase kinase)
MAPKAPK MAPK-activating protein kinase
MEK MAPK and ERK kinase
MMP matrix metalloproteinase
MNK MAPK-integrating kinase
MK-2/3 MAPKAPK-2/3
NF- κB nuclear factor κB
NO nitric oxide
PAF(-AH) platelet-activating factor (acetylhydrolase)
PAR protease activated receptor
PDGF platelet-derived growth factor
PG prostaglandin
PGI prostacyclin 2
PH pleckstrin homology
PIP phosphatidylinositol-4,5-bisphosphate 2
PI-3 K phosphoinositide-3 kinase
PL phospholipase
PMA phorbol 12-myristate 13-acetate
2+PMCA plasma membrane Ca -ATPase
PMNL polymorphonuclear leukocytes
PK(A/C) protein kinase A/C
PSGL P-selectin glycoprotein ligand
PT pentacyclic triterpene
PTx pertussis toxin
PtdCho/Ins phosphatidyl choline/inositol
p-tyr phospho-tyrosine
p12-LO platelet-type 12-lipoxygenase
RA rheumatoid arthritis
RGS regulators of G protein signalling
ROS reactive oxygen species
RTK receptor tyrosine kinase
SAPK stress-activated protein kinase Abbreviations 5
2+SERCA SR/ER Ca -ATPase
SH(2/3) src homology (2/3) domain
2+SOCE store-operated Ca entry
TA tirucallic acid
TF tissue factor
TGF transforming growth factor
TLR Toll-like receptor
TM transmembrane
TNF tumour necrosis factor
TRAP thrombin receptor-activated peptide
Tx thromboxane
vWf von Willebrand factor Introduction 6
2 Introduction

2.1. Inflammation
Inflammation is the physiological reaction of the immune defence system to a variety of
noxae, including internal stimuli (tissue injury, lesions) and external stimuli (pathogen
infection, bacterial toxins). On the one hand, inflammation consists of an exudative
component, resulting in blood vessel dilatation and enhanced capillary permeability
around the site of irritation or infection. As a consequence, redness, heat, swelling (edema)
and pain are caused. On the other hand, the second (cellular) component of inflammation
involves leukocytes, capable to enter the tissue through permeabilised capillary walls. At
the first stage, phagocytes attempt to eliminate the infectious stimuli, but if the
inflammatory stimulus persists, cytokines (interleukin-1 (IL-1), tumor necrosis factor
(TNF)) are released that activate the endothelium in order to upregulate the expression of
adhesion receptors (VCAM, ICAM, selectins). Other inflammatory cells such as
additional granulocytes, monocytes, T and B cells are recruited to the inflamed tissue.
Typically, neutrophilic granulocytes, also termed polymorphonuclear leukocytes (PMNL),
are found in acute states of inflammation, where they are responsible for phagocytosis and
chemokine/second messenger release so as to promote and finally terminate the
inflammatory process. At the infectious site, they can undergo a respiratory burst to attack
intruders, and secrete proteases to finally cleanse damaged tissue. In wound repair,
initially blood platelets are triggered by tissue collagen to release mediators, express
glycoproteins and aggregate, resulting in the formation of a fibrin/fibronectin-based clot,
that attracts other cells like PMNL. In subsequent processes, collagen deposition leads to
the re-formation of the extracellular matrix (ECM), and angiogenesis is induced (for
reviews, see [1, 2]).
In chronic or persisting inflammation, however, the short-lived neutrophils are gradually
substituted by monocytes from the peripheral blood. At the infected or inflamed tissue,
monocytes enter the tissue and mature into macrophages, which eliminate apoptotic
PMNLs after a few days [2, 3]. Macrophages are powerful producers of reactive oxygen
species (ROS), supposed as a defence mechanism but also causing destruction to the
surrounding tissue if the cells remain too long at the infectious site [4]. Thus, in many
diseases with chronic inflammation (arthritis, rheumatism), it is beneficial to downregulate
the activity of leukocytes by pharmacological agents in order to prevent progressive tissue
degradation.
Introduction 7
2.1.1. Inducers/Mediators of inflammation
In case of exogenous infections, the tripeptide N-formyl-methionyl-leucyl-phenylalanine
(fMLP) may be secreted by bacteria [5]. fMLP is a potent chemotactic for neutrophils,
where it binds to a specific G protein-coupled receptor (GPCR) [6, 7], causing neutrophil
2+activation via intracellular mobilisation of Ca and activation of protein kinases such as
the mitogen-activated protein (MAP) kinase cascade [8, 9] but also of cytosolic
phospholipase (cPL)A [10]. 2
Cytosolic phospholipase (cPL)A predominantly liberates AA which either functions as 2
mediator itself, inducing PMNL adhesion to the endothelium, ROS formation, chemotaxis
and MAP kinase activation [11-13], or serves as a precursor for bioactive lipid mediators
(platelet-activating factor, PAF) and eicosanoids. These include prostaglandins (PGD , 2
PGE ), prostacyclin (PGI ), and thromboxane (TxA), derived from cyclooxygenase (COX) 2 2
and PGE synthases, as well as hydroxyl-eicosatetraenoic acids (HETEs), leukotriene
(LT)B , cysteinyl (Cys)-LTs, and lipoxins, synthesised by lipoxygenases. PAF, derived 4
from lyso-PAF, attracts and stimulates granulocytes and monocytes/macrophages,
enhances vascular permeability and aids to maintain an inflammatory state [14]. PAF also
participates in platelet activation by binding to its corresponding G- and/or G -coupled i q
receptor [15]. Per se, PAF is a moderate stimulus for neutrophils and a strong stimulus for
. -eosinophils, that for instance causes substantial superoxide anion ( O ) formation and 2
generally shares many biological effects with fMLP [16]. COX products usually activate
specific GPCRs and have diverse physiological effects mainly related to pain sensation,
vascular homeostasis, and inflammation. Lipoxygenase (LO) products are discussed in a
later section. Taken together, eicosanoids act as local hormones with short half-life. In
contrast, cytokines share many features with “classical” hormones. They are polypeptides,
synthesised and released by many inflammatory and other cells, encompassing
interleukins (ILs), interferones (IFNs), colony stimulating factors (CSF), tumor necrosis
factors (TNFs), transforming growth factor β (TGF- β), and chemotactic cytokines
(chemokines) [17]. Cytokines bind to membrane-embedded cytokine receptors that induce
cytosolic responses but also modulate transcription factors via members of the MAP
kinase family, the c-Jun N-terminal kinases (JNKs). They have proinflammatory and
immunostimulant properties, modulate the haematopoietic system and control
proliferation, differentiation and cell survival [18]. Chemokines are the largest group of
cytokines (>40 human members), and act via specific GPCRs to mediate leukocyte
activation [19, 20]. Accordingly, leukocyte- or fibroblast-derived IL-8 is the most
important chemokine for neutrophil activation, inducing a respiratory burst [21].