Studies on the intracellular signalling pathways triggered by the anaphylatoxin c5a in human phagocytic cells 1

2.4 Transfection of cells with oligonucleotides 50 2.5 RNA extraction and reverse transcription-PCR 51 2.6 Isolation of primary human neutrophils 51 2.7 Isolation of primary human monocy

STUDIES ON THE INTRACELLULAR SIGNALING PATHWAYS TRIGGERED BY THE ANAPHYLATOXIN

C5a IN HUMAN PHAGOCYTIC CELLS

FARAZEELA BINTE MOHD IBRAHIM

ACKNOWLEDGEMENTS

I am extremely grateful and indebted to my supervisor, Dr Alirio J Melendez, for his

advice and guidance throughout the course of my research in the lab Regardless of

his commitments and busy schedule, he always found time to supervise my work and

was never short of comforting and motivating words for me, especially when the

going was tough Without his support and encouragement, this work would not have

been accomplished It was an eye-opening and wonderful experience to conduct

research under his supervision Thank you very much for introducing me to this

fascinating world of research

Special thanks belong to my lab colleagues who have given me excellent cooperation

and assistance throughout my stay in the Molecular and Cellular Immunology Lab in

the Department of Physiology I am honored to have had the opportunity to work with

each and every one of them in different aspects of my research In them, I have found

firm friends and I truly cherish the friendship we share

I wish to acknowledge my deepest gratitude and appreciation to my husband, who has

been my constant source of encouragement and moral support, my pillar of strength

and my confidante, without whom this journey would have been that much harder

I am thankful to my parents, sisters and family members for their support and love

throughout my life The knowledge of them being there has been of great

encouragement and importance to me

I would also like to thank Ms Anneke Melendez-Fraser for her helpful comments,

invaluable advice and the time spent proofreading this thesis

CHAPTER I INTRODUCTION

1.1.1 Complement proteins and nomenclature 2

1.1.2 Activation of the complement cascade 3

1.1.2.3 The mannan-binding lectin (MBL) pathway 5 1.1.3 Functions of the complement system 7

1.1.4 Regulation of the complement system 9

1.1.5.1 Biological properties of C5a 10

1.1.5.3 Complement 5a receptor (C5aR) 13

1.2 Important downstream events triggered by C5a 17

1.2.2 Nuclear factor kappa B 18

1.3.5 Role of SPHK and S1P in cellular processes 30

1.3.6 Role of SPHK and S1P in immune cells 32

1.4.5 Downstream signaling of PLD products 41

CHAPTER II MATERIALS AND METHODS

2.3 Cell culture and differentiation of cells 50

2.4 Transfection of cells with oligonucleotides 50

2.5 RNA extraction and reverse transcription-PCR 51

2.6 Isolation of primary human neutrophils 51

2.7 Isolation of primary human monocytes and differentiation to 52

macrophages

2.9 Measurement of sphingosine kinase activity in cell extracts 53

2.10 Measurement of sphingosine-1-phosphate generation in whole cells 53

2.18 Subcellular fractionation by differential centrifugation 58

2.19 Gel electrophoresis and western blotting analysis 58

2.22 Measurement of matrix metalloproteinase release 62

CHAPTER III RESULTS

3.1 Key role for sphingosine kinase in C5a signaling in human neutrophils 64

3.1.1 C5a stimulates SPHK activity in human neutrophils 67

3.1.2 Role of SPHK in C5a-triggered Ca2+signals 70

3.1.3 Role of SPHK in C5a-triggered degranulation 73

3.1.4 Role of SPHK in C5a-triggered chemotaxis 73

3.1.5 Role of SPHK in C5a-triggered NADPH oxidative burst 74

3.1.6 SPHK is the enzyme activated by C5a Antisense knockdown 78

of SPHK1 3.1.7 SPHK1 mediates C5a-triggered Ca2+ release, degranulation, 80

chemotaxis and NADPH oxidase activity 3.1.8 Effects of DMS and/or antisense oligonucleotides 83

3.1.9 Effects of sphingosine and sphingosine-1-phosphate on signaling 87

3.2 Key role for sphingosine kinase in C5a signaling in human macrophages 96

3.2.1 C5a stimulates SPHK activity in human macrophages 98

3.2.2 SPHK1 is the enzyme activated by C5a Antisense knockdown 100

of SPHK1 3.2.3 Role of SPHK1 in C5a-triggered Ca2+signals 103

3.2.4 Role of SPHK1 in C5a-triggered PKC activity 105

3.2.5 Role of SPHK1 in C5a-triggered degranulation 105

3.2.6 Role of SPHK1 in C5a-triggered chemotaxis 108

3.2.7 Role of SPHK1 in C5a-triggered cytokine production 108

3.3 Potential role for phospholipase D in C5a signaling in macrophages 116

3.3.1 C5a stimulates PLD activity in dbcAMP-differentiated U937 cells 119

3.3.2 C5a stimulates the translocation and redistribution of PLD1 119

isoform 3.3.3 Role of PLD in C5a-triggered Ca2+signals 122

3.3.4 Role of PLD in C5a-triggered NADPH oxidase activity 122

3.3.5 Role of PLD in C5a-triggered degranulation 125

3.3.6 Role of PLD in C5a-triggered chemotaxis 125

3.3.7 Role of PLD in C5a-triggered NF-κB translocation and activation 128

3.3.8 Role of PLD in C5a-triggered cytokine production 131

3.3.9 Role of PLD in C5a-triggered matrix metalloproteinase (MMP) 131

release 3.3.10 C5a induces Raf-1 translocation and phosphorylation of ERK1/2 135

and p38

3.3.11 C5a-triggered PLD activity is potentially upstream of SPHK, 138

ERK1/2, p38 MAPK and PKC

CHAPTER IV CONCLUSION 149

CHAPTER V REFERENCES 152

SUMMARY

Anaphylatoxins play a key role in inflammatory responses, and in many diseases they

contribute to the pathogenesis Inflammation is the body’s natural response to tissue

damage and injury, and is mediated by several interconnected enzymatic pathways

One such pathway is the complement cascade, through which the anaphylatoxin C5a,

a potent stimulator of mediators of chronic and acute inflammation, is generated

Although the actions of C5a are well established, the mechanisms regulating

C5a-triggered intracellular signaling pathways are poorly understood The

phospholipid-modifying enzymes, sphingosine kinase (SPHK) and phospholipase D (PLD), are

emerging as important signaling molecules, and have been suggested to function as

crucial players in the physiological responses triggered by activated immune-effector

cells

Hence, the objective of my study is to investigate the intracellular signaling pathways

triggered by C5a, particularly the roles of SPHK and PLD, in mediating

proinflammatory functions in human neutrophils and macrophages The ultimate goal

is to identify key molecules as candidates for novel therapeutic intervention

In this thesis, I provide evidence that demonstrate, for the first time, that the

anaphylatoxin C5a activates the intracellular signaling molecule SPHK, and present

data that support the role for SPHK in the proinflammatory responses triggered by

C5a in human neutrophils and macrophages, showing that inhibition of this enzyme

has potential anti-inflammatory properties We demonstrate that C5a receptor

activation stimulates SPHK activity in these cells Moreover, the inhibition of SPHK

by DMS inhibits C5a-stimulated Ca2+ mobilization, degranulation, chemotaxis, and

NADPH activation in these cells Furthermore, an antisense oligonucleotide specific

for SPHK1 also inhibited the C5a-induced responses, suggesting that SPHK1 is the

isoform triggered by C5a We also show here that C5a stimulation decreases cellular

sphingosine levels and increases the formation of sphingosine-1-phosphate (S1P),

suggesting a role for SPHK in removing a negative regulator (sphingosine), and

generating a positive regulator (S1P) We also studied the effects of exogenously

added sphingosine and S1P in the neutrophils We found that sphingosine has no

effect on C5a-triggered Ca2+ signals, chemotaxis and degranulation, but dual effect on

C5a-stimulated NADPH oxidase activation and minimal effect on C5a-triggered PKC

activity S1P by itself did not induce degranulation or chemotaxis, but it did

marginally induce Ca2+ signals and the oxidative burst However, S1P showed a

priming effect, enhancing all C5a-triggered responses

I also present data that suggest the potential role of PLD in C5a-induced

proinflammatory responses in macrophage-differentiated U937 cells In the presence

of a primary alcohol (butan-1-ol), C5a-triggered Ca2+ signals, NADPH oxidative

burst, chemotaxis, degranulation, NF-κB translocation and activation, cytokine

release and MMP release are significantly inhibited, suggesting a role for PLD in

triggering these responses I also show that C5a induces Raf-1 translocation, which

may activate MAPKs, and that PLD activity is potentially upstream of some signaling

enzymes

Thus, our data contribute not only to the understanding of the intracellular molecular

mechanisms utilized by C5a, suggesting that SPHK and PLD potentially play key

roles in C5a-triggered proinflammatory functions, but also point out SPHK and PLD

as novel candidates for potential therapeutic intervention to treat inflammatory and

autoimmune diseases

LIST OF FIGURES

Introduction:

Figure B Ribbon diagram of the human C5a molecule 12

Figure C Model for the interaction of C5a with C5aR 15

Figure E PLD-catalyzed hydrolysis and transphosphatidylation reactions 36

Figure G C5a-triggered intracellular signaling in human phagocytic cells 148

Results:

Figure 1 C5a triggers SPHK activity in primary human neutrophils and 68

differentiated HL-60 cells (neutrophil model)

Figure 2 C5a triggers S1P generation in primary human neutrophils and 69

differentiated HL-60 cells (neutrophil model)

Figure 3 C5a-triggered cytosolic Ca2+ signals in neutrophils are inhibited 71

by DMS

Figure 8 SPHK1 expression, subcellular localization and antisense 79

knockdown

Figure 9 SPHK1 mediates the physiological responses triggered by C5a 81

in differentiated HL-60 cells

Figure 10 Role of DMS and/or SPHK1 antisense oligonucleotide on cell 84

viability, PKC activity, calcium signals, S1P generation and sphingosine levels in neutrophils

Figure 11 Role of exogenously added sphingosine or S1P on neutrophil 88

functions

Figure 12 C5a triggers S1P generation and SPHK activity in human 99

macrophages

Figure 13 SPHK1 expression, subcellular localization and antisense 101

knockdown in the human monocyte-derived macrophages

Figure 14 S1P generation and SPHK activity in SPHK1 antisense 102

knockdown in human monocyte-derived macrophages

Figure 15 C5a-triggered cytosolic Ca2+ signals in macrophages are inhibited 104

by DMS, showing a role for SPHK

Figure 16 C5a-triggered PKC activity in the monocyte-derived macrophages 106

is not inhibited by the SPHK1 antisense

Figure 17 Degranulation triggered by C5a in macrophages is dependent on 107

SPHK activity

Figure 18 C5a-induced chemotaxis is inhibited in macrophages pretreated 109

with the SPHK1 antisense

Figure 19 TNF-α, IL-6, and IL-8 release triggered by C5a is inhibited in 110

macrophages pretreated with the SPHK1 antisense Figure 20 C5a triggers PLD activity in differentiated U937 cells 120

Figure 21 Only PLD1 translocates upon C5a trigger in macrophages 121

Figure 22 C5a-triggered cytosolic Ca2+ signals are inhibited by butan-1-ol: 123

a role for PLD Figure 23 NADPH oxidative burst is potentially PLD-dependent 124

Figure 24 C5a-induced degranulation is dependent on PLD activity 126

Figure 25 PLD potentially mediates C5a-induced chemotaxis 127

Figure 26 C5a-induced NF-κB translocation and activation is inhibited by 129

butan-1-ol

Figure 27 IL-6 and IL-8 release triggered by C5a in differentiated U937 132

cells is mediated by PLD Figure 28 PLD plays a potential role in C5a-induced MMP release 133

Figure 29 C5a induces the translocation of Raf-1 in the macrophage-like 136

Figure 30 C5a receptor stimulation activates ERK1/2 and p38 MAPK 137

Figure 31 C5a-induced PLD activity in differentiated U937 cells is 139

independent of the activation of SPHK, ERK1/2, p38 MAPK

ABBREVIATIONS

AP-1 Activating protein-1

APMA p-Aminophenylmercuric acetate

Arf Adenosine diphosphate-ribosylation factor

BAPTA Bis(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid

C5a Complement factor 5a

C5aR C5a receptor (CD88)

CGD Chronic granulomatous disease

dbcAMP Dibutyryl cyclic adenosine monophosphate

DHS DL-threo-dihydrosphingosine

DMS N,N-dimethylsphingosine

EDG Endothelial differentiation gene

EDTA Ethylenediaminetetraacetic acid

EGTA Ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic Acid

ELISA Enzyme-linked immunosorbent assay

ERK Extracellular signal-regulated kinase

FITC Fluorescein isothiocyanate

MAC Membrane attack complex

MAPK Mitogen activated protein kinase

MAPKK Mitogen activated protein kinase kinase

MAPKKK Mitogen activated protein kinase kinase kinase

MASP MBL-associated serine proteases

min Minutes

mRNA Messenger ribonucleic acid

NADPH Nicotinamide adenine dinucleotide phosphate

NF-κB Nuclear factor kappa B

NLS Nuclear localization sequence

NPB Nuclear preparation buffer

PAPH Phosphatidic acid phosphohydrolase

PBS Phosphate buffered saline

RLU Relative luminescence unit

ROS Reactive oxygen species

RPMI Roswell Park Memorial Institute

RT-PCR Reverse transcription-polymerase chain reaction

S1P Sphingosine-1-phosphate

SDS Sodium dodecyl sulfate

SDS-PAGE SDS-polyacrylamide gel electrophoresis

STAT Signal transducers and activators of transcription

TAD Transcriptional activation domain

TEMED N,N,N',N'-Tetramethylethylenediamine

TIMP Tissue inhibitors of metalloproteinase

TLC Thin layer chromatography

TMB 1,3,5-trimethylbenzene

TNF-α Tumor necrosis factor alpha

TRITC Tetramethylrhodamine isothiocyanate

VCAM Vascular cell adhesion molecule

WHO World Health Organisation

LIST OF PUBLICATIONS

Ibrahim FB, Pang SJ, Melendez AJ (2004) Anaphylatoxin signaling in human

neutrophils A key role for sphingosine kinase J Biol Chem 279(43):44802-11

Melendez AJ, Ibrahim FB (2004) Antisense knockdown of sphingosine kinase 1

in human macrophages inhibits C5a receptor-dependent signal transduction,

Ca 2+ signals, enzyme release, cytokine production, and chemotaxis J Immunol

173(3): 1596-603

LIST OF POSTERS AND ABSTRACTS PRESENTED

Ibrahim FB, Melendez AJ Phospholipase D mediates anaphylatoxin triggered

effector functions in macrophages

Abstract accepted for poster presentation at the Combined Science Meeting held at

Raffles Convention Centre, Singapore in November 2005

Abstract published in supplement issue of Annals, Academy of Medicine, Singapore

Ibrahim FB, Melendez AJ Intracellular signaling via anaphylatoxin C5a receptor

in macrophages: a key role for phospholipase D

Abstract accepted for poster presentation at the 2005 FASEB Summer Research

Conference ‘Immunoreceptors’ held in Tucson, Arizona, USA in July 2005

Ibrahim FB, Melendez AJ SPHK1 mediates anaphylatoxin triggered physiological

responses in phagocytic cells

Abstract presented for poster presentation in the Best Basic Science Poster Award at

the 8th NUH-NUS Annual Scientific Meeting held in NUS, Singapore in October

2004

Ibrahim FB, Pang SJ, Leung BP, Melendez AJ Sphingosine Kinase 1 mediates

intracellular Ca 2+ release, degranulation, cytokine production and chemotaxis in

response to anaphylatoxins

Abstract accepted for poster presentation at the 12th International Congress of

Immunology (ICI) and the 4th Annual Meeting of Federation of Clinical Immunology

Society (FOCIS) held in Montreal, Canada in July 2004

Abstract published in the supplement of Clinical Investigative Medicine

Tan Ryan, Ibrahim FB, Melendez AJ Study on the intracellular signaling

pathways triggered by C5a in macrophages

Abstract presented at the 16th Science Research Congress held in NUS, Singapore in

Mar 2004

Ibrahim FB, Melendez AJ Study on the intracellular signaling pathways triggered

by C5a in macrophages

Abstract presented at the Postgraduate conference on Immunology and Cancer

Biology held at the City University of Hong Kong, Hong Kong in February 2003

Ibrahim FB, Melendez AJ Studies on the intracellular signaling pathways

triggered by anaphylatoxin C5a on phagocytic cells: new targets for

inflammatory and autoimmune diseases

Abstract accepted for poster presentation in the 6th NUH-NUS Annual Scientific

Meeting held in NUS, Singapore in August 2002

SCHOLARSHIP

National University of Singapore Research Scholarship Jul 2002 – Jul 2006

CHAPTER I INTRODUCTION

1.1 The complement system

Traditionally, the complement system has been viewed as a major defense and

clearance system in the bloodstream that forms a central component of the innate

immune system However, in recent years, complement activation has been

implicated in the pathogenesis of many inflammatory and immunological diseases,

including sepsis (Ward, 2004), asthma (Hawlisch et al., 2004), rheumatoid arthritis

(Linton and Morgan, 1999), multiple sclerosis (Ffrench-Constant, 1994),

glomerulonephritis (Welch, 2002), adult respiratory distress syndrome (Robbins et al.,

1987) and ischemia-reperfusion injury (Arumugam et al., 2004)

The complement system was identified, based on the concept of bloodstream

clearance of micro-organisms It was Pfeiffer who first described complement as

being principally a heat-labile bactericidal activity in serum (Pfeiffer and Issaeff,

1894) Later in 1898, Bordet proved that complement was a substance, rather than

merely an activity found in the serum, which ‘complemented’ the effects of specific

antibodies in the lysis of bacteria and red blood cells (Bordet and Gengou, 1901)

Ehrlich then coined the term ‘complement’ to denote those factors in normal serum,

that were able to demonstrate the lysis of cells or micro-organisms, when functioning

together with antigen-bound antibodies (Ehrlich, 1996)

Over the years, it has been established that there is marked conservation of the

complement system between invertebrates and mammals, suggesting that they share a

common ancestry in host defense and tissue homeostasis (Dodds and Law, 1998;

Sahu and Lambris, 2001) However, while the complement system in invertebrates is

very simple and composed only of two or three proteins, that of mammals is highly

complex, and includes over 30 proteins Many of these proteins interact with each

other to form different functional complexes, eventually leading to the formation of a

membrane attack macromolecule with microbicidal properties (Smith et al., 2001)

1.1.1 Complement proteins and nomenclature

The mammalian complement system is composed of at least 35 proteins, which are

normally found as soluble components in plasma These include proteolytic

pro-enzymes, as well as non-enzymatic components that form functional complexes,

co-factors, regulators and receptors From the initial point of activation to the terminal

stage of cell lysis, complement proteins get sequentially activated in a cascade

fashion, thus providing tremendous amplification and activation of large amounts of

complement by relatively small initial signals (Lutz and Jelezarova, 2006)

The nomenclature of the complement system is often a significant obstacle to

understanding this system There are two distinct ways of characterizing the

complement proteins (IUIS-WHO, 1981; WHO, 1968) The plasma proteins that were

originally described are defined as ‘components’ and each is designated with a prefix

‘C’, followed by a number 1 to 9 Thus C1 to C9 (refer to Figure A), together with the

membrane attack complex (MAC), make up the classical pathway As for the

alternative pathway, the two proteins involved in the initiation and also the regulatory

proteins are termed ‘factors’ and given capital letters (Factor B and Factor D, Factor

H and Factor I, respectively) Fragments from the proteolytic cleavage of a certain

component are represented by lower case letters, such as C5a and C5b (coming from

C5), while the complement receptors are denoted by the symbol of the protein or

fragment to which they bind, followed by the capital letter R (C5aR)

1.1.2 Activation of the complement cascade

Three pathways of complement activation have been recognized: the classical,

alternative and mannan-binding lectin pathway (Figure A) They differ according to

the nature of recognition Early complement activation is characterized by a sequence

of proteolytic reactions, in which complement zymogens undergo successive cleavage

to generate two fragments The larger cleavage product is often the active serine

protease that remains covalently bound to the pathogen surface, to ensure the

activation of the next component zymogen The smaller liberated fragment is released

from the activation site and functions as a soluble mediator The early events

converge at the central component of the complement system C3, and from there, the

pathways take a final common pathway, which leads to the formation of a protein

complex on a complement-activating surface, the MAC

1.1.2.1 The classical pathway

The first complement pathway that was discovered, the classical pathway, is initiated

by the binding of C1q to the Fc regions of IgG or IgM within immune complexes, or

other structures such as HIV (Cooper et al., 1976), bacterial structures (Alberti et al.,

1993), double-stranded DNA (Jiang et al., 1992) or pentraxins such as C-reactive

protein (Jiang et al., 1991) This interaction promotes a conformational change in the

C1q molecule, consequently activating the serine proteases C1r and C1s Activated

C1s cleaves C4 into C4a and C4b, of which the latter binds covalently to the

complement-activating surface C4b binds C2, which is subsequently cleaved by C1s,

to form C2a and C2b While C2b is released, C2a remains bound to C4b and they

form the C4b2a complex Also known as the C3 convertase, this complex then

cleaves C3, generating C3a and C3b, which attaches itself to the C4b2a complex to

form C4b2a3b heterotrimeric complex, which has C5 convertase activity Cleavage of

C5 produces C5a and C5b, which functions as the initiator of the terminal phase of the

complement activation C5b binds to C6 followed by C7 This reaction leads to a

conformational change that exposes a hydrophobic site on C7, enabling the insertion

of the complex itself into the lipid bilayer of the cell membrane of the invading

pathogen C8 also undergoes similar changes Collectively, the complex C5678 forms

small pores in the membrane that may potentially lead to lysis However, the

perforin-like C9 is the fundamental component that does the damage C9 polymerizes around

the C5678 complex to form the MAC This is essentially a transmembrane channel

that allows the free passage of water and solutes across the lipid bilayer, leading to a

loss of cellular homeostasis The end result is lysis of the target cell (Guo and Ward,

2005; Seelen et al., 2005; Walport, 2001)

1.1.2.2 The alternative pathway

The alternative pathway was discovered as a second pathway for complement

activation after the classical pathway had been characterized (Gotze and

Muller-Eberhard, 1976) This pathway is activated by whole micro-organisms and their

products, such as lipopolysaccharides, zymosan and teichoic acid, and also certain cell

surfaces More importantly, the alternative pathway can be initiated through the

spontaneous hydrolysis of circulating C3, forming C3b, that can become covalently

attached to microbial surfaces The bound C3b then binds to a serum factor called

Factor B, forming a C3bB complex The complex is then further activated by another

serum factor, Factor D, which cleaves Factor B while it is still attached to C3b The

complex generated from this cleavage is C3bBb, which is the assembled C3

convertase of the alternative pathway Similar to that produced in the classical

pathway, the C3 convertase functions to cleave more C3 molecules, thus amplifying

the initial signal Due to the spontaneous nature of the hydrolysis of C3 to C3b, there

needs to be a regulatory thermostat that shuts down this amplification process

Properdin is one such serum protein that binds to and stabilizes the C3bBb complex

on microbial cells, while regulating the rapid dissociation of the C3bB complex on

host cells Other regulators include Factor H and Factor I, which inactivate the

released C3b With the formation of the C3 convertase, the alternative pathway takes

on the same route as the classical pathway, ultimately leading to the MAC formation

The two complement activation pathways do not function in isolation In fact, they are

highly interconnected because the amplification steps in the alternative pathway also

play a similar role in the classical pathway Thus the alternative pathway serves to

amplify both antibody-dependent and antibody-independent cleavage of C3 (refer to

Figure A) (Guo and Ward, 2005; Seelen et al., 2005; Thurman and Holers, 2006)

1.1.2.3 The mannan-binding lectin (MBL) pathway

The lectin pathway is mediated by plasma mannose-binding lectins that serve as

recognition molecules (Turner, 2003) MBL exists as a complex, with the MBL

associated serine proteases, MASP-1, -2 and -3, and will bind to mannose residues,

found in many proteins and polysaccharides, that are found uniquely in

micro-organisms but not in mammals Analogous to the C1q of the classical pathway, the

MBL proteins initiate the MBL pathway in a similar manner as the classical pathway

Activated MBL proteins trigger the MASPs, which are equivalent to the C1r and C1s

of the classical pathway, for the cleaving of C4 and C2, leading to the formation of C3

and C5 convertase of the classical pathway The pathway then takes a similar route as

the other two pathways (Fujita et al., 2004; Petersen et al., 2001)

C1 Activated C1

C4a

Antigen-Antibody (IgG or IgM) Complex

C5-9 C3

C3a

C3b

C3b C3

Spontaneously

occurring and after

contact with foreign

surfaces

C3a Factor B

C5a

C6 C7 C8 C9

Microbial surfaces

Polysaccharides

Classical pathway (C3 convertase)

Classical pathway (C5 convertase)

Alternative pathway (C3 convertase)

Alternative pathway (C5 convertase)

C5-9 C3

C3a

C3b

C3b C3

Spontaneously

occurring and after

contact with foreign

surfaces

C3a Factor B

C5a

C6 C7 C8 C9

Microbial surfaces

Polysaccharides

Classical pathway (C3 convertase)

Classical pathway (C5 convertase)

Alternative pathway (C3 convertase)

Alternative pathway (C5 convertase)

Factor D

Figure A Complement activation pathways

The complement system can be activated through three pathways: classical,

alternative, and mannan-binding lectin pathways The Classical Pathway is initiated

by the binding of C1 to antigen-antibody complexes or aggregated forms of

immunoglobulins The Alternative Pathway is initiated by the spontaneous hydrolysis

of C3 to C3b and the subsequent binding of C3b to various activating surfaces,

including microbial walls and complex polysaccharides The Mannan-binding Lectin

Pathway (MBL) is activated by contact of MBL, in serum, with repeating microbial

surface mannose residues The pathways converge at the C3 convertase step The final outcome of complement activation is the formation of the membrane attack complex (MAC)

1.1.3 Functions of the complement system

Activation of the complement system promotes four key effector functions which are

initiated by the terminal components, as well as the liberated complement fragments

These activities are, in a large part, mediated through the recruitment and activation of

many different cell types such as macrophages, neutrophils, mast cells, platelets and

endothelial cells, among others (Bohana-Kashtan et al., 2004)

Cell lysis is one effector function of the complement system The assembly of the

multimeric MAC, by the terminal components on the pathogen surface, results in a

massive influx of water, coupled with unregulated ionic movement This leads to

osmotic disequilibrium and proton gradient disturbances across the membrane The

invading cell swells, allowing the membrane to become permeable to

macromolecules, which can either escape from the cell or enter the cell, leading to

rapid cell lysis (Cole and Morgan, 2003; Ward, 2004)

The complement system also functions in opsonization, a process whereby invading

micro-organisms and other antigens become coated with complement particles that

will enhance their recognition, phagocytosis and killing by macrophages and

polymorphonuclear leukocytes (Nauta et al, 2004) The larger complement products

C3b, and to some extent C4b, can serve as opsonins These bind covalently to

glycoproteins scattered across the pathogen surface, tagging the invader These C3b-

and C4b-coated cells will then bind to their high-affinity receptor, the complement

receptor CR1, which is expressed mainly on erythrocytes, neutrophils, monocytes and

lymphocytes Phagocytes, such as neutrophils and monocytes, bind particles/antigens

and/or micro-organisms opsonized with C3b or C4b via CR1 and internalize the

opsonized complex, thus activating the phagocytic mechanisms that will eventually

result in the clearance of the toxins or pathogens

One other important biological function of the complement system is the activation

and mediation of the inflammatory response, mainly attributed to the smaller

bioactive fragments C3a, C4a and C5a (Gorski et al., 1979; Hugli and

Muller-Eberhard, 1978) Collectively known as the ‘anaphylatoxins’, the term was introduced

by Friedberger in 1910 who noticed that laboratory animals that were injected with

these components succumbed to anaphylactoid-like death (Freidberger, 1910)

Anaphylatoxins participate in inflammatory events either through direct cell

activation or by modulating the cytokines released by macrophages and monocytes

Briefly, they mobilize inflammatory immune cells, activate the production of oxidase

activity, enhance smooth muscle contraction and promote vascular permeability In

the subsequent section, emphasis will only be on the anaphylatoxin C5a, which is the

main player in this study

The complement system plays key roles in immune complex clearance and B cell

immunity, principally through the complement receptors CR1 and CR2, which are

expressed mainly on B cells and follicular dendritic cells The binding of complement

proteins to antigen-antibody complexes enhances the phagocytic clearance of these

complexes, which would otherwise get deposited in vessel walls and cause damage

As mentioned above, CR1 is important in clearance of opsonized particles by the

phagocytes CR1 on erythrocytes binds circulating immune complexes attached with

the opsonins, and transports the complexes to the liver and spleen where they are

removed by phagocytes, leaving the erythrocytes free to return back to the blood

circulation The ability of CR1 to induce phagocytosis is further enhanced when there

is simultaneous binding of IgG on microbial surfaces to Fcγ receptors In fact, they are more proficient as partners than each on their own in mediating the phagocytic

process This synergism seems to suggest cross-talk between the complement system

and the FcγRs (Schmidt and Gessner, 2005) CR2, on the other hand, is able to stimulate the humoral immune response by enhancing B cell activation by antigen and

by promoting the trapping of antigen-antibody complexes in the germinal centers of

lymphoid organs CR2 binds specifically to C3b and its cleaved products, such as

C3d, on pathogen surfaces Since the microbial antigen can bind to the B lymphocytes

via their Ig receptor, and the attached C3d on the antigen can simultaneously bind to

CR2 on the B lymphocytes, there is enhanced B cell activation due to this

co-engagement (Carter and Fearon, 1992) The complement system has also been

demonstrated to participate in the regulation of T cell immunity, thus explaining its

importance in bridging the innate and adaptive immunity (Barrington et al., 2001;

Carroll, 2004; Dempsey et al., 1996; Fearon and Carter, 1995)

1.1.4 Regulation of the complement system

Just like any other biological system in the body, there exist control mechanisms to

prevent the ongoing activation of the complement system Regulation is exceptionally

critical here because of the amplifying capacity of the complement system, and also

due to the production of various inflammatory mediators, that can cause significant

damage to the host tissue There are various complement regulators, both fluid and

membrane-bound, that are present to tightly regulate the complement system

(Liszewski et al., 1996) One such plasma protein is the C1 inhibitor, which inhibits

the proteolytic activity of C1s and C1r Factors H and I function to enzymatically

degrade cell-associated C3b and C4b, while membrane protein CD59 blocks C9

assembly and prevents MAC formation (Kirschfink, 2001; Morgan, 1995) These

inhibitory mechanisms are in place to ensure that complement activation is regulated

and does not cause adverse effects to the host

1.1.5 Complement component C5a

C5a is recognized as a unique and important mediator produced by complement

activation, because it is the most potent of all the anaphylatoxins, eliciting the

broadest responses (Guo and Ward, 2005) As described above, C5a is generated from

the cleavage of C5 by C5 convertase, generating C5a and C5b C5a acts on its

classical receptor C5a receptor (C5aR) Recently, a putative orphan receptor was

demonstrated to be the second receptor for C5a (Cain and Monk, 2002; Ohno et al.,

2000)

1.1.5.1 Biological properties of C5a

C5a exerts various proinflammatory effects on different types of cells, especially

immune cells It enhances the innate immune functions of phagocytic cells by

inducing superoxide anion production in neutrophils, granular enzyme release from

phagocytes and histamine release from mast cells C5a functions as a strong

chemoattractant involved in the recruitment of inflammatory cells such as neutrophils,

eosinophils, monocytes and T lymphocytes C5a ligand/receptor interaction on

phagocytes promotes the adhesion of these cells to the vascular endothelium, allowing

their infiltration through the basement membrane and chemotactic movement towards

an injury site C5a promotes smooth muscle contraction, vasodilation and increase in

vascular permeability; all of which are features of an acute inflammatory response In

this instance, local dilation of blood vessels causes stress on the endothelial lining at

the inflammatory site, allowing exudation of blood plasma into tissue which leads to

edema C5a exhibits immunoregulatory activities through the induction of cytokines,

as well as induces the synthesis and release of arachidonic acid metabolites, such as

eicosanoids, which will further amplify the effects of the anaphylatoxin (Cochrane

and Muller-Eberhard, 1968; Goldstein and Weissmann, 1974; Riedemann et al., 2003;

Sacks et al., 1978; Schumacher et al., 1991; Shin et al., 1968; Ward, 2004)

1.1.5.2 Structure of ligand C5a

Human C5a molecule is a compact 74-amino acid glycoprotein with a molecular

weight of 11 kDa It consists of four alpha helices in a complex anti-parallel topology

(Shin et al., 1968), connected by three peptide loops formed via six cysteine residues

(Figure B) The cysteine motif dominates the folding of C5a and provides great

stability to the molecule (Hugli, 1986; Vogt, 1986) Most of the biologic properties of

C5a can be attributed to a crucial arginine residue within the pentapeptide MQLGR in

the C-terminal region Removal of this residue was shown to decrease the

spasmogenic potency of C5a by about 1000-fold (Gerard et al., 1981; Gerard and

Hugli, 1981; Hugli, 1981) In fact, C5a is rapidly inactivated in vivo to the less potent

form, C5a des Arg, by serum carboxypeptidase N, which removes the arginine

residue Many studies using synthetic C-terminal fragments of C5a have demonstrated

that the C-terminal portion indeed contains the effector site of this molecule

(Chenoweth et al., 1979; Ember et al., 1992; Morgan et al., 1992) It was observed

that the N-terminal portion is not required for the biologic activity of C5a, but it

participates directly in receptor binding or in stabilizing binding sites elsewhere in the

native C5a conformation (Gerard et al., 1985) Furthermore, site-directed mutagenesis

studies have confirmed and extended early protein chemical studies on the model of

the C5a molecule, emphasizing the modulating role of the amino terminal region and

the importance of the carboxyl terminal pentapeptide (Mollison et al., 1989)

Huber-Lang et al (2003) J Immunol 170, 6115-6124

Copyright 2003 The American Association of Immunologists, Inc

Figure B Ribbon diagram of the human C5a molecule

This diagram shows the helices 1-4 as thick gray bands and the less well-defined interconnecting interhelical loop regions as colored narrow regions The selected inter-helical regions are surface residues of interest where possible C5a-C5aR interaction may potentially occur (D1 representing acid residues 12-20, D2 acid residues 28-33, and D3 acid residues 38-46, respectively The core is stabilized by disulfide linkages among residues 21-47, 22-57 and 34-55 (yellow)

1.1.5.3 Complement 5a receptor (C5aR)

Early characterization studies of the C5aR (CD88) on human neutrophils provided

evidence that the binding of C5a to its receptor is rapid, essentially irreversible and

occurs in nanomolar concentrations These studies also revealed that there are as

many as 105 copies of the C5aR on each cell and that some degradative products of

C5a, such as C5a des Arg, compete in binding with the intact C5a (Chenoweth and

Hugli, 1978) In late 1989, several groups reported that G-proteins are critical in the

signal transduction pathway of C5a activation (Bokoch and Gilman, 1984; Feltner et

al., 1986; Koo et al., 1983; Wilde et al., 1989) This led to the hypothesis that the

C5aR could be a GTP-binding protein-coupled receptor (GPCR) Not long after, in

1991, two separate groups cloned the C5aR using cell libraries from differentiated

leukocytic lines, U937 and HL-60 (Boulay et al., 1991; Gerard and Gerard, 1991)

Essentially, C5aR is a typical 45-kDa G-protein coupled rhodopsin-type receptor,

consisting of seven transmembrane-spanning regions and three extracellular loop

regions (Figure C) The effector binding site of C5a has been located on the

C-terminal half of C5aR, within the region containing the second and third extracellular

loops (Pease et al., 1994) Point mutation techniques have also revealed several

critical amino acid residues within the C-terminal, involved in the formation of the

primary effector-binding site of C5aR (DeMartino et al., 1994; Mery and Boulay,

1994; Monk et al., 1995) The N-terminal region of the receptor defines a secondary

non-effector binding site (Morgan et al., 1993) Features of the C5a molecule are

consistent with the two-site model proposed for the C5aR, where the non-effector site

on C5a binds to the N-terminal region of C5aR, while the C-terminal effector site of

C5a penetrates the ‘pore’ formed by the transmembrane regions of the C5aR

(Siciliano et al., 1994) Recent studies have revealed a more complex ligand/receptor

relationship between C5a and C5aR (Huber-Lang et al., 2003)

C5aR expression was originally described on myeloid cells, including neutrophils

(Chenoweth et al., 1979), eosinophils (Gerard et al., 1989), mast cells (Schulman et

al., 1988), basophils (Kurimoto et al., 1989), macrophages and monocytes

(Chenoweth and Goodman, 1983; Chenoweth et al., 1982) More recently, C5aR has

been found on non-myeloid cells including epithelial, endothelial and smooth muscle

cells in the human liver, lung, kidney, spleen, heart, intestines and regions of the brain

(Braun and Davis, 1998; Haviland et al., 1995; Osaka et al., 1999; Wetsel, 1995;

Zwirner et al., 1999a) It was also observed that the expression of C5aR is more

predominant in some tissues, suggesting that these tissues are more highly responsive

to C5a stimulation than others The wide-spread distribution of C5aR, thus, has

serious implications for C5a playing a significant role in vascular, pulmonary and

degenerative neurological diseases

In recent years, a second receptor for C5a, C5L2 has been described (Cain and Monk,

2002; Kalant et al., 2003; Ohno et al., 2000; Okinaga et al., 2003) From these reports,

it was noted that C5L2 is a high affinity receptor for C5a and C5a des Arg, with

varying expression on different cells and organs C5L2 has similar ligand binding and

activation domains as C5aR However, unlike C5aR, C5L2 is not coupled to

intracellular G protein signaling pathways It was also suggested that C5aR and C5L2

compete for C5a binding, and the latter is thought to be a scavenger or decoy receptor

for C5a, hence reducing the effective C5a levels that are available to act on the

classical C5aR The two receptors may cooperate to determine the strength of an

inflammatory response (Gao et al., 2005; Gerard et al., 2005)

Huber-Lang et al (2003) J Immunol 170, 6115-6124

Copyright 2003 The American Association of Immunologists, Inc

Figure C Model for the interaction of C5a with C5aR

The seven transmembrane helices (cylinders 1–7) of C5aR contain different charged loop regions; especially the amino-terminal and extracellular loop II with possible interaction sites for the D1 (acid residues 12–20) and D2 (acid residues 28–33) region

of C5a Electrostatic interactions with a possible generation of salt bridges (darker gray areas) in the extracellular loops of C5aR are shown, based on the computer-assisted structure analysis of the molecule

1.1.5.4 Role of C5a in diseases

It is a well established fact that C5a has a predominant and critical role to play in

mediating inflammation due to its powerful biological functions It is often the

inflammation that contributes to the underlying pathogenesis of a particular disease

Many inflammatory diseases are attributable to the effects of C5a It has potential

roles in the development of both acute and chronic inflammatory conditions such as

sepsis (Ward, 2004), ischemia-reperfusion injury (Arumugam et al., 2004), adult

respiratory distress syndrome (Bhatia and Moochhala, 2004), rheumatoid arthritis

(Grant et al., 2002), asthma (Karp et al., 2000), allergy (Kikuchi and Kaplan, 2002),

coronary artery disease (Kostner et al., 2006), glomerulonephritis (Bao et al., 2005),

psoriasis (Mrowietz et al., 2001) and Alzheimer’s disease (Farkas et al., 2003), among

others

Although the proinflammatory effects of C5a are indisputably beneficial in the

context of localized infections and injuries, its inappropriate and excessive generation

can lead to harmful, potentially life-threatening consequences due to severe

inflammatory tissue destruction Therefore, understanding the intracellular signaling

pathway triggered by C5a in phagocytic cells will potentially lead to the discovery of

novel therapeutic “targets” specific for inflammatory diseases

1.2 Important downstream events triggered by C5a

1.2.1 NADPH oxidase

In immune-effector cells, there is a plasma-membrane associated reduced

nicotinamide adenine dinucleotide phosphate (NADPH) oxidase multicomponent

enzyme, that catalyzes the generation of superoxide (O2-) from oxygen and NADPH

The reaction involves a one-electron reduction of oxygen using NADPH as the

electron donor The O2- produced acts as a precursor for the generation of other

reactive oxygen species (ROS), such as hydrogen peroxide, free radicals and singlet

oxygen; all of which are potent killing agents for use by phagocytes against invading

pathogens This process of generating ROS upon immune cell stimulation is also

known as respiratory or oxidative burst (Babior, 1999; Lambeth, 2004)

The multicomponent NADPH oxidase is present in various immune cells, such as

neutrophils, monocytes, macrophages, eosinophils and B lymphocytes It consists of

two membrane-bound components (gp91PHOX and p22PHOX), three cytosolic

components (p67PHOX, p47PHOX and p40PHOX) and the small GTPases (Rac1 or Rac2)

In a resting cell, the components are kept in distinct compartments to ensure that the

oxidase activity is inactive gp91PHOX and p22PHOX exist as a heterodimeric

flavocytochrome b558 and are located in the membranes of granules and vesicles

p67PHOX, p47PHOX and p40PHOX are distributed in the cytosol as a complex, while the

Rac proteins are kept inactive by their association with guanine nucleotide

dissociation inhibitor Activation of the NADPH oxidase, in response to stimuli,

involves the translocation of the cytosolic components to the plasma membrane, so

that the complete oxidase complex can be assembled (Babior, 2004)

gp91PHOX is the crucial component that serves as the electron transporter of the

NADPH oxidase, while p22PHOX stabilizes it and provides high-affinity binding sites

for the cytosolic NADPH components, and thus, functions to bring the whole

cytosolic complex to the membrane upon activation p67PHOX is an accessory protein

that regulates the electron transfer p47PHOX acts as an adaptor protein, forming a

bridge between p22PHOX and p67PHOX, and it binds to gp91PHOX to stabilize the

attachment of p67PHOX to the cytochrome b558 p47PHOX gets heavily phosphorylated

upon activation, resulting in conformational rearrangements that enable the interaction

between the cytochrome b558 and p67PHOX The function of p40PHOX is not very clear

but it may be needed for modulating the oxidase activity The guanine nucleotide

exchange of Rac-GDP to Rac-GTP is also a critical trigger for the initiation and

assembly of the NAPDH oxidase complex Hence the regulation of the NADPH

oxidase requires both spatial segregation and temporal orchestration (Nauseef, 2004;

Vignais, 2002)

The NADPH oxidase plays a pivotal role in microbial killing Absence of one of the

PHOX components that form the oxidase complex gives rise to chronic

granulomatous disease (CGD) Individuals with this inherited immune deficiency are

unable to make O2- and have a profound disposition to recurrent life-threatening

bacterial and fungal infections, as well as abnormal tissue granuloma formation (Segal

et al., 2000; Thrasher et al., 1994) The NADPH oxidase complex thus plays an

indispensable role in the innate immunity

1.2.2 Nuclear factor kappa B

NF-κB comprises a family of ubiquitously expressed inducible transcription factors, that serve as important regulators of immune and inflammatory response In addition,

NF-κB is important in the regulation of cellular proliferation, apoptosis and cell-cycle progression Dysregulation of the NF-κB pathway can thus lead to a variety of

inflammatory conditions, autoimmune diseases and carcinogenesis (Karin et al., 2002;

Yamamoto and Gaynor, 2001)

The mammalian NF-κB family consists of five members, Rel A (p65) Rel B, c-Rel, NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100) These are capable of forming homodimers and heterodimers with one another and

dimerization is necessary for their DNA-binding properties The most common and

classical NF-κB binding form is the p65-p50 heterodimer (Ghosh et al., 1998) All family members share an N-terminal domain, known as the Rel-homology domain

(RHD), which mediates their dimerization, nuclear translocation and DNA binding

Rel A, Rel B and cRel have a C-terminal transcriptional activation domain (TAD),

which can strongly activate transcription from NF-κB binding sites in target genes In contrast, p50 and p52 lack the TAD and have C-terminal ankyrin repeats instead and

thus homodimers of p50 and p52 can function as transcriptional repressors (Beinke

and Ley, 2004; Moynagh, 2005)

In unstimulated cells, NF-κB proteins exist in the cytoplasm in an inactive form, due

to their association with members of the inhibitory IκB family (IκB-α, IκB-β and IκB-ε) These interact via the RHD of the NF-κB proteins and the inhibitors’ ankyrin repeats (Ghosh et al., 1998) The current understanding is that IκB proteins sequester NF-κB in the cytoplasm by masking the nuclear-localization sequences (NLS) on the NF-κB subunits However, recent findings have indicated that the cytoplasmic localization of the inactive NF-κB is actually maintained by balancing continuous movement between the nuclear and cytoplasmic compartments, a process mediated

mainly by the shuttling of IκB, which functions to retain NF-κB in the cytoplasm, as well as remove it from the nucleus (Ghosh and Karin, 2002)

Classically, the activation of NF-κB depends on the phosphorylation and degradation

of IκB The phosphorylation of IκB proteins is mediated by IκB kinases (IKK) which phosphorylate IκB at two serine residues The phosphorylated IκB proteins are then ubiquitinated by the E3 ubiquitin ligase complex and targeted for degradation by the

26S proteasome Liberated κB translocates to the nucleus, where it binds to

NF-κB promoter elements, resulting in the activation of target gene expression A wide range of stimuli such as cytokines, chemokines, pathogens and stress signals that

activate the NF-κB pathway can induce the phosphorylation of IκB by IκB kinase (IKK) The IκB and IKK are thus key regulators in the activation of the NF-κB pathway (Moynagh, 2005)

It is well established that NF-κB plays a central role in coordinating the expression of many genes that control the immune response These genes include proinflammatory

cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases, vascular

endothelial growth factor, acute-phase proteins, receptors in immune recognition, and

enzymes that contribute to inflammation, such as inducible nitric oxide synthase,

cyclooxygenase-2, -5 and 12-lipoxygenase (Li and Verna, 2002) Due to the large

number of genes that it controls, the activation of NF-κB therefore, plays a critical role in the pathogenesis of many acute and chronic inflammatory diseases, including

asthma and rheumatoid arthritis (Yamamoto and Gaynor, 2001)

1.2.3 Matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a family of highly conserved zinc-dependent

endopeptidases, which collectively are capable of degrading components of the

extracellular matrix (ECM) (Nagase and Woessner, 1999; Sternlicht and Werb, 2001)

During development and morphogenesis, the ECM is indispensable as its components

constitute the building blocks that create cellular environments (Bosman and

Stamenkovic, 2003; Stamenkovic, 2003) Since MMPs are often implicated in ECM

remodeling, they are, thus, crucial in many biological processes such as

embryogenesis, angiogenesis, normal tissue remodeling, wound healing and tissue

repair (Stamenkovic, 2003) MMPs need to be precisely regulated under normal

physiological conditions because a loss of control can result in diseases like arthritis,

atherosclerosis, fibrosis, nephritis and cancer (Visse and Nagase, 2003) In fact, their

role in tumor invasion and metastasis has been extensively studied (Egeblad and

Werb, 2002)

Members of MMPs, otherwise also known as matrixins, share common structural and

functional elements and are products of different genes To date, there are 23 human

MMPs, which can be subdivided into six classes, based on their substrate specificity,

sequence similarity and domain organization: collagenases, gelatinases, stromelysins,

matrilysins, membrane-type MMPs and other MMPs MMPs are either secreted or

exist as inactive precursor zymogens that require activation to exert their matrix

degrading activity They can be activated by proteinases or in vitro by chemical

agents In addition, even though the individual members of the MMP family are

separately regulated, and their expression is highly tissue specific, the MMPs are

inextricably linked in the process of ECM degradation, with each MMP having a

unique yet slightly overlapping substrate specificity with another (Chakraborti et al.,

2003; Nagase and Woessner, 1999; Sternlicht and Werb, 2001; Visse and Nagase,

2003)

Because of their pivotal role in tissue architecture and homeostasis, there are

endogenous tissue inhibitors of metalloproteinases (TIMPs) These specific inhibitors

of MMPs function to control the local activities of MMPs in tissues There are at least

four distinct members of the TIMP family that are produced by a variety of cells Just

like the MMPs, the expression of TIMPs is highly regulated during development and

tissue remodeling Thus the balance between MMPs and TIMPs will determine the

eventual ECM remodeling in tissue (Brew et al., 2000)

1.2.4 Raf-Mitogen activated protein kinases

The MAPK signaling pathway is an important downstream target which is triggered

upon the activation of receptors It typically refers to a signaling module that is

organized in a three-kinase structure, consisting of a MAPK kinase kinase

(MAPKKK), MAPK kinase (MAPKK) and MAPK Signal transmission occurs via

the sequential phosphorylation and activation of the three different kinases, each of

which phosphorylates, then activates the next enzyme in the cascade The MAPKKKs

are serine/threonine kinases, which are commonly activated by phosphorylation

and/or via their association with a small GTP-binding protein of the Ras/Rho family,

in response to extracellular stimuli Activated MAPKKK phosphorylates and activates

MAPKK, which will then activate MAPKs through dual phosphorylation on threonine

and tyrosine residues The activated MAPKs will then phosphorylate target substrates

on serine and threonine residues The substrates include phospholipases, transcription

factors, cytoskeletal proteins and MAPK-activated protein kinases (MK) (Roux and

Blenis, 2004)

In mammalian systems, there are five distinguishable MAPK modules, which function

in coordination to regulate diverse extracellular stimuli and fundamental cellular

processes, such as gene expression, mitosis, metabolism, motility, differentiation,

survival and apoptosis The five are: extracellular signal-regulated kinases (ERKs) 1

and 2 (ERK1/2), p38 isoforms α, β, γ and δ, c-Jun amino-terminal kinases (JNKs) 1, 2

and 3, ERKs 3 and 4, and ERK 5 Even though MAPKs are activated by a wide range

of different stimuli, there is specificity in the system, because individual MAPK

modules are able to signal independently from each other, generating distinct

physiological responses; there are also scaffolding proteins that interact with the

MAPKs and thus organize the pathways into specific modules (Chen et al., 2001)

The classical MAPK pathway is the ERK1/2 module which consists of A-Raf, B-Raf

and Raf-1 as MAPKKKs, MEK1 and MEK2 as MAPKK, and ERK1 and ERK2 as

MAPKs ERK1 and ERK2 are mainly activated by growth factors, ligands of G

protein-coupled receptors and cytokines The Raf/MEK/ERK pathway involves the

activation of membrane-associated Ras, a small GTP-binding protein, which acts as a

molecular switch that is ‘on’ when bound to GTP and ‘off’ when bound to GDP

Activated Ras recruits the serine/threonine kinase Raf; activated Raf phoshorylates

and activates the dual specificity kinase MEK, which in turn phosphorylates and

activates ERK1/2 via the typical Thr-Glu-Tyr (TEY) motif found within the protein

ERK1/2 are expressed to varying extents in all tissues and, in unstimulated cells, they

are distributed throughout the cell, but upon stimulation, ERK1/2 predominantly

accumulate at the nucleus Substrates of ERK1/2 include membrane proteins, nuclear

substrates, cytoskeletal proteins and MKs The ERK MAP kinase pathway promotes

proliferation and differentiation by targeting transcription factors, such as c-Myc, Sap

and Elk-1 (Dal Porto et al., 2004) It is well established that ERK1/2 signaling is

critical in cell proliferation and it has often been implicated in oncogenesis and cancer

(Downward, 2003; Roux and Blenis, 2004)

The p38 module consists of several MAPKKKs, such as MEKKs 1 to 4, MAPKKs

MEK3 and MEK6, and the four isoforms of p38 MAPKs This pathway is strongly

activated by inflammatory cytokines, such as IL-1 and TNF-α, and physical and