Functional Studies of

Collagen-Binding Integrins

a2b1 and a11b1

Interplay between Integrins andPlatelet-Derived Growth Factor Receptors

BY

GUNILLA GRUNDSTRÖM

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Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in MedicalBiochemistry presented at Uppsala University 2003.

ABSTRACT

Grundström, G. 2003. Functional Studies of Collagen-Binding Integrins a2b1 anda11b1. Interplay between Integrins and Platelet-Derived Growth Factor-BB. ActaUniversitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations fromFaculty of Medicine 1295. 97 pp. Uppsala. ISBN 91-554-5769-X

Integrins are heterodimeric cell surface receptors, composed of an a - and a b -subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrinsmediate intracellular signals in response to extracellular stimuli, and co-operate withgrowth factor and other cytokine receptors. Cells execute their differentiatedfunctions anchored to an ECM. In this thesis functional properties of the twocollagen-binding integrins a2b1 and a11b1 were studied. In addition, the impact ofb1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.

The integrin a11b1 is the latest identified collagen-binding integrin. In this study,tissue distribution of a11 mRNA and protein during embryonal development wasexplored, and the first a11b1-mediated cellular functions were established. Both a11protein and mRNA were present in mesenchymal cells in intervertebral discs andaround the cartilage of the developing skeleton. a11 protein was also detected incornea keratinocytes. a11b1 mediated cation-dependent adhesion to collagen types Iand IV and localized to focal adhesions. In addition, a11b1 mediated contraction of acollagen lattice and supported cell migration through a collagen substrate. PDGF-BBand FBS both stimulated a11b1-mediated contraction and directed migration.

Expression of b1Y783,795F in b1-null cells, prevents activation of FAK in responseto fibronectin, and decreases cell migration. In this study, we investigated how thismutation affected a2b1-mediated functions in response to collagen. The b1 mutationimpaired collagen gel contraction and prevented activation of FAK, Cas and Src onplanar collagen, but not in collagen gels. PDGF-BB stimulated contraction via avb3,which also induced activation of Cas in collagen gels. The YY-FF mutation alsoabolished b1A-dependent down-regulation of b3.

In the final study integrin-crosstalk during collagen gel contraction wasinvestigated. In cells lacking collagen-binding integrins avb3 mediated contraction.Clustering of b1-integrins by antibodies and PDGF-BB stimulated avb3-mediatedcontraction in an ERK-dependent way. Expression of a2b1, but not a11b1, preventedavb3-mediated contraction. Contraction by a2b1 and a11b1 was ERK-independent.

Key words: integrin, collagen-binding integrin, a2b1, a11b1, avb3, collagen gelcontraction, PDGF-BB, FAK

Gunilla Grundström, Department of Medical Biochemistry and Microbiology, BiomedicalCentre, Box 582, SE-751 23 Uppsala, Sweden.

© Gunilla Grundström, 2003

ISSN 0282-7476ISBN 91-554-5769-X

Printed in Sweden by Universitetstryckeriet, Uppsala 2003

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In memory of my Father

You would have been sceptical but proud

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This thesis is based on the following papers, referred to in the text by their roman

numerals.

I Tiger, C. F., Fougerousse, F., Grundström, G., Velling, T., Gullberg, D. (2001).

a11b1 integrin is a receptor for interstitial collagens involved in cell migration

and collagen reorganization on mesenchymal nonmuscle cells. Dev Biol 237(1):

116-29.

II Grundström, G., Mosher, D.F., Sakai, T, Rubin, K (2003).

Integrin avb3 mediates PDGF BB-stimulated collagen gel contraction in cells

expressing signalling deficient integrin a2b1. Accepted, Exp Cell Res.

III Grundström, G., Lidén, Å, Gullberg, D, Rubin, K (2003).

Clustering of b1-integrins induces ERK1/2-dependent avb3-mediated

collagen gel contraction. Manuscript.

Published manuscripts have been reprinted with permission from the publishers.

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CONTENTS

ABBREVIATIONS 6

INTRODUCTION 7

INTEGRINS 8

Collagen-binding integrins 13

The multi-specific integrin avb3 16

PLATELET-DERIVED GROWTH FACTOR (PDGF) 18

PDGF isoforms and receptors 18

Biological functions of PDGF 21

FOCAL ADHESIONS 25

Integrin-associated proteins 26

Integrin-mediated signalling 28

PDGF-induced signalling 39

Actin-reorganization by Rho-GTPases 42

COLLAGENS 45

Collagen Type I 47

WOUND HEALING AND INFLAMMATION 50

Cell-mediated contraction of collagen matrices 51

AIMS AND RESULTS OF THE PRESENT INVESTIGATION 55

PAPER I 55

PAPER II 57

PAPER III 58

DISCUSSION AND FUTURE PERSPECTIVES 60

ACKNOWLEDGEMENTS 64

REFERENCES 66

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ABBREVIATIONS

Cas Crk-associated substrate

ECM Extracellular matrix

EGF Epidermal growth factor

ERK Extracellular-regulated kinase

FAK Focal adhesion kinase

GAP GTPase activating protein

GEF Guanosine nucleotide exchange factor

IAP Integrin-associated protein

IFP Interstitial fluid pressure

ILK Integrin-linked kinase

MAPK Mitogen-activated protein kinase

MEK MAPK/ERK kinase

MIDAS Metal ion-dependent adhesion site

MLC Myosin light chain

PAK p21-activated kinase

PDGF Platelet-derived growth factor

PI3-K Phosphatidylinositol 3-kinase

PIP2 Phosphatidylinositol-4,5-biphosphate

PIP3 Phosphatidylinositol-3,4,5-triphosphate

PKC Protein kinase C

PLC Phospholipase C

PTP Protein tyrosine phosphatase

TGF Transforming growth factor

SH2 Src homology 2

SH3 Src homology 3

VEGF Vascular endothelial growth factor

uPAR Urokinase plasminogen activator receptor

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INTRODUCTION

Connective tissues constitute the architectural framework of the vertebrate body and

are defined as the tissues that bind together and support the various structures of

the body, including other connective tissues. Within the term connective tissue

collagenous, elastic, mucous, reticular, osseous and cartilaginous tissues are

included, and according to some also blood. These tissues, with the exception of

blood, all contain plenty of extracellular matrix (ECM) and relatively sparsely

distributed cells. The structural elements characteristic of the connective tissue ECM

are a fibrous scaffold of rigid and elastic fibres with a filling of proteoglycans,

hyaluronan and structural glycoproteins, which controls the molecular flux through

the tissue. Hyaluronan and proteoglycans act as expanders, e.g. they can take up

water and swell and thereby create the mechanical tension needed for tissue

integrity. The rigidity of the fibrous scaffold is provided by collagen fibres, collagen

type I being the most abundant, and elasticity by elastin fibres and a micro-fibrillar

network built up by among others collagen type VI and fibrillin (reviewed in [1]).

Connective tissue is constantly under mechanical tension, preventing the tissue from

collapsing and is also exposed to mechanical challenges from the environment.

Maintenance of the structure, and hence the tension in response to mechanical or

inflammatory events is mediated by interactions between connective tissue cells and

the ECM (reviewed in [2], Figure 1). With the exception of blood cells, connective

tissue cells need to be attached to each other and to the ECM in order to survive. In

fact, all nucleated cells fulfil their differentiated functions anchored to an ECM. Four

major families of cell surface receptors, namely the immunoglobulin superfamily,

cadherins, selectins and integrins, mediate cell adhesion. Immunoglobulins,

cadherins and selectins are mediators of cell-cell adhesions while integrins mediate

cell-ECM interactions as well.

The aim of this study was to enlighten cellular mechanistic events mediated by

integrins in response to adhesion, and their interplay with platelet-derived growth

factor receptors.

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INTEGRINS

In multi-cellular organisms the minute control of cell anchorage and movement iscrucial for most biological processes, spanning from embryogenesis to immuneresponses via maintenance of tissue integrity and homeostasis. The integrin familyof adhesion receptors plays a central role in these functions by transducing signalsthat provide physical support, regulate migration and affect gene expression(reviewed in [3-6]). All known members of the integrin family are non-covalentlylinked heterodimers consisting of one a- and one b-subunit. To date 18 a-chains and8 b-chains combined into 24 different integrins are identified in mammalians (Figure2). However, in a recent screen of the human genome 24 a- and 9 b-chains wereidentified, suggesting 6 new a-subunits and 1 new b-subunit [7].

Both subunits fold into an N-terminal globular head, that combined create theligand-binding surface, a long stalk region connecting the ligand-binding head withthe transmembrane type I stretch and the C-terminal cytoplasmic tail (reviewed in[3, 8, 9]). The cytoplasmic tails are short (10-50 residues) and do not contain anycatalytic activity or consensus protein binding-motifs, except for the internalizationNPXY-motif in b-subunits. The only known exception is the b4-subunit with two

ProteoglycansCollagen fibers Hyaluronan

Fluid efflux

Fluid influx

Figure 1 Schematic illustration of how connective tissue cells regulate the tissue fluidcontent and thereby the interstitial fluid pressure (IFP).

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fibronectin type-III repeats present in its uniquely long (~1000 residues) cytoplasmic

domain. Several a- and b-subunits exist in different splice variants, and all known

alternative splicing of integrins occurs in the cytoplasmic tail.

Structural analyzes of the a-subunits have revealed seven homologous repeats in

their N-terminal part that fold into a seven-bladed b-propeller, similar to the

nucleotide-binding pocket of the trimeric G-protein b-subunit (a cytoplasmic

complex involved in GTP-mediated transmembrane signal transduction) [10].

Residues taking part in ligand binding are clustered on the upper surface of the b-

propeller while binding-motifs for regulatory divalent cations are exposed on the

lower surface in the three or four last repeats. Half of the a-subunits, e.g. the

collagen-binding a1, a2, a10 and a11 and the leukocyte specific aE, aD, aL, aM and

aX, contain an I-domain (also known as the von Willebrand factor A-domain) of

around 200 residues inserted between the second and third propeller repeat

(reviewed in [9]). The I-domain is exposed on the upper surface of the propeller and

is involved in direct recognition and binding of the ligand [11, 12]. Within the I-

domain there is a characteristic metal ion-dependent adhesion site (MIDAS), that

binds negatively charged residues in the ligand (reviewed in [13, 14]). In the a-

chains lacking an I-domain the ligand-binding surface of the b-propeller binds

directly to the ligand while it in I-domain containing chains either cooperate in the

binding of specific ligands, as in aM [15] or do not participate at all, as in aL [16, 17].

The I-domain, unlike the b-propeller is able to fold correctly without being

associated to the b-subunit [18]. C-terminal of the b-propeller the stalk region starts,

which comprises a large part of the extracellular domain and roughly can be divided

into three domains denoted thigh, calf-1 and calf-2. The thigh is an Ig-like domain

that together with the lower surface of the b-propeller creates an interface

encompassing two of the calcium binding-sites in the b-propeller. Calf-1 and -2 are

b-sandwich domains that together form hydrophobic interdomain contacts that lock

the structure. With the exception of a4 and a9, the stalk region of a-subunits lacking

the I-domain gets post-translationally cleaved within calf-2, creating an N-terminal

heavy chain and a C-terminal light chain joined by a disulfide bond. This post-

translational cleavage has been implicated in the activation of signalling pathways

mediated by av-integrins [19] and in the regulation of a6-integrin affinity [20].

10

The cytoplasmic tails of the a-subunits show very little homology except for the

transmembrane/cytoplasmic interface, where all a-subunits, with the exception of

a6B (DFFKR), a8 (GFFDR), a9 (GFFRR), a10 (GFFAH) and a11 (GFFRS), have a

GFFKR-motif. The different a-tails are, however, well conserved between species

and trigger specific cellular responses. Even though few proteins have been shown

to interact with the a-tails they seem to regulate both the availability of the b-tails

and the extracellular conformation, and thereby activity of the integrin. Truncations

of the a-tail lead to ligand-independent localization of the integrin to focal contacts

[21]. Furthermore, when chimeric constructs of the aIIb extracellular domain fused

with cytoplasmic tails from other a subunits were expressed in CHO cells some a-

tails led to an inactive integrin conformation and some to an active [22]. Mutations

within the a2-tail have been shown to suppress p38 mitogen activated protein kinase

(MAPK) activation and migration on collagen type I in response to epidermal

growth factor (EGF), and activation of the extracellular-regulated kinase (ERK)

MAPK and cell cycle progression in response to insulin [23].

The b-subunit has an N-terminal cystein-rich region homologous to other membrane

proteins called a PSI-domain (for plexins, semaphorins and integrins) [24]. The PSI-

domain co-operates with a more C-terminal cystein-rich region in restraining the

integrin in an inactive state via a long-range disulfide bond [25, 26]. The b-subunits

also contain an I-domain-like domain with a MIDAS and similar secondary structure

as the I-domain of the a-subunits. When associated with an a-subunit lacking an I-

domain this I-domain-like structure takes direct part in ligand binding while it

otherwise has a more indirect regulatory effect. This domain is also involved in the

actual association with the a-subunit and is mutually dependent on the b-propeller

for correct folding [27, 28]. Like in the a-subunits there is a stalk region in the C-

terminal part of the extracellular domain but, unlike the a-subunits this region

contains four EGF-like cystein-rich domains important in signal transduction and

activation of the integrin [29-32].

Six of the eight b-subunits show a high degree of similarity in their cytoplasmic tails

with three regions, called cyto-1, -2 and -3, important for focal contact localization

and a threonine/serine stretch involved in cell adhesion. The cyto-1 corresponds to a

highly charged membrane proximal part and is suggested to bind a-actinin [33]. The

cyto-2 and -3 consist of the NPXY- and NXXY-motif respectively and the tyrosines

11

within these motifs can get phosphorylated. Immortalized b1-null embryonal stem-

cells expressing the splice variant b1A with both cytoplasmic tyrosines replaced with

phenylalanines, Y783/795F, have impaired cell spreading and are unable to activate

focal adhesion kinase (FAK) in response to cell attachment [34]. Furthermore, a b3

Y747F mutation in platelets abolishes cell adhesion to vitronectin and clot retraction

[35]. The threonine/serine stretch has been implicated in the control of cell adhesion.

A naturally occurring S752P mutation in the b3-tail leads to a constitutively active

aIIbb3 in a variant of the bleeding disorder Glanzmann's thrombasthenia [36]. Cells

with a TT788/789AA double mutation in the b1A subunit are defective in cell

attachment and fibronectin fibril formation [37]. Finally the threonine/serine-motif

is suggested to be involved in the functional binding of a-actinin to the b2-subunit in

leukocytes [38].

On leukocytes and platelets, b2- and b3-integrins respectively, need to be activated

in order to perform their functions [38, 39]. Activation of integrins can occur by a

conformational change involving a straightening of the receptor that increases

integrin ligand affinity, but can also be accomplished by increased expression

and/or clustering of the integrins at the cell surface, which leads to increased

integrin avidity [40-42]. The generally accepted model of integrin activation is that

the cytoplasmic tail of the a-subunit somehow inhibits the b-subunit from

interacting with certain cytoskeletal components, and thereby restrains the integrin

in an inactive state (reviewed in [43]). The mechanism by which the a-subunit locks

the integrin in an inactive state is not known, but the conserved membrane proximal

sequences in both subunits (GFFKR for the a-chain and LLxxxiHDR for the b-chain)

has been implicated along with phosphorylation and proteolytic cleavage of the

cytoplasmic tails [37, 44-47]. This inactivation can be abrogated either by binding of

ligand or by the actions of agonists, like thrombin in the case of aIIbb3 in platelets,

and results in an opening or sliding of the cytoplasmic tails [48].

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Integrin activation by increased ligand affinity leads to a straightening of theintegrin and exposure of the ligand binding-site. To date two models are proposedfor this change in appearance, namely the switch-blade model where each subunithas a single bend in the stalk region bringing the head to face downwards [49], andthe angle-poise model where there is an additional bend, or sliding of the stalkregion close to the membrane [50]. The switch-blade model is the most commonlyaccepted and relies on data concerning long-range disulfide bonds within the b-subunit (reviewed in [9, 51]). The angle-poise model is based on data suggesting thatthe membrane and proxamembrane region of integrins allows for a tilting of theintegrin, with residues moving in and out of the membrane as a result [50]. Thismodel would leave the integrin "head" in a more favourable position and fits wellwith the high conservation of the proxamembrane domains. The activation into ahigh affinity conformation is followed by clustering and increased avidity of theintegrins that in turn leads to the formation of macromolecular signalling complexescalled focal adhesions (see FOCAL ADHESIONS).

b1

a3a

4a5

a6 a7a8

a9

av

b3a2a1 a

11 a10

aIIb

b8

b5

b6

aD

aLaMaX

b2

b8

b7

aE

Figure 2 The integrin family of cell surface receptors. Known ab-heterodimers areindicated by lines. I-domain containing a-subunits are shown in grey ovals.

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Collagen-binding integrins

To date there are four integrins known to bind triple-helical interstitial collagens,

namely a1b1, a2b1, a10b1 and a11b1. The a-subunits all have I-domains and

exclusively associate with the b1-subunit. Expression patterns and ligand

specificities of the four collagen-binding integrins point to both unique and

potentially overlapping physiological functions (reviewed in [52-54]). Both the a1-

and the a2-subunits were first identified as the a-components of the Very Late

Antigens (VLA-1 and -2 respectively) expressed on T-cells in various stages of

activation [55]. Later they were shown to belong to the integrin family of cell surface

receptors [56]. While a1b1 and a2b1 have been known for quite some time, and

hence have been thoroughly studied and characterized, a10b1 and a11b1 were only

recently identified. The a10b1 integrin was identified as a collagen type II-binding

integrin on chondrocytes [57] and a11b1 as a collagen type I-binding integrin on

cultured muscle cells [58]. Later studies though, have shown that a11b1 is not

expressed on muscle cells in vivo [59].

Tissue distribution of collagen-binding integrins both differs and overlaps. a1b1 and

a11b1 are predominantly expressed in mesenchyme [59, 60] while a2b1 is present

primarily on epithelial cells and platelets [61] and a10b1 mainly on chondrocytes

[62]. Along with a10b1 the other collagen binding integrins are also expressed by

chondrocytes, but to a lower degree and with less affinity for the cartilage specific

collagen type II [62-64]. In addition, a1b1 and a2b1 are present on fibroblasts,

activated T-cells and certain endothelial cells. In contrast to a1b1 and a2b1, the two

most recently characterized collagen-binding integrins a10b1 and a11b1 have not

been detected on cells of haemapoetic origin or on epithelial cells.

The four collagen-binding integrins differ with regard to ligand specificity. a1b1

mediates cell spreading on collagen types I, III, IV, V and XIII, with a preference for

type IV, but not on collagen type II. a2b1, on the other hand, mediates cell spreading

on collagen types I-V, but unlike a1b1 not on the transmembrane collagen type XIII

[65]. In the same study it was shown that recombinant a1-I-domain binds to collagen

type II, although cells expressing a1b1 do not spread on this substrate. This

difference suggests additional factors in the regulation of cell spreading than merely

attachment. Recombinant I-domains from all four collagen-binding integrins have

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been used in different binding-assays and have strengthened the idea that these

integrins differ in ligand specificity, and maybe in function. I-domains from the a10

subunit (a10-(I)) bound to collagens type I-VI while a2-(I) and a11-(I) preferred the

fibrillar collagens type I-III. a1-(I) showed a higher affinity for the basement

membrane specific collagen type IV and the beaded filament-forming collagen type

VI [11, 12, 66]. Point mutations in the a2-(I) that abolished cell-spreading on collagen

type I had no effect on spreading on collagen type IV while the I-domains of a1-(I),

a2-(I) and a11-(I) all bind to the same motif within interstitial fibrillar collagens,

namely the GFOGER or GFOGER-like domain [66, 67].

a1b1, a2b1, a10b1 and a11b1 do not exclusively bind collagens but also laminins

and other ECM proteins, and in contrast to the other collagen-binding integrins

a2b1, like aEb7 also mediates cell-cell adhesion by binding E-cadherin [68-70]. The

physiological relevance of this a2b1-mediated heterotypic cell-cell adhesion remains

to be explored but might very well be important for maintenance of tissue

architecture, both when it comes to wound contraction and during inflammatory

events.

The collagen-binding integrins have been shown to have a wide variety of specific

and vital biological functions, but while the b1-knockout leads to early embryonic

death at the stage of blastocysts, none of the existing a-subunit knockout animals

show any severe phenotype [71]. The a1-null mouse is viable and fertile and

develops normally during embryogenesis. In addition, liver functions, the immune

response, wound healing and general behaviour are all normal, and besides a slight

reduction in weight the animals are indistinguishable from wildtype mice [72].

Looking more specifically into the phenotypes of the a1-null mouse, a 20 % increase

in collagen turnover in the skin was detected [73]. The increased turnover is

probably due to abrogated down-regulation of collagen synthesis by a1b1 in

combination with increased up-regulation of the collagenase MMP1 by a2b1. Also

the integrin a2-null mice are viable, fertile and develop normally [74]. Even when

more detailed histological studies of different tissues were conducted, no

abnormalities were found. However, when analyzing branching morphogenesis in

mammary glands, a2-null animals showed markedly reduced branching complexity.

The a2b1 integrin has been suggested to be vital for fibroblast and keratinocyte

migration during wound healing. The only defects observed in the a2-null mice

15

regarding wound healing though, is that purified platelets fail to adhere to collagen

type I, and even if platelet aggregation in response to collagen do occur, it occurs

with lower kinetics and a longer lag-phase. Mice with a knocked out integrin a11

gene exist and are viable and fertile, and currently under investigation. The first

obvious phenotype discovered in a11-null animals was that they were much smaller

than their littermates. This defect could partly be overcome by giving the mice soft

food instead of pellets, suggesting a dental defect. Already at the age of two weeks

the a11-null mice showed increased teeth eruption and by the age of one year their

upper incisory teeth seemed to be out of their sockets (Gullberg, et al. unpublished).

In line with these observations, a11 is the only collagen-binding integrin expressed

on periodontal ligament fibroblasts. To date no phenotypes originating from a lack

of a10b1 have been published.

No consistent expression-pattern of collagen-binding integrin has been established

regarding cancer and tumour spreading. For instance, a1b1 has been shown to be

up-regulated in some melanomas [75] and down-regulated in others [76]. Likewise

a2b1 is up-regulated in anaplastic thyroid carcinomas [77] while the expression is

low to absent in breast adenocarcinomas [78, 79]. There have not yet been any

implications for the more recently identified a10b1 and a11b1 in tumours.

Several in vitro functions of the collagen-binding integrins a1b1 and a2b1 have been

suggested in the literature and the knowledge about a10b1 and a11b1 is constantly

growing. a1b1 has been shown to be a negative regulator of collagen synthesis [80]

and is down-regulated in scleroderma, a disease characterized by increased collagen

synthesis [81]. The a2b1 integrin has a positive effect on collagen gene expression,

but perhaps more as a competitor of a1b1 than a direct activator [82, 83]. The

regulation of collagenases seems to be more complex. The major collagenase, the

matrix metalloprotease 1 (MMP1) is not only up-regulated by a2b1 in response to

collagen [80, 83], but also binds the I-domain of the a2 integrin subunit, thereby

positioning it to its substrate [84]. Furthermore, in human skin fibroblasts seeded

within a collagen lattice a2b1 has been shown to induce another collagenase,

MMP13, in a p38 MAPK-dependent manner [85]. Together with the multi-specific

avb3, a2b1 is the integrin implicated in the most numerous and diverse

physiological and pathological conditions.

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The multi-specific integrin avb3

The av-integrin subunit associates with the b1-, b3-, b5-, b6- and b8-subunits. Like

the b1- and b2-integrins, the av-integrins are regarded as an integrin subfamily. The

multi-specific avb3 was first identified as a vitronectin receptor purified from

placenta [86]. Since then several additional ECM proteins, including fibrinogen,

fibronectin, thrombospondin and bone sialoproteins have been identified as ligands.

The key motif for avb3-binding is the Arg-Gly-Asp (RGD) sequence, that was first

identified as the binding-motif of the fibronectin-receptor, today called the a5b1

integrin [87]. Some ECM proteins, including collagens and laminins contain intrinsic

RGD-sequences that require conformational changes of the proteins to get exposed.

This may be of functional relevance in tissue repair and remodelling following

inflammation and injury. In addition to mediate cell-ECM interactions avb3 also

participates in cell-cell adhesions by binding PECAM-1/CD31 and L1/NILE, two

cell surface glycoproteins belonging to the immunoglobulin superfamily [88-90].

avb3 is expressed at low levels in most tissues, but high levels of expression is found

mainly in mature osteoclasts, activated macrophages, a subset of neutrophils,

angiogenic endothelial cells and migrating smooth muscle cells. This expression-

pattern well reflects the suggested involvement of integrin avb3 in bone resorption

and inflammation.

Being one of the major integrins expressed on osteoclasts, interference of avb3

functions by blocking ligand-binding leads to diminished bone resorption [91, 92].

Furthermore, in osteoclasts, as well as in endothelial cells and neutrophils, there is a

concomitant rise in intracellular levels of calcium in response to cell adhesion via

avb3, which in turn affects cell motility via the calcium dependent protease calpain

[93-96], important for not least inflammatory infiltration.

avb3 is up-regulated in several malignant melanomas, providing the malignant cells

with invasive properties by enabling them to attach to and migrate through several

ECM proteins. This occurs either directly or by regulating MMP activity, e.g. MMP2

[97, 98]. Disruption of the association between MMP2 and avb3 has been shown to

inhibit angiogenesis and tumour growth in experiments on melanoma cells [99].

Thus, high expression of the avb3 integrin in melanomas equals poor prognoses due

to an increase in metastases [100]. In addition, avb3 is up-regulated in certain

17

tumour vasculature and has, together with the closely related avb5 integrin, been

suggested as proangiogenetic. Inhibitors, monoclonal antibodies and blocking

peptides have been used in different experimental models with the attempt to

reduce tumour angiogenesis. Several reports show an anti-angiogenetic response to

agents blocking ligand engagement of avb3 and avb5 [101-104]. In fact a humanized

version of a monoclonal antibody (LM609) directed against avb3 has entered early-

phase clinical trials under the name Vitaxin [105].

The evidence implicating avb3 in the regulation of angiogenesis cannot be ignored

or disregarded. However, studies of genetically altered mice lacking any of the av,

b3 or b5 integrin subunits, or combinations of them, all show extensive angiogenesis.

b3-null animals (and b5- and b3/b5-null) are viable and fertile and with normal

vasculature, but suffer from bleeding disorders typical for Glanzmann

thrombasthenia that originates in defective platelet aggregation and clot retraction

[106]. In fact, mice lacking b3 not only supports tumour growth but the tumours

exhibit enhanced angiogenesis and angiogenetic response to hypoxia and VEGF

[107]. In addition, b3-null mice are defective in bone resorption and suffer from

osteosclerosis [108]. The av-null phenotype is more severe. Only about 20 % of the

animals are born alive, probably due to defective placentas, but all embryos develop

normally until E9.5 [109]. The av-null animals do have abnormalities in their

vasculature and suffer from intracerebral and intestinal haemorrhages and die soon

after birth. At least the defects in the brain though, seems to origin from defective

interactions between parenchymal cells and the vasculature, rather than the

vasculature itself [109, 110]. When characterizing b8-null mice, similar defects in the

intracerebral vasculature were seen, suggesting that these abnormalities originate

from the lack of avb8 [111].

Collagens contain intrinsic RGD-sequences and avb3 mediates cell adhesion to

denatured but not native collagen. avb3 is, however, involved in collagen-mediated

processes, such as remodelling of a collagen matrix [112-116]. The fact that avb3 is

involved in the regulation of collagenases as well [98, 117, 118], and is associated

with a hyper-phosphorylated fraction of the ligand-activated platelet-derived

growth factor b-receptor (PDGFR-b) [119] opens up for an interesting possibility of

crosstalk between not only integrins and growth factor receptors, but also between

integrins during tissue repair.

18

PLATELET-DERIVED GROWTH FACTOR (PDGF)

The platelet-derived growth factor (PDGF) family is a family of growth factors

acting as major mitogens for connective tissue cells, such as fibroblasts and smooth

muscle cells, but also for other cell types of mainly mesenchymal origin (reviewed in

[120-123]). As indicated by the name, PDGF was originally isolated from platelets,

but is produced by several other cell types as well, including macrophages,

fibroblasts and keratinocytes. PDGFs and their receptors are essential during

embryonic development, especially in the formation of the kidneys, blood vessels

and various connective tissues. Later in life, however, over-activity of PDGF has

been implicated in a variety of pathological conditions involving increased

proliferation, such as growth-stimulation of tumours, atherosclerosis and fibrotic

conditions. Due to its potent stimulation of growth and chemotaxis, a major role for

PDGF is as a promoter of healing of soft tissue injuries.

PDGF isoforms and receptors

Previously PDGF isoforms were thought to consist of homo- and heterodimers of the

well characterized and thoroughly studied PDGF-A- and -B chains. The family was,

however, expanded with the discovery of two new homodimer forming subunits,

PDGF-C and -D, resulting in five different active PDGF isoforms denoted PDGF-AA,

-AB, -BB, -CC and -DD (Fig. 3).

Both the A- and B-chains are synthesised as precursors and undergo proteolytic

modifications before secretion. Most cells that make PDGF make both PDGF-A and -

B, and although their regulation is independent and rather strict, it seems to be a

random process whether homo- or heterodimerization of the two occur. The A- and

B-chains appear as two variants, a longer and a shorter form, but while the PDGF-A

variants are generated through alternative splicing the PDGF-B chain is

proteolytically cleaved into its shorter form. Cells effectively secrete the shorter

forms of PDGF-A and -B while the longer chains are retained at the cell surface [124,

125]. The retention of the longer PDGF chains at the cell surface is, at least in part,

due to binding to glycosaminoglycans, especially heparan sulphate proteoglycans

[126, 127]. Furthermore, a recent study have shown that heparin is able to

specifically augment PDGF-BB mediated signalling via the PDGF a- but not b -

19

receptor (see below) by increasing receptor phosphorylation, resulting in a more

pronounced chemotactic response through increased activation of the mitogen-

activated protein kinase (MAPK) [128]. Although the A-chain contains a possible site

for N-glycosylation no data suggest that PDGF gets glycosylated.

The mature A- and B-chains show about 60 % amino acid similarity and have eight

completely conserved cysteins. Two of the cysteins are involved in interchain

disulphide bonds creating an anti-parallel dimer, and the other six in intrachain

disulphide bonds that create a characteristic knot-like structure. Similar cystein-rich

sequences are shared by the vascular endothelial growth factor (VEGF) family and

by the placenta growth factor.

The novel isoforms, PDGF-C and -D, share the PDGF/VEGF-domain defined by

eight conserved cysteins with the classical A- and B-chains, but have an extra three

amino acid sequence inserted, and four additional cysteins in PDGF-C and two in

PDGF-D ([129-131], reviewed in [123]). The novel PDGFs form a subgroup by having

an extra domain in their N-terminal part called the CUB-domain (complement

factor, urchin EGF-like protein, bone morphogenic protein). The CUB-domain needs

to be proteolytically cleaved off in order for the PDGFs to be activated, but its

function is unknown. Plasmin cleaves of the CUB-domain in vitro and activates the

PDGF-C and -D homodimers, but how they are activated in vivo is not yet known

[130, 131].

PDGFs act as disulfide bonded homo- or heterodimers and exert their cellular

functions by binding their two tyrosine kinase equipped receptors, the PDGF a- and

b-receptor. Structurally the two receptors are similar and consist of five extracellular

immunoglobulin (Ig) domains and an intracellular tyrosine kinase domain that is

interrupted by a characteristic inserted sequence [132, 133]. The three outer Ig-

domains are involved in ligand binding [134] and the fourth in the stabilization of

the receptor-ligand complex [135]. The different ligands bind to distinct motifs

within the same receptor that do not coincide. For example, PDGF-BB binds with

high affinity to Ig-domains 1-2 in PDGFR-a while PDGF-AA has the highest affinity

for Ig-domains 2-3 [136, 137].

20

Ligand binding induces dimerization of the receptor, but depending on whichreceptors are expressed, different ligands can induce either receptor homo- orheterodimerization. PDGF-A, -B and -C bind the a-receptor while PDGF-B and -Dbind PDGFR-b. Thus PDGF-AA, -BB and -CC can induce PDGFR-aa homodimers,PDGF-AB and -BB can induce PDGFR-ab heterodimers and finally PDGFR-bbhomodimers can be formed by PDGF-BB and -DD (Figure 3). PDGF-CC and -DDhave been reported to induce receptor heterodimerization as well [138, 139].

Ligand-induced receptor dimerization seems to be a common theme among tyrosinekinase receptors, including VEGF receptors and stem-cell factor receptor, and leadsto intracellular trans autophosphorylation. Autophosphorylation is a key event inPDGFR activation and serves two major purposes. First, the autophosphorylation ofa conserved tyrosine within the kinase domain, Y857 in the b-receptor and Y849 inthe a-receptor, leads to increased catalytic activity [140]. Second, phosphorylation oftyrosines outside the catalytic domains creates docking sites for SH2-domain

C-C A-A A-B B-B D-D

a a a b b b

TM

Extracellular

Intracellular

Ligand

Receptor

Figure 3 PDGF isoforms and their receptors

21

containing signalling molecules, enabling transduction of PDGF specific signals

(reviewed in [122] and discussed under PDGF-induced signalling). The direct

receptor-receptor interaction via extracellular Ig-domains suggests that if present at

high enough local concentrations, receptors might dimerize and autophosphorylate

independently of ligand. Apart from when over-expressing the receptors, ligand-

independent phosphorylation of PDGFR-b has been seen in normal fibroblasts as a

result of ligand-induced engagement and concomitant clustering of b1-integrins

[141].

The different receptor dimers mediate different signals. While the bb-homodimer is

a potent mediator of mitogenic signals in most cells the aa-receptor only transduces

mitogenic signals in certain cell types, e.g. cardiac fibroblasts [142-144]. Similarly the

PDGFR-aa both supports and inhibits chemotaxis, depending on cell type, whereas

the bb-receptor solely stimulates chemotaxis [145]. Furthermore, the heterodimeric

PDGFR-ab induces a higher mitogenic response, correlated with a lack of Ras-GAP

binding, than either the aa- or bb-homodimeric receptor [142, 146]. The increased

mitogenic response by PDGFR-ab depends on different autophosphorylation-

patterns between the isoforms, and results in a receptor with optimal kinase activity

and docking capacity for signal molecules that stimulate proliferation [147]. Binding

of PDGF to its receptors finally leads to deactivation by internalization and

degradation of the ligand-receptor complex [148]. Activated receptors undergo

ubiquitination and get targeted for degradation by proteosomes as well. The

internalization rate seems to be dependent on kinase activity and hence on

phosphorylation state [149]. An interesting set of observations is that PDGF

receptors can cluster independently of ligands [150] and are concentrated in caveolae

[151]. Caveolae are cell membrane structures implicated in endocytoses, suggesting

ligand-independent regulation of the PDGF receptor cell surface expression as well.

Biological functions of PDGF

Expression-patterns of PDGF isoforms and their receptors during embryonal

development indicate paracrine roles in the development of several connective

tissue compartments. Mice lacking the PDGF-B isoform show a similar phenotype as

those lacking the b-receptor, namely severe defects in the development of the

kidneys with complete absence of mesangial cells and poor glomerual filtration.

22

Defective blood vessels, probably due to incorrect growth of endothelial cells and an

inability to attract pericytes, lead to lethal bleedings at the time of birth [152-154].

Silencing of the PDGF-A gene in mice is lethal either during embryogenesis at E10 or

postnatally about three weeks after birth. The live-born mice eventually die from

lung emphysema due to absence of alveolar myofibroblasts and associated elastin

fibre deposits [155]. Animals lacking the PDGF-A chain also develop a defect

gastrointestinal tract with fewer and disfigured villi [156]. PDGFR-a knockout

animals have a more severe phenotype that includes malformation of the cranial

bones and alterations in vertebrae, ribs and sternum originating in a deficiency in

myotome formation [157]. A more severe phenotype for PDGFR-a than for PDGF-

A knockout animals is only logical since PDGFR-a binds PDGF-B and -C as well.

No knockout animals have been established for the most recently described PDGFs.

Both isoforms are widely expressed during embryonal development. PDGF-C is

expressed in mesenchyme around structures destined to become ducts and canals,

suggesting a role in ductal morphogenesis during development [158].

In adults PDGF plays a role in a variety of biological functions involved in tissue

homeostasis. Several cell-types engaged in wound healing of connective tissues

express, and in fact up-regulate their expression of PDGF receptors in response to

injury, e.g. during inflammation [159]. Fibroblasts and smooth muscle cells respond

both mitogenically and chemotactically to PDGF that also induces chemotaxis of

circulating neutrophils and macrophages. In addition, PDGF induces macrophages

to produce other growth factors involved in the different stages of wound healing.

Apart from acting as a mitogen and chemoattractant PDGF may play a role in the

formation of new tissue by inducing the production of several ECM components,

including collagen, fibronectin, proteoglycans and hyaluronan [160-162]. Finally,

PDGF may be important during the last stages of wound healing, namely wound

closure or contraction since it has been shown to stimulate contraction of cell-

populated collagen lattices in vitro (see Cell-mediated contraction of collagen matrices)

and has been shown to induce secretion of collagenases by fibroblasts [163].

Important to remember though is that in order to make a difference in vivo PDGF

has to be present at rather high concentrations at the actual wound site. Early on

PDGF was found to be secreted by activated platelets and macrophages but also

from endothelial cells stimulated by thrombin, activated fibroblasts and epidermal

keratinocytes and by smooth muscle cells in damaged arteries (reviewed in [164]), all

23

of which would place PDGF at the site of the wound. In addition, PDGF has been

found in wound fluid and in tear fluid during corneal wound healing [165, 166].

Addition of PDGF in vivo indeed increased healing, not by altering the healing

process but by increasing the rate of healing. Local application of PDGF leads to

quicker re-epithelization and neovascularization and an increase in fibroblast-rich

granulation tissue, as revealed from clinical trials [167].

Similar to the functions exerted by PDGF during wound contraction is its role in the

minute regulation of tissue pressure. The interstitial fluid pressure (IFP) is tightly

controlled to preserve the correct exchange of fluids and low molecular weight

molecules between the circulatory system and the surrounding tissues. The

generally slightly negative IFP is retained by interactions, mainly via integrins,

between connective tissue cells and the ECM, and since PDGF is able to affect these

interactions it also regulates IFP [168-170].

Although PDGF exerts a weaker angiogenic response than for example VEGF, and

has been shown not necessary for initial vessel formation, PDGF plays an important

role in angiogenesis in specific organs, e.g. the heart [171]. Furthermore, PDGF-B

synthesized by endothelial cells in the capillaries has been suggested as a major

attractant of pericytes, and thereby affects neoformation of vessels [172].

As for many essential molecules over-expression of PDGF leads to several severe

disorders, including malignancies. The well-known sis-oncogene translates into a

PDGF-like growth factor that transforms cells via an autocrine stimulation of growth

when expressed by cells also expressing PDGF receptors (reviewed in [120, 164]). In

the same way co-expression of PDGF-A and/or -B and their receptors results in an

autocrine stimulation of tumour growth in some sarcomas and glioblastomas, [173,

174]. Injection of glioblastoma cells treated with the PDGF receptor inhibitor STI571

into the brain of nude mice leads to decreased tumour growth compared to injection

of non-treated cells [175]. Moreover, chromosomal translocations leading to

constitutively active PDGF receptors have been identified in fibrosarcomas and

leukaemia (reviewed in [120, 164]). PDGF also act as a paracrine stimulator of

stromal cells. Over-expression of PDGF-B has been shown to create less necrotic and

faster growing tumours with a more vascularized and well-functioning stroma [176].

Expression of PDGF-C is enhanced by a protein specific for Ewing sarcoma [177],

24

but whether PDGF-C and -D are involved in tumourogenic mechanisms, autocrine

or paracrine, remains to be evaluated.

In the context of paracrine effects of PDGF on tumours its regulation of IFP should

be brought up. In general, solid tumours have an increased IFP that makes it harder

for circulating drugs to be taken up, and PDGF-BB produced by the tumour

contributes to the raise in IFP. In fact, chemical inhibition of the PDGF receptors

leads to a lowering of the IFP in the tumour and an increased permeability, making

PDGF an interesting target in the treatment of solid tumours [170].

Common for fibrotic conditions are proliferation of fibroblasts and accumulation of

extracellular matrix (ECM), processes that in many organs are stimulated by PDGF.

In fact, the development of various lung fibrosis can be deduced to overactivity of

PDGF. In idiopathic pulmonary fibrosis, an inflammatory and fibrotic disease, both

the alveolar macrophages and the alveolar epithelium produce PDGF [178]. An

increased level of PDGF also characterizes several forms of bronchiolitis and fibrosis

following hypoxic pulmonary hypertension, breathing of high concentrations of

oxygen or exposure to asbestos. In addition, elevated levels of PDGF are seen in

bronchial lavage from patients with Hermansky-Pudlak syndrome, a genetic form of

lung fibrosis (reviewed in [120, 164]). Experimental rat models have shown that

PDGF antagonists have beneficial effects on lung fibrosis and that intratracheal

injection of PDGF-BB caused proliferation of pulmonary cells and collagen

deposition [179]. Apart from lung fibrosis PDGF is implicated in different fibrotic

conditions of the kidney, e.g. in glomerulonephritis and tubulointerstitial fibrosis

(reviewed in [120, 164]). Forced over-expression of PDGF in fact induces

glomerulonephritis while antagonists have protective effects, and PDGF-BB induces

tubulointerstitial proliferation and collagen production [180-182]. Elevated levels of

PDGF also play a role in: liver cirrhosis, where fat-storing cells differentiate into

PDGF-responsive myofibroblast-like cells; scleroderma, an autoimmune disease

resulting in progressive fibrosis of the skin and internal organs; and finally in bone

marrow myelofibrosis where an increase of PDGF in serum and urine is

corresponded by a decrease in platelets [183-185].

Atherosclerotic diseases are another group of major maladies in which PDGFs and

their receptors play a central role. Whereas PDGF expression in arteries from healthy

adults is low, it is markedly up-regulated in response to the inflammatory and

25

fibroproliferative state characteristic of atherosclerosis. A "response to injury model"

has been proposed for atherosclerosis and according to that PDGF and other

cytokines are released when the endothelial cell layer of a blood vessel gets injured.

This recruits smooth muscle cells from the media layer to the intima layer of the

vessel and creates an intimal hyperplasia [186]. In agreement with this model

elevated levels of PDGF and PDGF receptors have been found in arteries injured by

naturally occurring as well as experimentally induced atherosclerosis. The

administration of neutralizing PDGF-antibodies, anti-sense oligonucleotides of

PDGFR-b, an antagonizing soluble form of PDGFR-b and PDGFR kinase inhibitors

all inhibit formation of an intimal hyperplasia in an experimental rat model

(reviewed in [120, 164]). PDGF-BB and the PDGFR-b seem to be of greater

importance regarding fibrosis than PDGF-AA and the a-receptor. Even so, inhibition

of PDGF or the receptors only gives a 50 % decrease in intimal hyperplasia

formation, suggesting other factors just as important.

Over-activity of the novel PDGF isoforms, CC and DD remains to be evaluated.

FOCAL ADHESIONS

Integrins primarily mediate cell-ECM interactions and following integrin attachment

and clustering, cytoskeletal and cytoplasmic proteins are recruited to the sites of

adhesion. The newly formed complexes are anchored to the actin cytoskeleton that

gets locally remodelled and form specialized structures called focal adhesions.

Besides forming a structural link between the ECM and the cytoskeleton, focal

adhesions serve as sites of signal transduction by recruiting and synchronizing a

large number of signalling molecules. Numerous cellular processes, like

proliferation and differentiation, are controlled by adhesion-dependent signalling

pathways (reviewed in [5, 187-189]).

Integrins are the major transmembrane receptors in these sites but other

transmembrane and membrane integrated proteins, such as tetraspanins, integrin-

associated protein (IAP), uPAR, the hyaluronan binding protein layilin and caveolin,

and growth factor receptors, like PDGFR-b, VEGFR-2 and EGFR, are recruited and

gathered at focal adhesions. Across the membrane focal adhesions contain a

meshwork of up to 50 different proteins. These can be divided according to their

26

biochemical activity into: cytoskeletal proteins, proteins that have a direct or indirect

association with actin and lack enzymatic activity, such as tensin, vinculin, a-actinin

and talin, adaptor proteins, signalling proteins without enzymatic activities, like

paxillin, Cas, Crk, Shc and Grb2, tyrosine kinases such as members of the Src family,

FAK, Pyk2, and Abl, serine/threonine kinases such as ILK, PKC and PAK, phosphatases

like SHP-2 and PTPs, other enzymes such as PI3-K and finally effectors of small GTPases

such as ASAP1 and different GAPs and GEFs. (The complexity of cell-matrix

interactions is reviewed in [190]).

Integrin-associated proteins

As mentioned above, besides binding to their ECM ligands integrins associate with

other molecules present at the cell surface. Lateral interactions at the cell surface can

influence integrin activity as well as help in the recruitment of cytosolic components

(reviewed in [191]). This creates a complex signalling organelle that can vary and be

modified depending on which challenges or stimuli the cells are exposed to.

Integrin associated protein (IAP), or CD47, is a transmembrane glycoprotein with an

Ig-like extracellular domain, five membrane-spanning stretches and a short

cytoplasmic tail. It has been shown to bind b3-integrins via the Ig-like extracellular

domain and is needed for avb3 binding of vitronectin [192]. Furthermore, IAP is a

receptor of thrombospondin, and avb3-mediated cell spreading on vitronectin is

stimulated by the binding of thrombospondin to IAP in human melanoma cells

[193]. Also a2b1 associates with IAP and the presence of a thrombospondin agonist

peptide increased a2b1-mediated cell migration of smooth muscle cells [194].

The tetraspanins (TM4) are a family of proteins with four membrane-spanning

domains, resulting in two extracellular loops and intracellular N- and C-termini.

More than 20 different tetraspanins have been identified and at least nine of them

have been reported to associate with integrins. The precise role of the tetraspanin

family is not known but its members have been implicated in cell motility,

metastasis and cell growth (reviewed in [195]). a3b1, a4b1, a6b1, a4b7 and aIIbb3

integrins have been identified in complex with tetraspanins, but so far no

associations with a2b1, a5b1, a6b4 or av- and b2-integrins have been reported. In

several cell lines all a3b1 expressed is associated with the tetraspanin CD151.

27

Neurite outgrowth was inhibited by anti-CD151 and -CD81 antibodies in human

NT2N cells seeded on the a3b1-specific substrate laminin-5, but not on laminin-1 or

fibronectin, [196]. CD151 also affects a6b1-dependent morphogenesis of fibroblasts

and endothelial cells [197], and participates in a6b4-mediated hemidesmosomes

formation in human skin [198].

Cell adhesion is essential for cell cycle progression and proliferation initiated by

growth factors. Adhesive interactions are also needed for growth factor-induced

chemotaxis. Whether all growth factor receptors are physically linked to integrins

are not known, but such arrangements do exist. The multi-specific integrin avb3 can

be co-immunoprecipitated with ligand-activated PDGFR-b, EGFR and VEGFR-2,

and with the insulin receptor [119, 199, 200]. Furthermore, engagement of b1-

integrins leads to ligand-independent phosphorylation of both the PDGF b-receptor

and EGF receptor [141, 199]. The b 1-induced EGFR phosphorylation occurs at

tyrosines distinct from those phosphorylated upon binding of the ligand EGF [199].

In general, growth factor-mediated signalling is enhanced by the engagement of

integrins. If this crosstalk depends on physical linkage of the receptors, and

concomitant clustering, or on the fact that integrins and growth factors activate and

recruit common downstream molecules, such as FAK, Src and PI3-K, is a matter of

debate.

Caveolin is a membrane-integrated protein and the main structural component of

caveolae. A direct binding of caveolin to integrins has not been shown but they co-

exist in signalling complexes and this association is dependent on the membrane-

proximal part of some integrin a-subunits. Ligation of some b1-integrins and avb3

leads to a caveolin-dependent recruitment of the adaptor protein Shc and

concomitant activation of the ERK MAPK pathway [201]. Integrins and caveolin

have also been seen in complex with urokinase-type plasminogen activator receptor

(uPAR) in kidney 293 cells [202]. Depletion of caveolin disrupts the uPAR/b1-

integrin complex in these cells and leads to a loss of focal contacts and cell adhesion.

Association of uPAR with integrins switches the ligand specificity of cell adhesion

from integrin-mediated fibronectin binding to uPAR-mediated vitronectin binding

[203]. These data suggest that some ligand-induced b1 signalling requires caveolin

and is regulated by uPAR. Although caveolin is vital for the uPAR/integrin

28

association, it also depends on direct binding of uPAR to the b-propeller of integrin

a-subunits [204].

uPAR is not the only ECM-degrading protein associated with integrins. Some of the

matrix metalloproteases (MMPs) are found to be cell-associated through interactions

with integrins. The collagenase MMP1 is associated with the collagen-binding I-

domain of the a2-subunit and is required for a2b1-mediated keratinocyte migration

on collagen type I [84, 205]. Another collagenase, MMP2, associates with avb3 via a

PEX-domain in its C-terminal part [206]. MMP2 activity is vital for avb3-mediated

migration and disruption of this interaction by a synthetic peptide inhibits

angiogenesis and tumour growth [99, 207].

Ligation of one integrin can influence the function of other integrins in processes

referred to as integrin crosstalk. The uPAR/a3b1 complex regulates the adhesive

functions of unoccupied b1-integrins through activation of the heterotrimeric G-

protein i-subunit [204]. In K562-cells the IAP/avb3 complex down-regulated a5b1-

mediated migration by suppressing the calcium- and calmodulin-dependent protein

kinase II (CaMKII) [208]. Binding of aLb2 to its ligand I-CAM decreases a4b1- and

to some extent a5b1-mediated T-cell adhesion to fibronectin [209]. Finally, work by

us indicate that while avb3-mediated collagen interactions are inhibited by the

presence of active a2b1, but not a11b1, clustering of b1-integrins has a stimulatory

effect [114, 115].

Integrin-mediated signalling

Cytoskeletal proteins. Across the membrane ligand-occupied integrins connect to the

actin cytoskeleton and participate in its reorganization. This not only couples

integrins to the actomyosin contractile apparatus essential for cell migration, but also

provides a surface where a number of signalling molecules are sequestered. Apart

from one in vitro study where F-actin bound a synthetic a2-tail [210], actin has not

been shown to bind integrins directly, but they are connected via several cytoskeletal

proteins, such as tensin, vinculin, a-actinin, filamin and talin.

Talin is a flexible dimeric actin-crosslinking protein that binds to the cytoplasmic tail

of integrins b2, b3, b1A and b1D in vitro, but not to b1B or b1C [38, 211, 212]. Talin

29

contains binding sites for both actin and the cytoskeletal protein vinculin in its rod-like and C-terminal domain [213, 214]. In addition, talin recruits FAK by a binding-site in the N-terminal globular region [215]. In the same study talin associates withlayilin, a transmembrane hyaluronan receptor, enabling for one more connectionbetween the ECM and the cytoskeleton. Cytoplasmic b1 and b3 domains have beenshown to bind both the head and rod-like domain of talin in platelet lysates [216].Mutations in the membrane proximal NPXY-motif of b-subunits abolish talinbinding and disrupt integrin localization to focal adhesions. In addition, over-expression of the talin globular head domain activated aIIbb3 [216]. Anotherindication of a role for talin in integrin activation is that it is proteolytically cleavedby calpain, a protease with positive effects on integrin activation and functions, suchas platelet aggregation and spreading [217]. Furthermore, while b2-integrins co-immunoprecipitate with talin in unstimulated leukocytes, they associate with a-actinin in stimulated cells [38].

a-actinin is an actin cross-linking protein that, besides b2-integrins, associates withthe b1 cytoplasmic tail [33]. Both talin and a-actinin associates with the actin-bindingadaptor protein vinculin. Filamin is another actin cross-linking protein that connectsintegrins to the actin cytoskeleton by interacting with the cytoplasmic tail of certain

babababa

babababa

ECM

Integrins

PM

IAPuPAR Caveolin

Actin

TM4

a-actinin

SrcFAK

Cas

Crk

JNK

Talin

VinculinPax

FAK

Grb2/SOS

Fyn

Shc

Ras

ERK

Ras

ERK

Figure 4 Schematic picture of focal adhesion complexes showing integrin interactionsand integrin mediated activation of MAPK pathways.

CalreticulinILK

FAK

PI3K

Tensin

30

b-subunits, e.g. b1 and b7 [211], and has been shown necessary for melanocyte

migration [218].

Vinculin consists of a globular head domain and an extended tail connected by a

proline-rich domain. The vinculin tail can form an intramolecular interaction with

the head domain. This creates a closed conformation that masks the binding-sites for

talin and a-actinin in the head, F-actin binding-site and protein kinase C (PKC)

phosphorylation-site in the tail, and VASP binding-site in the proline-rich domain

[219]. Binding of paxillin to the vinculin tail seems to occur independently of

vinculin conformation. The head and tail interaction is relieved by

phospatidylinositols, e.g. PIP2. Surprisingly, PIP2 has been reported to inhibit

binding of F-actin to vinculin [219]. VASP binding to vinculin might, in turn, recruit

profilin and G-actin and thereby increase the rate of actin polymerization at the

barbed ends exposed by PIP2-mediated uncapping (see Actin-reorganization by Rho-

GTPases). In fact, VASP associated with vinculin, but not with zyxin, is localized to

the tips of lamellipodia and filopodia [220]. Vinculin, unlike talin, is not vital for

focal adhesion assembly in mouse embryonic stem cells, but the adhesions formed

are fewer and have changed morphology [221]. Over-expression of vinculin in

fibroblasts results in more numerous and larger focal adhesions, while a down-

regulation is accompanied by fewer and smaller focal adhesions and increased cell

motility, suggesting a regulatory more than structural role for vinculin in focal

adhesions [222, 223].

Of the proteins interacting with actin at focal adhesions, tensin is thought to be

located closest to the ends of actin filaments because of its F-actin binding and

capping of the barbed ends [224]. Tensin gets phosphorylated upon integrin

engagement, stimulation by PDGF, thrombin or angiotensin and when cells get

transformed by oncogenes, such as v-Src or Abl [225-228]. Since tensin contains a

SH2-domain it also associates with certain tyrosine-phosphorylated proteins, such as

PI3-K and Cas [229]. These characteristics suggest that tensin might coordinate

signals involved in cytoskeletal changes. In addition, tensin shares sequence

homology with the tumour suppressor PTEN [230].

31

Tyrosine kinases. Many of the signals transduced in cell-matrix interactions upon

integrin activation involve tyrosine phosphorylation. Since integrins do not exhibit

any known kinase activity the presence of active tyrosine kinases is needed.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase that

localize to focal adhesions and gets activated in response to integrin ligation and

clustering. FAK plays a key role in integrin-mediated signalling by providing a

linkage to several intracellular signalling cascades that lead to actin reorganizations,

such as mitogenic responses, cell survival and cell migration [231-233]. Studies of

FAK-null cells have shown that FAK is vital for focal adhesion turnover and cell

migration, but not for integrin activation or focal adhesion formation [233].

The FAK protein contains three domains, the C-terminal domain, the kinase domain

and the N-terminal domain. Unlike many other tyrosine kinases FAK lacks SH2 and

SH3-domains, but have both phospho-tyrosines and proline-rich domains that

associate with SH2 and SH3 respectively. The C-terminal domain of FAK contains

the focal adhesion targeting (FAT) sequence to which both paxillin and talin bind

[234, 235]. A phospho-tyrosine (Y925) that mediates binding of the Grb2 adaptor

protein and two proline-rich domains that mediate binding of Src kinases, Cas and

Graf are also located in the C-terminal part of FAK [236, 237]. The FAT-region is

necessary for EGF-stimulated cell migration via its interaction with the EGF receptor

[238]. Interestingly, certain cells express a truncated form of FAK called FRNK (FAK-

related non-kinase), that is identical to the FAK C-terminal domain and thus without

catalytical activity. FRNK localizes to focal adhesions and exerts a dominant

negative effect on FAK-mediated functions [239]. The kinase domain contains

several tyrosine residues that get phosphorylated and participate in the regulation of

the kinase activity. The only tyrosine in the N-terminal domain is the major

autophosphorylation site Y397, that resides close to the kinase domain and has been

shown to bind Src kinases, Grb7, PLC-g and PI3-K [240-243]. The N-terminal domain

has been reported to contain a binding-site for integrin b-subunits, but whether this

interaction is important for focal adhesion localization is debated [244]. In cells

treated with tyrosine kinase inhibitors, however, antibody-clustered a5b1 co-

immunoprecipitate with FAK and tensin, but not with paxillin, talin or other

classical focal adhesion components, implicating a FAK/integrin association

independent of tyrosine phosphorylation [245]. In addition, FAK/b1-integrin

32

complexes that are dependent of an intact actin cytoskeleton, but independent of

FAK phosphorylation, has been shown to form in genestein-treated cells [246].

Even though activation of FAK might not be necessary for FAK/b-integrin subunit

association, activation of FAK is dependent on the b-subunit cytoplasmic tail [247].

Involvement of the b1-subunit in FAK activation has been shown to require the

tyrosines in the conserved NPXY- and NXXY-motifs, that could serve as binding-site

for a FAK-regulating protein [34, 114]. Activation of FAK is also dependent on an

intact actin cytoskeleton [248]. Integrin-mediated FAK activation has been shown to

require Rho-activity during the later phases of activation, while the initial

phosphorylation is independent of Rho-GTPases [248, 249]. In addition,

phosphorylated Y397 mediates binding of the regulatory subunit p85 of the lipid

kinase PI3-K. The FAK-PI3-K interaction has been implicated in cell survival-

signalling via the Ser/Thr kinase Akt and in cell migration [250] (see PDGF-induced

signalling and Actin-reorganization by Rho-GTPases). Finally, PKC is involved in the

activation of FAK, which would place PKC upstream of FAK in integrin-mediated

signalling [248].

Src and the related Yes and Fyn, are other tyrosine kinases that get activated during

integrin-mediated adhesion. Activation of Src-kinase involves dephosphorylation by

an unknown phosphatase and a subsequent conformational change exposing the

kinase domain. The kinase phosphorylating the inhibitory Y527 of Src is called Csk

[251]. Over-expression of Csk reduces FAK activity, cell attachment and cell

spreading [252]. Src phosphorylates five additional FAK tyrosines, some of which

influence FAK kinase activity, some creating new protein binding-sites, and some of

unknown functions [253]. Src phosphorylation of Y925 together with Y397 creates a

FAK binding-site for Grb2/Sos. The recruitment of Grb2/Sos links FAK-Src

signalling to the activation of the Ras-Raf-MEK-ERK MAP pathway that is important

in the control of cell contractility and migration [231, 250]. Re-expression of wildtype

FAK in FAK-null cells restores cell migration, while expression of FAK with mutated

Src-binding site fails to restore full haptotactic cell migration [254]. Furthermore, the

FAK/Src complex phosphorylates several other proteins localized at focal

adhesions, including paxillin and Cas (see Adaptor proteins).

33

Pyk2 (also called RAFTK, CAKb and CadTK) is a tyrosine kinase structurally similar

to FAK, and sometimes referred to as a second member of a FAK family [255].

Expression of Pyk2 is more restricted than FAK. Even though Pyk2 gets activated

upon integrin ligation it does not localize to focal adhesions to any higher degree

[256]. Similar to FRNK the C-terminal part of Pyk2 (PRNK) can be expressed by

itself. Both FRNK and PRNK localize to focal adhesions [257]. Pyk2 is up-regulated

in FAK-null cells where it seems to compensate for some FAK functions [258].

Elevated levels of intracellular calcium can activate Pyk2 but not FAK [259]. The

chemotactic effect of PDGF-BB on vascular smooth muscle cells is augmented by

angiotensin II in a process involving Pyk2 activation [260]. A role for Pyk2 in

functions dependent on actin reorganization is emphasized by a recent study, where

Pyk2 phosphorylates and inhibits ASAP1, an Arf-GTPase activating protein [261].

The tyrosine kinase Abl is present both in the cytoplasm and in the nucleus. The

nuclear pool act as a negative regulator of cell growth and is not dependent on

integrin-mediated cell attachment [262]. The cytoplasmic pool of Abl, on the other

hand, is activated and transiently localized to focal adhesions by integrin-mediated

adhesion [263]. Cytoplasmic Abl interacts with Grb2 and is, at least in part,

responsible for the transient activation of the ERK MAPK pathway in response to

cell adhesion [264]. Furthermore, Abl has been reported to bind and phosphorylate

paxillin in response to integrin ligation [265]. In complex with a protein called BCR

cytoplasmic Abl acts as an oncoprotein and transforms cells by inducing an

anchorage-independent, but growth factor-dependent cell growth [266]. Finally, Abl

tyrosine kinases exert an inhibitory effect on cell migration by disassembling the

Crk-Cas complex [267].

Adaptor proteins. Many of the tyrosine kinases localized at focal adhesions act by

creating binding-sites for other signalling proteins. Some of the recruited proteins

do not contain any enzymatic activity but act as coordinating adaptor proteins, or

scaffolds, within the signalling complex.

The primary function of paxillin is as a molecular adaptor that provides multiple

docking sites at the plasma membrane. Paxillin localizes to focal adhesions through

LIM-domains, possibly by a direct association with the b1-integrin cytoplasmic tail

[244]. It has also been shown to bind directly to the a4- and a9-tails in lymphoid

34

cells [268, 269]. Binding of paxillin to these integrin a-subunits leads to decreased

cell adhesion and increased cell migration [269, 270]. Binding of paxillin required

phosphorylation of the a4-tail [271]. Phosphorylation of paxillin recruits kinases,

such as FAK, ILK and Src [272, 273], and permits regulated recruitment of

downstream effector molecules such as Cas and Crk, which are important for cell

motility and gene expression by MAP kinase cascades [274, 275]. In addition,

negative regulators of these pathways, like the Src inhibitor Csk and PTP-PEST, a

phosphatase acting on Cas, bind directly to paxillin, thereby bringing them close to

their targets [276, 277].

Paxillin interacts with several proteins involved in actin reorganization and is vital

for processes dependent on cell motility, including embryonic development, wound

repair and tumour metastasis. Apart from structural proteins like vinculin that bind

actin directly, this includes proteins that regulate actin dynamics. Paxillin bind PKL,

an Arf-GAP that complexes with the exchange factor PIX and p21-activated kinase

(PAK). The PKL-PIX-PAK complex serves as an effector of Arf- and Rho-GTPases

that, like PTP-PEST, promote focal adhesion turnover and cell migration in complex

with paxillin [278]. Microtubule binding to paxillin may also contribute to the ability

of this filament system to destabilize focal adhesions and promote cell motility [279].

The paxillin bound serine/threonine kinase PAK is involved in activating the MAPK

cascades, as well as Rac- and Cdc42-stimulated reorganization of the actin

cytoskeleton ([280, 281] see Actin-reorganization by Rho-GTPases).

Another adaptor protein central in adhesion-mediated signalling is Cas. Cas was

first identified as a highly phosphorylated protein in transformed cells and later as a

tyrosine-phosphorylated protein localized to focal adhesions [282, 283]. The Cas

protein contains a SH3-domain, several tyrosines that when phosphorylated can

bind SH2-domains, and finally a proline-rich SH3-binding domain. The well-

established FAK/Cas interaction occurs via the SH3-domain of Cas, a domain that

also interacts with the R-Ras-GEF C3G and two protein tyrosine phosphatases, PTP-

PEST and PTP-1B [236, 284-286]. Src binds Cas via its SH2 and SH3-domains and

further phosphorylates the adaptor protein in complex with FAK, creating new

binding sites for among others, Crk and Nck [287, 288]. Localization of Cas to the

focal adhesions is dependent of its SH3-domain and requires the presence of Src,

although Src kinase activity is not needed [289]. A recent study implicates FAK and

35

Cas in a novel Src-independent pathway of focal adhesion formation, promoted by

R-Ras, in mammary epithelial. This novel mechanism is suggested to be distinct

from, but synergizing with, the integrin-mediated focal adhesion formation by a2b1

[290]. Cas has been shown to be important, if not vital, for FAK-mediated cell

migration. Expression of a FAK variant deficient in recruiting Cas fails to restore

haptotactic migration in FAK-null cells [254]. Furthermore, Cas appears to act as a

molecular switch for induction of migration by its recruitment of Crk. Cas-enhanced

migration is dependent on Cas phosphorylation and the binding of Crk [291].

Crk gets recruited to focal adhesions primarily by binding Cas and paxillin. The Crk

protein consists of one SH2-domain and two SH3-domains separated by an Abl

phosphorylation-site. Phosphorylation by Abl blocks binding to the SH2-domain of

Crk. Both C3G (a R-Ras-GEF) and DOCK180 (a Rac1-GEF) bind Crk and become

phosphorylated upon integrin-mediated cell adhesion [292, 293]. Co-expression of

DOC180 with Cas and Crk leads to membrane ruffling and increased cell spreading

[292]. Recently DOCK180 was shown to bind PIP3 and translocate to the plasma

membrane upon the activation of PI3-K [294]. Hence, DOCK180 might explain how

Cas/Crk enhances migration. Finally Crk has been shown to interact with Sos (a

Ras-GEF) in an association disrupted by EGF and insulin stimulation, which

implicates a link from Crk to Ras, and subsequently, ERK activation [295].

The adaptor proteinNck associates with Cas in a similar manner as Crk [288]. A

recently identified isoform of Nck, Nck-2, binds DOCK180 via its SH3-domains,

establishing yet another link between Cas and cell migration [296]. Interestingly,

Nck-2 has been shown to bind FAK via a SH2-domain, and while over-expression of

wildtype Nck-2 modestly decreased cell motility in 293 cells, over-expression of

Nck-2 lacking the DOCK180 binding-site significantly promoted cell motility [297].

Furthermore, Nck, but not Crk, interacts with PAK in human umbilical vein

endothelial cells [298]. VEGF stimulation of these cells recruits PAK to focal

adhesions and inhibition of Nck leads to the inhibition of both VEGF-induced focal

adhesion assembly and cell migration [298].

Zyxin is an adaptor protein proven to be important in cellular functions that are

dependent on actin reorganization. The zyxin protein contains an N-terminal

proline-rich domain, a nuclear export signal and three C-terminal LIM-domains.

36

Zyxin is connected to actin via binding of a-actinin, and also participates in its

remodelling by binding proteins like Vav and VASP [299-301]. Vav is a Rho-GEF

and VASP induces actin polymerization by recruiting the G-actin-binding protein

profilin to the growing end of actin filaments (see Actin-reorganization by Rho-

GTPases). In addition, a recently identified member of the zyxin family, TRIP6, was

shown to interact with Cas via its LIM-domains [302]. TRIP6 localizes to focal

adhesions and when over-expressed it slows down cell migration.

As described above the adaptor protein Grb2 links integrin- and Ras-mediated

activation of ERK MAPK pathways by binding of the Ras-GEF Sos and the integrin

signalling mediators FAK and Shc.

Shc is an adaptor protein consisting of a PTB-domain, a proline-rich domain

containing a tyrosine phosphorylation-site and a SH2-domain. The PTB and SH2-

domains localize Shc to focal adhesions, where it is phosphorylated and recruits

Grb2. Shc can get involved in integrin-mediated signalling via two pathways. Either

the FAK/Src complex phosphorylates Shc or Shc can associate with certain integrin

a-subunits and activate ERK independently of FAK. Association of Shc with integrin

a-chains is dependent on its association with caveolin. Caveolin binds Fyn via its

SH3-domain and Fyn, in turn, phosphorylates Shc. Not all a-subunits are able to

recruit Shc by associating with caveolin. For instance, fibroblasts from a1-null

animals proliferate less than wildtype cells when seeded on collagen since the

remaining collagen binding integrins, e.g. a2b1 and possibly a11b1, fail to activate

the caveolin/Fyn/Sch-ERK pathway [303].

Serine/threonine kinases. Even if tyrosine phosphorylation is the most studied event

in integrin-mediated cell signalling, serine/threonine kinases are engaged as well.

The role of ILK and PKC will be discussed here. The involvement of the Rho

Ser/Thr kinases, PAK and Akt, and CaMKII are discussed elsewhere in the text.

Integrin linked kinase (ILK) was identified as a b1-tail-binding protein in a yeast

two-hybrid screen [304]. In this study, ILK co-immunoprecipitated with both the b1-

and b3-subunit. N-terminal ankyrin repeats in ILK mediate interactions with the

phosphatase ILKAP, which negatively regulates ILK signalling, and the LIM-domain

protein PINCH. PINCH binds Nck-2 and thereby couples ILK to growth factor

37

receptor, PI 3-kinase and FAK/Cas signalling as discussed above. PINCH is also

required for localization of ILK to focal adhesions [305]. ILK, and thus integrins, is

connected to the actin cytoskeleton by interactions with paxillin and the ILK binding

proteins affixin and CH-ILKBP (ILK interactions are reviewed in [306]) . Activated

ILK phosphorylates PKB/Akt and GSK-3, thereby contributing to the suppression of

apoptosis and anoikis [307, 308]. Phosphorylation of GSK-3 results in its inhibition,

and subsequent stabilization of b-catenin. ILK over-expression prevents inactivation

of Erk during myogenic differentiation. ILK also phosphorylates myosin light chain

(MLC) in a calcium-independent manner, potentially regulating smooth muscle

contraction and cell motility [309]. ILK is activated by integrin-mediated cell

attachment and certain growth factors. In addition, PI3-K stimulates ILK activity via

its binding of the PI3-K product PIP3 (ILK regulation is reviewed in [310]). PTEN, a

tumour suppressor lipid phosphatase that dephosphorylates PIP3 to PIP2, and the

PP2C protein phosphatase ILKAP have been identified as negative regulators of ILK

activity. Over-expression of ILK in epithelial cells results in anchorage-independent

cell growth because of nuclear translocation of b-catenin, and loss of cell-cell

adhesions due to down-regulation of E-cadherin [311].

Protein kinase C (PKC) exists in several isoforms divided into three subclasses,

depending on how they are activated. The conventional PKCs (a , b and g) are

activated by calcium and diacylglycerol (DAG), the novel (e, d, h and q) are activated

independently of calcium and finally, the atypical (z and i/l) neither respond to

calcium nor DAG. Due to the many function-specific PKC isoforms their role in

integrin-mediated signalling is complex and available data somewhat confusing.

Three isoforms, PKC-a, -d and -e, have been shown to localize to focal adhesions.

Recently these isoforms were shown to be sequentially activated during integrin-

mediated muscle-cell spreading [312]. How PKCs get localized to focal adhesions

seems to vary and several mechanisms have been suggested. PKC-a can localize to

focal adhesions by binding talin and vinculin and it also associates with syndecan-4

and tetraspanins at focal contacts [313, 314]. In addition, in leukocytes PKC-b1 and -d

localize to the microtubule cytoskeleton in an integrin-dependent way [315]. The

activation of leukocytes and their integrins by phorbolester-induced PKC activation

has been known for quite some time, and PKC seems to generally promote integrin-

mediated adhesion [316]. Several reports also claim a role for PKC in cell spreading

and migration. For instance, it was recently shown that VEGF-stimulated migration

38

in multiple myeloma cells leads to the activation of PKC-a in a b1-integrin and PI3-K

dependent way [317]. PKC has been suggested to be a downstream effector of Rho

and inhibition of PKC leads to a decrease in focal adhesions and loss of actin stress

fibres [318].

Protein phosphatases. Considering the impact of tyrosine and serine/threonine

phosphorylations in integrin-mediated signalling, it is only logical that there is an

increasing amount of data regarding the role of protein phosphatases as well.

Protein tyrosine phosphatases (PTPs) reported to be involved in cell-ECM signalling

events include SHP-1 and -2, PTP-PEST, PTP1B, PTPa, PTEN and LAR and then

there are the Ser/Thr phosphatases including calcineurin and PP1. Here selected

phosphatases will be briefly described, while some others are mentioned elsewhere

in the text.

The cytoplasmic PTP-PEST has been shown to bind a number of focal adhesion

components, including paxillin, Shc, Grb2, Crk and Cas, but only seems to

dephosphorylate Cas [286]. Over-expression of PTP-PEST does not affect cell

adhesion or spreading but reduces cell migration by inhibiting the formation of

Cas/Crk complexes [319]. In addition, PTP-PEST reduces integrin-mediated

migration by inactivating the Rho-GTPase Rac-1 at the tips of membrane protrusions

[320]. PTP1B is another cytosolic PTP that binds Cas and exerts its functions by

reducing Cas binding of Crk [285]. The tumour suppressor PTEN has been

implicated both as a tyrosine protein phosphatase and as a lipid phosphatase. Over-

expression of PTEN inhibits stress fibre formation and thus cell spreading and

migration, correlated with reduced activation of FAK and ERK [321]. As mentioned

earlier, PTEN has also been shown to dephosphorylate PIP3 in vivo and antagonizes

tumour necrosis factor (TNF) induced PI3-K/Akt/NF-KB and PI3-K/Akt/mTor

pathways [322, 323]. Another cytoplasmic PTP implicated in both integrin and

growth factor signalling is SHP-2. SHP-2 binds ligand-activated PDGFR-b, and

expression of receptors that lack SHP-2 binding-motifs compromise Ras and ERK

activation in response to PDGF-BB [324]. The mitogenic response to PDGF-BB in

these cells was unaffected whereas the chemotactic response was impaired. SHP-2

might also be responsible for the increased mitogenic effect exerted by the PDGFR-

ab heterodimer compared to homodimeric receptors (see PDGF isoforms and

receptors) by modulating the recruitment of Ras-GAP and subsequently signalling

39

via the Ras/Raf/ERK pathway [325]. Furthermore, SHP-2 has been suggested in the

control of cell motility by regulating RhoA activity [326]. Finally, integrin a2b1 has

been shown to mediate negative cell survival signals by inducing dephosphorylation

and thus deactivation of Akt and GSK3. Cell adhesion to collagen type I led to an

a2b1-mediated activation of the protein Ser/Thr phosphatase PP2A and subsequent

dephosphorylation of Akt and GSK3 [327].

PDGF-induced signalling

The PDGF receptors get intracellularly autophosphorylated upon dimerization (see

PDGF isoforms and receptors). This regulates their catalytic activity and creates

docking-sites for a variety of signal-transduction molecules. More than ten

molecules containing SH2-domains have been shown to bind activated PDGF

receptors (reviewed in [122]). Some of them contain enzymatic activities of their

own, such as phosphatidylinositol 3' kinase (PI3-K), phospholipase C-g (PLC-g), Src

tyrosine kinases and the tyrosine phosphates SHP-2, while others are adaptor

molecules, like Grb2, Shc, Nck and Crk. Finally, also signal transducers and

activators of transcription (Stats) have been shown to bind. The different SH2-

domain containing molecules bind to individual phosphates and it is not known

how many can bind at the same time. Cellular effects mediated by activation of

PDGF receptors and downstream effector molecules include cell growth,

chemotaxis, actin reorganization and prevention of apoptosis (Figure 5).

Members of the PI3-K family are lipid kinases that mediate 3' phosphorylation of

phosphatidylinositols. Their preferred substrate is phosphatidylinositol-4,5-

biphosphate (PIP2), which is phosphorylated into phosphatidylinositol-3,4,5-

triphosphate (PIP3). The PI3-K isoform that binds PDGF receptors consist of a p110

catalytic subunit in complex with a regulatory p85 subunit. The p85 subunit contains

two SH2-domains that interact with Y740 and Y751 in the activated PDGF b-receptor

[328]. Y740 and Y751 get autophosphorylated upon receptor dimerization and are

conserved in the PDGFR-a. Binding of p85 to the phosphorylated receptor induces a

conformational change and subsequent increase of enzymatic activity of the

associated catalytic subunit p110. PI3-K plays a central role in several intracellular

signalling events and is involved, directly or indirectly, in most of the PDGF-

induced cellular responses, including actin reorganization, migration, proliferation

40

and antiapoptotic signals (reviewed in [329]). Exactly how PI3-K work in thedifferent responses is not fully understood, but it prevents apoptosis by activatingthe Ser/Thr kinase Akt/PKB, that in turn phosphorylates the Bcl-2 protein Bad[330].

Furthermore, PI3-K activates the Rho-GTPases shown to be important for both actinreorganization and directed migration (chemotaxis) [331, 332]. Activation of thePDGF b-receptor induces transient loss of stress fibres and induction of membraneruffles in fibroblasts [333]. The activation of Rho-GTPases, in particular Rac, involvesa mechanism where PIP3 recruits Rho-GTPase activators containing pleckstrinhomology (PH) domains, such as Tiam-1 and Vav [334, 335]. Other moleculesinvolved in cell survival and proliferation, such as certain PKC isoforms andphospholipase C-g (PLC-g ), also bind to PIP3 via their PH-domains. Actinreorganization is further discussed in Actin-reorganization by Rho-GTPases.

Src

Grb2/Sos PI3K

PLC-g

Raf-1

MAPK

Rho-GTPases

PKCfamily

Akt

Bad

MEK

Ras

Proliferation Cell migration Apoptosis

GAP

Figure 5 Schematic picture of selected signalling pathways induced by PDGFR-b

41

PLC-g prefers PIP2 as substrate and the products are inositol-1,4,5-triphosphate (IP3)

and diacylglycerol (DAG), which leads to an increase in cytoplasmic Ca2+ and

activation of some members of the PKC family, respectively [336]. PLC-g isoforms

bind to phosphorylated Y1009 and Y1021 of the b-receptor and phosphorylated Y988

and Y1018 of the a-receptor. Upon binding PLC-g gets phosphorylated and its

catalytic activity is increased. In order to reach full activity, however, PLC-g needs to

bind the PI3-K product PIP3, possibly leading to a translocation of the enzyme to the

plasma membrane. The dynamic interactions of cells with a collagen lattice, both in

vivo and in vitro, are stimulated by PDGF-BB and have been shown to depend on

PI3-K. This dependence can be overcome by chemically inducing an elevation of

cytosolic Ca 2+ [337]. The role of PLC-g in PDGF-induced signalling is very complex,

and while it in most cell types appears not to be important for stimulation of cell

growth and motility, it has been shown to affect these responses in certain cells.

Activation of the small GTPase Ras is of major importance for several cellular

responses. In its activated form Ras binds the Ser/Thr kinase Raf-1 that in turn

initiates activation of the ERK MAPK pathway, implicated in stimulation of cell

growth, migration and differentiation (reviewed in [338]. Ras is activated by the

nucleotide exchange factor Sos1 which complexes with the adaptor protein Grb2.

Binding of the Grb2/Sos1 to the receptor juxtaposes it to the Ras molecules

associated with the inner leaflet of the plasma membrane. Grb2 binds directly to

autophosphorylated PDGF receptors but can also be indirectly attached by

associating with other molecules, like the adaptor Shc or the tyrosine phosphates

SHP-2 [324, 339]. By presenting a binding-site for Grb2 activated SHP-2 indirectly

contributes to the activation of Ras. In addition, SHP-2 dephosphorylates the Ras-

GAP binding-site on the b-receptor, preventing it from binding [325]. The Ras-GAP

is a negative regulator of Ras and PI3-K. Being a phosphatase SHP-2 is also involved

in negative regulation of the receptor activity. Interestingly, there is a crosstalk

between the small Ras GTPase and PI3-K in that they physically interact and are able

to activate each other [340].

Three members of the Src family of tyrosine kinases, namely Src, Yes and Fyn bind

phosphorylated PDGF receptors that contribute to their activation. The Src family

members contain one SH2- and one SH3-domain in addition to the kinase domain

and are regulated by a phosphorylated tyrosine in its C-terminal part. This tyrosine

42

binds the SH2-domain and restrains the enzyme in an inactive state. Src binds to

phosphorylated Y579 and Y581 in the PDGF-b-receptor and Y572 and Y574 in the a-

receptor and gets activated. Activation of Src kinases includes dephosphorylation of

the regulatory tyrosine and phosphorylation of other tyrosine. Src has been reported

to be important for PDGF induced mitogenic responses, illustrated by the fact that

administration of blocking antibodies, or a dominant negative Src, inhibited PDGF-

mediated proliferation of NIH 3T3 cells [341]. Contradictory to these findings, Src,

Yes and Fyn are dispensable for PDGF-induced signalling, but required for specific

signalling regulated by ECM- binding proteins, e.g. integrins [342].

Actin-reorganization by Rho-GTPases

Many of the above described interactions and signals mediated by integrins and

PDGF receptors result in the activation of the Rho family of GTPases and subsequent

remodelling of the actin cytoskeleton.

More than ten different Rho-GTPases belonging to the Ras superfamily of

monomeric GTP-binding proteins have been identified. Of these Rho, Rac and Cdc42

are the most extensively characterized. GTPases act as molecular switches and cycle

between an active GTP-bound and an inactive GDP-bound state, and can interact

with the membranes via a posttranslational geranylgeranyl lipid modification. The

active GTP-bound form interacts with effector molecules to initiate downstream

responses. An intrinsic GTPase activity then brings the proteins back to their GDP-

bound inactive state and terminates signal transduction. This cycling between a

GTP- and a GDP-bound state is mediated by guanosine nucleotide exchange factors

(GEFs), which facilitate the exchange of GDP for GTP, and GTPase activating

proteins (GAPs), which increase the intrinsic rate of GTP hydrolysis. All Rho-GEFs

contain a Dbl-homology (DH) domain containing the catalytic activity and an

adjacent pleckstrin homology (PH) domain that mediates membrane localization

through lipid binding. More than 30 Rho-GEFs, including Sos, Trio and Vav, and

about 20 Rho-GAPs have been identified.

Rho-GTPases are known to get activated by ligands of G-protein-coupled receptors

(GPCRs), like lysophosphatidic acid (LPA), sphingosine-1-phosphate, bombesin,

thrombin and endothelin. These ligands induce dissociation of the receptor-

associated heterotrimeric G-protein into its Ga and Gbg subunits, that both can

43

initiate downstream signalling. Growth factor receptors and integrins however, are

able to activate certain Rho-GTPases by pathways involving tyrosine kinases and/or

PI3-K as well. How the actin cytoskeleton dynamics are regulated is important for

cell migration and cell mediated regulation of tissue homeostasis (Rho-GTPase

reviews [343-346]).

To date around 30 different potential effectors for Rho, Rac and Cdc42 have been

identified and they all interact specifically with the GTP-bound form of the GTPases.

The most common mechanism for Rho-GTPase-mediated activation of their effectors

seems to involve a disruption of intramolecular inhibitory interactions and

subsequent exposure of functional domains within the molecule. As an example,

Rac- and Cdc42-mediated activation of p21-activated kinases 1-3 (PAK1-3) involves

the displacement of a regulatory domain of the kinase and concomitant exposure of

the Ser/Thr kinase domain [347]. Some of the best characterized GTPase effectors

involved in actin reorganization are summarized in Figure 6.

Rho-associated kinase (ROK) and Dia are two effectors that appear to be required for

Rho-induced stress fibre formation and focal adhesion assembly. ROKs are Ser/Thr

kinases that get activated by binding Rho-GTP [348]. Over-expression of

constitutively active ROK induces stellate actin-myosin (actomyosin) filaments in

Swiss3T3 and HeLa cells, whereas a ROK inhibitor caused loss of serum- and Rho-

induced stress fibres, indicating that ROK is necessary but not sufficient for Rho-

induced stress fibre formation [349-351]. However, in combination with activated

Dia, a member of the formin-homology (FH) proteins that binds to the G-actin

binding protein profilin, ROK is able to induce stress fibre formation [352]. ROK

binds to and phosphorylates both MLC itself and the myosin-binding domain of

MLC phosphatase. Both actions result in direct or indirect activation of MLC and

subsequent assembly of contractile actin-myosin filaments [353, 354]. ROK also

activates LIM kinase (LIMK) that phosphorylates and inactivates cofilin, that in turn

leads to a stabilization of the actin filaments [355].

Few unique Rac effectors are implicated in actin reorganization. Truncation

constructs of POR-1 (partner of Rac-1) act as dominant negative constructs in Rac-

induced lamellipodia formation, and a specific Rac-1 associated protein (p140Sra-1)

co-sediments with F-actin, but little is known about these proteins cellular functions

[356, 357]. A better-known association is the GTP-independent direct interaction of

44

Rac with phosphatidylinositol-4-phosphate 5-kinase (PI-4-P5K) [358]. Rac activationof PI-4-P5K provides the PIP2 needed for the uncapping of actin filaments requiredfor thrombin-induced actin filament assembly in platelets [359]. PIP2 is suggested tobe involved in actin-reorganizing events mediated by Rho as well. A WASP-likeprotein called WAVE (also known as Scar) can be precipitated with Rac but whetherthis is a direct interaction is not known. Both the adaptor protein Nck and the insulinreceptor substrate IRSp53 have been suggested as the missing link [360, 361]. WAVE,like WASP, interact with and activate Arp2/3, and Rac in complex with Nckactivates WAVE and initiates actin nucleation [360]. Over-expressed wildtypeWAVE causes clustering of actin bundles, whereas mutations in the actin-bindingdomain inhibits Rac- and PDGF-induced membrane ruffling but has no effect onCdc42-induced micro spikes [362].

Cdc42 binds and activates WASP and the more ubiquitously expressed N-WASP,and over-expression of N-WASP together with Cdc42 induces long cellularmicrospikes, suggesting the involvement of N-WASP in Cdc42-mediated filopodiaformation [363, 364]. Upon Cdc42-mediated activation N-WASP/WASP interactsdirectly with both actin monomers (G-actin), profilin and the Arp2/3 complex, andthus acts as a scaffold for the machinery needed for new actin polymerization [363,365, 366]. Cdc42 also interacts with two Ser/Thr kinases thought to be involved inactin reorganization and filopodia formation, MRCK-a and -b. MRCKs are Cdc42-

Rho-GTP Rac-GTP Cdc42-GTP

ROKDia

MLC- PMLCLIMK- P

Cofilin- P

G-actin

Actomyosinassembly

and contraction

Actin filamentstabilization

Increased actinpolymerization

WAVEprofilin

G-actinG-actin Arp

2/3

PI-4-P5K

PIP2

Release ofcappingproteins

PAKN-WASP

LIMK- P

Cofilin- P

Actin filamentstabilization

Decreasedactomyosincontraction

?

Actin nucleation andpolymerization

Actin nucleation andpolymerization

Stress-fibre formation

MLCK- P

Lamellipodia and membrane ruffles Filopodia microspikes

Figure 6 Schematic picture of actin reorganization mediated by Rho-GTPases

profilin profilin

G-actinG-actin Arp

2/3

45

specific effector proteins with a ROK-like kinase domain which can phosphorylate

MLC [367]. Unlike PAK1-3 a new member of the PAK family, PAK4, binds only

Cdc42 and is not activated by GTPase binding. PAK4 and Cdc42 synergize in the

formation of large filopodia-like structures [368].

Both Rac and Cdc42 utilize the traditional PAKs 1-3 in lamellipodia and filopodia

formation, respectively. It is hard to elucidate whether all three PAKs are targets of

both Rac and Cdc42. Conflicting reports about the involvement of PAK1 in actin

reorganization exist. On one hand activated mutants of PAK1 induced similar effects

as constantly active Rac and Cdc42, in that they induce filopodia and membrane

ruffling in Swiss3T3 [369]. On the other hand over-expression of PAK1 failed to

induce any changes in the actin cytoskeleton [370]. Established PAK-mediated

pathways for affecting the actin cytoskeleton involve LIMK and MLC. PAK has been

reported as a negative regulator of actin-myosin assembly by the phosphorylation

and inactivation of MLC kinase, leading to decreased MLC phosphorylation and a

concomitant reduction in actin-myosin assembly [371]. In addition, PAK1 binds and

activates LIMK, and induce actin filament stabilization via the recruitment and

phosphorylation of cofilin [372].

COLLAGENS

The extracellular matrix (ECM) is traditionally defined as the space between cells in

connective tissues and consists of proteins, proteoglycans and glycosaminoglycans.

Among the proteins, collagens are the most abundant. Collagen fibrils together with

proteoglycans are the only truly ubiquitous components of the ECM and are present

in all connective tissues. In its most primitive form the ECM can be described as an

isotropic meshwork of entangled collagen fibres of variable length, keeping together

a highly hydrated proteoglycan matrix. This basic structure can still be found but

collagen fibrils with distinct properties besides the tensile strength have evolved.

Collagens are homo- or heterotrimeric proteins built up from type-specific a-chains.

Common for all collagens are stretches of triple-helical domains where the a-chains

are wound around each other in a coiled-coil conformation. These tight structures

are known as collagenous domains. The triple-helix conformation is made possible

by that every third amino acid in the a-chains is a glycine, which due to its small size

46

allows narrow turns. The most common amino acids in collagens, besides glycine,

are proline and lysine that can get hydroxylated and form intra- as well as interchain

hydrogen bonds, keeping the tight structure together.

The collagen family is a large heterogeneous family of proteins that besides the

collagenous domain contain a variety of other domains, which often mediate protein

binding. Non-collagenous protein-binding domains include the fibronectin type III

repeat and the A-domain of von Willebrand factor. Collagens are in fact, known to

form supramolecular structures, both with other collagens and other ECM

components. To date more than 20 different collagens have been described of which

the most well known are summarized in Table 1. Depending on their capability to

form organized fibres collagens can be divided into two major classes, the fibril

forming collagens and the non-fibrillar collagens (reviewed in [1, 373, 374]).

The fibril forming collagens form a rather homogenous group, consisting of

collagens type I, II, III, V and XI, structurally characterized by that almost the entire

molecule consists of one single collagenous domain. By intermolecular interactions

these collagens form fibrils, fibres and finally fibre bundles that are the main

providers of mechanical support and resistance in the ECM (reviewed in [375]).

Fibrillar collagens are synthesized as precursors called procollagens. Procollagens

have N- and C-terminal pro-petides that are cleaved off after secretion into the

extracellular space, resulting in helical rod-like molecules ending with short non-

collagenous telopeptides. Collagen monomers assemble into fibrils by a longitudinal

head-to-tail association followed by a quarter-staggered lateral aggregation. There

are two major tissue-specific collagen fibrils: the collagen type II-containing fibrils,

typical of cartilage, and the collagen type I-containing fibrils present in most other

interstitial connective tissues. The collagen type I fibrils have a core of collagen type

V surrounded by polymerized collagen type I and III, whereas collagen type II fibrils

instead seem to have a core of collagen type XI surrounded by collagen type II

(reviewed in [376, 377]). The abundant collagens type I-III provide the fibril with its

mechanical strength while the core collagens contribute to the fibril morphology and

thereby tissue-specificity. In addition to their scaffolding functions, these collagens

serve as attachment sites for other constituents of the ECM and cell surface receptors

like the integrins.

47

The non-fibrillar subgroup is more heterogeneous and houses collagens consisting of

a variable number of triple-helices interrupted by non-collagenous motifs. This

subgroup can be further subclassed with regard to their structure and are divided

into; 1) network-forming collagens (types IV, VIII and X) where collagens type IV

together with laminin networks constitute the main supportive components of the

basement membranes, type VIII is a component of the tissue surrounding vessels

and type X is present in hypertrophic cartilage, 2) micro-fibrillar collagen (type VI) that

bind glycosaminoglycans (GAGs) and thereby can interact with other ECM

polymers, 3) fibril-associated collagen with interrupted triple helix (FACIT) collagens (type

IX, XII, XIV, XVI and XIX-XXI) that bind fibrillar collagens either directly or via

small leucine-rich proteoglycans and actually contain a GAG-chain of their own, and

hence can be described as proteoglycans, 4) multiple triple-helix domain and

interruptions (multiplexin) collagens (type XV and XVIII) that also are localized to the

basement membranes, 5) transmembrane collagens (type XIII, XVII and XXIII) that are

a type II transmembrane protein and finally 6) bundle-forming collagen type VII that is

vital for anchoring basement membranes underlying stratified epithelia (reviewed in

[378-380]).

Collagen Type I

Collagen type I belong to the fibrillar collagens and is the most abundant protein in

the vertebrate body. In concert with other collagens and proteoglycans, e.g.

fibromodulin and decorin, collagen type I give a variety of tissues their optimal

functional structure. Apart from providing tissues tensile strength and integrity,

collagen type I also induces specific signals upon cell attachment, vital for several

cellular functions including migration and proliferation [381].

Although it is a major component of most connective tissues collagen type I is

synthesized only by a subset of cell types, e.g. by fibroblasts in the loose connective

tissue and by osteoblasts and odontoblasts in bone and dentin matrices, respectively.

However, judging from several reports it seems likely that under certain conditions

any mesenchymal cell has the potential to change into a collagen producing

phenotype. For instance, smooth muscle cells change into a proliferative and

synthetic mode in vivo in atherosclerosis [382] and cultured chondrocytes start

producing collagen type I when allowed to spread on plastic dishes [383].

48

SUBFAMILY TYPE CHAINS ISOFORMS

Fibril-forming collagens I a1(I), a2(I) [a1(I)]2a2(I)

II a1(II) [a1(II)]3

III a1(III) [a1(III)]3

V a1(V), a2(V), a3(V) [a1(V)]2a2(V)[a1(V)a2(V)a3(V)]

XI a1(XI), a2(XI), a3(XI) [a1(XI)a2(XI)a3(XI)]and a(V)/a(XI) chimeras

Network-forming collagens IV a1(IV), a2(IV), a3(IV) [a1(IV)]2a2(IV)a4(IV), a5(IV), a6(IV) [a3(IV)a4(IV)a5(IV)]

[a1/a2(IV),a5/a6(IV)]?

VIII a1(VIII), a2(VIII) [a1(VIII)]2a2(VIII)

X a1(X) [a1(X)]3

Micro-fibrillar collagen VI a1(VI), a2(VI), a3(VI) [a1(VI)a2(VI)a3(VI)]

FACIT collagens IX a1(IX), a2(IX), a3(IX) [a1(IX)a2(IX)a3(IX)]

XII a1(XII) [a1(XII)]3

XIV a1(XIV) [a1(XIV)]3

XVI a1(XVI) [a1(XVI)]3

Multiplexin collagens XV a1(XV) [a1(XV)]3 ?

XVIII a1(XVIII) [a1(XVIII)]3 ?

Transmembrane collagens XIII a1(XIII) [a1(XIII)]3

XVII a1(XVII) [a1(XVII)]3

Bundle-forming collagens VII a1(VII) [a1(VII)]3

Table 1 The collagen family. Collagenous domains are shown as thick grey lines.

Collagen type I consists of two a1(I) chains and one a2(I) chain, encoded by two

different genes. Generation of transgenic mice harbouring segments of the collagen

type I promotors has shown that different elements within the promotors are

responsible for collagen expression in different cell types (reviewed in [384]). For

example, one element located within 800 bp from the proximal a1(I) gene promotor

induced expression exclusively in skin fibroblasts while another element, ~1.7 kb

upstream, induced expression in osteoblasts and odontoblasts [385].

49

Several in vitro experiments have shown that collagen type I expression is induced

by a variety of soluble factors including cytokines such as interleukin-1 (IL-1) and -4

(IL-4), growth factors like TGF-b and PDGF-BB and vasoactive peptides like

endothelin-1 (reviewed in [386]). TGF-b is one of the most potent stimulators of ECM

production in cultured cells and its ability to increase collagen synthesis and inhibit

collagen degradation is well established. TGF-b stimulates both a1(I) and a2(I) and a

"TGF-b responsive element" has been identified in the proximal region of the a2(I)

promotor [387]. PDGF-BB, on the other hand, could replace cell spreading as a

prerequisite for collagen synthesis in human dermal fibroblasts cultured on

substrates that induced distinct morphologies [160]. Other soluble factors such as

tumour necrosis factor-a (TNF-a), interferon-g (IF-g), IL-10 and prostaglandin E2

(PGE2) inhibit collagen type I production (reviewed in [386]).

The disruption of collagen type I expression leads to embryonic lethality associated

with ruptured blood vessels and mesenchymal cell death [388]. Other mutations of

the collagen type I genes lead to a variety of disorders, mainly affecting the structure

of bone, skin and tendons. Two of the most studied disorders are osteogenesis

imperfecta and Ehler-Danlos syndrome (reviewed in [389]). Osteogenesis imperfecta

is a heterogeneous group of hereditary diseases characterized by brittle bones where

90 % of the cases origin in a1(I) and a2(I) mutations leading to structurally altered a-

chains. Ehler-Danlos syndrome on the other hand is characterized by joint

hypermobility and fragile skin. The type III Ehler-Danlos syndrome results in

accumulation of partially processed collagen and is caused by mutations of the a(I)

genes affecting the processing of procollagen into collagen.

While collagen type I is essential for maintaining the mechanical strength of almost

any tissue, and de novo formation is vital during wound healing, over-expression of

collagen type I is a common theme among a variety of fibrotic disorders. These

disorders however, origin in defective regulation of collagen synthesis rather than

defects in the collagen per se.

50

WOUND HEALING AND INFLAMMATION

Wound healing is a complex process that requires the cooperative actions of several

cell types in different tissues, and it can to some extent serve as a model for

inflammatory reactions. The wound healing process is pursued in different stages,

overlapping in time, as well as in function and can roughly be described by clotting,

inflammation, tissue deposition and finally tissue remodelling or contraction

(reviewed in [390-392]).

Upon tissue injury blood leaks out from the damaged vessels and forms a clot

consisting of a fibrin fibre mesh enclosing activated platelets. The mesh acts as a

provisional matrix for recruited cells to adhere to and migrate through during the

wound repair. Activated platelets within the clot degranulate and release

chemotactic factors, e.g. cytokines and growth factors including PDGF (see Biological

functions of PDGF) and TGF-b. Fibroblasts and keratinocytes from the surrounding

tissues and circulating inflammatory cells thereby get attracted to the wound site

(reviewed in [142, 393-395]). Neutrophils arrive within minutes after injury and

besides clearing the wound from foreign matter they release pro-inflammatory

factors like IL-1 and TNF-a [396]. Recruited monocytes get activated into

macrophages that accumulate in the wound where they are essential not only by

cleansing of the wound but also by amplifying inflammatory signals from platelets

and neutrophils (reviewed in [142, 393-395]).

After a few days fibroblasts, along with blood vessels, appear in the wound clot and

start to replace the damaged tissue by depositing a new collagen rich matrix. Finally,

the newly formed granulation tissue is organized and contracted into a more dense

tensile structure by myofibroblasts (reviewed in [391, 392]). Myofibroblasts,

although they express a-smooth muscle actin, are not smooth muscle cells but

believed to be derived from activated fibroblasts [397, 398]. However, other reports

have suggested pericytes as the precursors of not only myofibroblast but also of

wound fibroblasts [399]. If the wound has cut through the skin reepitheliazation of

the epidermis by activated keratinocytes occurs before de novo formation and

contraction of granulation tissue (reviewed in [390]). When matrix remodelling is

complete cell density in the new tissue is decreased, mainly by apoptosis of

myofibroblasts, creating a poorly populated scar [400, 401].

51

Contraction of a fibroblast-populated collagen gel is used as an in vitro model of cell-

mediated tissue remodelling, including wound contraction and maintenance of

tissue homeostasis ([402, 403], reviewed in [392, 404]).

Cell-mediated contraction of collagen matrices

When cultured within, or on top of, a three-dimensional collagen gel fibroblasts will

reorganize the collagen fibrils and contract the gel in a process referred to as

collagen gel contraction [405]. Cell-mediated collagen gel contraction is considered

an in vitro opportunity to study in vivo events that influence loose connective tissue

homeostasis.

Two major experimental setups are used when studying collagen gel contraction,

namely contraction of a relaxed, free-floating gel or a stressed, attached gel. When

cells are cultured in gels attached to a rigid surface they generate a mechanical load

and develop an isometric tension, resulting in flattened cell morphology and

prominent actin stress fibres ([406], reviewed in [404]). This morphology is

accompanied by a synthetic mode where cells start to produce new matrix, like

collagen and fibronectin [407, 408]. In contrast, cells grown in relaxed matrices

become quiescent and take on a dendritic morphology ([406, 407, 409-411], reviewed

in [404]).

Fibroblasts grown in collagen gels under tension respond differently to growth

factor stimulation than fibroblasts cultured in relaxed gels. Transforming growth

factor-b (TGF-b) is able to up-regulate the putative contractile marker a-smooth

muscle actin (a-SMA) and the integrin subunits a2 and b1 in fibroblasts cultured in

anchored, but not free-floating collagen gels [412-415]. Furthermore, TGF-b has been

shown to act stimulatory on cell-mediated contraction of relaxed floating gels both

directly as agonist and indirectly by pre-activation of the fibroblasts, while it only

acts indirectly on cells cultured in stressed gels [412]. Another example is that while

PDGF and lysophosphatidic acid (LPA) stimulates contraction of a free-floating gel

in an additive manner and with similar kinetics, LPA exerts an immediate effect on

stressed gels whereas the effect of PDGF is delayed [414]. In the same study it was

shown that pertussis toxin inhibits the LPA-induced signalling in relaxed but not

52

stressed collagen gels, indicating that depending on mechanical load receptors ofLPA couple to different G-proteins, or alternatively different receptors get activated.

Contraction of a free-floating gel is believed to be a consequence of cell-mediatedreorganizing of collagen fibres into bundles by traction forces [416] similar to thosegenerated during cell migration. Since the matrix is non-attached it offers littleresistance and the collagen fibrils are organized at random. This results in a locallydenser collagen network in close vicinity of the cells. In fact, it has been shown thatthe concentration, or density, of collagen fibres surrounding the cells in a contracted

Actomyosin drives MT retrograde flowcreating a gradient in MT dynamics

MT shortening in the cell activates RhoAMT growth at the edge activates Rac1

Growing MTShortening and pausing MT

Actin retrograde flowHigh Rac1 activity

Contractile actin bundlesHigh RhoA activity

Figure 7 Feedback interactions between the microtubule and actin cytoskeletondynamics during cell migration. Black lines represent microtubules (MT),white arrows show net MT shortening and black arrows net MT growth.Grey lines and areas represent actin structures and grey arrows actinretrograde flow. The dashed line defines the leading lamella.

Direction ofcell migration

Direction ofcell migration

53

floating gel resembles that found in the dermis. In contrast remodelling of an

attached gel results in collagen fibrils organized parallel with the restraining surface

[406].

Contraction, as well as migration, is dependent on an intact actin cytoskeleton and

an active turnover of the microtubule cytoskeleton [417-420]. There is a positive

feedback between actin and microtubule dynamics during cell migration that might

be important during collagen gel contraction as well (Figure 7, reviewed in [420]). In

this model actin-myosin (actomyosin) drives the microtubule retrograde flow.

Growing microtubule at the leading edge of the cell activates Rac1 that promotes

actin assembly in the lamellipodia, whereas microtubule shortening centrally in the

cell activates RhoA that induce stress fibre formation. The fact that collagen gel

contraction reportedly is dependent on myosin light chain kinase (MLCK) supports

this idea [421]. Two recent studies showed that while calcium-induced MLCK

activation was sufficient for smooth muscle cell-mediated contraction, fibroblasts

depended on Rho-mediated inhibition of MLC phosphatase as well [422].

Contraction is mediated by collagen-binding b1-integrins, mainly a2b1 and

a11b1[59, 423, 424]. However, a role for the promiscuous vitronectin integrin

receptor avb3 in collagen gel contraction has been suggested, despite the fact that

avb3 is unable to mediate cell attachment to native collagen [112-116]. With this in

mind a recent study showing b1-dependent and -independent cell migration

through collagen gels becomes even more interesting [425]. In this study spindle-

shaped invasive tumour cells migrated through a collagen gel using b1-integrins and

left behind a tube-like proteolytic degradation track. Inhibition of MMPs and other

proteases, however, induced an amoeba-like morphology of the cells that by

squeezing through pre-existing matrix gaps continued to migrate. The amoeboid

morphology was accompanied with loss of b1-integrins at adhesion sites and a

diffuse cortical distribution of the actin cytoskeleton. Unfortunately the authors only

looked for b1-integrins and not for avb3.

Several inflammatory factors influence fibroblast-mediated collagen gel contraction.

PDGF-BB and TGF-b are known to stimulate the process [412, 426-429] whereas

PGE1 and IL-1 have an inhibitory effect [430-432]. These effects correspond with

their in vivo functions during processes like wound closure and increased fluid

54

influx in tissues during inflammation. The mechanisms behind growth factor-

mediated stimulation of collagen gel contraction are poorly understood, and could

origin in both their effects on actin dynamics and their chemotactic potentials. It has

been shown that PDGF-BB stimulates contraction via an PI3-K dependent elevation

of intracellular calcium [169, 337]. Interestingly, stimulation with PDGF-BB also

leads to increased mobility of the PDGF b-receptor in the membrane mediated by an

increase in intracellular calcium [433].

Collagen gel contraction activates both the ERK and the p38 MAPK pathways [85,

434, 435] but the impact of this and how the signals are mediated remains unclear to

a large extent. Fibroblast-mediated contraction of a free-floating gel decreases ERK

signalling compared to contraction of a restrained gel [409]. If the decrease in ERK

activity in relaxed compared to stressed gels is a result of general cellular activity or

the difference between traction forces and isometric tension remains to be evaluated,

along with effects of integrin expression pattern.

In addition to serving as a model of wound closure, collagen gel contraction has also

been implicated as an vitro assay for the regulation of the interstitial fluid pressure

(IFP) in several physiological and pathological processes, including acute

inflammation and concomitant oedema formation [430, 436]. Collagen gel

contraction has also been used in cancer research since solid tumours often display

an interstitial hypertension obstructing the uptake of anticancer drugs [169].

Lowering of the IFP increases the transport of low molecular weight compounds

into a solid tumour [437, 438] and hence, this model could serve as an important in

vitro set up for anticancer drug treatment.

55

AIMS AND RESULTS OF THE PRESENT INVESTIGATION

Integrins are heterodimeric cell surface receptors involved in most anchorage-

dependent cellular processes. Of special interest for this investigation are the

migratory and contractile events involved in cellular functions like tumour invasion

and metastasis, inflammation and tissue homaestasis. These processes are

dependent on integrin ligation but are often induced by soluble factors, like growth

factors. This study has focused on the functions of collagen-binding integrins and

how they are affected by platelet-derived growth factor (PDGF). More specific aims

are stated in each study.

PAPER I

a11b1 Integrin is a Receptor for Interstitial Collagens Involved in Cell

Migration and Collagen Reorganization on Mesenchymal Nonmuscle Cells

The collagen binding integrin a11b1 is the most recent addition to the integrin

family and hardly anything was known about its functions and distribution. Here

we investigated the presence of both a11 mRNA, using in situ hybridization and a11

protein, using immunohistochemistry during human embryonic development. We

also looked into in vitro cellular processes mediated by a11b1 in response to

collagen.

To establish the distribution of a11 during embryonic development we used

morphologically normal human embryos, 4-8 postovulatory weeks old, from

induced abortions. After determining the developmental stage the embryos were

collected and frozen in dry ice within the first 24 h post-mortem. Cryostat sections of

7 microns were prepared and mounted on pretreated slides. In the case of in situ

hybridization the sections were fixed with paraformaldehyde, rinsed and then

dehydrated in ethanol. To detect a11 mRNA an antisense riboprobe to the 3'-end of

the human integrin a11 cDNA was used [58], and for detection of the expressed

protein a polyclonal rabbit antibody towards the cytoplasmic tail of human a11 [58]

was used. We show in this work that a11 protein and mRNA are present in

mesenchymal cells in intervertebral discs and around the cartilage of the developing

56

skeleton. Furthermore, a11 is expressed on keratinocytes in the cornea. These

expression-sites all comprise a highly organized collagen network that suits well

with the idea of a11b1 as a receptor for interstitial collagens in vivo. Neither a11

mRNA nor protein could be detected in any type of myogenic cells. Distribution of

the related collagen binding integrins a 1b1 and a2b1 has been thoroughly

investigated and both a 1 and a2 are widely expressed during embryonic

development [64, 439]. Postnatally, besides from being expressed on fibroblasts

throughout the body, a1 is expressed on pericytes, smooth muscle cells and the

endothelium of microcapillaries [440] and a2 is found on platelets, haematopoietic

cells and most epithelia [64, 441]. The differences in expression patterns, and

functional studies of collagen binding integrins implicate unique biological roles

(reviewed in [53]) but the lack of major defects in knockout mice [72, 74, 442]

suggests that collagen binding integrins can overlap in function.

To establish whether a11b1 is a collagen receptor in vitro we stably transfected the

murine satellite cell line C2C12, lacking endogenous collagen receptors, with full-

length human a11 cDNA. Expressed in these cells a11b1 mediates adhesion to

collagens type I and IV in a cation-dependent way typical of integrin receptors.

Adhesion was more efficient on collagen type I than on collagen type IV. We also

show that in normal cultured fibroblasts (AG 1518) seeded on collagen type I and

allowed to attach and spread for 1 h, a11b1 is present in focal adhesions.

Furthermore, a11b1 mediates collagen gel contraction in serum free medium in a

way similar to a2b1. Both PDGF-BB and FBS stimulate a11b1-mediated contraction.

Finally, a11b1 supports cell migration through a collagen substrate and responds

chemotactically to both PDGF-BB and FBS. Noticeable is that PDGF-BB has a bigger

impact on both a11b1-mediated collagen gel contraction and cell migration than

does FBS, while the opposite is true for a2b1 mediated processes.

The biological importance of a11b1 remains to be elucidated but the results in this

work strongly supports the idea of a11b1 as a functional receptor of interstitial

collagen.

57

PAPER II

Integrin avb3 mediates PDGF BB-stimulated collagen gel contraction in cells expressing

signalling deficient integrin a2b1

The two tyrosines (Y783 and Y795) in the cytoplasmic tail of the b1-subunit are

important for cell migration [37] and adhesion-induced activation of FAK, but not

Cas in the GD25 cell system [34]. Furthermore it has been shown that the expression

of full-length b1A in these cells down-regulates the integrin b3-subunit while other

b1 splice variants, or intracellularly truncated b1A, do not [443]. GD25 cells do not

express collagen receptors even after re-expression of the integrin b1-subunit and

have been shown to utilize the integrin avb3 in collagen gel contraction [113]. Here

we investigated the impact of the b1 cytoplasmic tyrosines in collagen-induced

signalling by expressing the integrin a2-subunit in GD25-cells expressing wildtype

or mutated b1A.

.GD25 cells, expressing wild-type b1A or b1A with the YY783,795FF double mutation

(b1Amut), were transfected with full-length human integrin a2 cDNA. This resulted

in cells expressing only one collagen receptor, a2b1A and a2b1Amut respectively,

enabling us to study in vitro cellular processes, as well as intracellular signalling, in

response to collagen. We found that cells expressing a2b1Amut adhered to collagen

to a similar degree as cells expressing a2b1. Furthermore, the mutation did not affect

cell migration through a collagen substrate or the chemotactic response to PDGF-BB.

The ability to contract a collagen gel was however, significantly reduced in cells

expressing a2b1Amut compared to cells expressing a2b1A. PDGF-BB significantly

stimulated contraction mediated by a2b1Amut and was able to rescue contraction to

some extent. However, the stimulatory effect mediated by PDGF-BB on a2b1Amut-

mediated contraction, as well as the chemotactic response by these cells, were

exerted by the integrin avb3.

When cells were seeded on 2D collagen substrates cells expressing a2b1A, but not

a2b1Amut, supported activation of FAK, Cas and Src. However, when cells were

cultured in 3D collagen gels the levels of activation were the same between the cells.

If the cells were seeded in gels in the presence of blocking antibodies towards the b3-

subunit cells expressing a2b1Amut failed to induce phosphorylation of Cas.

58

Interestingly, expression of b1Amut did not down-regulate b3 expression but b1A

did. b1A has been shown to down-regulate b3-expression by affecting its mRNA-

stability [443] and our data pinpoint this effect to the two tyrosines in the

cytoplasmic tail of b1A.

These data suggest that a fully functional integrin a2b1 shuts off the ability of cells

to use avb3 as a mediator of collagen-induced signals. Furthermore they implicate

avb3 as a main mediator of the crosstalk between integrins and the PDGF receptors.

Finally, this work in combination with other reports [34, 37, 444] put forward unique

ligand-dependent signalling and functions of b1-integrins, and also indicate

integrin-crosstalk in collagen-induced signalling and integrin expression.

PAPER III

Clustering of b1-integrins induces an ERK1/2-dependent avb3-mediated collagen gel

contraction

Cell-mediated collagen gel contraction is an in vitro model of wound contraction and

tissue maintenance. Collagen-binding integrins are the main mediators of collagen

contraction but work by us (see Paper II) and others have shown that the multi-

specific integrin avb3 can take part in this process [59, 112-114, 116, 423, 424].

Culturing of fibroblasts in a collagen matrix induces MAPK signalling [435] and

growth factors like PDGF are known to stimulate fibroblast-mediated contraction

[426, 428]. In this work we aimed to establish the impact of and interplay between

different integrins and PDGF-BB in collagen gel contraction.

The murine satellite cell C2C12, lacking endogenous collagen receptors, wildtype or

transfected with either full-length human integrin a2- or a11-cDNA were used.

Wildtype C2C12 mediated collagen gel contraction via the RGD-specific integrin

avb3. This contraction was delayed compared to contraction mediated by a2b1 or

a11b1, and significantly stimulated by PDGF-BB. Whereas anti-b3 integrin IgG

completely blocked C2C12-mediated contraction anti-b1 integrin IgG, to our

surprise, significantly stimulated the process. Clustering of b1-integrins and PDGF-

BB additatively stimulated avb3-mediated contraction. We were able to block the

avb3-mediated contraction by inhibiting the ERK1/2 pathway. Also the stimulatory

59

effect seen after clustering of b1-integrins was mediated via the ERK1/2 pathway.

Contraction mediated by C2C12 cells expressing a2b1 was completely blocked by

antibodies against the b1-subunit, which also blocked the stimulatory effect of

PDGF-BB. a2b1-mediated contraction was unaffected by antibodies against the

integrin b3-subunit. a11b1-mediated contraction was not inhibited by neither b1- nor

b3-integrin specific antibodies, but completely blocked by the combination of anti-b1

and -b3 integrin IgG. The stimulatory effect exerted by PDGF-BB on a11b1-mediated

contraction was prevented in the presence of anti-b3 IgG. Neither a2b1- nor a11b1-

mediated contraction was ERK-dependent. Inhibition of the p38 MAPK pathway did

not affect contraction mediated by any of the integrins studied. By blocking

signalling through the PDGF receptors we could reduce but not block avb3-

mediated contraction. Collagen gel contraction mediated via non-collagen receptors,

e.g. avb3 has been proposed to occur via induction of collagenases and concomitant

exposure of cryptic RGD-sites. In our system however, inhibition of collagenases

had no effect on contraction mediated by either a2b1, a11b1 or avb3.

Taken together these data implicate that while crosstalk exist between avb3 and

a11b1 during remodelling of a collagen matrix, a2b1 shuts off the use of avb3 in

collagen gel contraction. Furthermore, our results indicate that a different subset of

signalling events is elicited dependent on whether integrins are engaged in ligand

binding or merely physically clustered.

60

DISCUSSION AND FUTURE PERSPECTIVES

It has been thrilling to take part in the characterization of a new integrin. a11b1 has

turned out to be a collagen-receptor with specialized functions and rather restricted

expression in vivo. An a11-null mouse exist and is currently under investigation, but

the work has only begun and the possibilities are uncountable. Future functional

studies of interest are dependent on the phenotypes of these animals.

During the course of my studies words like crosstalk, interplay and network have

become high fashion in the biochemical field of science. This investigation is no

exception, and I think we have only seen the tip of the iceberg. A lot of thoughts and

questions pop into mind.

What are the implications of integrin crosstalk during dynamic interactions with the

ECM? Work from our group and co-workers have shown that by blocking a2b1

ligand binding the interstitial fluid pressure can be lowered. By the addition of

PDGF-BB the pressure is raised again. What about avb3? Does a11b1 have any role

in the regulation of tissue homeostasis? What happens during inflammation and

oedema formation? Which inflammatory signals make integrins let go of their ligand

and stay unoccupied? And what happens intracellularly? Is it substrate-dependent

or solely receptor-dependent which signalling pathways that get activated?

Clustering versus binding of ligand? In Paper III we show that clustering of b1-

integrins induce avb3-mediated collagen interactions, while ligand-activated a2b1

blocks these interactions. a11b1, on the other hand, functions in parallel with avb3.

How can one integrin exclude another from binding a ligand?

Like for all other PhD-students there have been failed projects. Some I want to forget

but some I still believe in but was unable to prove. When this work was initiated

only two collagen-binding integrins, a1b1 and a2b1, were identified. However,

function-blocking antibodies raised against the a subunits failed to completely block

b1-integrin mediated functions like collagen gel contraction and cell migration, one

by one or in concert. It was also known that a1b1 and a2b1 mediate different signals

in response to the same ligand, e.g. collagen type I, and that a1 but not a2 associates

with caveolin which in turn recruits Fyn and Shc leading to the activation of ERK.

61

These data led us to firstly, search for a new I-domain containing integrin a-subunits

using: 1) RT-PCR with primers specific for homologous regions within the a1 and a2

I-domain and the well conserved proxamembrane sequence GFFKR; and 2)

screening of human fibroblast cDNA phage libraries using polyclonal antibodies

raised against recombinant a1 and a2 I-domains, and secondly to look for proteins

associating with the a2 cytoplasmic tail using yeast the two hybrid system. While

performing these experiments a10 and a11 were characterized, neither containing

the conserved GFFKR-motif, and the HUGO project was completed and did not

reveal any additional I-domain containing integrins. Hence the search for an

additional collagen-binding integrin was abrogated but the fishing for binding

partners to the cytoplasmic tail of the integrin a2 subunit continued. We used a

GAL4-based yeast two-hybrid system where we created the bait by fusing the

cytoplasmic tail, starting directly after the transmembrane sequence, in frame with

the DNA-binding motif of the GAL4 promotor. The bait was screened against a

cDNA library derived from human normal lung fibroblasts WI38.

One specific clone passed all negative controls possible to perform and still turned

out to be of interest, namely the 8 kD cytoplasmic dynein light chain 1 (cdlc-1 or

LC8). Dyneins, together with kinesins are ATP-driven massive molecular motors

associated with the microtubule cytoskeleton and are responsible for the transport

and positioning of membrane associated organelles and protein complexes. Dyneins

transport the cargo towards the minus end, e.g. the centromeres and kinesins

towards the plus end. Furthermore dyneins interact with the myosins that are the

actin motor proteins. Dyneins consist of two heavy chains responsible for binding to

the microtubule and the motor activity, and several intermediate and intermediate

light chains involved in cargo specificity, and finally light chains that are suggested

to be involved in the tight control of motor and cargo-binding activity (reviewed in

[445, 446]). Cdlc-1 is a highly conserved 8 kD protein, 94 % homology between man

and drosophila, common for many multimeric complexes. The cdlc-1 knock out in

drosophila is lethal and partial loss of function leads to female sterility due to

disrupted cellular shape and organisation of the actin cytoskeleton [447]. Apart from

belonging to the dynein macromolecule cdlc-1 is also associated with myosin-V

[448], which could mean, if our finding is true, that a2 interacts with the actin

cytoskeleton instead, or as well.

62

Connecting the integrins to the microtubule, and not only the actin cytoskeleton is a

very exciting thought. Indirectly a lot of data supports this interaction. Microtubules

(MT) are connected to the focal adhesions and involved in their regulation, e.g. de-

polymerization of MT leads to stress fibre formation and focal adhesion assembly,

with concomitant phosphorylation of FAK and paxillin [449]. Furthermore, MT play

an active role in the assembly and/or delivery of signalling complexes [450]. MT are

also required for polarized cell movement during for example wound healing. De-

polymerization of the MT in the minus end activates RhoA and induces formation of

contractile actin bundles, and new MT formation in the leading lamella induces Rac1

and hence, lamellapodia formation (reviewed in [420, 451]). To summarize, MT are

essential for modifications of the actin cytoskeleton both regarding Rac- and Cdc42-

mediated formation and rearrangement of lamellapodias and filapodias, and

regarding Rho-mediated stress fibre formation. This crosstalk has been suggested to

take place at sites of focal adhesions (reviewed in [452, 453]). In a recent study

fibroblasts were shown to take on a neuronal-like dendritic morphology in relaxed

collagen gels, where the dendritic cell extensions contained a tubulin core with actin

localized cortically and concentrated in the tips in a manner resembling a growth

cone [411]. Another exciting work shows that the TGF-b receptor interacts with

another cytoplasmic dynein light chain, LC7 and that over-expression of the LC7

induces TGF-b specific responses including JNK MAPK activation [454]. This

becomes even more interesting by the fact that cdlc-1 has been shown to bind the

NF-KB inhibitor IK-B a [455]. NF-KB is a transcription factor involved in the

activation of several MAPK pathways that recently was shown to be necessary for

melanoma cell migration on collagen type IV [456].

Unfortunately we were unable to confirm this interaction by other methods than the

yeast two-hybrid system. Our first attempt was to raise antibodies against a cdlc-1

specific peptide in order to perform co-immunoprecipitations and immunostainings.

The antibodies obtained specifically recognized recombinant cdlc-1 in Western

blotting of bacterial lysates but failed to detect the protein in cell lysates derived

from human foreskin fibroblasts (AG1518). Nor could any protein be detected after

immunoprecipations of either the cdlc-1 or the a2 integrin subunit. When

performing immunofluorescence studies the antibody raised against cdlc-1 was

unable to detect the protein. Antibodies raised against a GST- cdlc-1 fusion did not

detect cdlc-1 at all. Next affinity chromatography was tried and a full-length a2

63

cytoplasmic peptide was successfully immobilized on Sepharose beads and exposed

to recombinant cdlc-1, but no interaction could be detected. The cdlc-1 has been

reported to dimerize in vivo [457]. Since GST dimerizes by itself, GST-cdlc-1 fusions

as well were analyzed for binding but without results. The reverse setup was also

used, with GST-cdlc-1 immobilised and exposed to cell lysate from AG1518 but

again, no binding could be detected. SDS-PAGE followed by immunoblotting, using

our cdlc-1 specific anti-sera, of eluted fractions either directly or after ethanol

precipitation, was used to try to detect binding. We also tried to detect bound

peptide by measuring protein concentration (A280) in the fractions but perhaps we

should have gone further and sent the gels to amino acid analyzes after binding, or

at least have boiled the sepharose and analyzed the supernatant in case we failed to

elute bound peptide. Finally the Biacore approach was tried, by immobilizing both a

full-length a2 cytoplasmic peptide for direct detection and collagen for "sandwich"

detection. Neither recombinant cdlc-1 nor cell lysates from AG1518 showed any

distinct detectable binding. Binding of recombinant cdlc-1 to the a2 cytoplasmic tail

did give a reproducible, but very small, positive signal (~20 RU). Regeneration of the

surface was however very hard and it could not be excluded that the signal was

background noise. If our lack of result is due to an unfortunate combination of a

small highly conserved cytoskeletal protein with an increased avidity as dimer, and

a transmembrane heterodimeric receptor existing in an active and an inactive form,

time will tell.

64

ACKNOWLEDGEMENTS

The first author of this work is I, but several persons, both inside and outside the labhave helped me complete it. Hereby I express my sincere gratitude to these persons.If it feels like you have contributed, you most definitely have. Tank you all!

Kristofer Rubin, my supervisor. Thanks for taking in a "dark-horse" like me, that wasbrave. I also have to express my admiration of the amount of basic chemistry andbiology you keep stored in your head. You really do learn for life.

Lars Rask, my examiner. Thanks for not only keeping track of my scientificaccomplishments but also taking the time to ask about my family, e.g. my son Linus.Most appreciated.

Donald Gullberg, for letting me have a bite at the unexplored smörgåsbord of a11 andfor attacking my projects from a different point of view, sometimes annoying butalways useful. (Thanks for noticing my glasses)

All people who make our lives easier at the department. Kerstin, Olav, Pjotor, Tonyand Ylva for assisting us with whatever practical things we can't do ourselves - fromcomputers to dishes. Erika, Maud, Liisa and Barbro for keeping track of andadministrating us. Most of all, thanks to all of you for doing this with a smile!

I would like to thank former and present technicians of the Rubin group. Eva, Ann-Marie and Maria - life would have been so much more difficult without you! I have toadd special thanks to Eva, without whom this thesis hardly would have existed.Thank you for teaching me about collagen gel contraction, cell migration and life ingeneral. I couldn't have asked for a better supervisor or friend. To Maria, who tookcare of me and taught me the methods used in the lab when I arrived, you aremissed. And finally, thanks to Ann-Marie for amazing food and plenty of goodlaughter.

I don't want to think about what my PhD-time would have been like without all thefantastic people I've had the pleasure to get to know in our group. Here they come inalphabetical order: Alan, Alexei, Anna-Karin, Bianca, Christian, Ellen, Karina, Kia, Ihor,Marcin, Mikael, Patrik, Sorin, Therésè and Åsa. I especially thank Ellen, Åsa, Karina andTherésè for fruitful conversations about anything but science, and for being not onlygood co-workers but also great friends. Åsa and Alexei, thanks for proofreading thiswork.

Working side by side with us, the Rydén group has been just as important. So plentythanks to Cecilia, Lisa, Lena and Christian as well.

For several years we have shared corridor with the Rask group and I thank you all forpleasant company, good party-tradition and many laughs. It has been a pleasure. I'msorry Mårten, but being who I am I can't help but thanking you for letting me finishfirst.

Finally, I would like to thank the Gullberg, Robinson and Sundberg groups for beingsuch good neighbors.

65

Badminton on Tuesdays at 9 pm is what has kept me from living a life completelydevoid of exercise. Thanks to Ellen, Kia, Sorin, Maria, Gunbjörg, Jonas and all the restfor making physical activity fun.

Standing ovations to the Soup-Group. You all made Thursdays at 11:30 am the mostimportant time of the week.

To all my friends back home and in the Uppsala-area. Thanks for not abandoningme even if I never call, forget birthdays etc etc etc. I'm so lucky to have you! I willonly mention Camilla and Annika by name because they are special, and because Idon't want to forget anyone. You know who you are.

To my Mother. Tack för att du alltid har stöttat mig oavsett (nästan) vad jag velatgöra. Det har betytt oerhört mycket för mig, speciellt eftersom det innebar att jaglämnade dig under en mycket svår tid. En lysande mor och underbar mormor.

To my sister and her family. Tack Emelie och Mattias för att ni kommit och hälsat på ergamla moster år efter år efter...... Det har betytt jättemycket för mig. Samma tack tillKina och Bengt, ni har ett oerhört tålamod med mig.

Last but definitely not least - my guys:

Tack Staffan för att du är den du är och för att du inte bara låter mig vara den jag ärutan dessutom verkar uppskatta det. Du är BÄST och oerhört viktig för mig. Påminnmig om att visa och säga det oftare.

Linus, min lille älskling och busunge. Du är det bästa som har hänt mig och definitivtvärd att bli vuxen för. Tack för att du ger mig perspektiv på vad som är viktigt påriktigt.

66

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