Functional Studies of Collagen-Binding Integrins A2b1 and A11b1

Functional Studies of Collagen-Binding Integrins a2b1 and a11b1

Interplay between Integrins and Platelet-Derived Growth Factor Receptors

GUNILLA GRUNDSTRÖM Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medical Biochemistry presented at Uppsala University 2003.

ABSTRACT

Grundström, G. 2003. Functional Studies of Collagen-Binding Integrins a2b1 and a11b1. Interplay between Integrins and Platelet-Derived Growth Factor-BB. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from Faculty of Medicine 1295. 97 pp. Uppsala. ISBN 91-554-5769-X

Integrins are heterodimeric cell surface receptors, composed of an a-andab- subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and co-operate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins a2b1 and a11b1 were studied. In addition, the impact of b1 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 was explored, and the first a11b1-mediated cellular functions were established. Both a11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. a11 protein was also detected in cornea keratinocytes. a11b1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, a11b1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated a11b1-mediated contraction and directed migration. Expression of b1Y783,795F in b1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected a2b1-mediated functions in response to collagen. The b1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar 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 also abolished b1A-dependent down-regulation of b3. In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins avb3 mediated contraction. Clustering of b1-integrins by antibodies and PDGF-BB stimulated avb3-mediated contraction in an ERK-dependent way. Expression of a2b1, but not a11b1, prevented avb3-mediated contraction. Contraction by a2b1 and a11b1 was ERK-independent.

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

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

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

Printed in Sweden by Universitetstryckeriet, Uppsala 2003

2 In memory of my Father

You would have been sceptical but proud

3 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.

4 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

5 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

6 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.

7 Fluid efflux

Fluid influx

Collagen fibers Proteoglycans Hyaluronan

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

INTEGRINS

In multi-cellular organisms the minute control of cell anchorage and movement is crucial for most biological processes, spanning from embryogenesis to immune responses via maintenance of tissue integrity and homeostasis. The integrin family of adhesion receptors plays a central role in these functions by transducing signals that provide physical support, regulate migration and affect gene expression (reviewed in [3-6]). All known members of the integrin family are non-covalently linked heterodimers consisting of one a-andoneb-subunit. To date 18 a-chains and 8 b-chains combined into 24 different integrins are identified in mammalians (Figure 2). However, in a recent screen of the human genome 24 a-and9b-chains were identified, suggesting 6 new a-subunits and 1 new b-subunit [7].

Both subunits fold into an N-terminal globular head, that combined create the ligand-binding surface, a long stalk region connecting the ligand-binding head with the 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 any catalytic activity or consensus protein binding-motifs, except for the internalization NPXY-motif in b-subunits. The only known exception is the b4-subunit with two

8 fibronectin type-III repeats present in its uniquely long (~1000 residues) cytoplasmic domain. Several a-andb-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 a4anda9, 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 posttranslational cleavage has been implicated in the activation of signalling pathways mediated by av-integrins [19] and in the regulation of a6-integrin affinity [20].

9 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

10 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].

11 aIIb a1 2 a11 a a b3 E 3 10 b a a 5 4

b a 1 v

b a b

X 9 a b

8 a

a 6

8 a a 7 M 2

a b

8 b

D a

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

Integrin activation by increased ligand affinity leads to a straightening of the integrin and exposure of the ligand binding-site. To date two models are proposed for this change in appearance, namely the switch-blade model where each subunit has a single bend in the stalk region bringing the head to face downwards [49], and the angle-poise model where there is an additional bend, or sliding of the stalk region close to the membrane [50]. The switch-blade model is the most commonly accepted 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 that the membrane and proxamembrane region of integrins allows for a tilting of the integrin, with residues moving in and out of the membrane as a result [50]. This model would leave the integrin "head" in a more favourable position and fits well with the high conservation of the proxamembrane domains. The activation into a high affinity conformation is followed by clustering and increased avidity of the integrins that in turn leads to the formation of macromolecular signalling complexes called focal adhesions (see FOCAL ADHESIONS).

12 Collagen-binding integrins

To date there are four integrins known to bind triple-helical interstitial collagens, namely a1b1, a2b1, a10b1anda11b1. 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 a1b1anda2b1 have been known for quite some time, and hence have been thoroughly studied and characterized, a10b1anda11b1 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 a11b1isnot expressed on muscle cells in vivo [59].

Tissue distribution of collagen-binding integrins both differs and overlaps. a1b1and 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, a1b1anda2b1 are present on fibroblasts, activated T-cells and certain endothelial cells. In contrast to a1b1anda2b1, the two most recently characterized collagen-binding integrins a10b1anda11b1 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

13 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 a1b1in 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

14 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 a1b1anda2b1 have been suggested in the literature and the knowledge about a10b1anda11b1 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.

15 The multi-specific integrin avb3

The av-integrin subunit associates with the b1-, b3-, b5-, b6- and b8-subunits. Like the b1-andb2-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

16 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-andb3/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.

17 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-

18 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].

19 Ligand C-C A-A A-B B-B D-D

Extracellular

Intracellular

Receptor aa ab bb

Figure 3 PDGF isoforms and their receptors

Ligand binding induces dimerization of the receptor, but depending on which receptors are expressed, different ligands can induce either receptor homo- or heterodimerization. PDGF-A, -B and -C bind the a-receptor while PDGF-B and -D bind 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-bb homodimers can be formed by PDGF-BB and -DD (Figure 3). PDGF-CC and -DD have been reported to induce receptor heterodimerization as well [138, 139].

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

20 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-orbb-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.

21 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

22 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],

23 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

24 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

25 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.

26 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 b1-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

27 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 avb3viaa 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, b1Aandb1D in vitro, but not to b1Borb1C [38, 211, 212]. Talin

28 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 with layilin, a transmembrane hyaluronan receptor, enabling for one more connection between the ECM and the cytoskeleton. Cytoplasmic b1 and b3 domains have been shown 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 talin binding and disrupt integrin localization to focal adhesions. In addition, over- expression of the talin globular head domain activated aIIbb3 [216]. Another indication of a role for talin in integrin activation is that it is proteolytically cleaved by calpain, a protease with positive effects on integrin activation and functions, such as 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].

ECM

a b a b a b a b a b a b a b Integrins a b IAP TM4 uPAR Caveolin

PM Src Fyn FAK ILK Calreticulin Talin a-actinin Pax Cas Shc Vinculin FAK Crk FAK Grb2/ SOS PI3K Tensin Actin Ras Ras

ERK JNK ERK

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

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

29 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].

30 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

31 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).

32 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-anda9-tails in lymphoid

33 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

34 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.

35 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

36 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

37 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

38 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

39 and antiapoptotic signals (reviewed in [329]). Exactly how PI3-K work in the different responses is not fully understood, but it prevents apoptosis by activating the 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 actin reorganization and directed migration (chemotaxis) [331, 332]. Activation of the PDGF b-receptor induces transient loss of stress fibres and induction of membrane ruffles in fibroblasts [333]. The activation of Rho-GTPases, in particular Rac, involves a mechanism where PIP3 recruits Rho-GTPase activators containing pleckstrin homology (PH) domains, such as Tiam-1 and Vav [334, 335]. Other molecules involved in cell survival and proliferation, such as certain PKC isoforms and phospholipase C-g (PLC-g), also bind to PIP3 via their PH-domains. Actin reorganization is further discussed in Actin-reorganization by Rho-GTPases.

Src PLC-g

GAP Grb2/Sos PI3K Akt

Ras Rho- PKC GTPases family Bad Raf-1

MEK

MAPK

Proliferation Cell migration Apoptosis

Figure 5 Schematic picture of selected signalling pathways induced by PDGFR-b

40 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

41 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

42 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

43 Rac with phosphatidylinositol-4-phosphate 5-kinase (PI-4-P5K) [358]. Rac activation of PI-4-P5K provides the PIP2 needed for the uncapping of actin filaments required for thrombin-induced actin filament assembly in platelets [359]. PIP2 is suggested to be involved in actin-reorganizing events mediated by Rho as well. A WASP-like protein called WAVE (also known as Scar) can be precipitated with Rac but whether this is a direct interaction is not known. Both the adaptor protein Nck and the insulin receptor 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 Nck activates WAVE and initiates actin nucleation [360]. Over-expressed wildtype WAVE causes clustering of actin bundles, whereas mutations in the actin-binding domain inhibits Rac- and PDGF-induced membrane ruffling but has no effect on Cdc42-induced micro spikes [362].

Rho-GTP? Rac-GTP Cdc42-GTP

WAVE ROK profilin G-actin Arp Dia N-WASP PAK G-actin 2/3 profilin profilin G-actin Arp G-actin 2/3 G-actin PI-4-P5K MLC MLC- P LIMK- P MLCK- P LIMK- P PIP2

Release of Cofilin- P capping Cofilin- P proteins

Actomyosin Actin filament Increased actin Actin nucleation and Decreased Actin filament Actin nucleation and assembly stabilization polymerization polymerization actomyosin stabilization polymerization and contraction contraction

Stress-fibre formation Lamellipodia and membrane ruffles Filopodia microspikes

Figure 6 Schematic picture of actin reorganization mediated by Rho-GTPases

Cdc42 binds and activates WASP and the more ubiquitously expressed N-WASP, and over-expression of N-WASP together with Cdc42 induces long cellular microspikes, suggesting the involvement of N-WASP in Cdc42-mediated filopodia formation [363, 364]. Upon Cdc42-mediated activation N-WASP/WASP interacts directly with both actin monomers (G-actin), profilin and the Arp2/3 complex, and thus 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 in actin reorganization and filopodia formation, MRCK-a and -b. MRCKs are Cdc42-

44 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

45 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.

46 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].

47 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].

48 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.

49 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].

50 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 a2andb1 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

51 stressed collagen gels, indicating that depending on mechanical load receptors of LPA couple to different G-proteins, or alternatively different receptors get activated.

Shortening and pausing MT Growing MT

Direction of cell migration

MT shortening in the cell activates RhoA Actomyosin drives MT retrograde flow MT growth at the edge activates Rac1 creating a gradient in MT dynamics

Direction of cell migration

Contractile actin bundles Actin retrograde flow High RhoA activity High Rac1 activity

Figure 7 Feedback interactions between the microtubule and actin cytoskeleton dynamics 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 actin retrograde flow. The dashed line defines the leading lamella.

Contraction of a free-floating gel is believed to be a consequence of cell-mediated reorganizing of collagen fibres into bundles by traction forces [416] similar to those generated during cell migration. Since the matrix is non-attached it offers little resistance and the collagen fibrils are organized at random. This results in a locally denser collagen network in close vicinity of the cells. In fact, it has been shown that the concentration, or density, of collagen fibres surrounding the cells in a contracted

52 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 a2b1and 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

53 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.

54 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

55 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 1b1anda2b1 has been thoroughly investigated and both a1anda2 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.

56 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.

57 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 a2b1or 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

58 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 avb3and a11b1 during remodelling of a collagen matrix, a2b1 shuts off the use of avb3in 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.

59 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.Ana11-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, a1b1anda2b1, 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 a1b1anda2b1 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.

60 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 a1anda2 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.

61 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

62 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.

63 ACKNOWLEDGEMENTS

The first author of this work is I, but several persons, both inside and outside the lab have 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 was brave. I also have to express my admiration of the amount of basic chemistry and biology you keep stored in your head. You really do learn for life.

Lars Rask, my examiner. Thanks for not only keeping track of my scientific accomplishments 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 and for attacking my projects from a different point of view, sometimes annoying but always useful. (Thanks for noticing my glasses)

All people who make our lives easier at the department. Kerstin, Olav, Pjotor, Tony and Ylva for assisting us with whatever practical things we can't do ourselves - from computers to dishes. Erika, Maud, Liisa and Barbro for keeping track of and administrating 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 to add 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 in general. I couldn't have asked for a better supervisor or friend. To Maria, who took care of me and taught me the methods used in the lab when I arrived, you are missed. And finally, thanks to Ann-Marie for amazing food and plenty of good laughter.

I don't want to think about what my PhD-time would have been like without all the fantastic people I've had the pleasure to get to know in our group. Here they come in alphabetical order: Alan, Alexei, Anna-Karin, Bianca, Christian, Ellen, Karina, Kia, Ihor, Marcin, Mikael, Patrik, Sorin, Therésè and Åsa. I especially thank Ellen, Åsa, Karina and Therésè for fruitful conversations about anything but science, and for being not only good co-workers but also great friends. Åsa and Alexei, thanks for proofreading this work.

Working side by side with us, the Rydén group has been just as important. So plenty thanks to Cecilia, Lisa, Lena and Christian as well.

For several years we have shared corridor with the Rask group and I thank you all for pleasant company, good party-tradition and many laughs. It has been a pleasure. I'm sorry Mårten, but being who I am I can't help but thanking you for letting me finish first.

Finally, I would like to thank the Gullberg, Robinson and Sundberg groups for being such good neighbors.

64 Badminton on Tuesdays at 9 pm is what has kept me from living a life completely devoid of exercise. Thanks to Ellen, Kia, Sorin, Maria, Gunbjörg, Jonas and all the rest for making physical activity fun.

Standing ovations to the Soup-Group. You all made Thursdays at 11:30 am the most important time of the week.

To all my friends back home and in the Uppsala-area. Thanks for not abandoning me even if I never call, forget birthdays etc etc etc. I'm so lucky to have you! I will only mention Camilla and Annika by name because they are special, and because I don'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 velat göra. Det har betytt oerhört mycket för mig, speciellt eftersom det innebar att jag lämnade 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 er gamla moster år efter år efter...... Det har betytt jättemycket för mig. Samma tack till Kina 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 är utan dessutom verkar uppskatta det. Du är BÄST och oerhört viktig för mig. Påminn mig om att visa och säga det oftare.

Linus, min lille älskling och busunge. Du är det bästa som har hänt mig och definitivt värd att bli vuxen för. Tack för att du ger mig perspektiv på vad som är viktigt på riktigt.

65 REFERENCES

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