Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 688

_____________________________ _____________________________

The Laminins and their Receptors

BY

MARIA FERLETTA

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2002

Dissertation for the degree of Doctor of Philosophy in Molecular Cellbiology presentedat Uppsala University 2002

ABSTRACTFerletta, M. 2002. The Laminins and their Receptors. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 688. 67 pp. Uppsala. ISBN 91-554-5239-6

Basement membranes are thin extracellular sheets that surround muscle, fat andperipheral nerve cells and underlay epithelial and endothelial cells. Laminins are one ofthe main protein families of these matrices. Integrins and dystroglycan are receptors forlaminins, connecting cells to basement membranes. Each laminin consists of threedifferent chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and isthe most frequently studied. Despite this, it was unclear where its α1 chain wasexpressed. A restricted distribution of the α1 chain in the adult epithelial basementmembranes was demonstrated in the present study. In contrast, dystroglycan was foundto have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins werealso shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2-laminins.

The biological roles of different laminin isoforms have been investigated. Differenceswere found in the capacity of various tested laminins to promote epithelial cell adhesion.The α5-laminins were potent adhesive substrates, a property shown to be dependent onα3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesionto α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splicevariant could be linked to laminin-mediated activation of a mitogen-activated proteinkinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activatedextracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant,but not in cells expressing α6B. A new role for dystroglycan as a suppressor of thisactivation was demonstrated. Dystroglycan antibodies, or recombinant fragments withhigh affinity for dystroglycan, decreased ERK activation induced by integrin α6antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers.Cell attachment to α5-laminins was found to facilitate growth factor induced cellproliferation. This proliferation could be blocked by antibodies against integrin αvβ3 orby an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.

Keywords: Basement membrane, laminin, integrin, dystroglycan, epithelial cells, signaltransduction

Maria Ferletta, Department of Cell and Molecular Biology, Biomedical Center, UppsalaUniversity, Box 596, SE-751 24 Uppsala, Sweden

© Maria Ferletta 2002

ISSN 1104-232XISBN 91-554-5239-6

Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2002

LIST OF PAPERS

This thesis is based on the following papers, referred to in the text by the theirRoman numerals I-V:

I. Falk, M.*, Ferletta, M.*, Forsberg, E. and Ekblom, P. (1999).Restricted distribution of laminin α1 chain in normal adult mouse tissues. Matrix Biol. 18, 557-568. (*Shared first authorship).

II. Durbeej, M., Henry, M., Ferletta, M., Campbell, K.P. and Ekblom, P. (1998). Distribution of dystroglycan in normal adult mouse tissue. J. Histochem. Cytochem. 46, 449-457.

III. Ferletta, M and Ekblom, P. (1999). Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line. J. Cell Sci. 122, 1-10.

IV. Ferletta, M., Kikkawa, Y., Yu, H., Talts, J.F., Durbeej, M., Campbell, K.P., Ekblom, P. and Genersch, E. 2002. Opposingroles of integrin α6Aβ1 and dystroglycan in laminin mediatedERK activation. Manuscript.

V. Genersch, E., Ferletta, M., Virtanen, I., Haller, S. and Ekblom, P.2002. Integrin αvβ3 binding to α5-laminins facilitates FGF-2 andVEGF induced proliferation of human ECV304 cells. Manuscript.

Reprints were made with permission from the publishers.

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CONTENTS

ABBREVIATIONS 6

INTRODUCTION 7

BASEMENT MEMBRANE 7

THE LAMININ FAMILY 8Structure 8The laminins based on their α chains 11

Laminins containing the α1 chain 11Laminins containing the α2 chain 13Laminins containing the α3 chain 15Laminins containing the α4 chain 15Laminins containing the α5 chain 16

BASEMENT MEMBRANE COLLAGENS 16

NIDOGENS 18

LAMININ RECEPTORS 19Dystroglycan 19

Structure of dystroglycan 19Function and ligand binding of dystroglycan 19

The integrin family 22Integrin structure 22Alternative splice variants 23Integrin interactions and functions 24

CELL SIGNALING 26The MAP kinase family 27Basic regulation of the MEK/ERK pathway 27Integrin and MAP kinase signaling 29What is known about laminins and MAP kinase signaling? 30

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PRESENT INVESTIGATIONS 33Specific aims of the thesis 33

RESULTS AND DISCUSSION 33The laminin α1 chain has a restricted distribution in adult mouse tissues (I) 33Broad distribution of dystroglycan in adult mouse tissues (II) 34Laminin-10/11 is a strong adhesive substrate for epithelial cells and is mediating ERK activation through integrin α6Aβ1 (III, IV) 36Laminin α5 containing laminins are ligands for integrin αvβ3 and are involved in FGF and VEGF stimulated proliferation of ECV304 cells (V) 40

CONCLUDING REMARKS AND FUTURE PERSPECTIVES 43

POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA 45Cellers samspel med omgivande proteiner 45

ACKNOWLEDGEMENT 47

REFERENCES 48

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ABBREVIATIONS

BM Basement membraneCCN Cyr61, CTGF, NovDGC Dystrophin-glycoprotein complexE Embryonic dayECM Extracellular matrixEGF Epidermal growth factorERK Extracellular signal regulated protein kinaseES Embryonic stem cellFAK Focal adhesion kinaseFGF Fibroblast growth factorGrb2 Growth-factor-receptor-bound protein 2JEB Junctional epidermolysis bullosaLCMV Lymphocytic choriomeningitisLE Laminin EGF-like repeatsLFV Lassa fever virusLG Laminin globular domainMAP kinase Mitogen-activated protein kinaseMEK MAPK/ERK kinaseNMJ Neuromuscular junctionPI3K Phosphoinositide 3-kinasePKB Protein kinase BPMA Phorbol 12-myristate 13-acetateRGD Arg-Gly-AspRTK Receptor tyrosine kinasesSAPK/JNK Stress activated protein kinase/Jun N-terminal kinaseSOS Son of sevenlessVEGF Vascular endothelial growth factor

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INTRODUCTION

Cells continuously send and receive information, either from cell-cell contacts,from the surrounding extracellular matrix (ECM) or from circulating solublehormones and growth factors. The ECM is a complex network surrounding cells,serving as a structural element in tissues. The ECM also provides the cells withinformation about their environment and thus influences tissue development.The ECM is composed of glycoproteins such as laminins, collagens andproteoglycans, which are secreted by the cells and assembled into networks towhich cells can adhere. The ECM is present from the two-cell stage inmammalian embryos. The composition of the ECM varies and depends on thetissue and the stage of development. The matrix can be calcified and form hardteeth and bone structure, or it can be very tensile. In this thesis I will focus onthe protein family laminin and its cell receptors. The laminin family is acomponent of the basement membrane, a specialized form of ECM. Theadhesion of cells to basement membranes influences the tissue formation inmany ways. In addition to stabilizing the tissue, basement membranes regulatecell behavior through different signaling cascades, which can startdifferentiation, movements, proliferation, growth and cell survival. It is knownthat disturbances or mutations in basement membrane proteins can lead todiseases such as muscular dystrophies, epidermolysis bullosa, or cancer.Thickened basement membranes are seen in later stages of diabetes. Further,some bacteria and viruses use the basement membrane as a link to infect cells.The basement membrane components are well conserved throughout theevolution, indicating the importance of correct basement membranes fordevelopment of a multicellular organism.

BASEMENT MEMBRANES

Basement membranes are thin extracellular sheets that surround muscle, fat, andperipheral nerve cells, and underlay all epithelial and endothelial cells. Basementmembranes also provide tissue compartmentalization in many tissues, acting as abarrier for cells. Different basement membranes are quite heterogeneousmolecularly and vary from tissue to tissue, as well as in the same tissue atdifferent developmental stages (Durbeej et al., 1996). A few protein families arethe main components of basement membranes. These include laminins and

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collagen IVs, nidogens and the proteoglycans agrin and perlecan. In addition,there are several associated proteins including collagen XV, fibulins, andcollagen XVIII (Erickson and Couchman, 2000; Timpl, 1996).

The basement membrane contains two networks that can self assembleindependently of each other. Laminin isoforms form one of the networks byinteracting with their N-terminal modules of the three short arms (Timpl andBrown, 1996). The second network consists of different type IV collagens. Thecollagen network is considered to stand for the mechanical stability and thelaminin network for the dynamic flexibility in the basement membrane (Timpland Brown, 1996; Yurchenco and O´Rear, 1994). The two networks are believedto be connected to each other by nidogens (Timpl, 1996; Timpl and Brown,1996; Yurchenco and O´Rear, 1994).

THE LAMININ FAMILY

Structure

Laminins are large cross- or T-shaped glycoproteins. Each laminin is composedof one α, one β and one γ chain. So far 5 α, 3 β, and 3 γ chains are known, givingrise to at least 15 laminin heterotrimers (Fig. 1.) (Colognato and Yurchenco,2000; Libby et al., 2000; Brunken, presentation at the 10th ISBM meeting). Allchains are different gene products and a few of the individual polypeptide chainscan be alternatively spliced (Miner et al., 1997), while some undergoextracellular proteolytic processing (Miner et al., 1997; Talts et al., 1998; Taltset al., 2000). Laminin trimers are assembled intracellularly, mediated byinteractions of domain I of the C-terminal part of the chains (Beck et al., 1993)(Nomizu et al., 1994). All laminins share the same domain structure (fig. 2.).The α chains have five homologous laminin globular (LG) domains, namedLG1-5, at the C-terminal end. Many cell surface receptors such as dystroglycanand integrins bind to the LG domains (Hohenester et al., 1999; Talts et al., 1999;Talts and Timpl, 1999). Domains I and II make up the triple α-helical coiled-coilstructure forming the rod-like part of the long arm. Domain III and V in all threeshort arms of the laminin molecule consist of three to eight laminin EGF-like

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Laminin-1

α1

β1 γ1

Laminin-2

α2

β1 γ1

Laminin-3

α1

β2 γ1

Laminin-4

α2

β2 γ1

Laminin-5

α3

β3 γ2

Laminin-6

α3

β1 γ1

Laminin-7

α3

β2 γ1

Laminin-8

α4

β1 γ1

Laminin-9

α4β2 γ1

Laminin-10

α5

β1 γ1

Laminin-11

α5

β2 γ1

Laminin-12

α2

β1 γ3

Laminin-14

α4β2 γ3

Laminin-15

α5

β2 γ3

Fig.1. The laminin family. Chain composition and schematic structure of each laminin isoform.

Laminin-13

α3β2 γ3

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Laminin-1

α1

β1 γ1

C-terminus

N-terminus

LG1-5

I

II

IIIb

V

IVa

IVb

IV

IIIa

IIIIIIIVV VVI

VI

VI

P1

E8 E3

N-terminusN-terminus

Integrin α1β1Integrin α2β1

Integrin α6β4Integrin α7β1 Dystroglycan

Nidogen

Agrin

PerlecanHeparinFibulin-1

Integrin α6β1

Sulfatides

Fig. 2. Structural model for laminin-1 composed of the α1, β1 and γ1 chains. Domains are indicated by roman numbers. The E3 and E8 (elastase) fragments are indicated by dashed boxes, the P1 (pepsin) fragment is indicated with a box. Binding sites for cell receptors and other matrix proteins are indicated with arrows.

Integrin α3β1(hLN-1)

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(LE) repeats. They are rod-like and separate the globular domains IVa, IVb andVI.

The globular domain VI, also called the LN module, terminates theshort arms and is the most conserved part of the laminins. It has been shown thatlaminin isoforms can self-assemble into a network when the three short arms areinteracting via the N-terminal of the LN modules. In agreement with this onlylaminins with three full length chains, such as laminin-1, -2 and -4, have beenreported to stabilize such a network (Cheng et al., 1997; Yurchenco and O´Rear,1994). The α5 containing laminin-10 and -11 can probably also self-assemble.

Elastase and pepsin digestion of laminin-1 give some characteristiclaminin fragments called E3, E8 and P1 (fig. 2), which all are recognized byintegrin receptors (Paulsson et al., 1985; Rohde et al., 1980; Sonnenberg et al.,1990). The E3 fragment corresponds to the globular domains LG4 and LG5 ofthe C-terminal end of the α1 chain to which dystroglycan and heparin bind(Andac et al., 1999).

The laminins based on their α chains

At present 15 laminins are known and it is still possible that new chaincombinations will be discovered (Colognato and Yurchenco, 2000; Libby et al.,2000; Brunken, presentation at the 10th ISBM meeting). They are here groupedbased on their α subunit, and some characteristics of each laminin will bediscussed. The phenotypes of mice lacking laminin chains have beensummarized in table 1.

Laminins containing the α1 chain

Laminin-1 (α1β1γ1), the first found laminin (Timpl et al., 1979) has been themost studied so far. It seems to be a major laminin during early embryogenesis.Laminin β1 and γ1 chains are expressed already at the two-four-cell stageintracellularly, and the α1 chain is expressed at the 16-cell stage. β1 and γ1chains are expressed in almost all basement membranes and can assemble withmost known α chains. The α1 chain is largely limited to the epithelial basementmembrane during embryogenesis (Ekblom et al., 1990; Sorokin et al., 1997;Thomas and Dziadek, 1993), but there has been conflicting data about its

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Lamininsubunit

Laminin deficient phenotypes References

α1 Deletion of the E3 fragment of the α1 chain isembryonic lethal, the mouse dies shortly afterimplantation.

(Schéele et al.,2001)

α2 Deficient mice display growth retardation and severemuscular dystrophic symptom and die at 5 weeks ofage. Spontaneous mutant mice dy2J and dy sufferfrom merosin-deficient congenital musculardystrophy.

(Miyagoe et al.,1997; Sunada et al.,1994; Xu et al.,1994)

α3 The mouse dies 2-3 days after birth. It developsprogressive blistering of forepaws, limbs andmucosa.

(Ryan et al., 1999)

α4 The deficient mouse develops normally and is fertilebut have uncoordinated movements and changes inNMJ.

(Patton et al., 2001)

α5 The deletion is embryonic lethal at E13.5-16.5. Themouse has defects in limb and placenta development,exencephaly, and defects on internal organs, lung,heart, intestine and kidney.

(Miner et al., 1998;Miner and Li, 2000)

β1 N.D.β2 The mutant has postnatal lethal defects in glomerula

filtration and in NMJ.(Noakes et al.,1995a; Noakes etal., 1995b)

β3 Mutations are found in the LAMB3 gene in patientssuffering from JEB.

(Kivirikko et al.,1996)

γ1 The deficient mice die before E5.5. Complete lack ofbasement membranes.

(Smyth et al., 1999)

γ2 Mutations in the LAMC2 gene are found in patientswith the disease JEB.

(Pulkkinen et al.,1994)

γ3 N.D.

Table 1. Summary of the phenotypes of deleted laminin chains. N.D., no data.

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distribution in the adult body. Some data suggested a broad expression patternfor the laminin α1 chain at adult stage (Engvall et al., 1990; Sanes et al., 1990;Virtanen et al., 1995), but this was based on studies with the monoclonalantibody 4C7 (Engvall et al., 1986), now known to recognize the laminin α5chain (Tiger et al., 1997).

Mouse laminin-1 can strongly stimulate cell attachment, migration anddifferentiation (Colognato and Yurchenco, 2000; Ekblom, 1996). In vitro studiessuggest that laminin α1 chain plays a role in branching epithelial morphogenesisand in neurite outgrowth (Edgar, 1996; Kadoya et al., 1995). Tumor growth innude mice was significantly increased by transfecting the α1 chain in coloncancer cells, suggesting that laminin α1 chain is a potent activator of cell growth(De Arcangelis et al., 2001). Mice with targeted deletion of the laminin γ1 chaingene die before day E5.5, and no covalently bound laminin subunits were found,which was coupled with complete lack of basement membrane structures (Smythet al., 1999). Mice with a targeted disruption of the C-terminal domain E3 of thelaminin α1 chain die shortly after implantation (Schéele et al., 2001). Hence,binding of the cell receptor dystroglycan for the E3 fragment of laminin α1could be important for cell survival. These few examples from variousapproaches performed during the past 20 years suggest a significant role forlaminin-1 in epithelial cell differentiation and survival.

The α1 chain is also found in laminin-3 (α1β2γ1), which can be foundin human placenta (Champliaud et al., 2000), and possibly also in developinghuman muscle in myotendinous junctions, since laminin α1 chain is co-distributed with the β2 chain there (Pedrosa-Domellöf et al., 2000). Thedistribution of the β2 chain could be broader than often assumed, so it could beof interest to study laminin-3 in more detail.

Laminins containing the α2 chain

The laminin α2 chain appears in three different laminins, laminin-2 (α2β1γ1),laminin-4 (α2β2γ1) and laminin-12 (α2β1γ3). The laminin α2 chain ispresumably not expressed before day 11 of mouse development, but then itbecomes predominantly expressed in skeletal and heart muscle, as well as inperipheral nerves. Further it has been identified in lung, intestinal crypts, testis,kidney, liver, skin, and brain vasculature (Leivo et al., 1988 Lefebvre et al.,1999; Miner et al., 1997; Sasaki et al., 2002). The laminin-12 heterotrimer hasbeen identified in human placenta and was the first laminin not to be localized

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within the basement membrane. The γ3 mRNA expression was very broad, withhigh levels in spleen, testis, lung and placenta. The γ3 chain was also found onthe apical surface of ciliated epithelial cells of lung, oviducts, and epididymis. Inhuman skin, γ3 was found along the dermal-epidermal junction (Iivanainen etal., 1999; Koch et al., 1999).

Mutations in the laminin α2 chain in humans have been found inpatients suffering from congenital muscular dystrophies (McGowan andMarinkovich, 2000). There are several mouse models with muscular dystrophieswith defective laminin α2 chain. The dyW and dy3 mice have engineeredmutations, while dy and dy2J mice have spontaneous mutations in the α2 chain(Kuang et al., 1998b; Miyagoe et al., 1997; Sunada et al., 1994; Xu et al., 1994).Disruption of the LamA2 gene in embryonic stem cells did not abrogate myotubedevelopment, but the myotubes failed to contract and underwent degradation andapoptosis, suggesting that laminin α2 ensures stable attachment of muscle fibersduring contraction (Kuang et al., 1998a). To experimentally study this issuefurther, the mutant dyW mouse with complete lack of laminin α2 was mated withtransgenic mice carrying the human LAMA2 gene, regulated by a muscle specificpromoter. The human laminin α2 protein became localized in skeletal and heartmuscle, and the life span increased in these mice. However, from 3 weeks ofage, these mice became increasingly paralyzed and developed musclecontractures. Most likely, this is a neurological problem because the humanlaminin α2 chain was not re-expressed in nerves (Kuang et al., 1998b).

Based on immunofluorescence data with a monoclonal antibody, theβ2 chain has been suggested to have a fairly restricted expression as comparedwith the β1 chain. It was reported to be expressed in skeletal neuromuscularjunctions, basement membrane surrounding Schwann cells, glomerular basementmembrane, and in some arterioles and vessels (Durbeej et al., 1996; Miner andPatton, 1999). However, this apparent restricted expression could be due tomasking of epitopes (Durbeej et al., 1996), and the β2- laminins could thus becommon laminins in many locations. Mice missing the laminin β2 chain die 2-4weeks after birth possibly because of defects in the renal glomerulus, causingfiltration problems. In addition, they have defects in the neuromuscular junction(Noakes et al., 1995a; Noakes et al., 1995b).

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Laminins containing the α3 chain

In laminin-5 (α3β3γ2) all three chains are truncated. Laminin-5 is expressed inthe basement membrane of stratified epithelia as one of the molecules making upthe hemidesmosomal complex. Laminin-5 is the main form of laminin in skinand bladder, and is also found in oesophagus and large intestinal epithelium(Miner et al., 1997; Tunggal et al., 2000). Mutations in laminin α3, β3, or γ2chains have been found in patients suffering from junctional epidermolysisbullosa (JEB), characterized by skin fragility. Blisters can also occur on mucousmembranes and may affect internal organs (Christiano and Uitto, 1996;Kivirikko et al., 1996; Pulkkinen et al., 1994). Laminin α3 deficient mice die 2-3days after birth and develop progressive blistering of the forepaws, limbs andoral mucosa (Ryan et al., 1999). The epidermis of these mice contained clustersof cells with an undifferentiated morphology. A similar pathology was seen inβ4-integrin null mice (Dowling et al., 1996; van der Neut et al., 1996). The skinphenotype in mouse is comparable with the human disease JEB. The sameblistering phenotype was also seen in mice with a spontaneous disruption in thelaminin β3 subunit (Kuster et al., 1997).

Laminin-6 (α3β1γ1) is expressed at the same places as laminin-5(Miner et al., 1997; Tunggal et al., 2000), and laminin-7 (α3β2γ1) is found in theamnion (Champliaud et al., 1996). Laminins, which lack one or more LNdomains can not self-aggregate into a network, but both laminin-6 and -7 canassociate with laminin-5 and co-polymerize to a stable basement membraneformation (Champliaud et al., 1996).

Laminins containing the α4 chain

Laminin-8 (α4β1γ1), laminin-9 (α4β2γ1) and laminin-14 (α4β2γ3) are thelaminins containing the α4 chain. The laminin α4 chain is widely expressed inbrain, intestine, heart, kidney, liver, lung, muscle and skin (Miner et al., 1997;Talts et al., 2000). Libby et al recently found the combination for the laminin-14heterotrimer in the retinal matrix (Libby et al., 2000).

Laminin α4 chain deficient mice develop normally and are fertile, buthave uncoordinated movements of the hindlimbs. Analyses at ultra structurallevels showed that the active zones in the nerve terminal of the neuromuscularjunction (NMJ) were not positioned directly opposite each other. Studies of thelaminin α4 -/- mouse suggest that the α4 chain regulates the localization of the

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active zones to each other in the nerve terminals and that something else controlsthe formation (Patton et al., 2001). In addition, β2 deficient mice have defects inthe NMJ (Noakes et al., 1995a; Noakes et al., 1995b).

Laminins containing the α5 chain

Laminin α5 chain has a broad distribution in the embryo, but it is absent frommost embryonic blood vessels (Durbeej et al., 1996; Sorokin et al., 1997). Theα5 chain is found in developing epithelial sheets known to produce very smallamounts of the α1 chain, as shown for the embryonic kidney (Durbeej et al.,1996). In the adult stage the laminin α5 chain is a major α chain of mostbasement membranes, where it forms the laminin-10 and -11 trimers (Miner etal., 1995; Miner et al., 1997; Sorokin et al., 1997). Laminin-11 (α5β2γ1) ispresumably present in the glomerular basement membrane, neuromuscularsynaptic cleft in skeletal muscle, in placenta and lung. Motorneurons andSchwann cells recognize laminin-11 and stop growing upon contact (Miner andPatton, 1999; Patton et al., 1997). Laminin-l5 (α5β2γ3) was recently found in theretinal matrix (Libby et al., 2000). The laminin-10/11 complex has been shownto stimulate cell adhesion for various cell types (Ferletta and Ekblom, 1999; Guet al., 1999; Kikkawa et al., 1998; see results).

The β2 deficient mice, which have defective glomerular filtration anddefects in the NMJ also show a loss of the α5 chain in the synaptic basementmembrane (Noakes et al., 1995a; Patton et al., 1997). Mouse embryos lackingthe laminin α5 chain die late during embryogenesis between E13.5 and 16.5.Defects were seen in many different tissues, for example the limbs failed to formseparate digits (syndactyly) and the brain was enlarged, misshaped and notcovered by skin in 60% of the mice. Also, the placental labyrinth wasmalformed. Additional defects were seen in some internal organs such as lung,heart, intestine, and kidney (Miner et al., 1998; Miner and Li, 2000).

BASEMENT MEMBRANE COLLAGENS

The collagen super family consists of at least 24 protein members found in theECM, and the super family is divided into different families depending on theirassembly and on other features (Myllyharju and Kivirikko, 2001). Collagens areformed from three α chains, arranged in a triple-helix. The type IV collagen

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family is a very common component of the basement membrane. There are sixknown α chains of the collagen type IV family; the most common collagen typeIV isoform consists of two α1 chains and one α2 chain (Brown and Timpl, 1995;Kuhn, 1995). Collagen type IV triple helices forms a covalently stabilizedpolymer network through amino-terminal and carboxy-terminal bonds, andinteracts laterally with their triple-helical domains. Collagen IV belongs to thenon-fibrillar, network-forming collagens (Kuhn, 1995; Yurchenco and O´Rear,1994). Collagen type I and IV bind to the globular domain 2 of nidogen-1, andnidogen binds closely to the C-terminal end of collagen type IV (Aumailley etal., 1989; Kohfeldt et al., 1998). Interestingly, collagen IV does not bindlaminin, but supramolecular complexes are formed in solutions containinglaminin-nidogen and collagen type IV (Aumailley et al., 1989). Collagen type IVis involved in interactions with cells and this is mediated among others by theintegrins α1β1, α2β1 and α11β1 (Kuhn, 1995; Tiger et al., 2001). Integrin α1β1and α2β1 bind to distinct epitopes of collagen within the triple helix (Kuhn,1995).

Collagen XVIII is the second most abundant basement membranecollagen. Three different forms of collagen XVIII with various tissuedistributions have been found in mouse. Among other places, expression wasseen in the vascular system. Collagen type XV is a structurally homologous totype XVIII collagen and has a widespread tissue expression. Collagen XVIII hasbeen shown to be a heparan sulfate proteoglycan and collagen XV is a disulfide-bounded chondroitin sulfate proteoglycan (Erickson and Couchman, 2000;Marneros and Olsen, 2001). Both collagens bind the laminin-nidogen complex,but collagen XVIII binds with stronger affinity (Sasaki et al., 2000).Recombinant proteins of the C-terminal part of collagen XVIII, calledendostatin, bind to α5- and αv- integrins on the surface of endothelial cells(Marneros and Olsen, 2001; Rehn et al., 2001). Endostatin has also been shownto have tumor-suppressing activity and to inhibit endothelial proliferation(Erickson and Couchman, 2000; Marneros and Olsen, 2001).

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NIDOGENS

Nidogens, also called entactins, are thought to connect the laminin and thecollagen type IV network. So far two nidogens have been identified (Durkin etal., 1988; Kimura et al., 1998; Kohfeldt et al., 1998; Mann et al., 1989). The twonidogens have a similar shape and consist of three globular domains connectedwith a flexible link and a rod-like domain (Fox et al., 1991; Kohfeldt et al.,1998; Mayer et al., 1995). Nidogen-1 has been shown to bind several basementmembrane proteins such as laminins, collagen type I, IV, XV and XVIII,perlecan and fibulins (Kohfeldt et al., 1998; Mayer et al., 1993; Sasaki et al.,2000). The nidogen-1 binding site on laminin has been mapped to the laminin γ1chain and more precisely to the LE module γ1III4 (fig. 2) (Mayer et al., 1993).Since nidogen-1 binds to the γ-chain of laminin, it can bind 10 out of the known15 laminin trimers. Human nidogen-2 binds collagen I, IV and perlecan, but thebinding to laminin-1 and the γ1III4 fragment is only moderate compared to thatof nidogen-1 (Kohfeldt et al., 1998).

Nidogen-2 may be more cell adhesive than nidogen-1, but theresponsible receptors for nidogen-2 are not yet known. An RGD site has beenidentified in the rod-like region of nidogen-1, indicating that the binding couldbe mediated by integrins (Kohfeldt et al., 1998; Mann et al., 1989). Based onorgan cultures studies, it has been suggested that the laminin-nidogeninteractions are important for epithelial morphogenesis. Antibodies against thenidogen-1 binding site on laminin inhibited branching epithelial morphogenesisof embryonic kidney, lung and submandibular gland in vitro. Epidermal growthfactor (EGF) increased expression of nidogen-1, and also counteracted the effectof the blocking antibodies (Ekblom et al., 1994; Kadoya et al., 1997).Surprisingly, the nidogen-1 deficient mice are viable and fertile. This could bedue to a compensatory effect of nidogen-2, since the expression patterns of thetwo nidogens are very similar in mice (Murshed et al., 2000; Salmivirta et al.,1999). In mice lacking the nidogen binding site on laminin γ1 chain, lamininheterotrimers are formed but nidogen-1 is absent from basement membranes.The mice die postnatally due to severe kidney and lung defects (Halfter et al.,2001). This confirms that the laminin γ1 nidogen-binding site is crucial fordevelopment of some epithelia and that proper binding of nidogen to laminin γ1is required.

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LAMININ RECEPTORS

Interactions between ECM and cells are mediated by transmembrane cell surfacereceptors, which link the ECM to intracellular signaling pathways. Dystroglycanand integrins have been shown to be main receptors for laminins. Figure 3 showsa schematic model of laminin and the receptors.

Dystroglycan

Structure of dystroglycan

The protein complex dystroglycan is derived from one single transcript that isdivided into two subunits, α- and β-dystroglycan, as a result of post-translationalmodifications of the precursor protein (Ibraghimov-Beskrovnaya et al., 1992). α-Dystroglycan is noncovalently attached to β-dystroglycan. β-Dystroglycan inturn spans through the plasma membrane and binds to cytoskeletal proteins atthe inside of the cell (Ervasti and Campbell, 1991). Dystroglycan was first foundin skeletal muscle as a component of the dystrophin-glycoprotein complex(DGC) (Ervasti et al., 1990) but has later been shown to have a much broaderdistribution and is found in many non muscle cells (Ibraghimov-Beskrovnaya etal., 1992). In particular, dystroglycan is prominent on the basal side of epithelialcells (Durbeej et al., 1998a; Durbeej et al., 1998b).

In muscle, dystroglycan associates with the sarcoglycan-sarcospancomplex (α-, β-, γ- and δ-sarcoglycan, and sarcospan). α-Dystroglycan binds tothe laminin α2 chain and β-dystroglycan links the complex to dystrophin(Ervasti et al., 1990; Sunada et al., 1994). In epithelial cells, the dystroglycancomplex is not associated with any of the known sarcoglycans or sarcospan andit seems that dystroglycan forms distinct complexes depending on the tissue(Durbeej and Campbell, 1999).

Function and ligand binding of dystroglycan

α-Dystroglycan binds to ECM proteins such as laminin-1 and -2, perlecan andagrin. For laminin-1 it has been shown that the E3 fragment, or more preciselythe α1LG4 domain, is a major binding site for α-dystroglycan. Also for laminin-2 and perlecan, the binding sites for α-dystroglycan are within the LG domains

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α β

α

Dystroglycan

Integrin

Laminin

α

β γ

Epithelial cell

Signal transduction

Basment membrane

Nidogen

Fig. 3. A schematic model showing the interaction between laminins and their main receptors integrins and dystroglycan, linking epithelial cells to the basement membrane.

β

21

(Andac et al., 1999; Talts et al., 1999). Laminin-1, -2 and perlecan all shareoverlapping binding sites on α-dystroglycan (Talts et al., 1999). The binding ofα-dystroglycan to the E3 fragment is calcium-dependent and can be inhibited byheparin (Gee et al., 1993; Pall et al., 1996). A recombinant fragment α5LG1-5 ofthe laminin α5 chain has been shown to interact with dystroglycan, but thisfragment was made in bacteria and could lack important post-translationalmodifications (Shimizu et al., 1999). In the present study, dystroglycan-laminin-10/11 interactions were therefore studied more in detail.

Muscle β-dystroglycan binds dystrophin, but it has also been shown tobind the growth factor receptor binding protein 2 (Grb2), an adapter protein thatcan mediate signal transduction through the Ras/Raf pathway (Yang et al.,1995a). This suggests that dystroglycan could be involved in signal transductionas well.

In organ cultures, antibodies that block dystroglycan binding tolaminin-1 perturb epithelial development (Durbeej et al., 1995; Durbeej et al.,2001). The phenotype of dystroglycan null mice also indicates that dystroglycanis important for basement membranes during early embryogenesis. These nullmice die already at E6.5, due to disruption of Reichert’s membrane (Williamsonet al., 1997). Targeted deletion of the E3 domain of laminin α1 chain, containingthe binding site of dystroglycan, also display an early phenotype (Schéele et al.,2001). Further, embryoid bodies that lack dystroglycan cannot organize abasement membrane (Henry and Campbell, 1998). Recently it was shown that α-dystroglycan is the cell receptor that arenaviruses use for infecting cells (Cao etal., 1998). Mycobacterium Leprae binds both α-dystroglycan and laminin-2,which together form a bridge for Mycobacterium Leprae to invade Schwanncells and thereby cause damage to peripheral nerves (Rambukkana et al., 1998).Mutations in dystrophin, sarcoglycans or laminin α2 chain all give rise tomuscular dystrophies, but no dystrophy has been linked directly to dystroglycan,even though decreased expression of dystroglycan has been noticed. However,chimaeric mice lacking dystroglycan in skeletal muscle do develop musculardystrophy (Cote et al., 1999).

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The Integrin family

The large family of integrins connects cells to the surrounding ECM, and theseinteractions initiate cell signaling, which mediate a wide variety of biologicaleffects such as cell growth, death, differentiation and movements.

Integrins are integral membrane glycoproteins and they areheterodimers composed of one α and one β chain, which are non-covalentlylinked to each other. To date, 18 α chains and 8 β chains are known and theyassociate into 24 different integrins (Hynes, 1999). Many α chains associate withonly one β chain, but a few α chains bind to different β chains. For example, α6can associate with both the β1 and the β4 chains. The integrins are often dividedinto subfamilies depending on the β subunit. The β1 subunit can combine with12 different α chains. It is widely expressed and binds many extracellular matrixproteins, such as different laminins. In addition, laminins also bind α6β4 and tosome extent three of the integrins containing the αv subunit (αvβ3, αvβ5 andαvβ8) (Hynes, 1992; Hynes, 1999).

Many cells express multiple integrins, each capable to interact withspecific extracellular proteins. The α/β pairing specifies the ligand bindingabilities of the integrin. Differences in adhesiveness are dependent not only onthe affinity to the ligand, but also on cell surface clustering and cytoskeletalassociations. Binding to the ligands also requires divalent cations such as Mg2+

and Ca2+, and alterations in the divalent cation composition can influence celladhesion (Humphries, 2000; Plow et al., 2000). Most integrins bind more thanone ECM protein, and one ligand can recognize several integrins.

Integrin structure

The N-terminal parts of both the α and the β subunits are important for ligandbinding. The α integrin subunit contains a large N-terminal extracellular domainwith 7 conserved repeats, of which 3 repeats has putative divalent cation-bindingsites (Loftus et al., 1994; Corbi et al., 1987). These seven repeats are folded intoa seven-bladed β-propeller, each containing 4 β-sheets (Springer, 1997). Someintegrins contain an I-domain of 200 amino acids (also known as the A-domain),which is inserted between the second and the third N-terminal repeat. This I-domain has been shown to contain a metal ion-dependent adhesive site andrecognizes binding ligands to the integrin (Kern et al., 1994; Lee et al., 1995).The transmembrane part of the integrin subunits is highly conserved and the

23

transmembrane and cytoplasmic domains are not necessary for ligand binding(Dana et al., 1991). The extracellular parts have been suggested to be involved inthe assembly of the heterodimer (Humphries, 2000), but in one study deletionsin the integrin α6A cytoplasmic domain prevented association with the β1subunit (De Melker et al., 1997). A number of integrin subunits (α3, α5, α6,α7, α8, αv and αIIb) are posttranslationally cleaved in the extracellular partclose to the membrane spanning region, resulting in two chains (one N-terminalheavy chain and one C-terminal light chain) which are hold together by adisulfide bridge (Corbi et al., 1990; Hynes, 1992). Uncleaved α6 integrin canstill dimerize with the β4 subunit (Lehmann et al., 1996) as well as with the β1subunit (Delwel et al., 1996). Further, the cells could attach to laminin-1 afterstimulation with antibodies against the β1 subunit and thus outside/in signalingwas not affected. On the contrary, PMA-induced inside/out signaling wasdisturbed, since the cells could not bind to laminin-1 after PMA stimulation(Delwel et al., 1996). This suggests that the proteolytic cleavage is important forintegrin activation.

The integrin cytoplasmic domain connects the extracellularenvironment with the intracellular structures and interacts directly or indirectlywith the cytoskeletal-signaling network. Both the α and the β cytoplasmicdomains have been shown to be involved in signaling. Several cytoplasmicproteins such as talin, α-actinin and focal adhesion kinase (FAK) bind directly tothe integrin β1 cytoplasmic domain (Burridge and Chrzanowska-Wodnicka,1996; Yamada and Geiger, 1997). The cytoplasmic domain is usually very shortand does not contain more than 50 amino acids, except for the β4 subunit, whichhas a larger cytoplasmic domain with more than 1000 amino acids (Hynes,1992).

Alternative splice variants

Some integrin subunits undergo alternative splicing of their extracellular orcytoplasmic domain in a tissue-type specific manner. The alternative splicingincreases the integrin diversity even more. Alternative splicing of thecytoplasmic domains suggests specific intracellular functions as well as abilityto mediate different signals. A few of the laminin binding integrins includingα3β1, α6β1 and α7β1 undergo alternative splicing. Both the α3 and the α6subunit are spliced into A and B variants. This results in a frameshift, whichleads to two different cytoplasmic domains (Hogervorst et al., 1991; Tamura et

24

al., 1991). The α7 subunit undergoes alternative splicing in the extracellular aswell as in the cytoplasmic domain. In addition to A and B splice variants, α7also has a C cytoplasmic variant and two extracellular variants named X1 andX2 (Ziober et al., 1993).

Five different splice variants of the β1 subunit have been found. Theβ1A variant is the most widely expressed and found in most tissues and cells, butin cardiac and skeletal muscle, for example, the β1A subunit is displaced by theβ1D variant (de Melker and Sonnenberg, 1999).

Integrin interactions and functions

There is a lot of information available about integrin expression, function andtheir different binding partners. Here I will concentrate on a few integrins, whichare main receptors for laminins. Some of these integrins bind only one specificligand but other integrins as for example integrin α6β1 is a receptor for manydifferent ligands. Phenotypes of mice deficient in different integrin subunits aresummarized in table 2.

Integrin α6β1 is a main receptor for laminin-1 and the E8 fragment hasbeen shown to be the binding domain, but integrin α6β1 also binds laminin-2, -4,-5, -8 and -10/11 (Colognato and Yurchenco, 2000; Gu et al., 1999; Kortesmaaet al., 2000). Further, integrin α6β1 binds other types of ligands such as theangiogenic factor Cyr61 and CTGF (Chen et al., 2001). Surprisingly, integrin α6deficient mice survive to birth, but die shortly thereafter with severe blistering ofthe skin and of other epithelia (Georges-Labouesse et al., 1996). The integrin α6subunit also dimerizes with the β4 subunit, and the α6β4 integrin has beenshown to be a major receptor for laminin-5 in hemidesmosomes (Marchisio etal., 1993). The β4 null mice also die shortly after birth with detachment of theepidermis and no hemidesmosomes were found (van der Neut et al., 1996).Patients with mutations in either of the laminin-5 chains suffer fromepidermolysis bullosa. A very similar phenotype is seen in laminin α3 deficientmice, which develop blisters and die at 2-3 days after birth (Kivirikko et al.,1996; Pulkkinen et al., 1994; Ryan et al., 1999). This supports the idea thatintegrin α6β4 is a receptor for laminin-5 and that they are important componentsof the hemidesmosomes. Another integrin subunit that binds laminin-5 is theintegrin α3 subunit. Integrin α3 deficient mice show less severe blisters, but inaddition they have abnormal epithelia in kidneys and lungs (DiPersio et al.,1997; Kreidberg et al., 1996). The sequence similarity of the integrin α3

25

Integrinsubunit

Integrin deficient phenotypes in mice Reference

α1 The mice are viable, but have proliferation defects and adisturbed regulation of collagen synthesis in skin fibroblasts.

(Gardner et al., 1999;Gardner et al., 1996;Pozzi et al., 1998)

α2 The mutant is viable and fertile, but has defects in mammarygland branching, and platelet adhesion to collagen isdefected.

(Holtkotter et al.,2002)

α3 The mutant mice survive birth but die neonatally. Thekidneys and lung developed abnormally, there was reducedbranching morphogenesis and disorganized basementmembrane in the skin.

(DiPersio et al., 1997;Kreidberg et al., 1996)

α4 Embryonic lethal, the mutant had defects in the placenta andthe heart.

(Yang et al., 1995b)

α5 The mutant embryo dies around E10-11, the embryo haddefects in the posterior trunk and the yolk sac mesodemalstructures.

(Yang et al., 1993)

α6 The mice developed normally before birth but die shortlythereafter with severe blisters of the skin and other epithelia.The phenotype was similar to epidermolysis bullosa andhemidesmosomes were absent from the mutant.

(Georges-Labouesse etal., 1996)

α7 The mutant is viable and fertile, but histological analysisshows a progressive muscular dystrophy starting soon afterbirth.

(Mayer et al., 1997)

α8 Most mice die soon after birth due to kidney defects. Manyof the surviving mice have difficulty balancing, consistentwith the structural defects of the inner ear.

(Muller et al., 1997)(Littlewood Evans andMuller, 2000)

α9 The mutant dies 6-12 days after birth with respiratoryfailure.

(Huang et al., 2000)

α10 No apparent phenotype. (Bouvard et al., 2001)α11 To be further characterized. C. Tiger and D.

Gullberg unpublisheddata.

αv 80% of the mutants die in midgestaion, probably because ofplacental defects. 20% are born alive with intracellebralhemorrhages.

(Bader et al., 1998)

β1 Preimplantational lethal did not gastrulate. (Fassler and Meyer,1995)

β4 The homozygous mice die shortly after birth withdetachment of the epidermis and other squamous epithelia.No hemidesmosomes were found.

(van der Neut et al.,1996) (Dowling et al.,1996)

Dystro-glycan

Embryonic lethal dies around E6.5 with a disruptedReichert’s membrane.

(Williamson et al.,1997)

Table 2. Summary of the phenotypes of the laminin receptors.

26

and α6 subunits suggests that they could compensate for each other. Indeed,mice lacking both integrin α3 and α6 died at E16.5 with more severeabnormalities, they seem to have synergistic activities in limbs, lungs and theurogenital system (De Arcangelis et al., 1999). The preferred ligands for integrinα3β1 seems to be laminin -5, -10, and -11, however integrin α3 is also seen infocal contacts on other matrix components such as laminin-1, fibronectin andcollagen IV. In the latter cases it could be that integrin α3β1 is not the main celladhesion receptor but is recruited to mediate secondary responses to variousextracellular matrices (DiPersio et al., 1995).

Integrin αvβ3 is known to bind ligands such as vitronectin, fibronectin,Von Willebrand factor, Cyr61 and denatured collagens in a RGD dependentmanner (Chen et al., 2001; Plow et al., 2000). Recently it was shown thatintegrin αvβ3 also binds recombinant fragment of laminin α5 in a RGDdependent fashion (Sasaki and Timpl, 2001). This suggests that α5 containinglaminin trimers might be ligands for integrin αvβ3 (see results). Integrin αvβ3 issuggested to be involved in angiogenesis and tumor growth (Eliceiri andCheresch, 2001). Deletion of the αv gene is lethal, and the mice exhibitintracerebral and intestinal hemorrhages (Bader et al., 1998). In contrast, micedeficient in integrin β3 subunit develop apparently normal blood vessels(Hodivala-Dilke et al., 1999). There is some evidence for cross talk betweengrowth factor receptors and integrin αvβ3, and integrin αvβ3 co-precipitateswith VEGF receptor 2 as well as with PDGF receptor (Eliceiri and Cheresch,2001).

CELL SIGNALING

The integrin-dependent anchorage of cells to the ECM can activate intracellularsignaling pathways. The composition of the ECM together with the receptorsexpressed on the cell surface decides whether a cell will survive, proliferate orexit the cell cycle and differentiate. Integrins can recruit a number ofintracellular proteins to specialized focal adhesion complexes. Some proteins inthese complexes are structural elements while others mediate signals. There aremany known signaling pathways such as MAP kinase, PI3 kinase, Akt, p38MAPkinase, FAK, SAPK/JNK, and Cytokine/JAK/Stat (Aplin et al., 1998). Here Iwill concentrate on the highly conserved mitogen-activated protein kinase (MAPkinase) cascade and especially on the MEK/ERK pathway.

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The MAP kinase family

MAP kinases are a family of serine/treonine kinases found in all eukaryoticorganisms. They are involved in many cell programs such as cell proliferation,differentiation, cell movements and death. MAP kinase cascades can be dividedinto three classical subfamilies (ERK, p38MAPK, SAPK/JNK). The ERK(extracellular signal regulated protein kinase) family plays a critical role inregulation of cell growth and differentiation. The p38MAPK family is activatedby different types of environmental stress such as ionizing radiation, heat shock,oxidative stress, osmotic shock, UV-light, inflammatory cytokines and growthfactors. p38MAPK regulates proliferation, apoptosis, and cell differentiation.SAPK/JNK (stress activated protein kinase/Jun terminal kinase) family is alsoactivated through environmental stress including UV and γ radiation,inflammatory cytokines and in some instance growth factors. SAPK/JNKpathway has been shown to regulate transcription (Cobb, 1999; Robinson andCobb, 1997).

Each MAP kinase cascade consists of at least three enzymes that areactivated in series (Fig. 4): the MAP kinase or ERK is activated by MAPK/ERKkinase or MEK, which in turn is activated by a MEK kinase or MEKK. TheMEK kinase is activated by interactions with small GTPases and/or other proteinkinases connecting the MAP kinase module to the cell surface receptor orexternal stimuli (Cobb, 1999; Robinson and Cobb, 1997).

Basic regulation of the MEK/ERK pathway

The incoming signal, which can be a hormone, growth factor, differentiationfactor or a matrix protein, stimulates integrins or receptor tyrosine kinases(RTK). Integrins have no intrinsic kinase activity, whereas RTK becomeautophosphorylated when activated. The archetypal Ras exchange factor, Sos(son of sevenless), is brought to the cell membrane by the Grb2 adaptor protein.Grb2 recognizes tyrosine phosphate sites on the receptor or on the receptorsubstrate proteins. Sos enhances GDP release and GTP binding to Ras, whichconverts Ras into its active form (Li et al., 1993; McCormick, 1993). The GTPactivation of Ras functions as an adaptor that binds to Raf and brings it to theplasma membrane where its protein kinase activity is activated and the kinasecascade can start (fig. 5) (Morrison and Cutler, 1997). MEK is activated byphosphorylation of two serine residues. Phosphorylated MEK1/2 in turn

28

phosphorylates MAP kinases ERK1 and ERK2 (also called p42 and p44 MAPkinase). ERK1 and 2 must be phosphorylated on both their threonine andtyrosine residues for full activation. (Cobb and Goldsmith, 1995). Inunstimulated cells ERK1 and 2 are located in the cytoplasm. Phosphorylation ofERK1 and 2 induces dimerization, which seems to be required for translocationto the nucleus (Khokhlatchev et al., 1998). After the nuclear translocation, ERKis thought to phosphorylate a number of transcription factors such as Elk, c-Fosand c-Myc, which in turn serve as important regulators of transcription (Aplin etal., 1998). Phosphorylated ERK has also been found in newly formed focalcontacts, suggesting a role for activated ERK in cell adhesion/cytoskeletalnetwork (Fincham et al., 2000). It is also possible that ERKs are involved innegative feedback mechanisms for upstream components by disassembly of theSos complex and termination of Ras activity (Kolch, 2000).

RafPP

P

MEK1/2P P

MAPK/ERK1/2

P P

Growth DifferentiationDevelopment

Growth factors Mitogens

Receptor

Cytosolic substrates

Elk gene

Nucleus

TranscriptionP

Membrane

Cytoplasm

Fig. 4. Schematic picture over the ERK/MAPK pathway.

29

The scheme outlined above is simplified, since a large number ofcomponents can regulate the ERK pathway at many levels of the cascade. Thisfine-tuning is probably crucial for proper signaling, but is still poorlyunderstood. A further complication is the accumulating evidence of extensivecross talk between different pathways. For example, PI3K can also mediateMAP kinase activation. PI3Ks have been found to have both lipid kinase activityand serine/threonine kinase activity. When domains responsible for PI3K lipidkinase activity were mutated, the mutant could still activate MAP kinase but notAkt/PKB (protein kinase B) (Bondeva et al., 1998).

Integrins and MAP kinase signaling

Although both integrins and RTK can activate ERK, there are some notabledifferences. Signals from growth factors usually lead to an immediate strong butshort response lasting for a few minutes. The signals produced by integrins areas a rule of lower amplitude, and activated after 30-60 minutes and thensustained for a few hours (Aplin et al., 1998). Interestingly, a third type of ERKactivation was recently demonstrated for the CCN growth factor family. Twomembers of this family were shown to bind integrins α6β1 or αvβ3, which leadto a sustained activation of ERK lasting for up to 12 hours (Chen et al., 2001).At present, it is unclear why ECM components do not stimulate a similar longlasting activation through integrins. One possibility is that some domains of thelarge ECM components bind other receptors that act as inhibitors of ERKactivation. Another possibility is a cross talk between integrins and growthfactor receptors.

Activation of the MAP kinase pathway by integrins differs at somepoints from the basic regulation of the MEK/ERK pathway. Integrins transducesignals by associating with adaptor proteins that connect the integrins to thecytoskeleton, cytoplasmic kinases or transmembrane growth factor receptors.Integrin signaling and cytoskeleton assemblies are tightly linked. When integrinsbind to an ECM protein, they become clustered in the plane of the cellmembrane and associate with a cytoskeletal and signaling complex thatpromotes the assembly of actin filaments (Giancotti and Ruoslahti, 1999).

Some integrin-transduced signals are mediated by activation of FAK.FAK might interact directly with the cytoplasmic part of the integrin β subunit orindirectly by cytoskeletal proteins such as talin and paxillin (Akiyama et al.,1994; Aplin et al., 1998; Miyamoto et al., 1995). When FAK is phosphorylated,

30

a binding site for Src or Fyn is creating a stable complex (Schaller et al., 1994;Schlaepfer and Hunter, 1997). Src then phosphorylates a number of focaladhesion components. Besides this, Src can phosphorylate FAK, which creates abinding site for the Grb2-Sos complex, linking FAK to the MAP kinase pathway(Calalb et al., 1995; Schlaepfer and Hunter, 1997). FAK has also been shown toactivate the MAP kinase pathway via Shc, which associates with Grb2(Schlaepfer and Hunter, 1997; Schlaepfer et al., 1998). In addition, integrins canalso mediate MAP kinase activation without FAK involvement. In this case theintegrin α subunit is apparently involved. Ligation of the integrin activates Fyn,which associates with caveolin-1, leading to phosphorylation of Shc andformation of the Shc-Grb2 complex, which finally activates the MAP kinasepathway (fig. 6.) (Wary et al., 1996; Wary et al., 1998; Mainiero et al., 1997).

What is known about laminins and MAP kinase signaling?

Laminin-integrin interactions can in some settings activate ERK, but due to thelarge number of ligands and receptors, information is only available for someinteractions. Further, the cell types used differ and that makes it even moredifficult to draw any clear conclusions, and the studies some times contradicteach other.

Laminin-5 was reported to activate the Ras-ERK and Rac-Jnksignaling pathways in keratinocytes and the signaling was mediated throughintegrin α6β4 via Shc and Grb2. It was proposed that the major lamininreceptors integrin α6β1 and α3β1 do not belong to the integrins coupled to theERK pathway (Mainiero et al., 1997; Wary et al., 1996). In addition, Wary et alreported that endothelial cells attached to laminin-1 or laminin-4 were not ableto stimulate ERK, through the presumed adhesion receptor, integrin α2β1 (Waryet al., 1996).

The results above are in contradiction to what other investigator report.It has been shown that laminin-5 activates ERK phosphorylation in epithelialcells through integrin α3β1 leading to cell proliferation, however laminin-1 wasnot able to activate ERK in these cells (Gonzales et al., 1999). In macrophages,laminin-1 did not activate ERK, but a shorter laminin α1 peptide did. Theinvolved receptor has in this case not yet been identified (Khan and Falcone,2000). Further, it has been shown that laminin-1 can activate ERK in fibroblasts(Chen et al 2001; Fincham et al 2000). Interestingly, macrophages expressingeither integrin α6A or α6B variant did attach to laminin-1, but only cells

31

Grb2SOS

Ras

RafP

P

P

P P

ERK1/2

P P

Elk gene

Pax

Tal

Nucleus

Matrix

Transcription

Membrane

Cytoplasm

β

α

FAK

Tal Pax

ShcGrb2

SOS

Src

Fyn

Shc

Fyn Yes

Integrinβ

α

Cav-1

Integrin

ERK1/2ERK1/2

P P P

P

ERK1/2ERK1/2

P P PP

P

Fig.5. Schematic outline of some of the signaling molecules activated by integrins involved in the MEK/ERK signaling pathway. Arrows indicate activation. See text for details.

MEK1/2

32

expressing the integrin α6A were able to phosphorylate MEK and ERK (Shaw etal., 1995; Wei et al., 1998). However, both α6Aβ1 and α6Bβ1 expressingmacrophages were able to induce ERK activation when attached to fibronectin(Wei et al., 1998). Dystroglycan has been reported to have an interplay withintegrin α6 on β-cells attached to laminin-1, where integrin α6 promotesproliferation through MEK/ERK signaling and dystroglycan cell differentiation(Jiang et al., 2001). In another report, β-casein expression was inhibited whenintegrin α6 or β1 subunit was blocked on mammary epithelial cells adhering tolaminin-1. When E3 fragments and heparin were added, both cell shape changesand β-casein expression induced by laminin-1 were inhibited. This indicates thatthe heparin-binding region within the laminin E3 fragment could be one of theparticipating parts in the interaction of laminin-1 with mammary epithelial cells(Muschler et al., 1999). The receptor involved could be dystroglycan since itbinds to the E3 fragment of laminin-1 (Andac et al., 1999; Gee et al., 1993).How dystroglycan could be involved in regulation of ERK is unknown, but it isknown that β-dystroglycan interacts with the Grb2 adaptor protein (Russo et al.,2000; Yang et al., 1995a). These conflicting data with laminins involved in ERKsignaling are suggesting that ERK activation in response to laminins could beregulated at many levels.

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PRESENT INVESTIGATIONS

Specific aims of the thesis

• To compare the distribution of the laminin α1 chain and dystroglycan inadult mouse tissues.

• To study epithelial cell binding to different laminin isoforms• To identify adhesion receptors for laminin-10/11 • To study signal transduction mediated by laminin receptors

RESULTS AND DISCUSSION

The laminin α1 chain has a restricted distribution in adult mouse tissues (I)

Even though laminin-1 was the first discovered laminin, there have beenconflicting data about the expression of its α1 chain. To study the distribution ofthe laminin α1 chain, many investigators have used the monoclonal antibody4C7 (Engvall et al., 1986; Engvall et al., 1990; Virtanen et al., 1995). However,it was shown that 4C7 instead reacted with the laminin α5 chain (Kikkawa et al.,1998; Tiger et al., 1997). This was in agreement with reports that showed abroad expressed of the laminin α5 chain at adult stage (Miner et al., 1995; Mineret al., 1997). The aim of this study was to clarify the expression pattern of the α1chain in adult mouse tissues. We used a well-characterized rat monoclonalantibody (mAb 200) against the E3 fragment of the laminin α1 chain inimmunofluorescence of frozen sections (Sorokin et al., 1992). We found strongreactivity for the α1 chain in several but not all organs. The reactivity wasrestricted to epithelial basement membranes.

The reproductive system both in male and female mice stained positivefor the α1 chain. Prominent staining was seen in testis, epididymis and prostate.In the female reproductive organs the ovary, placenta and the mammary glandsexpressed laminin α1. As previously reported, a prominent expression of thelaminin α1 chain was seen in the proximal tubes of the kidney (Ekblom et al.,1990; Sorokin et al., 1992) and a faint staining of the urinary bladder. In thecentral nervous system, larger blood vessel walls expressed the laminin α1chain. Astrocytes or invaginated epithelial cells rather than endothelial cells arethe probable sources of laminin α1 in these tissues (Sixt et al., 2001). Laminin

34

α1 expression was also seen in the pia mater covering the brain. In sensoryorgans the vitreous chamber and the lens of the eye expressed the α1 chain.

In the gastrointestinal tract the α1 chain was detected at the basal sideof the dentine of unerupted tooth. Yet, no staining was detected with apolyclonal antibody against all three laminin-1 chains (α1β1γ1). This couldindicate the presence of a novel laminin isoform, masking of the epitope for β1and γ1 chains, or unspecific reactivity of mAb 200 in this location. In addition totooth, Brunners’s gland was the only tissue expressing the α1 chain in thegastrointestinal tract. In endocrine organs, the α1 chain was expressed in thecortex of the adrenal gland. No staining was seen in muscle, heart, cartilage, fat,or Schwann cells.

The study established that the distribution of the laminin α1 chain isrestricted to some epithelial basement membranes in the adult mouse body. Avery similar distribution of the α1 chain was recently demonstrated for adulthuman tissues by novel monoclonal antibodies against the human laminin α1chain (Virtanen et al., 2000). Our findings are also strongly supported by recentquantitative measurements, which demonstrate substantial amounts of lamininα1 chain in those adult mouse tissues that stain positively with mAb 200.However, a few other adult mouse tissues that show no reactivity with mAb 200(esophagus, intestine, liver, spleen and stomach) nevertheless express the α1chain. This could be demonstrated by using a polyclonal antibody against the N-terminal part of the α1 chain. Apparently, the C-terminal part of the laminin α1chain is masked by other ECM components in these tissues (Sasaki et al., 2002).Taken together, current data provide strong support for the view that laminin α1chain is almost exclusively expressed in epithelial basement membranes both inembryonic and adult tissues. This distribution is conserved between mice andman, suggesting conserved functions for epithelial cells. In these locations,several β and γ chains might associate with the α1 chain, and the trimercomposition should be experimentally proven for each case.

Broad distribution of dystroglycan in adult mouse tissues (II)

Integrins and dystroglycan are main laminin receptors. Dystroglycan is encodedby a single RNA transcript but is later posttranslationally cleaved into two partsresulting in an extracellular part, α-dystroglycan (156kDa), and atransmembrane subunit, β-dystroglycan (43kDa) (Ervasti and Campbell, 1991;Ibraghimov-Beskrovnaya et al., 1992). Dystroglycan was first shown to bind

35

laminin-2 in muscle, connecting the cells to the ECM (Ervasti et al., 1990), butnorthern blot analyses showed that dystroglycan is expressed in non-muscletissues as well (Ibraghimov-Beskrovnaya et al., 1992; Ibraghimov-Beskrovnayaet al., 1993). Further, dystroglycan was shown to bind laminin-1 (Ibraghimov-Beskrovnaya et al., 1992). Immunofluorescence and in situ hybridization ofembryonic tissues identified epithelial cells as major sources of non-muscledystroglycan. In many embryonic tissues, laminin α1 chain and dystroglycan arecoexpressed (Durbeej et al., 1995), suggesting that the binding of the α1LG4module to dystroglycan is involved in epithelial development (Andac et al.,1999). This is supported by data showing that branching epithelialmorphogenesis can be perturbed in vitro by antibodies that block binding ofdystroglycan to α1LG4 (Durbeej et al., 1995; Durbeej et al., 2001; Kadoya et al.,1995). The early lethality of dystroglycan null mice at E6.5 also points to thispossibility (Williamson et al., 1997). All these data suggest an important role fordystroglycan in basement membrane assembly and mouse development. It wastherefore of interest to analyze dystroglycan expression in various adult mousetissues.

Western blot analysis of the β-dystroglycan subunit showed a broadtissue distribution of the 43kDa protein. Expression was seen in all tested tissuessuch as in kidney, liver, stomach and uterus. Two different antibodies were usedfor immunofluorescence; FP-B detecting α/β-dystroglycan and AP83recognizing only the β subunit. Both antibodies stained the sarcolemmalmembrane in skeletal muscle as expected and previously shown (Ibraghimov-Beskrovnaya et al., 1992). At the sarcolemmal membrane dystroglycan was co-localized with the laminin α2 chain (Schuler and Sorokin, 1995). Dystroglycanwas broadly expressed in the digestive tract. Expression was seen at the surfaceepithelium and in the smooth muscles of the following tissues, salivary gland,pancreas, intestinal glands, villi, liver, trachea and kidney. Laminin α2 chain wasco-localized in salivary gland, pancreas, intestinal glands, and trachea. In liver, apolyclonal antibody against laminin-1 stained the sinusoidal linings and largerblood vessels but no staining was seen with antibodies against the laminin α1 orα2 chains.

Dystroglycan was also expressed in the reproductive organs at theepithelial cells lining the basement membranes. Expression was detected in themammary glands, uterus and testis. In mammary gland and testis both lamininα1 and α2 chain co-localized with dystroglycan. In addition, α/β-dystroglycanwas expressed in all epidermal layers of the skin. In a few locations such as in

36

the intestinal glands, spermatogenic cells and Sertoli cells in the testis and in allepidermal layers of the skin dystroglycan staining was seen over the entire cellsurface. Dystroglycan has previously been detected at cell-cell contacts byBelkin and Smalheiser (Belkin and Smalheiser, 1996). In a few locationsdystroglycan was only detected by one of the antibodies, such as in the tubularbasement membrane of the kidney and at the surface epithelial basementmembrane in the villi of the small intestine. Here, only the FP-B antibody wasreactive, indicating that α-dystroglycan is expressed in the absence ofβ−dystroglycan, or that the AP83 epitope is masked.

In conclusion, dystroglycan has a broader expression pattern than thelaminin α1 or α2 chains in adult mouse. It is expressed at the basal side of mostepithelial cells in several analyzed tissues and this suggests that dystroglycan is acell receptor linking the epithelial cells to the basement membrane. Apericellular expression of dystroglycan was seen in epidermis, which could pointto a function for dystroglycan even in hemidesmosomes, but this needs to befurther investigated. It is also possible that α-dystroglycan is present without β-dystroglycan at some places, as in the villi of the small intestine and tubularbasement membrane of the kidney. It has been suggested that α-dystroglycan isexpressed in rat cerebellum without β-dystroglycan (Tian et al., 1996). However,it could be a problem of epitope masking. In most tissues dystroglycan is co-localized with laminin α1 or α2 chain, which are known dystroglycan ligands,but laminin α5 chain has an even broader distribution and could be a ligand aswell (Miner et al., 1995; Miner et al., 1997). Recently it was shown that arecombinant fragment of the laminin α5 globular domain produced in bacteriawas able to bind α-dystroglycan (Shimizu et al., 1999). In paper IV we thereforecompared the binding affinity of laminins to α-dystroglycan.

Laminin-10/11 is a strong adhesive substrate for epithelial cells andmediates ERK activation through integrin α6Aβ1 (III, IV)

Cells are probably differentially influenced by adhesion to different laminins,but this has not yet been extensively studied. The monoclonal antibody 4C7detecting the laminin α5 chain (Tiger et al., 1997) has been used to isolatelaminins from placenta. Such commercially available laminins have been widelyused. In some reports this laminin is still incorrectly, referred to as humanlaminin-1.

37

To evaluate findings obtained with laminins isolated with the 4C7antibody, it is useful to characterize them in further detail. We thereforeidentified the chain composition of human placental laminin purified with the4C7 antibody. Western blot analysis showed that this preparation contained theα5, β1, β2 and the γ1 chains and no α1 chain. Based on these Western blots weassumed that this preparation was containing laminin-10 (α5β1γ1) and -11(α5β2γ1). It will here be referred to as laminin-10/11. Under reduced conditionsthe α5 chain seemed to be slightly degraded, but posttranslational cleavage hasbeen reported to give rise to bands sized 380, 350 and 210kD of the laminin α5chain (Miner et al., 1997). To our knowledge, this is the only commerciallyavailable laminin-10/11. Our characterization of this laminin was important forthe identification of integrin α6β1 (Gu et al., 1999) and Lutheran glycoprotein(Parsons et al., 2001) as receptors for laminins containing the α5 chain.

We compared the ability of two epithelial like cell lines to adhere todifferent laminin isoforms. The WI-26 VA4 lung epithelial cell line attachedwell to laminin-10/11 and to fibronectin. Adhesion to laminin-1 and a mixture oflaminin-2 and -4 was slightly less effective. A similar binding profile wasestablished for the WCCS-1 cell line, derived from Wilm’s tumor (Talts et al.,1993). Laminin-10/11 has also been shown to be adhesive for other cell typesand was shown to be inhibited by antibodies against either integrin α3β1(Gehlsen et al., 1989; Kikkawa et al., 1998), integrin α6β1 (Gu et al., 1999) orα6β4 (Kikkawa et al., 2000). Yet, in our initial attempts adhesion of WCCS-1 orWI-26 VA4 cells to laminin-10/11 could not be inhibited with antibodies againstintegrin β1, α3 or α6 in one-hour assays (study III). However, in subsequentassays carried out at 10 minutes after attachment, a combination of antibodiesagainst integrin α3 and α6 completely inhibited WCCS-1 or WI-26 VA4 cellbinding to laminin-10/11. In these assays, blocking with integrin β1 gave a totalinhibition of WCCS-1 cell adhesion to laminin-10/11, whereas a residualadhesion of 10% was still seen with WI-26 VA4 cells. Antibodies against eitherα3 or α6 still failed to inhibit adhesion of these cell types, even in these short-term assays. This suggests that one of these receptors is enough to promote celladhesion to laminin-10/11, as reported for other cell lines (Gehlsen et al., 1989;Gu et al., 1999; Kikkawa et al., 1998). The most likely reason why laminin-10/11 binding of certain carcinoma cells could be inhibited by antibodies againstintegrin α3β1 (Kikkawa et al., 1998) and to primitive hematopoietic cells byantibodies integrin α6β1 (Gu et al., 1999) is that these cell lines express only oneof these receptors.

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Laminins can in some cases activate ERK phosphorylation in cells.Since both laminin-1 and -10/11 promoted adhesion of WCCS-1 and WI-26VA4 cells, it was of interest to compare the ability of laminin-1 and -10/11 toactivate ERK in these cell lines. The laminin receptors expressed by WCCS-1 orWI-26 VA4 cell were analyzed in more detail by immunoprecipitation andFACS analyses. This revealed that both cell lines lacked integrin α6β4 butexpressed several β1 integrins including integrin α6β1 and α3β1. Althoughlaminin-10/11 was a slightly stronger adhesion complex than laminin-1 for bothcell lines, both laminins activated ERK only in the WI-26 VA4 cell line. Thesefindings clearly revealed that adhesion to a laminin does not invariably lead toactivation of a signaling cascade, even though the same cellular receptors seemto be used for attachment. This suggested that activation of ERK could becritically dependent on the expression of a proper receptor, with capacity toinitiate a signaling cascade.

Three different assays were used to identify the receptors involved inERK activation in the responding WI-26 VA4 cell line. First we tested theability of antibodies against integrin α3 and α6 to influence ERK activation incells attached to laminins. In this assay antibodies against the integrin α6 subunitreduced laminin-induced ERK activation. To confirm this finding, we tested theability of the different antibodies to influence ERK activation. Cells wereallowed to attach to the antibodies, or were incubated with the antibodies insolution. In both these assays, antibodies against the α6 subunit activated ERK,whereas the other antibodies failed to do so. These findings are not in conflictwith the results showing that the α6 antibody reduced ERK activation in cellsallowed to attach to laminin substrates. A reasonable assumption, supported byour data, is that the natural ligands, the laminins, are more efficient activators ofERK than the integrin antibodies.

To clarify why adhesion to the laminins only activated ERK in one ofthe cell lines when both expressed integrin α6, we analyzed the expression of theintegrin α6 splice variants (α6A and α6B) which have different cytoplasmicdomains. The responding cell line expressed both splice variants but α6A moreprominently, whereas the non-responding cell line expressed only α6B. This isin complete agreement with a report suggesting that α6A but not α6B isinvolved in ERK activation (Wei et al., 1998). We conclude that laminin-1 and -10/11 share the ability to activate ERK via the α6β1 integrin in epithelial cells,and that this response is regulated by the cytoplasmic domains of the α6 subunit.

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Laminins could be more efficient activators of ERK than the α6antibodies, possibly since the natural ligands stimulate several receptors.However, we found that dystroglycan antibodies reduced ERK activationinduced by integrin α6 antibodies. This raised the interesting possibility thatnatural ligands for dystroglycan might act as physiological negative regulators ofERK activation. Cells attached to wells coated with integrin α6 antibodies weretherefore incubated with recombinant laminin fragments known to bind α-dystroglycan with varying affinities (Andac et al., 1999; Talts et al., 1999). Onlythe fragments with high affinity to α-dystroglycan reduced ERK activationinduced by the integrin α6 antibodies. Based on these observations, we suggest anovel role for dystroglycan as a suppressor of ERK activation. Differentdomains of the large laminins can apparently have opposing roles in signaltransduction. In this context, it is notable that the CNN family of growth factorscan promote a much more sustained integrin-mediated activation of ERK thanthe laminins. One possibility is that this is due to the opposing influence ofdiscrete domains of the large extracellular matrix components. Our findingscould also be relevant for the interpretation of organ culture studies carried outwith function blocking antibodies. It has been clearly demonstrated that bothintegrin α6 or dystroglycan antibodies influence epithelial cell development inorgan cultures of embryonic tissue in distinct ways (Durbeej et al., 1995; Falk etal., 1996).

Since binding of dystroglycan from an endothelial cell line torecombinant fragment α5LG1-5 has been reported, we compared binding ofpurified laminin-1 and -10/11 to purified dystroglycan from muscle andepithelium in overlay assays, and tested dystroglycan binding to these lamininsin solid phase assays. In solid phase assays some binding to the α5 containinglaminin-10/11 was noted, but compared with the much stronger binding to theα1 containing laminin-1, it was of modest affinity as also shown for LG domainsof the laminin α4 chain (Talts et al., 2000). Overlay assays also demonstratedbinding of laminin-10/11 to dystroglycan isolated both from muscle and a tissuerich in epithelium (kidney). Our quantitative binding studies show a clearhierarchy among laminin isoforms for α-dystroglycan binding. This is inreasonable agreement both with structural predictions (Hohenester et al., 1999;Timpl et al., 2000) and the report that α5LG1-5 fragment can interact withdystroglycan (Shimizu et al., 1999).

The biological significance of the differential binding of laminins todystroglycan remains to be clarified. Recently, dystroglycan has been identified

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as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) andLassa fever virus (LFV). LCMV and laminin-1 use, in part, an overlappingbinding site on α-dystroglycan, and the ability of an LCMV isolate to competewith laminin-1 for receptor binding is determined by its binding affinity to α-dystroglycan. This competition of the virus with ECM molecules for receptorbinding might explain the recently found correlation between the affinity ofLCMV binding to α-dystroglycan, tissue tropism, and pathological potential(Kunz et al., 2001). Taken together, our current observations on the distributionof laminin-1 in adult tissues and the binding studies with dystroglycan raise thepossibility that LCMV binding to dystroglycan could occur more efficiently inlocations lacking laminin-1. Although this remains to be shown, it provides oneexample that mapping the expression patterns of the laminins and measurementsof binding affinities to cellular receptors could be relevant for seeminglydifferent issues in biology and medicine.

In conclusion, different laminin isoforms have varying capacities tomediate cell adhesion and different laminin receptors are involved. Bothlaminin-10/11 and laminin-1 have the ability to activate ERK signaling. Onlyone of the two epithelial cell lines were able to signal in contact with theselaminins even though both cell lines used integrin α3β1 and α6β1 for adhesion.The signaling was mediated by integrin α6Aβ1 and the non signaling cell line,WCCS-1, only expressed the integrin α6B splice variant compared to WI-26VA4, which expressed both splice variants. We also propose a novel role fordystroglycan as a suppressor of ERK activation and we show that α5 containinglaminins are ligands for dystroglycan.

Laminin α5 containing laminins are ligands for integrin αvβ3, and areinvolved in FGF and VEGF stimulated proliferation of ECV304 cells (V)

The human ECV304 cell line is particularly useful for analyses of three-dimensional tube formation mediated by basement membrane components(Hughes, 1996). However, receptors involved in such processes have not beencharacterized, and may differ for distinct laminins. It was therefore of interest toanalyze the attachment of these cells to distinct laminins, and to identify theinvolved receptors. ECV304 cells adhered to all tested laminin isoformsincluding laminin-1, -2/4 and -10/11. The potent adhesive capacity of laminin-10/11 (Ferletta and Ekblom, 1999) was confirmed. Unexpectedly, these cells didnot adhere at all to fibronectin in our hands despite previous data that fibronectin

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can influence signaling in these cells. Antibody inhibition studies showed thatlaminin-1 binds via integrin α6β1, and laminin-2/4 via the α6β1 and α3β1combination, as could be expected from previous data. ECV304 attachment tolaminin-10/11 was more complex. Antibodies against integrin α3 blocked celladhesion by 80%. A total inhibition was seen with the integrin α6 and α3antibody combination, but also by combining the α3 antibody either withantibodies against αvβ3 or RGD peptides. For the β-subunits a completeinhibition was only seen when antibodies against β1, β3 and β4 were combined.This raised the possibility that laminin-10/11 use integrin αvβ3 as a co-receptor,which was confirmed in solid phase assays. Both the commercial laminin-10/11complex and a laminin-10 trimer bound αvβ3 in a cation-dependent fashion.This could be inhibited by certain RGD peptides. While our work was inprogress, another study demonstrated that a recombinant fragment of mouselaminin α5 chain can bind integrin αvβ3 (Sasaki and Timpl, 2001). Our dataclearly show that an intact laminin trimer can interact with integrin αvβ3. This isin contrast to intact laminin-1, which cannot bind integrin αvβ3, even tough theP1 fragment of laminin-1 can do so (Aumailley et al., 1990; Pfaff et al., 1994).Integrin αvβ3 has earlier been shown to bind in a RGD dependent manner toother ligands such as vitronectin and fibronectin (Plow et al., 2000) and in aRGD independent manner to matrix metalloproteinases (Eliceiri and Cheresch,2001). Both mouse and human laminin α5 chain have been shown to contain twoRGD sites each (Miner et al., 1995; Nagase et al., 1998).

It is known that growth factors together with laminins can enhanceproliferation (Drago et al., 1991). To test this possibility, we analyzedproliferation of ECV304 cells attached to laminins in the presence or absence ofVEGF or FGF. Both FGF and VEGF enhanced proliferation on cells attached tolaminin-10/11, but not on the other substrates. This suggests a specific role forthe adhesive receptors unique for laminin-10/11. To identify this receptor,proliferation assays were carried out in the presence of integrin antibodies. Theantibody against integrin αvβ3 decreased the additional proliferation induced bythe presence of FGF and VEGF, whereas the other tested antibodies failed to doso. In addition, proliferation also decreased when an inhibitor of MEK wasadded, suggesting that the MEK/ERK signaling pathway is necessary for theproliferation. The phosphorylation of MEK and ERK was very weak on gelatinand on laminin-1, but considerable phosphorylation occurred when cells wereattached to laminin-2/4 and laminin-10/11. Phosphorylation was also seen whenECV304 cells adhered to antibodies against integrin α6 and αvβ3. We conclude

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that the combined influence of growth factor receptors and integrin αvβ3enhances proliferation of ECV304 cells. One necessary signaling pathway forthis seems to be αvβ3-dependent ERK activation. Interestingly, integrin α6antibodies were equally potent activators of ERK as integrin αvβ3 antibodies,and yet integrin α6 could not be linked to enhanced proliferation. This indicatesthe presence of biologically distinct signaling pathways that can be activated bythe discrete integrins. The nature of these pathways remains to be explored.

It is well known that integrin αvβ3 can be associated with growthfactor receptors. Such interactions are emerging as possible physiologicallysignificant signaling platforms (Eliceiri and Cheresch, 2001). This associationmay initially occur in the extracellular space (Miyamoto et al., 1996),emphasizing the importance to define ligands for integrin αvβ3 in locations werethis integrin plays a major role. αvβ3 is considered to be important for cancerprogression, and has also received considerable attention in angiogenesisresearch. However, it is unclear whether it acts as an inhibitor or stimulator ofangiogenesis (Reynolds et al., 2002). Further advances may require a betterknowledge of the many physiological ligands expressed at sites of angiogenesis.Several basement membrane components can interact with αvβ3. This includesangiogenesis inhibitors such as endostatin (Rehn et al., 2001) and tumstatin(Maeshima et al., 2001), which are fragments of basement membrane collagens.Our data show that an intact laminin trimer can interact with αvβ3. It willtherefore be of interest to analyze the role of α5-laminins and their integrinreceptors in angiogenesis.

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CONCLUDING REMARKS AND FUTURE PERSPECTIVES

This thesis has mainly been focusing on the behaviors of epithelial cells incontact with laminins containing either the α1 or the α5 chain. The receptorsinvolved in these interactions have also been investigated. Figure 6 summarizesthe main conclusions and is based on the results from different types of celladhesion assays and solid phase results.

α β

Integrin

Signal transduction

Fig. 6. Schematic model over the interactions between laminin-10/11 and their integrins receptors and dystroglycan, linking laminin-10/11 to the epithelial cell and to the MAP kinase pathway.

Dystroglycan

α

β

α3β1α6β1α6β4

αv β3

α5

γ1

β1/2

MEK1/2P P

ERK1/2

P P

gene

Nucleus

TranscriptionP

transcriptionfactor Proliferation

(αvβ3)

VEGFFGF-2

Laminin-10/11

Membrane

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Recently it was shown that LCMV and LCM, in order to enter the hostcell, use the same binding domain on dystroglycan as laminin-1. The receptorbinding affinity between viruses for α-dystroglycan vary, some can displacelaminin-1 and others not (Kunz et al., 2001). Here we show that dystroglycanhas a much broader tissue distribution compared to laminin α1 chain. Thissuggests that viruses are competing with other ligands than laminin-1 in mosttissues. α5 containing laminins could be these ligands. The tissue distribution ofthe α5 chain is as broad as that of dystroglycan (Miner et al., 1995; Miner et al.,1997). We have also shown that dystroglycan binds laminin-10/11 with loweraffinity than laminin-1. This suggests that it could be even easier for viruses todisturb the laminin α5-dystroglycan interaction. With this in mind, it would bevery interesting to study which domains are involved in the laminin α5-dystroglycan interaction and if viruses use the same domains.

Integrin αvβ3 is suggested to be involved in angiogenesis and tumorgrowth (Eliceiri and Cheresch, 2001) and integrin αvβ3 interacts withangiogenic inhibitors such as endostatin and tumstatin (Maeshima et al., 2001;Rehn et al., 2001). We show that integrin αvβ3 bind α5 containing laminins. Itwould therefore be very interesting to investigate the role of laminin α5fragments versus αvβ3 in angiogenesis.

Laminins are able to signal through the MAP kinase pathway and wehave suggested integrin α6Aβ1 as the mediating receptor. In addition, wepropose an opposing role for dystroglycan. Target deletion of the C-terminaldomain, E3, of laminin α1 chain in mice are embryonic lethal (Schéele et al.,2001), which also indicate an important role of the E3 receptor dystroglycan. E3null cells would not only show the importance of the E3 fragment, they couldalso serve as an excellent tool for investigating dystroglycans role in signaling inembryoid bodies and during early development. Integrin α6 has been targetingdeleted in mice and these mice die at birth (Georges-Labouesse et al., 1996). Tocompare the ability of cells from these null mice to signal would also beinteresting. In addition, the branching morphogenesis in kidney organ cultures isinhibited when integrin α6 or dystroglycan are blocked (Durbeej et al., 1995;Falk et al., 1996). It would therefore be of interest to analyze the MAP kinaseactivation in these cultures.

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POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA

Cellers samspel med omgivande proteiner

En vävnad består av celler men en stor del av vävnadens volym är ocksåextracellulärt utrymme som fylls ut av något som kallas bindväv. Bindvävenbestår av olika proteiner och polysackarider som utsöndras av cellerna. Dessaproteiner sätts ihop till ett nätverk i nära kontakt med cellerna som producerardem. Man kan säga att bindväven utgör byggnadsstommen i kroppen. Beroendepå vilket organ och i vilket utvecklingsstadium organet befinner sig varierarandelen bindväv och dess proteinuppsättning. Förutom att bindväven stabiliserarvävnaden så har den en mycket aktiv och komplex roll, och reglerar cellernasuppträdande, påverkar cellernas utveckling, förflyttning, delning, form ochfunktion.

Basalmembran är en specialiserad typ av bindväv. Basalmembran ärett tunt nätverk som omger muskel-, fett-, och nervceller, men finns också underandra celltyper som epitel- och endotelceller. Basalmembranet kan även delaupp vävnaden i olika delar och fungerar då som en barriär eller som ett filter förcellerna. Även basalmembranen innehåller olika proteiner beroende påvävnadstyp och i vilket utvecklingsstadium de är. Basalmembran består ihuvudsak av två proteinfamiljer, laminin och kollagen typ IV, och det är dessaproteiner som bildar det tunna nätverket. Jag har främst studeratlamininfamiljen, som består av 15 medlemmar numrerade 1-15. Varje laminin äruppbyggd av tre proteinkedjor en α-, en β- och en γ-kedja, och tillsammansbildar de ett större korsliknande protein. Förändringar i generna för dessa kedjorleder till olika muskeldystrofier och flera olika hudsjukdomar, cancer ochtroligen också till nervsjukdomar. Det har även visat sig att bakterier och viruskan använda basalmembranet som en länk för att infektera celler. När man hartagit bort generna för olika lamininkedjor i möss, man har gjort en så kalladknockout vilket innebär att genen som kodar för ett visst protein är borta ochproteinet kan inte bildas längre, har det visat sig att nästan alla kedjorna ärlivsviktiga för att mössen ska kunna överleva. Saknar de en av kedjorna dörmössen oftast redan innan födseln.

I avhandlingens första del har jag bland annat studerat var laminin α1-kedjan är uttryckt i fullvuxna möss. Det visade sig att den endast fanns på ettfåtal ställen i kroppen medan den i musfoster var något mer uttryckt. I tidigarestudier av laminin α1-kedjan har man använt en antikropp som istället kände

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igen en annan lamininkedja, nämligen laminin α5-kedjan. Detta har gjort att manlänge trodde att laminin α1-kedjan fanns i stort sett i alla basalmembran vilket vinu visat att så är det inte. Laminin-1, som består av kedjorna α1, β1 och γ1,använder sig ofta av en mottagare eller receptor på cellen som kallas fördystroglycan. Vi har också studerats var dystroglycan finns i den vuxnamuskroppen. Dystroglycan visade sig finnas i flera organ, i vissa organ fannsden tillsammans med laminin α1-kedjan och i andra organ tillsammans med α2-kedjan. Dystroglycan verkar vara en mycket viktig receptor som binder cellernatill basalmembranet.

I andra delen har vi studerat hur celler i odling reagerar när de träffarolika sorters laminin. Vi kunde se att cellerna fäste sig olika bra till de olikalamininerna och att de fick olika utseenden beroende på vilken lamininsort defäste sig till. Vidare har vi studerat vilka receptorer cellerna använde för att fästasig till respektive laminin och vi upptäckte att de använde olika receptorerberoende på vilket laminin de satte sig på.

Sista delen i avhandlingen handlar om hur laminin kommunicerar medcellerna, dvs. vilka signaler laminin skickar till dem. Man har länge trott attbindväv eller basalmembran endast stabiliserar cellen men så är det inte.Receptorerna på cellen kopplar nämligen ihop proteinerna på utsidan av cellenmed proteinerna på insidan. Vi kunde se att flera lamininer startadecellsignaleringen via vissa av cellernas receptorer som i sin tur aktiveradeproteiner inuti cellen. Gav vi dessutom cellerna tillväxtfaktorer (vitaminer ochaminosyror är exempel på tillväxtfaktorer) så påverkade dessa tillsammans medlaminin cellernas delning. Det gick också att stoppa signaleringen om mantillsatte kända blockerande ämnen.

Cellernas kommunikation med sin omgivning är förmodligen mycketmer komplex än vi kan tänka oss, men med en ökad förståelse för hur cellerna ikroppen fungerar och interagerar med sin omgivning kommer vi bättre kunnaförstå sjukdomsmekanismer och förhoppningsvis då också finna nya botemedel.

47

ACKNOWLEDGEMENTS

This work has been carried out at “Zoofys” (later ICM) at Uppsala University and at thesection of Cell and Developmental Biology at Lund University. I would like to expressmy warmest thanks to my co-authors and all present and past colleges for all the nicetimes in and outside the lab, thank you all!

In particular I would like to express my sincere gratitude to:

Peter Ekblom, my supervisor and mentor, for excellent guidance and support, and yourscientific enthusiasm. You always manage to put everything into an interestingperspective.

Madde for help and guidance in the beginning and for introducing me to the lab.

Elke for your excellent guidance into the protein and signaling world, and for a nicefriendship.

My roommates, Susanne and Mange, for making the Lund period as good as it became.Good luck!

All the people at the lab in Lund; Marja, Maria, Tord, Hao, Yu Chen, Jan, Ulrika, Siw,Jang, and Alexander for nice collaborations and friendship.

The Cartela people and Emma for creating such a nice atmosphere, for pub evenings andparties.

The people from “zoofys”, which remained at Uppsala; Erik, Mats, Anne-Marie, Calle,Teet, Agnetha, Maria W., Olle and Sigrid. Special thanks to Maria R. and Katta, for“taking care” of me during my “visits” at Uppsala University and to Donald for all theanswers.

All my friends outside the lab, for reminding me of the world outside the lab and for allthe fun things we have done together. No one mentioned, no one forgotten.

Robban for his patience, love and support during this time. Finally!

My parents and my brother Martin, for always believing in me, and for your endlesssupport and love.

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