CELLULAR LOCALISATION OF TYPE XIII COLLAGEN, AND ITS INDUCED EXPRESSION IN HUMAN NEOPLASIAS AND CORNEAL DISEASES

TIMOVÄISÄNEN

Faculty of Medicine,Department of Medical Biochemistry

and Molecular Biology,Collagen Research Unit,

Biocenter Oulu,University of Oulu

OULU 2005

TIMO VÄISÄNEN

CELLULAR LOCALISATION OF TYPE XIII COLLAGEN, AND ITS INDUCED EXPRESSION IN HUMAN NEOPLASIAS AND CORNEAL DISEASES

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in Auditorium F101 of the Department ofPhysiology (Aapistie 7), on December 2nd, 2005, at 1 p.m.

OULUN YLIOPISTO, OULU 2005

Copyright © 2005University of Oulu, 2005

Supervised byProfessor Taina Pihlajaniemi

Reviewed byDocent Varpu MarjomäkiDocent Asta Pirskanen

ISBN 951-42-7909-3 (nid.)ISBN 951-42-7910-7 (PDF) http://herkules.oulu.fi/isbn9514279107/

ISSN 0355-3221 http://herkules.oulu.fi/issn03553221/

OULU UNIVERSITY PRESSOULU 2005

Väisänen, Timo, Cellular localisation of type XIII collagen, and its induced expressionin human neoplasias and corneal diseases Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology, CollagenResearch Unit, Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu,Finland 2005Oulu, Finland

AbstractType XIII collagen belongs to the group of transmembrane collagens. In this thesis the plasmamembrane localisation and function of type XIII collagen have been studied using cell biologicalmethods.

Type XIII collagen was found to reside in focal adhesions. It appeared in these structures at a veryearly stage of their assembly and disappeared from them concurrently with focal adhesion proteinstalin and vinculin. Insect cells expressing type XIII collagen showed an enhanced adhesion to certainmatrix components. These localisation and adhesion data suggested that the function of type XIIIcollagen is related to cell adhesion. Supporting this, in tissues type XIII collagen was found to localiseto cell-matrix and cell-cell adhesion structures.

Type XIII collagen was found to be partly present in cholesterol-enriched membranemicrodomains. With other membrane proteins this localisation has been shown to be linked toectodomain shedding. The connection between the membrane microdomain localisation and theectodomain shedding of type XIII collagen was also characterised, and it was demonstrated thatmanipulation of the cellular cholesterol level affected the efficiency of the ectodomain shedding.Additionally, insights into intracellular shedding of type XIII collagen in the Golgi apparatus wereobtained.

The study of type XIII collagen expression in human cancers revealed that it was enhancedespecially in the desmoplastic cancer stroma. Since the increased expression of type XIII collagenwas detected during the dysplastic stages, type XIII collagen may be involved in the earlypathogenesis of cancer. The result indicated that type XIII collagen is involved in the matrixremodelling. In support of this, the cell culture experiments showed that the soluble type XIIIcollagen ectodomain altered the vitronectin-rich matrix unfavourable for cell adhesion and spreading.This may enhance cancer metastasis.

Type XIII collagen expression was also induced in the remodelled stroma of keratoconus andcorneal wounds. Data suggested that myofibroblasts were responsible for the increased expression oftype XIII collagen in these situations. Therefore both in cancer and in the corneal pathologies studied,type XIII collagen expression was induced by the activated stromal cells.

Keywords: cancer, collagen, extracellular matrix, keratoconus, raft, vitronectin

Acknowledgements

This work was carried out at the Department of Medical Biochemistry and Molecular Biology, University of Oulu.

I would like to thank Professor Taina Pihlajaniemi for the opportunity to work in her group. Also her input in building the Biocenter Oulu organisation into the forefront research unit in Finland is greatly appreciated. I would also like to acknowledge Research Professor emeritus Kari Kivirikko for his groundbreaking resesearch in the collagen field and the invaluable work he has done by creating the Collagen Research Unit at our university. Professor emeritus Ilmo Hassinen, with his extensive work on energy metabolism, Professor Leena Ala-Kokko, Professor Seppo Vainio, Acting Professor Peppi Karppinen and Docent Johanna Myllyharju have been instrumental in creating the scientific community of the department.

I wish to express my sincere gratitude to Docent Varpu Marjomäki and Docent Asta Pirskanen for their thorough review of this thesis and the valuable suggestions. Aaron Bergdahl is acknowleged for the careful revision of the language.

I owe a great debt of gratitude to my scientific collaborators who have been involved in this thesis. I thank Ph.D., MD Pasi Hägg for being my first collaborator in type XIII collagen research. His initial guiding through the maze of corridors in the department and the numerous para-scientific discussions are warmly remembered. Ph.D., MD Marko Määttä is acknowledged for providing the opportunity to work with cornea diseases. My sincere and deep gratitude goes to Docent Helena Autio-Harmainen for her collaboration in the cancer study. Her scientific input and continuous encouragement were crucial for the completion of the work.

I want to thank all the colleagues and staff at the department, especially the members of type XIII collagen group who have shared the difficulties and occasional frustrations of working with such an uncooperative protein. Your help with scientific and technical issues has made life a lot easier in the lab. My warm and sincere thanks go to the members of the “Happy Lounge” in the former L120. Your friendship, help and support have taken me through thick and thin during these years. It has been a priviledge to know and work with you.

This work would not have been possible whitout the the technical assistace of Maija Seppänen and Ritva Savilaakso. They have managed to decipher the scribbles of my

instructions and, despite of them, perform the tasks brilliantly. Tuulikki Moilanen and Heli Auno from the Department of Pathology have also been important assets. Pertti Vuokila has been important in organizing the numerous practical things in the lab and also deserves many thanks for the countless enjoyable discussions and interest on this thesis project. Marja-Leena Kivelä and Auli Kinnunen have helped with many issues starting with patiently telling me numerous times whether I should put a zero before the fax numbers. Risto Helminen, Marko Kervinen and Antti Viklund are acknowledged for their excellent help with computers and more than once retrieving me from horrifying data disasters. Seppo Lähdesmäki has helped a lot by keeping equipment up and running.

I want to express my gratitude to my mother Vieno Väisänen for her love and support during these years. My brother Juha Väisänen and his family have provided me joyable moments and everlasting belief in reaching this goal. I wish also to thank all the members of the Keränen and Kircher families. My deepest thanks go to my wife and the most important collaborator Phil.Lic., M.D. Marja-Riitta Väisänen. Without her invaluable scientific contributions, support and love, this thesis would not have been completed. Sharing my life with her has been the greatest reward of these years. PP/M.

This thesis has been supported by grants from Biocenter Oulu, The Academy of Finland Center of Excellence Programme, The Sigrid Jusélius Foundation, The European Commission Framework 6 Programme (507743), The Cancer Society of Finland and The Osthrobothnia Cancer Society.

Oulu, November 2005 Timo Väisänen

Abbreviations

ADAM a disintegrin and metalloprotease BFA brefeldin A BSA bovine serum albumin cDNA complementary deoxyribonucleic acid CHAPS 3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propane-sulfonate COL collagenous DME Dulbecco’s modified Eagles medium EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EGTA ethyleneglycol-bis(β-aminoethyl)N,N,N’,N’-tetraacetic acid FBS fetal bovine serum FGF fibroblast growth factor GPI glycosyl-phosphatidylinositol HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IGF insulin-like growth factor MCD methyl-β-cyclodextrin MMP matrix metalloproteinase mRNA messenger ribonucleic acid NC non-collagenous PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PMSF phenylmethylsulphonylfluoride SDS sodium dodecyl sulphate TACE TNF-alpha converting enzyme TGF transforming growth factor TMA tissue microarray Tris tris(hydroxymethyl)aminomethane TX-100 or 114 Triton X-100 or 114 X (in Gly-X-Y) any amino acid Y (in Gly-X-Y) any amino acid

List of original articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Hägg P, Väisänen T, Tuomisto A, Rehn M, Tu H, Huhtala P, Eskelinen S & Pihlajaniemi T (2001) Type XIII collagen: a novel cell adhesion component present in a range of cell-matrix adhesions and in the intercalated discs between cardiac muscle cells. Matrix Biol 19: 727-742.

II Väisänen T, Väisänen M-R & Pihlajaniemi T (2005) Modulation of the cellular cholesterol level affects shedding of the type XIII collagen ectodomain. Manuscript.

III *Väisänen T, *Väisänen M-R, Autio-Harmainen H & Pihlajaniemi T (2005) Type XIII collagen expression is induced during malignant transformation in various epithelial and mesenchymal tumours. J Pathol 207: 324-335. *Equal contribution.

IV Määttä M, Väisänen T, Väisänen M-R, Pihlajaniemi T & Tervo T (2005) Altered expression of type XIII collagen in keratoconus and scarred human cornea – increased expression in scarred cornea is associated with myofibroblast transformation. Cornea, in press.

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ...................................................................................................................15 2 Review of the literature .................................................................................................16

2.1 The collagen group of proteins ...............................................................................16 2.1.1 Structure and synthesis of collagens................................................................16 2.1.2 Subgroups of the collagen family ....................................................................17 2.1.3 Transmembrane collagens ...............................................................................18

2.1.3.1 Type XIII collagen....................................................................................19 2.1.3.2 Type XVII collagen ..................................................................................21 2.1.3.3 Type XXIII and XXV collagens ...............................................................22

2.2 Cell adhesion ..........................................................................................................22 2.2.1 Focal adhesion .................................................................................................22

2.3 Cell membrane........................................................................................................24 2.3.1 Membrane microdomains (Rafts) ....................................................................25 2.3.2 Membrane microdomains and protein shedding..............................................26

2.4 Tissue stroma ..........................................................................................................27 2.4.1 Stroma in cancer initiation and progress..........................................................28 2.4.2 Myofibroblasts.................................................................................................29

2.5 Cornea ....................................................................................................................30 2.5.1 Structure of the cornea.....................................................................................30 2.5.2 Collagens of the cornea ...................................................................................30 2.5.3 Keratoconus.....................................................................................................31 2.5.4 Corneal wound healing....................................................................................33

3 Outlines of the present study .........................................................................................35 4 Materials and methods...................................................................................................36

4.1 Analysis of type XIII collagen localisation (I)........................................................36 4.1.1 Cells and tissue samples (I) .............................................................................36

4.1.2 Microinjections (I)...........................................................................................36 4.1.3 Production of the NC1 domain antibody (I) ....................................................37 4.1.4 Immunofluorescence staining (I) .....................................................................38 4.1.5 Insect cell adhesion experiment (I)..................................................................38 4.1.6 Immunoprecipitations and preparation of protein samples (I).........................39

4.2 Cholesterol dependence of type XIII collagen ectodomain shedding (II) ..............39 4.2.1 Analysis of membrane domains by density gradient ultracentrifugation (II)...39 4.2.2 Whole-cell shedding assay (II) ........................................................................40 4.2.3 Determination of cellular cholesterol (II) ........................................................40 4.2.4 Filipin staining (II) ..........................................................................................40 4.2.5 Cell-surface biotinylation and streptavidin pull-down assay (II).....................40 4.2.6 Vesicle secretion assay (II)..............................................................................41 4.2.7 TX-114 phase partitioning (II).........................................................................41 4.2.8 Cell fractionations (II) .....................................................................................41

4.2.8.1 Golgi fractionation (II) .............................................................................41 4.2.8.2 Sucrose density gradient fractionation (II) ...............................................42

4.3 Type XIII collagen expression during malignant transformation (III)....................42 4.3.1 Collection of the tumor material (III) ..............................................................42 4.3.2 In situ hybridisation of tumour samples (III, IV).............................................43 4.3.3 Antibody production and immunohistochemistry of tumour samples (III, IV)..............................................................................43

4.3.3.1 Production of the NC4 domain antipeptide antibody (III, IV)..................43 4.3.3.2 Immunohistochemistry of tumour samples (III, IV).................................44

4.3.4 Conditioned medium experiment (III).............................................................44 4.3.4.1 Preparation of conditioned medium and treatment of cells (III)...............44 4.3.4.2 Whole-cell shedding assay (III)................................................................45 4.3.4.3 CM cell proliferation assay (III) ...............................................................45

4.3.5 Expression of type XIII collagen in cancer cell lines (III)...............................45 4.3.6 TGF-β1 and PMA treatments (III)...................................................................46 4.3.7 Cell adhesion and spreading assays (III) .........................................................46

4.4 Expression of type XIII collagen in cornea, keratoconus and corneal scarring (IV).......................................................................................46

4.4.1 Collection of the cornea material (IV).............................................................46 4.4.2 In situ hybridisation of the cornea samples (IV)..............................................47 4.4.3 Immunohistochemistry of the cornea samples (IV).........................................47

5 Results ...........................................................................................................................48 5.1 Production of a polyclonal antibody against the mouse type XIII

collagen NC1-domain and human NC4-domain (I,III) .........................................48 5.2 Subcellular localisation of type XIII collagen (I) ...................................................49

5.2.1 Type XIII collagen is a membrane-linked focal adhesion protein in cultured human cells (I)...............................................................................49

5.2.2 Type XIII collagen dynamics during assembly and disassembly of focal adhesions resemble that of talin, vinculin and phosphotyrosine (I).................49

5.3 Cell surface expression of type XIII collagen changes adhesiveness of Sf9 insect cells (I)..............................................................................................50

5.4 Localisation of type XIII collagen on tissue level (I) .............................................50

5.4.1 Type XIII collagen localises to the adherens junctions in skeletal muscle and the intercalated disks in heart (I) .....................................50

5.4.2 Type XIII collagen in other tissues (I).............................................................51 5.5 Type XIII collagen is partly localised in membrane microdomains (II).................52 5.6 Analysis of type XIII collagen release from cells (II).............................................53

5.6.1 Type XIII collagen ectodomain shedding is sensitive to cholesterol deprivation (II) ..............................................................................53 5.6.2 Extracellular Golgi-derived vesicles contain cleaved ectodomain (II) ............54 5.6.3 Type XIII collagen found in the Golgi is of full length (II).............................55 5.6.4 Inhibition of proprotein convertases removes ectodomain from the Golgi-derived vesicles (II) .........................................................................55

5.7 Type XIII collagen in human cancers (III)..............................................................56 5.7.1 Type XIII collagen mRNA expression is upregulated in tumour stromal cells and in mesenchymal tumours (III) .............................................56 5.7.2 Type XIII collagen expression is upregulated in the invasive fronts of

neoplastic foci and in the placenta (III) ...........................................................56 5.7.3 Induction of type XIII collagen expression and phenotypic cellular transition of fibroblasts by cancer cell-derived factors (III) ............................57 5.7.4 TGF-β1 upregulates COLXIII expression (III) ...............................................57 5.7.5 The soluble ectodomain of type XIII collagen reduces cell adhesion and

spreading on vitronectin (III)...........................................................................58 5.8 Type XIII collagen in pathological cornea (IV)......................................................58

5.8.1 Immunohistochemistry of normal and pathological cornea (IV).....................58 5.8.2 In situ hybridisation of normal and pathological cornea (IV)..........................59

6 Discussion .....................................................................................................................60 7 Future perspectives........................................................................................................66 References

1 Introduction

Creation of the extracellular matrix (ECM) has been a culmination point in the evolution of multicellular organisms. The ECM is the platform which cells can attach to and move along when they assemble various tissues and organs that together form a complete organism. The ECM has been shown to be a cornucopia of various proteins with differing functions, one of the major components being the family of collagens. The concept of collagens has over time evolved from a pure structural one to a highly dynamic, versatile and complex group of proteins. The members of this group have been associated with an increasing number of varying functions. A valid example of this trend is type XIII collagen, which differs greatly from the classical collagens in terms of structure and function.

The goal of this thesis is to explore the cell biology of type XIII collagen, including its involvement in certain pathological processes. The literature review provides an overview of the collagen superfamily in general, and more detailed discussions of the transmembrane collagens, cell adhesion and focal adhesions as well as cell membranes and their constituent microdomains. The role of stroma in the pathogenesis of cancer and certain pathological processes of cornea is also addressed.

2 Review of the literature

2.1 The collagen group of proteins

2.1.1 Structure and synthesis of collagens

The collagen family of proteins, which at present contains 27 members in humans (Koch et al. 2004), is a highly versatile group of proteins involved in structural functions but also in a number of other processes such as cell adhesion and migration, morphogenesis, differentiation and tissue remodelling. The importance of collagens is emphasised by the fact that in some tissues 90% of the protein content is composed of collagens, and also by the fact that various pathological conditions are known to result from either mutations or excessive or deficient synthesis of collagens. The structural hallmark of collagens is the presence of one or more triple-helical collagenous domains, which consist of three α-helical polypeptides. These α chains are composed of repeating Gly-X-Y sequences, which during synthesis wind around each other to form rod-like right-handed trimeric structures. Glycine residues are essential for this folding since they allow packing of the three collagen monomers into a triple helix by being placed at the centre of the coil. At X and Y positions any other amino acid can be found. However, X is most often proline and Y 4-hydroxyproline. The latter is essential for the stability of the triple helix. Collagens can also contain non-collagenous domains. (van der Rest & Garrone 1991, Brodsky & Shah 1995, Gelse et al. 2003, Myllyharju & Kivirikko 2001, Kleinman et al. 2003, Myllyharju & Kivirikko 2004.)

The biosynthesis of mature collagens is a multistep process involving extensive post-translational modifications. Fibril-forming collagen polypeptides are synthesised in the membrane-bound ribosomes, and are subsequently transferred into the lumen of the endoplasmic reticulum, where the signal peptides are cleaved, and certain residues are hydroxylated and glycosylated. Collagen polypeptides associate by interaction between the C-terminal recognitions sequences, followed by formation of intra- and interchain disulfide bridges. The formation of the triple-helix proceeds from the C-terminal association site(s) to the N-terminus in a zipper-like fashion. The triple-helical

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procollagen molecules are transported through the Golgi apparatus, packed into transporting vesicles, and secreted into the extracellular space where the N- and C-terminal propeptides are enzymatically cleaved. Subsequently, collagen molecules assemble into fibrils, which are stabilised by covalent crosslinks. In addition to fibrillar collagens, this chain of events also applies to other collagens as well. (Prockop & Kivirikko 1995, Myllyharju & Kivirikko 2001.) However, at least in the case of transmembrane type XIII and XVII collagens the direction of trimerisation is the opposite (Areida et al. 2001, Snellman & Pihlajaniemi 2003). Canty et al. have also recently provided data suggesting that the propeptide cleavage and fibrillogenesis may occur intracellularily, at least in the embryonal tendon (Canty et al. 2004, Canty & Kadler 2005).

Several collagens are encoded by more than one gene. Therefore, in a particular triple-helical collagen molecule, the polypeptide chains can be either identical (a homotrimer) or dissimilar (a heterotrimer). Alternative splicing also creates additional structural variability to this protein group. (Kielty & Grant 2002, Myllyharju & Kivirikko 2004.) Triple-helical structures are resistant to proteases. However, if a given collagen protein contains non-collagenous domains or is partially denatured, it is more susceptible for enzymatic digestion by some proteases, like matrix metalloproteinase (MMPs). Recent data have shown that the resulting proteolytic fragments are not simply discarded end products, but can have important biological regulatory functions during physiological and pathological tissue remodelling. (Ortega & Werb 2002.)

2.1.2 Subgroups of the collagen family

Based on their supramolecular structure, the members of the collagen family can be divided into subgroups. Fibril-forming collagens (I-III, V, XI, XXIV and XXVII) assemble into extracellular fibres. These fibres function as a supporting scaffold of high mechanical strength for cells and tissues. A characteristic feature for the fibres is a cross-striation resulting from a staggered positioning of the collagen molecules. Type I and II collagens are the major collagen types in the body. Type III, V and XI collagens are involved in the regulation of type I and type II collagen fibril formation. The exact functions of type XXIV and XXVII collagens, expressed primarily in cartilage (type XXVII) and in testis and the ovaries (type XXIV), are not yet fully known. (Sato et al. 2002, Boot-Handford et al. 2003, Koch et al. 2003, Pace et al. 2003.)

FACIT (fibril-associated collagens with interrupted triple helices) collagens (IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI) share some structural homology with each other, and some of them have been shown to associate with the fibrillar collagens. Type IX collagen, which associates with type II collagen in cartilage and vitreous body, is proposed to mediate interactions between collagen fibrils and other matrix components. (Olsen 1997, Tuckwell 2002.) Type XII and XIV collagens, which resemble type IX collagen, colocalise with type I collagen in several tissues (Gelse et al. 2003). Type XIX collagen is expressed in basement membrane zones (Myers et al. 1997, Myers et al. 2003). Type XX collagen is expressed in low amounts in several connective tissues, especially in the corneal epithelium, and type XXI collagen in smooth muscle cells in

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vessel walls (Fizgerald & Bateman 2001, Koch et al. 2001). Type XXII collagen localises in tissue junctions. It does not directly associate with fibrillar collagens, instead it may interact with microfibrils (Koch et al. 2004). Type XVI collagen has dual association properties. Namely, in papillary dermis it localises with microfibrils and in cartilage with type II collagen (Kassner et al. 2003). Type XXVI collagen has structural features similar to the FACIT collagens but it lacks association with the fibrillar collagens (Sato et al. 2002).

Type X and VIII collagens form a subgroup which is characterised by their ability to form hexagonal lattices. Type X collagen localises in hypertrophic cartilage, and type VIII collagen in subendothelial matrix and Descemet’s membrane. (Gelse et al. 2003, Kvansakul et al. 2003.)

Type VI collagen is a heterotrimer of three different α-chains. In ECM type VI collagen molecules assemble into microfibrillar structures called beaded filaments with a wide presence in the body (Gelse et al. 2003). Type VII collagen is a highly specialised protein which localises in the epithelial-basement membrane zone attaching basement membranes to the stroma (Gelse et al. 2003, Myllyharju & Kivirikko 2004). Type IV collagen is one of the most important structural components of the basement membrane separating epithelial cells from the underlying tissue. Actually, type IV collagens comprise their own protein subfamily consisting of three collagen heterotrimers variably composed of six different α subunit chains, α1 to α6. In addition to the structural role in the basement membranes, type IV collagens regulate cell adhesion and migration. (Ortega & Werb 2002, Gelse et al. 2003, Myllyharju & Kivirikko 2004.) Furthermore, proteolytic fragments of α1(IV), α2(IV) and α3(IV), called arresten, canstatin and tumstatin respectively, have shown anti-angiogenic properties (Colorado et al. 2000, Kamphaus et al. 2000, Maeshima et al. 2000). Type XV and XVIII collagens, also called multiplexins (multiple triple helix domains with interruptions), have mutually homologous structures and are both located in basement membrane zones (Oh et al. 1994, Pihlajaniemi & Rehn 1995, Saarela et al. 1998, Muona et al. 2002). Also with the multiplexins, proteolytic cleavage can create C-terminal non-collagenous fragments that have been shown to have anti-angiogenic activity (Olsen & Folkman et al. 1997, Ramchandran et al. 1999).

Over 1000 different mutations have been described for the different collagen genes in humans. Most of the mutations have been identified as the cause of relatively rare heritable diseases, including osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplacias, Alport syndrome and epidermolysis bullosa. In addition, collagen mutations may contribute to some common diseases like osteoporosis, aneurysms, osteoarthrosis and intervertebral disc disease. Mutations have also been detected in the genes encoding collagen modifying enzymes. The severity of the defects in patients ranges from a very mild phenotype to perinatal lethality. (Myllyharju & Kivirikko 2001, Myllyharju & Kivirikko 2004.)

2.1.3 Transmembrane collagens

A common feature for the collagens (types XIII, XVII, XXIII, XXV) belonging to this group is that they are integrally linked to the cell surface by a single hydrophobic

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membrane spanning domain. There are also other transmembrane proteins, namely ectodysplasin (EDA), macrophage scavenger receptors I-III, macrophage receptor (MARCO) and colmedins (collagen repeat plus olfactomedin-containing proteins), which contain collagenous sequences, but are not classified as collagens. (Franzke et al. 2003, Snellman & Pihlajaniemi 2003, Franzke et al. 2005.) The presentation of this latter group of proteins is excluded from the scope of this thesis.

Fig. 1. Schematic figure of the structures of the transmembrane collagens. Black boxes depict transmembrane domains, white boxes depict non-collagenous domains and triple helixes collagenous domains. Structural data is compiled from the articles in the text.

2.1.3.1 Type XIII collagen

Type XIII collagen is a homotrimer consisting of three collagenous domains (COL1-3), four noncollagenous domains (NC1-4), and a hydrophobic transmembrane segment close to the aminoterminal end of the protein. Type XIII collagen is classified as a type II transmembrane protein. (Hägg et al. 1998, Snellman et al. 2000b.) The gene encoding type XIII collagen is almost 140 kb long and it is localised on chromosome 10 (Pajunen et al. 1989, Shows et al. 1989, Kvist et al. 1999, Sallinen et al. 2001). According to sequence analysis, the longest possible type XIII collagen molecule is 726 amino acids long in humans. However, as type XIII collagen is spliced extensively at the COL1, NC2 and COL3 domains, size variants ranging in size between 85-95 kDa are observed in denatured samples of cultured cells. These proteins are likely to represent the splice variants. Type XIII collagen makes multimeric disulfide-bonded complexes of 180 kDa in size. (Pihlajaniemi et al. 1987, Tikka et al. 1988, Pihlajaniemi & Tamminen 1990, Hägg et al. 1998, Snellman et al. 2000b, Tu et al. 2002.) Rotatory shadowing analysis has shown that type XIII collagen is a rod-like protein which can bend at noncollagenous domains (Tu et al. 2002). The N-terminal NC1 and the C-terminal NC3 domains of type XIII collagen contain conserved sequences that are predicted to form coiled-coil domains. These are widely occurring protein oligomerisation domains important for the trimerization of collagneous sequences. Presense of such domains and biochemical data have suggested that type XIII collagen is trimerised starting from the N-terminus to the

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C-terminus, i.e. in an opposite direction to most other collagens. (Kammerer 1997, Snellman et al. 2000a, Latvanlehto et al. 2003, McAlinden et al. 2003.)

Type XIII collagen is expressed in many tissues like skin, muscle, bone, cartilage, intestine, lung, nervous tissue, eye and placenta (Sandberg et al. 1989, Juvonen et al. 1992, Juvonen & Pihlajaniemi 1992, Juvonen et al. 1993, Sandberg-Lall et al. 2000). The expression of type XIII collagen begins early during development and increases towards the final stages of organogenesis and foetal growth. However, in mature tissues its expression is low (Sandberg et al. 1989, Sund et al. 2001a). In cell type XIII collagen localises in plasma membrane and in tissues in cell-cell and cell-matrix contacts such as intercalated discs, myotendinous junctions and adherens junctions (Hägg et al. 1998, Peltonen et al. 1999, Sandberg-Lall et al. 2000, Sund et al. 2001b). These cellular and tissue localisations have indicated a role in cell adhesion and adhesion related functions for type XIII collagen.

The ectodomain of type XIII collagen can be cleaved at the stem of the ectodomain and released to the extracellular space (Peltonen et al. 1999, Snellman et al. 2000a, Väisänen et al. 2004). The extracellular portion of the NC1 domain contains a proprotein convertase recognition sequence (Thomas 2002), and the furin family of mammalian proprotein convertases were found to be responsible for ectodomain shedding (Snellman et al. 2000a, Väisänen et al. 2004). Other proteinases are not involved in the cleavage. There seems to be cell type-specific differences in the regulation of shedding. In addition to the cell surface, recent data suggests that type XIII collagen is also cleaved intracellularly in the Golgi apparatus. Cells may use shedding as a mechanism to maintain a stable amount of type XIII collagen on the plasma membrane when attached and/or to release excess type XIII collagen during cell detachment. (Väisänen et al. 2004.)

The recombinant ectodomain binds to several ECM and plasma membrane proteins, including fibronectin, vitronectin, nidogen-2, heparin, perlecan, fibulin-2, type VI collagen and the I-domain of α1 integrin (Nykvist et al. 2000, Tu et al. 2002). The released ectodomain is a biologically active molecule, as it can associate with the fibronectin matrix and interfere with its assembly. This association is mediated by the C-terminus of type XIII collagen and the N-terminus of fibronectin (Väisänen et al. 2005). The ectodomain itself has a low capacity to perform as an adhesion or migration substrate (Väisänen et al. 2004), although the I-domain of the collagen binding integrin α1 has been shown to bind to it (Nykvist et al. 2000). On vitronectin the recombinant type XIII collagen ectodomain can affect cell behaviour as it reduces cell adhesion, spreading, migration and proliferation (Väisänen et al. 2004).

Transgenic mice expressing mutated type XIII collagen molecules present a variety of phenotypes. Replacement of the intracellular and transmembrane domains with a short nonsense peptide results in a progressive myopathy. In these mice the ultrastructure of the skeletal muscles and the organisation of the cytoskeleton are distorted, the sarcolemma has an undulating appearance and the basement membrane is partially detached from the muscle cells. Fibroblasts isolated from the dermis of these animals show a reduced cell adhesion in the presence of serum and to type IV collagen in serum-free conditions. (Kvist et al. 2001.) Dominant negative mice with a partial deletion of the COL2 domain and the whole NC4 domain show an increased lethality during embryonic development due to abortions resulting either from a placentation failure or from defects in the

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cardiovascular system (Sund et al. 2001b). Mice overexpressing type XIII collagen develop high bone mass postnatally because of increased bone formation (Ylönen et al. 2005).

2.1.3.2 Type XVII collagen

Type XVII collagen, also known as bullous pemphigoid antigen (BP180), is a component of a cellular adhesion structure, called hemidesmosome, which mediates the attachment of epithelial cells to the underlying basement membrane. The N-terminus of type XVII collagen is located at the hemidesmosomal plaque, whereas the rest of the molecule reaches to the basement membrane zone. Compared to other transmembrane collagens, type XVII collagen has a long intracellular domain, which has been shown to interact with β4 integrin, BP230 and plectin. The extracellular domain is divided into 15 collagenous and 16 non-collagenous domains. (Schäcke et al. 1998, Areida et al. 2001, Franzke et al. 2003.) The ectodomain interacts with laminin 5 and α6 integrin (Hopkinson et al. 1995, Koster et al. 2003). The juxtamembranous linker region between the transmembrane domain and the first collagenous domain contains a coiled-coil domain, which is proposed to serve as a starting point for the trimerisation (Areida et al. 2001).

In addition to being a membrane-integral collagen, type XVII collagen also exists as a soluble ECM component. It is constitutively shed from cells so that the soluble form contains almost the entire ectodomain (Hirako et al. 1998, Schäcke et al. 1998). Ectodomain has been isolated from the epidermis and the amniotic fluid, indicating a role in physiological functions (Schumann et al. 2000). Cleaving occurs near the transmembrane domain by members of ADAM family of proteinases, ADAM-17 (TACE) being the major cleaving enzyme (Franzke et al. 2002, Franzke et al. 2004). Recently type XVII collagen, but not TACE, was shown to co-localise at the cell surface with a membrane raft marker protein placental alkaline phosphatase, suggesting a membrane raft localisation. Depletion of the cellular cholesterol and resulting raft disruption concomitantly induced type XVII ectodomain shedding. This suggests that rafts are involved in the regulation of type XVII collagen ectodomain shedding by sequestration effect. (Zimina et al. 2005.) However, the roles of shedding and the shed form of type XVII collagen are not fully known. Recombinant COL15 domain of type XVII collagen has been shown to promote cell adhesion and the shed full-length ectodomain to decrease keratinocyte motility. This indicates a role in cell adhesion, differentiation and migration for the released ectodomain. (Tasanen et al. 2000, Franzke et al. 2002.)

The diseases involving type XVII collagen manifest themselves with symptoms related to the cell adhesion function in hemidesmosomes. Junctional epidermolysis bullosa is a hereditary disease in which type XVII collagen is mutated or is completely lacking. Because of this, the hemidesmosome structure is disturbed. Patients display variable phenotypes caused by decreased epithelial adhesion leading to blister formation in skin and mucous membranes. Symptoms vary from mild to severe, depending on the mutation in the type XVII collagen gene. (Zillikens & Giudice 1999, Franzke et al. 2003, Franzke et al. 2005.) Type XVII collagen is also a target protein in a group of

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autoimmune diseases called pemphigoids, in which autoantibody binding to type XVII collagen leads to skin blister formation and skin detachment (Hirako et al. 1998, Zillikens & Giudice 1999, Schumann et al. 2000).

2.1.3.3 Type XXIII and XXV collagens

Type XXIII collagen resembles type XIII and XXV collagens by having a short intracellular domain and an extracellular domain divided into three collagenous domains. The function of type XXIII collagen is unknown. It is expressed in heart, retina and metastatic cancer cells. The ectodomain of type XXIII collagen contains a potential furin cleavage site and has been shown to be released from cells by furin. (Banyard et al. 2003.) Type XXV collagen (CLAC, collagen-like Alzheimer amyloid plaque component) was originally found to be a component of the amyloid plaques in the brains of Alzheimer’s disease patients. CLAC is a proteolytically cleaved ectodomain of the membrane-bound type XXV collagen precursor (CLAC-P), and is released from the membrane by furin proteinase. Both the shed form and the membrane form bind specifically to amyloid β peptide (Aβ) fibres found in Alzheimer’s plaques. (Hashimoto et al. 2002.) The exact role of CLAC in Alzheimer’s disease and plaque formation is unknown. Osada et al. have proposed that CLAC binding inhibits Aβ fibrillisation and Söderberg et al. have suggested that CLAC makes fibrils more resistant to proteases (Osada et al. 2005, Söderberg et al. 2005).

2.2 Cell adhesion

Adequate cell adhesion is of critical importance to cell survival, but is also important in a wide variety of other processes such as cell migration, proliferation, differentation, and regulation of gene expression. Cell structures mediating adhesion to the ECM are essential in the assembly of cells into multicellular organisms. The cell adhesion structures not only mediate physical contact, but function as signalling units as well. (Petit & Thiery 2000, Geiger & Zamir 2001.) Cell adhesion has been studied mostly with cells grown on two dimensional surfaces. Under these conditions cells form focal adhesions which link cells to the surrounding ECM.

2.2.1 Focal adhesion

Focal adhesions are located on the basal side of cells. At these sites, where plasma membrane is in close contact with the ECM, a group of transmembrane receptor proteins called integrins mediate cell adhesion to the ECM. These receptors use their extracellular ends to bind to the ECM components and their intracellular ends to bind to the actin cytoskeleton, thereby linking these two entities. (Zamir & Geiger 2001, Cukierman et al. 2002, Wozniak et al. 2004.) Focal adhesions are highly complex structures containing at

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present more than 50 proteins having both structural and signalling functions. The actual linkage of actin cytoskeleton to integrins has been proposed to occur via several different pathways involving either one or more multifunctional adaptor proteins (Figure 2.). (Weis & Pokutta 2002, Brakebusch & Fässler 2003.)

Fig. 2. Schematic figure of a focal adhesion structure. Figure depicts proteins shown or suggested to link actin cytoskeleton to integrins. Focal adhesion proteins involved in other functions are not included. The figure is compiled from the articles in the text.

Adhesion structures of cultured cells can be divided into four types based on their size, subcellular localisation, and composition: focal complexes, focal adhesions, fibrillar adhesions and three-dimensional (3-D) matrix adhesions. These structures also depict the maturation process of these adhesion complexes. (Wozniak et al. 2004.) The earliest structures, focal complexes, are located in the periphery of migrating or spreading cells, and some of them mature to focal adhesions. These can be found at the cell periphery and also more centrally, where they associate with actin stress fibres. Fibrillar adhesions are elongated focal adhesions and contain α5β1 integrin and tensin. The three-dimensional matrix adhesions are present in cells, when the surrounding matrix is also three-dimensional. Structurally, focal adhesions are complex assemblies made of a large number of proteins, which include adhesion receptors at the cell surface, GTPases, enzymes and various adaptor proteins linking the intracellular actin cytoskeleton to the receptors and mediating signalling functions. (Nobes & Hall 1995, Charzanowska-Wodnicka & Burridge 1996, Pankow et al. 2000, Cukierman et al. 2002.)

Focal adhesions are dynamic structures that are assembled and disassembled to meet the functional requirements of the cell. In migrating cells new focal adhesions are assembled at the leading edge, and at the same time are disassembled at the trailing edge of the cell. The assembly of focal adhesions is a hierarchical process. It has been proposed that the first contact between cells and the matrix is not mediated by integrins but rather by cell surface glycosaminoglycans. (Hanein et al. 1993, Hanein et al. 1994, Zimmerman et al. 2002, Cohen et al. 2003.) However, this initial contact is rapidly replaced by integrin-containing contacts. At the leading edge (lamellipodia), cell

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membrane protrusions are stabilised by early adhesive foci containing αvβ3 integrin, talin and paxillin. These foci are also heavily tyrosine-phosphorylated. Shortly after, these early focal complexes mature by gaining α-actinin, FAK, VASP, vinculin and Arp2/3. (Zaidel-Bar et al. 2003.) The function of focal complexes is not fully known, but they may attach the actin network of the cell edge to the matrix and thus enable the polymerisation driven lamellipodium protrusion (Laukaitis et al. 2001, Zaidel-Bar et al. 2003, von Wichert et al. 2003). Some of the focal complexes disassemble while the cell edge protrudes further. A subset of complexes starts to grow and their composition changes by the addition of zyxin, and by the assembly of the cytoskeletal actin stress fibres, which attach to the adhesion site. (Zaidel-Bar et al. 2003.)

The maturation of focal adhesions also requires cytoskeleton-mediated contractility, which applies mechanical force to the contact site, integrin clustering and occupancy (Riveline et al. 2001). When matured, focal adhesions translocate towards the cell centre by a polarised assembly/disassembly process. Fibrillar adhesions are fibronectin matrix-associated adhesion structures, probably derived from focal adhesions by centripetal translocation of α5β1 integrins. These structures contain α5β1 integrin and tensin, and their formation requires mechanical force. It has been suggested that fibrillar contacts are involved in matrix reorganisation and fibronectin fibrillogenesis. (Smilenov et al. 1999, Katz et al. 2000, Zamir et al. 2000, Cukierman et al. 2002.) Once the cells have assembled a three-dimensional matrix, the adhering structures are adapted into 3D-adhesions of a distinct composition. This change also influences cell behaviour. The delicate evolution of the adhering structures from focal complexes to 3D-adhesions suggests that focal adhesions are not merely a cell culture artefact but a functional adaptation to the properties of the surrounding matrix. (Cukierman et al. 2002.)

The cyclical formation, maturation and turnover of focal adhesions is a complex process where GTPases Rac, Cdc42, Rho as well as FAK and Src play a central role. Focal complex formation in the membrane protrusions is controlled by Rac and Cdc42, after which Rho activation is sequentially needed for the maturation of focal adhesions. (Rottner et al. 1999, Wozniak et al. 2004.) Rho induces actin stress fibre formation, integrin clustering and cytoskeleton contraction (Charzanowska-Wodnicka & Burridge 1996, Wozniak et al. 2004). Integrin clustering, in turn, activates FAK, which binds to and activates Src. These two are important for focal adhesion turnover and disassembly. Namely, Src phosphorylates focal adhesion components, including FAK and integrin cytoplasmic domains, which then promote the adhesion site turnover. (Parsons & Parsons 1997, Katz et al. 2003, Wozniak et al. 2004.) Calcium-dependent protease calpain, which cleaves focal adhesion components, is also needed. The activity of calpain is controlled by FAK, which associates with calpain and localises it to the focal adhesions, and also by Src, which induces calpain-mediated FAK cleavage. (Huttenlocher et al. 1997, Carragher et al. 1999, Dourdin et al. 2001, Bhatt et al. 2002, Carragher et al. 2003.)

2.3 Cell membrane

All biological membranes have essentially the same basic structure. They are composed of phospholipid bilayers which are symmetrical structures with the polar head groups of

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phospholipids exposed to the aqueous medium and the nonpolar fatty acyl side chains towards the centre of the bilayer. Cell membranes also contain proteins, which are largely responsible for the various functions of each membrane type, cholesterol and glycolipids. The localisation of proteins, both lateral and integral, and glycolipids create asymmetry, making the two faces of the membrane dissimilar. The components are also laterally mobile and can move relatively freely. Membranes establish boundaries, separating cells from the surrounding milieu, and allow compartmentalisation of the cell interior into functional units. (Munro 2003, Simons & Vaz 2004.)

2.3.1 Membrane microdomains (Rafts)

According to the Singer-Nicholson fluid mosaic model, the cell membrane can be regarded as a lipid bilayer that behaves like a neutral two-dimensional solvent with little influence on membrane proteins. However, in model membranes, lipids have been found to exist in different phases. These include gel, liquid ordered and liquid disordered phases, the latter being the most fluid of the three. However, these phases have been difficult to observe in cellular membranes. (Brown & London 1998, Simons & Toomre 2000.) The lipid raft hypothesis postulates that the cell membranes contain dynamic assemblies of cholesterol and glycosphingolipids in the exoplasmic leaflet of the membrane bilayer. Sphingolipids in cellular membranes contain mainly saturated hydrocarbons. This allows cholesterol to pack tightly between hydrocarbon chains in a manner that is similar to the liquid ordered state. Since the concentration of these components is lower in other parts of the membrane, membrane microdomains form lipid platforms in the cell membrane, hence the name rafts. These differ biophysically from their surroundings, the raft membranes being less fluid. They are also more resistant to non-ionic detergents at low temperatures and have a lower buoyant density compared to other parts of the membrane. These properties have been used to characterise, isolate and to study membrane rafts. (Brown & London 2000, Maxfield 2002.)

Cholesterol, which is enriched in rafts 3-5-fold compared to the rest of the membrane, is a key factor in the determination of raft stability and organization. Depletion of cholesterol from the cells leads to a loss of detergent-resistance and disassembly of rafts. (Ilangumaran & Hoessli 1998, Simons & Toomre 2000, Munro 2003, Pike 2004, Silvius 2003, Simons & Vaz 2004.) The exact size of the rafts is not known, since the direct visualisation of rafts in living cells has been difficult by conventional light microscopy due to their very small size. However, if rafts are clustered, they separate into patches large enough to be directly visualised. In general, rafts are most abundant at the plasma membrane, but rafts can also be found in several other membranes along e.g. the secretory pathway and endocytic compartments. (Simons & Toomre 2000, Fielding & Fielding 2003.)

The distribution of the microdomains at the cell surface is cell type-dependent. For example, in polarised epithelial cells rafts are most abundant at the apical surface, but in fibroblasts the distribution is more even (Simons & Ikonen 1997, Vogel et al. 1998, Simons & Toomre 2000). It has been proposed that rafts assemble in the Golgi apparatus from which they move to the plasma membrane and other destinations. Rafts are also

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endocytosed, and recycled back to the cell surface. Some of them have been proposed to be linked to the cytoskeleton. (Brown & London 1998, Mukherjee & Maxfield 2000, Rodgers & Zavzavadjian 2001, Nichols 2003.) The only morphologically identifiable raft-like membrane microdomain in cells is the caveola. It is a small plasma membrane invagination with a diameter of 60-80 nm. The general function of caveolae is not fully known, but they have been associated with endocytosis, transcytosis, T-tube formation in myocytes and signal transduction. (Schnitzer et al. 1994, Parton et al. 1996, Fielding & Fielding 2003, Nichols 2003.)

Based on the proposed model, rafts function as lateral sorting areas for the various membrane proteins. Since rafts can either include or exclude different proteins, they can influence their functions by facilitating or inhibiting interactions. In general, rafts are relatively deficient in transmembrane proteins whereas some proteins are enriched within them. Several different mechanisms have been reported to be responsible for protein targeting to rafts. These include protein modifications such as GPI-anchors and palmitate groups. In the case of transmembrane proteins, targeting may require the presence of certain amino acids or the correct length of the transmembrane domain. Also, domains other than transmembrane domains may be essential, suggesting that protein-protein and protein-lipid interactions can be important. (Lucero & Robbins 2004, Pike 2004.)

Rafts are most likely heterogeneous in protein and lipid composition, size, detergent resistance and stability, because of selective incorporation of the lipids, GPI-anchored and transmembrane proteins. This heterogeneity probably affects the responses rafts have on cell functions. (Pike 2004.) Nonetheless, lipid rafts have been proposed to be involved in signal transduction, membrane trafficking of proteins, cell adhesion, migration, cytoskeletal organisation and pathogen entry (Simons & Toomre 2000, Ikonen 2001, Munro 2003, Upla et al. 2004).

The methods, which are mostly used to define the association of a given protein with rafts, are the detection of the presence of a protein in detergent-resistant low density membrane (DRM) fractions and co-patching of a protein with raft markers. However, all these methods contain potential sources of error in the isolation of the DRMs. Combined with the difficulties assiciated with raft visualisation in living cells, evidence of their in vivo existence and their biological significance has remained somewhat inconclusive. (Munro 2003, Lai 2004, Lagerholm et al. 2005.)

2.3.2 Membrane microdomains and protein shedding

Proteolysis is an effective regulatory mechanism that cells use to activate, inactivate or modulate proteins and their functions. Proteolysis can occur at the plasma membrane or in the extracellular space, as a response to external or internal stimuli. Cleavage of the ectodomains of transmembrane proteins changes the assortment of proteins at the cell surface. The released fragments themselves can have functional roles in growth, morphogenesis and tissue repair. On the other hand, inappropriate proteolysis can result in pathological conditions. The enzymes responsible for ectodomain shedding belong mainly to the ADAM and MMP families, also some serine proteases have been implicated. (Werb 1997, Werb & Yan 1998, Blobel 2000.)

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Recently, a number of studies have linked ectodomain shedding to membrane rafts. These studies have shown that the shedding of a number of transmembrane proteins is sensitive to cellular cholesterol content and raft localisation. Neuregulin-1 is cleaved at its extracellular domain by ADAM19, the localisation of which, in rafts, is crucial for cleavage to occur (Wakatsuki et al. 2004). Depletion of the cellular cholesterol with methyl-β-cyclodextrin increases IL-6R shedding by ADAM10 and ADAM17 (TACE) (Matthews et al. 2003). CD30, a lymphoid activation marker, resides partially in lipid rafts, whereas its main cleaving enzyme, ADAM17, is mainly in the non-rafts compartment. Depletion of cholesterol in CD30-positive cells leads to an increase in the formation of the soluble CD30. Disruption of rafts by the lowering of cellular cholesterol level may thus lead to dynamic interactions between these two proteins, causing enhanced shedding. (von Tresckow et al. 2004.) The down-regulation of lymphocyte L-selectin by metalloproteinases may involve membrane rafts as signalling platforms (Phong et al. 2003). The selective cleavage of the amyloid precursor protein (APP) by α-, β or γ-secretases is a determining factor in the development of Alzheimer’s disease. Based on the Ehehalt et al. study, the availability of APP to β-secretase, leading to the production of senile plaque amyloid β-peptide (Aβ), or alternatively, to α-secretase, preventing Aβ formation, may be dependent on whether APP is present in rafts. (Ehehalt et al. 2003.) Zimina et al. have shown that the shedding of transmembrane type XVII collagen is dependent on cellular cholesterol levels. Namely, a majority of type XVII collagen co-localises with a raft marker protein whereas its cleaving enzyme, TACE, does not. Because the lowering of cellular cholesterol disrupts rafts and concurrently increases type XVII collagen ectodomain shedding, the results suggest that the rafts crucially restrict the accessibility of type XVII collagen to its cleaving enzyme and hence the efficiency of ectodomain shedding. (Zimina et al. 2005.)

2.4 Tissue stroma

In multicellular organisms most cells are in continuous contact with a complex three-dimensional structure of the ECM. The matrix consists of collagens, proteoglycans and glycoproteins, together with growth factors, chemo- and cytokines. The function of the ECM is to give the tissues mechanical strength and cushioning, and to serve as a platform that cells can attach to, migrate along, proliferate and use to determine their polarity. It stores growth factors, chemokines and cytokines that, when released, can function as signalling molecules in tissues. The complexity of the matrix is further increased by protein modifications and proteolysis, the latter of which can reveal functional domains and release cryptic fragments with biological activities. Biological responses of cells to the ECM are conducted by cell-surface receptors such as integrins, proteoglycans, receptor tyrosine kinases, dystroglycans and immunoglobulin superfamily receptors. The ECM is also a dynamic structure which has both temporal and spatial variation. The dynamic nature and complexity of the matrix is essential during morphogenesis and differentiation, where cells, via their cellular receptors, receive developmental cues from their surroundings. When combined with resident cells, such as fibroblasts, immune defence cells and adipose cells, the matrix forms an entity called stroma (Figure 3.). It is

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in constant interaction with various epithelia. Correct interactions between these two compartments are crucial for the ordered development and maintenance of tissue homeostasis. Changes in these interactions are needed during normal physiological processes and trauma recovery, but when persistent or otherwise derailed, can lead to pathological changes like cancer. (Werb 1997, Bissel & Radisky 2001, Kleinman et al. 2003.)

Fig. 3. Schematic representation of the tissue stroma, containing various cell types, for instance fibroblasts and adipocytes, and the fibrillar extracellular matrix.

2.4.1 Stroma in cancer initiation and progress

Epithelial cancers, i.e. carcinomas, undergo certain cellular changes favourable for malignant growth. These include loss of growth control, resistance to apoptosis, limitless replicative capability, ability to induce angiogenesis and ability to metastasise (Hanahan & Weinberg 2000, Krtolica & Campisi 2002). Traditionally, the initiation and progression of cancer has been thought to be caused by accumulative mutations of the epithelial cells. Also, for a long time it has been well known that carcinoma cells cause histological changes to the surrounding stroma. The resulting desmoplastic stroma is characterised by increased deposition of the ECM components, especially collagens, increased levels of MMPs, angiogenesis, increased proliferation of fibroblasts and recruitment of inflammatory cells. However, desmoplasia has been considered as a reaction of the stroma in response to cancer cell influence on the stroma, which earlier was considered to play a more passive role in tumorigenesis. (Weaver & Gilbert 2004.)

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The premise of the more recent tumour microenvironment concept of malignancy holds that there are always genetically primed epithelial cells in the tissues that have a low innate pre-disposition to develop into cancer. However, if these cells receive exogenous stimuli and reside in a favourable microenvironment, the likelihood of cancer initiation and progression in these cells drastically becomes higher through the stimuli that they experience. (Bissell & Radisky 2001, Sung & Chung 2002, Kenny & Bissell 2003, Unger & Weaver 2003.) Epithelial cancer cells can induce genotypic and phenotypic changes in stromal cells. These activated stromal cells release factors that can support malignant transformation, survival and progression of tumour cells. This creates a cycle in which the epithelial and stromal compartments influence one another so that eventually cancer progression is greatly enhanced. This means that the stroma plays a far more active role in the initiation of the cancer that has so far been thought. (Berking et al. 2001, Bissel & Radisky 2001, Sung & Chung 2002, Bhowmick et al. 2004, Weaver & Gilbert 2004.) An increasing number of soluble factors have been implicated as autocrine and paracrine factors involved in cancer initiation and progression. These include the FGF-, IGF-, EGF-, HGF- and TGF-β-families (Bhowmick et al. 2004). Most of the factors involved are proliferation stimulants. Members of the TGF-β family, expressed by tumour cells, fibroblasts and inflammatory cells, have both tumour promoting and suppressing effects. Increased TGF-β signalling has been shown to inhibit tumour formation by reducing cancer cell proliferation. On the other hand, loss of sensitivity to TGF-β is linked to increased tumorigenicity. TGF-β also reduces the amount of adherens junctions and thus induces the migration of epithelial cancer cells. This can have pro-invasive and metastatic effects. (Akhurst & Derynck 2001.) In stroma TGF-β induces a desmoplastic reaction with increased collagen expression, and the release of stromal cell-derived cytokines, which affect cancer cell behaviour (Löhr et al. 2001, Sung & Chung 2002).

2.4.2 Myofibroblasts

Myofibroblasts are defined as fibroblast-like cells with features resembling smooth muscle cells. A notable feature is the ability of these cells to contract and, in most cases to express α-smooth muscle actin. Therefore, myofibroblasts can be considered as intermediates between fibroblasts and smooth muscle cells. Myofibroblasts are involved in various processes like organogenesis, inflammation, tissue repair, fibrosis and oncogenesis. In desmoplasia, myofibroblasts secrete a number of ECM components, including collagens, other fibrillar proteins, proteinases and a wide variety of growth factors leading to the remodelling of the matrix. Neoplastic growth of myofibroblasts themselves leads to angiosarcomas, fibrosarcomas, histiocytomas and mesotheliomas. Myofibroblasts most likely transdifferentiate from tissue fibroblasts, although they may originate from other cells, too. Cancer cells have been shown to induce this transdifferentation by secreting soluble factors, most notably TGF-β1. (Powell et al. 1999, Tomasek et al. 2002.)

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2.5 Cornea

2.5.1 Structure of the cornea

The cornea forms the outermost layer of the eye (Figure 4.). It functions as a part of the optical path in the eye. Together with the sclera it forms the protective shield around the inner structures of the eye. In general, tissues contain blood vessels to receive nourishment, oxygen and protection against infection. However, the cornea is an exception to this rule since it normally does not contain any vessels. Instead, it receives its nourishment from tears and the aqueous humor that fills the chamber behind it. The reason for this is that cornea must remain transparent to refract light properly. (Fini 1999, Kanski 2003.)

The outermost structure in the cornea is epithelium, which has two primary functions. It blocks the entrance of foreign material such as dust, water and bacteria into the eye. It creates a smooth surface that absorbs oxygen and nutrients from tears and supplies these to the inner layers of the cornea. The corneal epithelium is stratified, squamous and non-keratinised with a strong regenerative capacity. The epithelium also contains a dense network of nerve endings that make the cornea extremely sensitive to pain. Under the epithelial cells is basement membrane, which is situated next to the Bowman’s layer, which is composed of collagen fibres. If injured, the Bowman's layer can form a scar.

The largest part of the cornea is the stroma, which comprises as much as about 90 percent of the thickness of the cornea. The corneal stroma is an extremely hydrated structure, and it contains large amount of collagens, the presence of which gives the cornea strength, elasticity and form. The stromal collagen fibres have a unique lamellar arrangement and spacing which gives the cornea its light-conducting transparency. Corneal cells, called keratocytes, reside between the 50-100 collagen lamellas. The innermost parts of the cornea are Descemet's membrane and the endothelium. Descemet's membrane is a thin strong sheet that serves as a protective barrier against infection and injuries. It is composed of collagen fibres and it is made by the endothelial cells. Endothelial cells collectively function as a pumping machine, keeping the fluid balance of the cornea stable by pumping the excess fluid out of the stroma. Without this action, the stroma would swell and become opaque. Endothelial cells do not have the capacity to regenerate themselves after trauma or disease, so if the loss of these cells is sufficient, corneal edema and loss of sight ensues. (Fini 1999, Kanski 2003.)

2.5.2 Collagens of the cornea

The eye is a complex structure and a large number of collagen proteins have been detected in the various parts of the eye. So far at least 16 collagen types have been detected in maturing or adult corneas. These include type I, II, III, IV, V, VI, VII, VIII, IX, XII, XIII, XIV, XVII, XVIII, XX and XXIV collagens (Ihanamäki et al. 2004); this section describes only those that have mutations linked to corneal abnormalities.

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The importance of collagens in the cornea is exemplified by the fact that 70% of the dry weight of cornea is composed of collagens (Tseng et al. 1982). Stromal collagens are multimerised into highly organized lamellar structures. These structures are composed of type I, III and V collagens (Tseng et al. 1982), the latter of which is proposed to be involved in the regulation of collagen fibril diameter, presumed to be essential for transparency (Birk et al. 1990). Mutations of type V collagen have been shown to cause corneal abnormalities (Giunta & Steinmann 2000). Type IV collagen localises in the basement membranes. In Alport syndrome, with defects in COL4A3, A4 and A5 genes, a variable degree of corneal defects has been observed (Pajari et al. 1999). Type VIII collagen is the major component of the Descement’s membrane, and mutations in type VIII collagen cause endothelial dystrophies in the cornea (Biswas et al. 2001, Hopfer et al. 2005). Type VII collagen localises to the basement membrane zone of the corneal epithelium where it forms the anchoring fibrils (Tuori et al. 1996, Fukuda et al. 1999). In dystrophic forms of the epidermolysis bullosa, mutations in type VII collagen cause corneal clouding and blisters under the basal lamina (Tong et al. 1999).

2.5.3 Keratoconus

Keratoconus is a progressive eye disease that affects the cornea and disturbs its tissue architecture. This condition usually starts at puberty, continuing to worsen until the patient has reached 30-40 years of age. It is, however, possible that the disease may begin later in life or it may stop earlier. In rare cases it is congenital. The incidence of the disease is 1 per 2000 individuals. (Rabinowitz 1998.)

In clinical inspection the keratoconus cornea has a conical shape as a result of a non-inflammatory thinning of the corneal stroma. This abnormal curvature changes the refractive power of the cornea, producing moderate to severe distortion of vision. Symptoms usually begin in one eye but ultimately both eyes are affected. In severe cases scarring develops on the cornea leading to severe impairment of sight. In most cases symptoms are milder, however. (Kanski 2003.)

Histopathological signs of the disease are breakages in the Bowman’s layer, degeneration of the basal epithelial cells, deposition of iron in the basal epithelium and subepithelial scarring. The stroma becomes thinner and acquires structural changes. As it becomes more compact, the arrangement of the anterior collagen fibrils deteriorates and the number of lamellae decreases. In addition, granular and fibrillar material starts to accumulate in the stroma. In advanced stages, all corneal layers may be affected but in most cases Descemet’s membrane and endothelium are normal. (Rabinowitz 1998.)

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Fig. 4. Schematic picture of the structure of the eye, cornea and the perpendicular corneal collagen fibre lamellae. The various layers of cornea are not depicted in scale relative to each other. BM indicates basement membrane.

The ethiology of keratoconus is not fully known. It has been suggested that keratoconus could be a disease of the epithelial-to-stromal communications. An injured corneal epithelium could stimulate keratocyte apoptosis and a greater turnover of the keratocytes, which in turn could increase degradative enzymes in diseased corneas. Supporting this the keratoconus corneas have apoptotic keratocytes, which are not present in normal corneas, and an increased level of matrix-modifying enzymes. In a majority of the cases, keratoconus is a sporadic disease with no associated systemic or ocular disease. However, in about 7% of cases there is a family history of keratoconus. There are also reported associations of keratoconus with osteogenesis imperfecta, Ehlers-Danlos syndrome, joint hypermobility and Down’s syndrome, for example. Also, mechanical trauma and a prolonged use of hard contact lenses have been implicated as causative factors. Based on this, the initiation of keratoconus may be a combination of mechanical trauma and genetic predisposition. (Rabinowitz 1998, Kanski 2003, Wilson et al. 2003.)

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2.5.4 Corneal wound healing

The post-trauma or -infection wound healing processes of the cornea resemble in some respects those of the skin. In the case of the cornea, the healing process must restore the mechanical integrity of the cornea as a whole and the protective function of the epithelium. This has to be achieved while maintaining optical properties. For the healing process to succeed, the interactions between the stromal and epithelial cells, corneal nerves and epithelial cells, corneal and immune cells, and those between the stromal cells and the endothelium, must occur in an appropriate and timely manner. These interactions are mainly mediated by growth factors, cytokines and chemokines, but also direct cell-cell interactions are involved. The overall wound healing process can last from months to years. (Wilson et al. 2001, Wilson et al. 2003.)

The first step in corneal wound healing is a fast initiation of keratocyte apoptosis in the most apical stroma. The apoptotic effect can cover 20-50% of the stromal thickness (Wilson et al. 1996, Mohan et al. 2003). Apoptosis is a cytokine-mediated process, where interleukin-1(IL-1) and tumour necrosis factor α (TNF-α) play a central role. IL-1 is expressed constitutively in the epithelium but is decificient from the stroma. Instead, keratocytes express the IL-1 receptor which can bind to IL-1 if the epithelial barrier is compromised. (Wilson et al. 1997, Wilson et al. 2001.) In the remaining keratocytes IL-1 induces its own production creating an autocrine loop which eventually induces the expression of other growth factors, cytokines and matrix remodelling enzymes (West-Mays et al. 1995, Weng et al. 1996).

Immediately after injury, epithelial cells next to the affected area start to migrate to form a monolayer over the defect. The migrated cells and also cells from further away begin to proliferate in order to increase the number of cells and restore the full thickness of the epithelium. Later these cells start to differentiate and develop the layered structure of the epithelium. (Suzuki et al. 2003.) Normally keratocytes, but not epithelial cells, express low levels of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), which are responsible for the homeostasis of stromal-epithelial interactions. These factors regulate epithelial cell growth, migration and differentiation. The expression of these factors in the keratocytes is, however, markedly upregulated after injury. (Wilson et al. 1994.) Immediately after corneal trauma there is also an increase in the HGF level in tears. This response is probably autonomously mediated by the extensive innervation of the corneal epithelium to the lacrimal glands. The cytokines in tears may have a role in the regulation of cell behaviour in the trauma area. (Wilson et al. 1999.)

The early loss of keratocytes in the apical stroma is followed by keratocyte proliferation in other parts of the stroma. These cells migrate to the trauma area where they most likely differentiate into myofibroblasts. The myofibroblast transformation is at least partly induced by the epithelium-derived TGF-β. (Jester et al. 1999, Jester et al. 2003, Suzuki et al. 2003.) Myofibroblasts express a variety of growth factors and cytokines, together with matrix remodelling enzymes and different matrix components, including collagens. This leads to an extensive remodelling of the stroma. Myofibroblasts are also responsible for wound contraction. Epithelium- and stroma-derived cytokines and chemokines also attract inflammatory cells to the trauma area where their likely

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function is to remove pathogens and cellular debris. (O’Brien et al. 1998, Wilson et al. 2001.) After the initial trauma has been repaired and the epithelium is intact, inflammatory cells and myofibroblasts are eliminated by apoptosis. This stromal remodelling can continue for a long period of time, during which the original optical qualities of the cornea gradually improve. However, in some cases the lamellar structure of the stroma remains at least partially unorganised and clinically visible scarring remains. (Wilson et al. 2001.)

3 Outlines of the present study

Previous work on type XIII collagen has revealed details concerning its biosynthesis, the existence of a soluble proteolytic form of the protein, information about the association of the soluble ectodomain with some matrix components, and its effects on cell behaviour and matrix structure. Data from transgenic animals has suggested that type XIII collagen may be involved in human diseases. To gain an insight on the function of a protein, information about its cell and tissue localisations is essential. The focus of this thesis has therefore been to study the cell biology of type XIII collagen. Additionally, so far practically no information about the expression levels of this collagen in human diseases has existed. Therefore, the aims of this thesis were determined as:

1. To study the subcellular and tissue localisation of type XIII collagen. 2. To study whether type XIII collagen associates with membrane microdomains, and if

this localisation has relevance to the shedding of the ectodomain. 3. To study the expression of type XIII collagen in some human diseases, e.g. human

neoplasias and corneal pathologies.

4 Materials and methods

4.1 Analysis of type XIII collagen localisation (I)

4.1.1 Cells and tissue samples (I)

HT-1080, MG-63, WI-38 and WI-38-VA13/3 cells were cultured according to the suppliers protocol (ATCC). Primary human umbilical vein endothelial cells were obtained from the Department of Pharmacology, University of Oulu. Endothelial cells were cultured on rat tail collagen type I-coated plastic dishes or glass cover slips in 10% FBS in DME medium (Becton Dickinson). Normal human skin fibroblasts were established locally from skin biopsies taken from healthy individuals by standard methods. The human muscle sample was obtained from a neck operation on a healthy male individual with an informed consent of the patient and an approval of the Ethical Committee of the Oulu University Hospital. The mouse samples were derived from NMRI mice and frozen in liquid nitrogen immediately after excision. Mice were obtained from the Experimental Animal Center, University of Oulu.

4.1.2 Microinjections (I)

The microinjections were performed using an Eppendorf micromanipulator 5171 and a microinjector 5246 installed on an Axiovert 405M inverted microscope with a heating stage (Zeiss). C3 exoenzyme was dissolved in 100 mM KCl buffer (pH 7.25) and used at a concentration of 0.15 mg/ml. Injections into cells were done by applying a pressure of between 93-150 hPa for 0.3 seconds (Eskelinen & Lehto 1994). Intracellular pH was maintained normal during the injections by culturing the cells in Eagle's minimal essential medium with Hank's salts (pH 7.4). After the injection, the cells were returned to the normal growth medium and incubated for the desired time. Approximately 20–40 cells were injected within 15–30 minutes for each experiment. Cells were then analysed by immunofluorescence staining as described in the section 4.1.4.

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4.1.3 Production of the NC1 domain antibody (I)

Antigens were produced as follows: two mouse type XIII collagen cDNA fragments, corresponding to the cytoplasmic (Q47) and extracellular (Q67) regions of the NC1 domain, were amplified from a previously isolated cDNA clone (Hägg et al. 1998) by PCR. The products were subcloned into the pQE-41 vector (Qiagen) in frame with the dihydrofolate reductase (DHFR) gene, with an aminoterminal His tag in the vector. After transformation into E. coli strain M15, the correct subcloning of the cDNA fragments was verified by nucleotide sequencing, followed by a large-scale protein expression in the bacteria according to the instructions of the vector supplier (Qiagen). The bacterial cultures were suspended and lysed in 6 M guanidine–HCl, 0.5 M NaCl, 20 mM Tris–HCl (pH 7.9), followed by centrifugation at 12 000 g for 20 minutes to clarify the lysate. After sonication, the supernatant was supplemented with 1 mM imidazole and applied to ProBond columns (Invitrogen) pre-equilibrated with 8 M urea, 0.5 M NaCl, 20 mM Tris–HCl (pH 7.9). The bound polypeptides were eluted with imidazole gradient. Collected fractions were analyzed by SDS-PAGE and Coomassie staining. The fractions containing type XIII collagen-derived polypeptides were dialysed against PBS and concentrated by ultrafiltration for the immunisation. Rabbits were immunised subcutaneously with 300 μl of the purified fusion protein solutions combined with complete Freund's adjuvant. Booster injections, with incomplete Freund's adjuvant, were administered at 14 day intervals.

The affinity resin was prepared for the purification of the crude rabbit antisera as follows: the intracellular and extracellular cDNA fragments of the NC1 domain (i.e. the NC1 domain without the transmembrane domain) of type XIII collagen were amplified by PCR, and subcloned into pQE-31 vector (Qiagen), with a vector-derived aminoterminal His tag. Transformation, sequencing and the protein expression in the bacteria were done as described above. One clone (Q610.22) was selected for the large-scale protein expression and purification, performed as above, except that the bound polypeptides were renatured for 2 hours by a stepwise decrease in the concentration of urea, followed by stabilisation in 0.5 M NaCl, 20 mM Tris–HCl buffer (pH 7.9) before the elution. Subsequently, the collected fractions were analysed by SDS-PAGE and Coomassie staining. Fractions containing type XIII collagen polypeptide were pooled, dialysed against 50 mM sodium phosphate buffer (pH 6.9) with 5% glycerol, and applied into a HiTrap SP column (Pharmacia Biotech). Elution was performed using a stepwise gradient of NaCl from 0 to 2M in 50 mM sodium phosphate buffer (pH 5.5) with 5% glycerol. The bound polypeptides were analysed by SDS-PAGE and Coomassie staining, and by partial aminoterminal amino acid sequencing using Edman degradation chemistry with an Applied Biosystems 477A Protein Sequencer (Department of Medical Biochemistry and Molecular Biology, University of Oulu). The purified polypeptide Q610.22 was dialysed against 0.5 M NaCl in 0.2 M NaHCO3 buffer, pH 8.6, and immobilised on CNBr-Sepharose 4B (Pharmacia Biotech) according to the manufacturer's instructions. The rabbit anti-sera were diluted 1:1 with PBS, and applied to the CNBr-Sepharose 4B columns. After washing with 2 M NaCl in PBS, bound antibodies were eluted with 250 mM glycine–HCl (pH 2.5) and 100 mM triethylamine (pH 11.0). Fractions containing antibodies were detected by UV absorbance, neutralised

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with 2 M Tris–HCl (pH 7.5), pooled and concentrated by ultrafiltration. The affinity purified antibody was named Q610.

4.1.4 Immunofluorescence staining (I)

Cells cultured on glass cover slips and 5 μm cryosections were fixed with methanol at −20°C for 5 or 15 min, respectively. Samples were blocked with 1% BSA and incubated with the primary antibodies for 30 minutes to overnight. After washing in PBS, the samples were incubated with the secondary antibodies for 30 minutes under the same conditions. Stained samples were embedded with Immu-Mount mounting medium (Shandon) and viewed with an epifluorescence microscope (Leitz Aristoplan). Phosphotyrosine was stained identically, except that the HEPES-buffered saline was used instead of PBS in all steps and the blocking was omitted.

Sf9 insect cells were infected as described in section 4.1.5 Cells were blocked with 0.2% BSA and incubated with 10 μg/ml anti-type XIII collagen/NC3 antibody for 30 minutes at +4°C. After post-fixing with methanol at −20°C for 5 minutes, the cells were incubated with the secondary antibody for 30 minutes, followed by embedding. The stained cells were viewed with an epifluorescence microscope.

Immunofluorescence stainings were performed with the following antibodies: mouse monoclonal antibodies against phosphotyrosine (Sigma), talin (Serotec), vinculin (BioGenex), human collagen IV (Dako), α-catenin (Dr S. Tsukita, University of Kyoto, Japan), desmoplakins I and II (Boehringer Mannheim) and fluorescein-conjugated α-bungarotoxin (Molecular Probes). Actin filaments were stained with Bodipy Phallacidin (Molecular Probes), as suggested by the manufacturer.

4.1.5 Insect cell adhesion experiment (I)

Viruses encoding full-length human type XIII collagen (virus wt humanXIII; Snellman et al. 2000b) (m.o.i.=5) and the α and β subunits of prolyl 4-hydroxylase (virus 4PHαβ; Nokelainen et al. 1998) (m.o.i.=1) were used to infect SF9 insect cells (7.6×104 cells/cm2) (Invitrogen) cultured in serum-free HyQ CCM3 medium (Hyclone Laboratories) at +27°C for 48 hours. Culture medium was renewed daily with 80 μg/ml ascorbate. Infected cells were suspended in serum-free HyQ CCM3 medium, and 3×104 cells per well were seeded on 96-well coated plates (Nunc MaxiSorp). Prior to cell seeding, the wells had been coated with 2 μg/cm2 human vitronectin, rat tail collagen type I or poly-D-lysine (Becton Dickinson Collaborative Biomedical Products) and blocked with 10 mg/ml BSA (Serva). Adheration was allowed to proceed for 20 minutes at +27°C, after which the number of adhered cells was measured with CyQuant Cell Proliferation Assay Kit (Molecular Probes).

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4.1.6 Immunoprecipitations and preparation of protein samples (I)

For immunoprecipitations, cells were scraped and lysed into ice-cold PBS supplemented with 1 mM PMSF, 1 mM N-ethylmaleimide and 1% TX-100. Nuclei were removed by centrifugation. Protein concentrations of the supernatants were measured using the BioRad Protein assay kit. Preclearing of 1 mg of protein per sample was done with 100 μl of 10% w/v protein A-Sepharose (Pharmacia Amersham). Immunoprecipitation was performed overnight at +4°C with 10 μg of the antibody of interest. Immunocomplexes were recovered by incubation with 100 μl of 10% w/v protein A-Sepharose. After washing and boiling in sample buffer containing 100 mM β-mercaptoethanol, equal volumes of samples were analysed by SDS-PAGE and immunoblotting using standard protocols. Sodium bicarbonate extraction of the cell membranes has been described previously (Hägg et al. 1998).

4.2 Cholesterol dependence of type XIII collagen ectodomain shedding (II)

4.2.1 Analysis of membrane domains by density gradient ultracentrifugation (II)

Equal numbers of HT-1080 cells were treated for 30 minutes with either 20 mM MCD or 20 mM MCD-cholesterol complex (1:1) (Leitinger & Hogg 2002) or with 5 μg/ml cytochalasin D for 20 minutes before detachment with 5 mM EDTA. Cells were resuspended in 20 mM Tris-HCl, pH 8.2 with 2 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mM PMSF and 1% TX-100 or 1% CHAPS as a detergent. In detergent-free experiments cells were resuspended in 150 mM Na2CO3, pH 11, containing 2 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin and 1 mM PMSF (Song et al. 1996). All cell suspensions were centrifuged at 12300x g for 30 minutes after homogenisation, sonicated and sheared by passing through hypodermic needles. Equal volumes of supernatants were mixed with lysis buffer containing 90% sucrose. The obtained supernatant samples with 45% sucrose were assembled into gradients with 35% sucrose and 5% sucrose lysis buffers. Gradients were centrifuged at 150000x g for 22 hours in a Beckman SW50.1 rotor. (Claas et al. 2001, Maile et al. 2002). All the steps mentioned above were carried out at +4ºC. After centrifugation, fractions were collected from the top of the gradients and prepared for SDS-PAGE by adding sample buffer with β-mercaptoethanol, and boiled. Equal sample volumes were analysed by immunoblotting with type XIII collagen, furin (Santa Cruz Biotechnology), CD71 (Santa Cruz Biotechnology) and caveolin-1 (BD Transduction Laboratory) antibodies.

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4.2.2 Whole-cell shedding assay (II)

Equal numbers of HT-1080 cells were cultured in serum-free medium supplemented with MCD, MCD-cholesterol complex (1:1), or Lovastatin–mevalonate-MCD for 30 minutes. The Lovastatin-mevalonate-MCD treatment was preceded by preincubation of the cells with Lovastatin (2 or 4 μM) and 0.25 mM mevalonate in 2% fatty acid-free BSA in DME medium for 24 hours. After an overnight shedding phase, the shed ectodomain in the media was precipitated by ice-cold methanol at -20°C, collected by centrifugation, suspended in a reducing sample buffer, and analysed by Western blotting using type XIII collagen antibody. (Hooper et al. 1997, Väisänen et al. 2004).

4.2.3 Determination of cellular cholesterol (II)

Cellular cholesterol was isolated from equal cell numbers using 3x106 cells per assay. Cells were disrupted by three freeze-thaw cycles followed by sonication. Cholesterol was extracted from the cell lysates with chloroform-methanol (1:1) by rotation for 15 minutes at room temperature. After centrifugation, the organic solvent was evaporated under a nitrogen flow. The residual cholesterol was dissolved in 100 μl ethanol, of which 40 μl was used for the determination of cholesterol content by means of the enzymatic colorimetric test (Roche). (Folch et al. 1957, Danthi et al. 2004).

4.2.4 Filipin staining (II)

Before staining, cells were treated with 5 mM MCD, 5 mM MCD-cholesterol complex (1:1) or a combination of 2 μM Lovastatin, 0.25 mM mevalonate and 5 mM MCD for 30 minutes, followed by fixation with 4% paraformaldehyde for 30 minutes on ice. The cellular cholesterol was visualised by staining the cells with 125 μg/ml filipin for 15 minutes. (Keller & Simons 1998, Simons et al. 1998). The cells on the cover slips were mounted with ImmuMount™.

4.2.5 Cell-surface biotinylation and streptavidin pull-down assay (II)

Cell surface biotinylation was performed for equal cell numbers prior to the shedding assay with a FluoReporter Cell-Surface Biotinylation Kit (Molecular Probes). Subsequently, cells were treated with either 5 mM MCD or 100 ng/ml phorbol 12-myristate 13-acetate (PMA). Culture media were centrifuged and subjected to streptavidin pull-down with avidin-conjugated protein A Sepharose. (Arribas et al. 1996, Baldwin & Ostergaard 2002, Väisänen et al. 2004). The residual media were precipitated with ice-cold methanol as described in section 4.2.2 The samples were dissolved in a reducing sample buffer and analysed by SDS-PAGE and immunoblotting with type XIII

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collagen, GM130, calnexin (BD Transduction Laboratory), γ-adaptin (BD Transduction Laboratory), and Rab-5 (Abcam) antibodies, using standard protocols.

4.2.6 Vesicle secretion assay (II)

Equal numbers of HT-1080 cells were either biotinylated, as described in the section 4.2.5, or left unbiotinylated, depending on the assay. Following that, cells were treated with 5 mM MCD, 100 ng/ml PMA or 100 μM dec-RVKR-chloromethylketone (Garten et al. 1994). After the shedding phase, the media were cleared of detached cells by centrifugation. The vesicles were collected by ultracentrifugation in a Beckman Ti55.2 rotor at 74000x g for 3 hours. Pellets containing the vesicles were recovered in 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1% TX-100 and 20 mM Tris, pH 7.6. (Gutwein et al. 2002). In the case of the biotinylated cells, the vesicle suspensions were directed to the streptavidin pull-down, as described in section 4.2.5 Cell lysates were produced by scraping the cells from the plates and suspending them in the lysis buffer. The samples were analysed by SDS-PAGE and immunoblotting, with type XIII collagen, GM130, calnexin, γ-adaptin and Rab-5 antibodies, using standard protocols.

4.2.7 TX-114 phase partitioning (II)

The cell culture media were cleared of detached cells by centrifugation and chilled to +4°C, before dissolving in TX-114 to a final concentration of 1%. TX-114 was condensed at +37°C for 15 minutes, followed by collection of the aqueous phase by centrifugation at 1700x g for 20 minutes. After cooling, partitioning was repeated with fresh TX-114. (Bordier 1981, Holm et al. 1986). The aqueous and detergent phases were pooled and precipitated with ice-cold methanol or acetone, respectively. Precipitated proteins were dissolved in a reducing sample buffer, separated in SDS-PAGE and analysed by immunoblotting with type XIII collagen antibody, using standard protocols.

4.2.8 Cell fractionations (II)

4.2.8.1 Golgi fractionation (II)

HT-1080 cells were homogenised in 0.25 M sucrose, 5 mM MgCl2 and 25 mM HEPES, pH 7.3, with a glass-teflon homogenizer, centrifuged in a Sorvall SS-34 rotor at 10000x g for 10 minutes. The postnuclear supernatant was collected and centrifuged with a Beckman Ti55.2 rotor at 100000x g for 45 minutes. The resulting pellet, containing the total membrane fraction, was resuspended in the homogenisation buffer, the sucrose content of which was adjusted to 1.25 M. This was centrifuged in a discontinuous sucrose gradient in a Beckman SW40 rotor at 100000x g for 2.5 hours. Materials at the 0.25/1.1

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M and 1.1/1.25 M interfaces were defined as Golgi and smooth endoplasmic reticulum (sER) fractions. The pellet representing the rough endoplasmic reticulum (rER) was resuspended in the homogenisation buffer. The sucrose concentration of the Golgi fraction was diluted to 0.25 M, followed by centrifugation in a Beckman Ti55.2 rotor at 100000x g for 45 minutes. The resulting pellet was incubated in 10 mM EGTA for 30 minutes on ice, centrifuged as before and the supernatant representing peripheral Golgi proteins was collected. The pellet was resuspended in 150 mM Na2CO3, pH 11.5, incubated on ice for 30 minutes and centrifuged as before. The supernatant contained the luminal Golgi proteins and the pellet the Golgi membranes. All procedures were carried out at +4oC. (Howell & Palade 1982, Subramamiam et al. 1992, Yan et al. 2001). When treated with brefeldin A (BFA), the cells were preincubated in 30 μM BFA for 6 hours prior to fractionation. Fractions were analyzed by SDS-PAGE and immunoblotting with type XIII collagen and calnexin antibodies using standard protocols.

4.2.8.2 Sucrose density gradient fractionation (II)

For the sucrose density gradient fractionation, HT-1080 cells were homogenised in 0.25 M sucrose, 10 mM magnesium acetate and 10 mM Tris, pH 7.4, and centrifuged at 620x g for 10 minutes. The postnuclear supernatant was centrifuged in a discontinuous sucrose gradient in a Beckman Ti55.2 rotor at 74000x g for 2.5 hours. Fractions of 1 ml were collected from the top of the gradient, precipitated at -20oC with an equal volume of ice-cold methanol, resuspended in reducing sample buffer with β-mercaptoethanol, and boiled. All the samples and fractions were analysed by SDS-PAGE and immunoblotting with type XIII collagen, GM130, calnexin, γ-adaptin and Rab-5 antibodies.

4.3 Type XIII collagen expression during malignant transformation (III)

4.3.1 Collection of the tumor material (III)

All the tissue samples used were originally collected for diagnostic purposes. Suitable cases were selected on the basis of patient records and original pathological diagnosis. Hematoxylin-eosin-stained archival tissue sections obtained from the Department of Pathology, University of Oulu, were inspected by a pathologist and the representative foci for the core biopsies (normal, dysplastic, tumour) were marked. 1 mm core needle samples from the donor blocks were re-assembled into 96-sample tissue micro array (TMA) blocks as described elsewhere (Kononen et al. 1998). Final arrays consisted of normal, dysplastic and tumour samples of colon (sample number n=109), cervix (n=113), urinary bladder (n=64), endometrium (n=86), ovary (n=63) and various mesenchymal tumours (n=89). Array blocks were cut into 4-μm sections for analyses. The study was approved by the Ethical Committee of the Northern Ostrobothnia Hospital District

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(85/2002), and it followed the Declaration of Helsinki for experiments involving human tissues.

4.3.2 In situ hybridisation of tumour samples (III, IV)

Type XIII collagen probes were constructed by ligating the BamHI-SmaI fragment (bases 1538-2505) of the human type XIII collagen cDNA into the pSP72 expression vector (Promega). The specificity of the probe was assayed with Northern blotting using standard protocols. For this, COS-1 cells were transiently transfected with a plasmid encoding the human full-length type XIII collagen, using FuGENE transfection reagent (Boehringer). Total RNA was isolated with the RNeasy Mini Kit (Qiagen). In order to be used as a template in the in situ hybridisations, the probe plasmid was linearised with HindIII or EcoRI to obtain anti-sense or sense templates. These templates were transcribed with T7 or SP6 RNA polymerases (Promega), respectively, to produce digoxigenin-11-UTP-labelled (Roche Diagnostics) RNA probes. Unspecific hybridisation was detected using the sense probe. Deparaffinized TMA sections were treated with 0.2 M HCl and rinsed in water, followed by proteolysis with proteinase K (Roche). The reaction was stopped with 0.2% glycine, after which sections were fixed in 4% paraformaldehyde and acetylated. This was followed by prehybridisation for 2 hours and hybridisation overnight, both at +60ºC. After post-hybridisation washes, the sections were blocked with 2% FBS and incubated with alkaline phosphatase-conjugated anti-digoxigenin Fab-fragments (Roche). Bound antibody was detected by a colour reaction using Fast Red substrate (Roche). Finally, the sections were counterstained with methyl-green and mounted. The TMA in situ hybridisation results were semi-quantified using a four-step grading score for the overall staining intensity (score 0: none, score 1: faint positive, score 2: moderate positive, score 3: strong positive) (Bachmeier et al. 2000). Differences in sample numbers between groups were taken into account by expressing the results as means of overall signal intensity score.

4.3.3 Antibody production and immunohistochemistry of tumour samples (III, IV)

4.3.3.1 Production of the NC4 domain antipeptide antibody (III, IV)

The cyclic synthetic peptide corresponding to the NC4 domain of human type XIII collagen was synthesized with Applied Biosystems 433A peptide synthesizer (Department of Biochemistry, University of Oulu) and purified using reversed-phase high pressure liquid chromatography. 5 mg of the purified peptide was coupled to cBSA (Pierce) by standard procedures using glutaraldehyde. For the immunisation, the coupled peptide with complete Freund's adjuvant was injected subcutaneously into rabbits. Booster injections with incomplete Freund's adjuvant followed at intervals of 14 days. Rabbit

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anti-serum was purified by affinity chromatography by coupling the peptide antigen to epoxy-activated Sepharose 6B according to the manufacturer (Amersham Pharmacia Biotech). Serum was diluted 1:5 with 20 mM K2HPO4, 0.1 M NaCl, pH 7.0, and applied

to the affinity column, which was subsequently washed with 20 mM K2HPO4, 0.5 M NaCl, pH 7.0, and eluted with 30 mM glycine-HCl, pH 2.9, and thereafter with 100 mM triethylamine, pH 11.0. The protein-containing fractions were detected by UV absorbance at 280 nm, immediately neutralised with 0.2 volumes of 2 M Tris-HCl, pH 7.5, pooled, and concentrated to a final concentration of 0.5 mg/ml (Microsep 30; Filtron Technology Corp.). Antibody was analysed with immunoblotting using purified type I, III, IV, V and VI collagens, lysates of HT-1080 cells or K562 cells transiently expressing either full-length type XIII collagen or the NC4 domain deletion variant as samples (Väisänen et al. 2005). The recognition specificity of the antibody was tested by a 4-fold peptide antigen competition.The purified antibody was named 777B.

4.3.3.2 Immunohistochemistry of tumour samples (III, IV)

TMA sections were deparaffinised and treated with 0.3% H2O2 in methanol to remove tissue-derived peroxidase activity. The antigen retrieval was done by boiling in 1 mM EDTA, pH 8.0 for 15 minutes, followed by blocking with 1% BSA. Sections were incubated with the primary antibodies overnight at +4ºC, followed by incubations with the biotinylated secondary antibodies and avidin-biotin-horseradish peroxidase H complex (Vectastain ABC Kit, Vector Laboratories), as instructed by the manufacturer. Bound antibodies were visualised with an enzymatic reaction using diaminobenzidine as a color substrate. Sections were counterstained with hematoxylin. In a separate control experiment for the exclusion of the unspecific staining, 777B antibody was preincubated with a molar excess of the antigen before use in immunostaining. Anti-cytokeratin CK-PAN antibody (Dako) was used to mark the cells of epithelial origin.

4.3.4 Conditioned medium experiment (III)

4.3.4.1 Preparation of conditioned medium and treatment of cells (III)

Conditioned media (CM) were collected from HT-1080 and HeLa cell cultures after an overnight incubation in serum-free DME medium. After ultrafiltration, media were supplemented with 10% FBS and diluted 1:1 with fresh serum-containing growth medium. (Löhr et al. 2001, Arnold et al. 2002). Primary skin fibroblasts were cultured in CM throughout the experiment, and the cell lines eventually obtained were named CM(HT-1080) and CM(HeLa) cells, depending on the CM used. For comparison, fibroblasts were also cultured in normal DME medium with 10% FBS for the same length of time. When studying the possible reversibility of transdifferentiation of the cells, the CM cell cultures were deprived of the CM and cultured in normal growth medium instead. The effect of TGF-β1 blocking antibody was tested by supplementing the

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undiluted CM of HT-1080 cells with 200 ng/ml anti-human TGF-β1 antibody (R&D Systems). For the analysis of protein expression, cells were suspended in the lysis buffer of 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1% TX-100 and 20 mM Tris, pH 7.6. The protein concentrations of the lysates were determined using BCA Protein Assay Kit (Pierce). Protein samples were separated in SDS-PAGE and analysed by immunoblotting using standard protocols with the following antibodies: anti-fibronectin (Sigma), anti-PCNA, anti-actin, anti-cadherin, anti-α4, anti-α5, anti-αV, anti-α2 and anti-β1 integrins (all from Santa Cruz Biotechnologies), anti-vimentin and anti-α-smooth muscle actin (α-SMA) (Abcam).

4.3.4.2 Whole-cell shedding assay (III)

CM cells were plated to sub-confluency. After an overnight incubation in a serum-free medium, precipitatation of the shed ectodomain was done with methanol at -20oC. The precipitated material was suspended in a lysis buffer of 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1% TX-100 and 20 mM Tris, pH 7.6. The protein concentrations of the lysates were determined using BCA Protein Assay Kit (Pierce). Samples were analysed by SDS-PAGE and immunoblotting with type XIII collagen antibody, using standard protocols. (Väisänen et al. 2004).

4.3.4.3 CM cell proliferation assay (III)

Equal numbers of primary fibroblasts and CM cells in serum-containing DME medium were plated in quadruplicate on 96-well plates (MaxiSorp™; NuncNalge International) and grown for 4, 24, 48 and 72 hours with daily changes of the culture medium. DNA content of the adhered cells was measured using CyQuant Cell Proliferation Kit (Molecular Probes) to determine the cell proliferation rate.

4.3.5 Expression of type XIII collagen in cancer cell lines (III)

HeLa (cervix adenocarcinoma), MG-63 (osteosarcoma), HT-1080 (fibrosarcoma), SW-480 (colon adenocarcinoma), FHC (normal colon fibroblasts), HT-29 (colorectal adenocarcinoma), T24 cells (transitional carcinoma of the urinary bladder), HEC-1-A (endometrial adenocarcinoma), MCF-7 (mammary gland adenocarcinoma), T-47D (mammary gland ductal carcinoma), OVCAR-3 (ovarial adenocarcinoma) cells were grown according to the supplier´s (ATCC) instructions. Primary dermal fibroblasts were cultured in DME medium. All media were supplemented with 10% FBS (EuroClone), except that for the OVCAR cells with 20% FBS. Cell samples were prepared for analysis as in section 4.3.4.1, and they were analysed by immunoblotting using type XIII collagen antibody.

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4.3.6 TGF-β1 and PMA treatments (III)

Serum-starved primary human fibroblasts were incubated in 0.1% BSA in DME medium, supplemented with 1 or 5 ng/ml TGF-β1 (Chemicon) or 100 ng/ml PMA (Sigma) for 24 hours (Kissin et al. 2002, Kubota et al. 2003). Untreated cells served as a control. mRNA isolation was done with the MicroFastTrack® 2.0 mRNA Isolation Kit (Invitrogen). Equal amounts of RNA samples were analysed by Northern blot hybridisation according to standard protocols using type XIII collagen cDNA (bases 1538-2505) as a probe. Loading of mRNAs was controlled by hybridisation with a β-actin control probe (Clontech).

4.3.7 Cell adhesion and spreading assays (III)

Equal numbers of primary dermal fibroblasts, HT-1080 or HeLa cells were plated in serum-free DME medium to 96-well plates that were coated with 2 μg/cm2 fibronectin or vitronectin, alone or double-coated with 20 μg/ml type XIII collagen ectodomain. All wells were blocked with 2% BSA. Cells were allowed to adhere for 30 minutes, after which unbound cells were removed. The number of adhered cells was measured with CyQuant Cell Proliferation Kit (Molecular Probes) with BSA-coated wells used to measure background adhesion. In the spreading assay, cells were allowed to adhere and spread for up to 24 hours on single or double-coated cover slips, during which time they were photographed at regular intervals.

4.4 Expression of type XIII collagen in cornea, keratoconus and corneal scarring (IV)

4.4.1 Collection of the cornea material (IV)

18 human corneas originating from penetrating keratoplasties (the Oulu University Hospital) were collected, including eight keratoconus and six scarred corneas, which had suffered previous incisional corneal wound injuries involving the corneal stroma within a mean of two years beforehand. Additionally, four cadaver corneas were excised and used as normal corneas for the comparison. The diagnoses of the diseased corneas were based on clinical history, typical clinical features and histopathology. Specimens were fixed in 10% neutral formalin and embedded in paraffin. The research was performed according to the tenets of the Declaration of Helsinki and approved by the Ethical Committee of the Northern Ostrobothnia Hospital District.

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4.4.2 In situ hybridisation of the cornea samples (IV)

Preparation of the type XIII collagen anti-sense and sense probes and the in situ hybridisation procedure have been described in section 4.3.2.

4.4.3 Immunohistochemistry of the cornea samples (IV)

The production of the 777B antibody has been described in section 4.3.3.1. The immunohistochemical staining protocol is described in section 4.3.3.2. The only exception here is replacement of the primary antibodies with phosphate-buffered saline in order to control α-SMA and CD34 antibody (Dako) staining specificity.

5 Results

5.1 Production of a polyclonal antibody against the mouse type XIII collagen NC1-domain and human NC4-domain (I,III)

Escherichia coli were used to produce mouse type XIII collagen NC1 domain polypeptides lacking the transmembrane segment. Immunisation of rabbits with the polypeptides produced an antiserum, which recognised human and mouse type XIII collagens in cell lysates from insect cells infected with recombinant viruses encoding prolyl 4-hydroxylase and human or mouse type XIII collagens, respectively (Infected insect cells were provided by A. Snellman, Department of Medical Biochemistry and Molecular Biology, University of Oulu). No immunopositivity was observed in the control lysates of cells infected with only recombinant prolyl 4-hydroxylase virus (not shown). The antiserum was affinity-purified using a column in which the recombinant type XIII collagen fragment was coupled to Sepharose beads. The antibody specifically detected a protein of the same size as the previously described anti-type XIII collagen NC3 domain antibody (Hägg et al. 1998).

A novel polyclonal antibody against the conserved NC4 domain of human type XIII collagen was also produced. This antibody was named 777B. In immunoblotting of type XIII collagen expressing K562 cells, the new antibody recognised a protein of the same size as the type XIII collagen NC3 domain antibody (Hägg et al. 1998). Blocking of the antibody with the antigen peptide ruled out an unspecific reaction both in immunoblotting and in IHC.

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5.2 Subcellular localisation of type XIII collagen (I)

5.2.1 Type XIII collagen is a membrane-linked focal adhesion protein in cultured human cells (I)

A previous study has shown that type XIII collagen is an integral plasma membrane protein in HT-1080 fibrosarcoma cells (Hägg et al. 1998). Type XIII collagen from normal skin fibroblasts was compared with that derived from HT-1080 fibrosarcoma cells, and immunoblotting with a specific antibody revealed a group of protein variants in the size range of 85–95-kDa in reduced conditions. This indicated that type XIII collagen protein variants with similar sizes exist in both cell types. However, when compared to HT-1080 cells, the expression level in the human skin fibroblasts was very low. To assess the association of the primary fibroblast-derived type XIII collagen with the cellular membranes, total membrane preparation was extracted with 0.1 M NaHCO3 (pH 11.5) as described earlier (Hägg et al., 1998). Detected by immunoblotting, the presence of 85–95-kDa proteins in the membrane pellet indicated that type XIII collagen is an integral membrane protein in normal human skin fibroblasts.

For the determination of the subcellular localisation of type XIII collagen, human cells were grown on glass cover slips for 18–24 hours and stained with a specific antibody against type XIII collagen. In normal human skin fibroblasts type XIII collagen was detected mostly as round or slightly elongated peripheral spots, which were not present in pre-immune serum controls or in antigen-blocked controls. In double immunofluorescence stainings, focal adhesion marker proteins vinculin and talin co-localised with type XIII collagen at these sites. These sites were also the endpoints of the phallacidin-stained actin stress fibres. In fully spread cells, centrally located type XIII collagen-positive fibrillar structures were also observed.

5.2.2 Type XIII collagen dynamics during assembly and disassembly of focal adhesions resemble that of talin, vinculin and phosphotyrosine (I)

The turnover of cell adhesion structures is a highly regulated process. The behaviour of type XIII collagen in this process, such as its appearance and/or disappearance in focal complexes and/or mature adhesions, may give information about the association of type XIII collagen with focal adhesions.

To study this process, human skin fibroblasts were plated on glass cover slips and observed by immunofluorescence staining. Cells formed lamellipodia-like round formations around the cell centre shortly after the initial attachment. These structures contained circular stainings at the cell edges, which were positive for type XIII collagen, vinculin and talin. At a later point, the staining pattern turned spot-like and subsequently elongated into focal adhesion-like stripes. These structures were localised at the end of the actin stress fibres. Closer to the cell centre, the type XIII collagen antibody also stained fibrous structures resembling fibrillar adhesions. Notably, type XIII collagen was

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also identified at the outer edge of the leading lamellae in the migrating cells co-localising with vinculin.

ADP-ribosyltransferase C3 (C3 exoenzyme) causes the inactivation a small GTPase Rho by selective ADP-ribosylation (Aktories & Frevert 1987, Sekine et al. 1989), leading to the dissociation of actin stress fibres and focal adhesions (Nobes & Hall, 1995). C3 toxin was microinjected into the human primary fibroblasts, and the disappearance of type XIII collagen from the focal adhesions was compared to phosphotyrosine and vinculin. After C3 exoenzyme injection, the disintegration of actin stress fibres and focal adhesions was seen, whereas the control cells injected with KCl buffer or the uninjected cells exhibited well-developed, unaffected stress fibres. After 90 minutes an almost complete disintegration of the focal adhesions and their components, including type XIII collagen, had taken place, and the components had been relocated to the central parts of the injected cells. The few remaining cellular adhesions which were still detectable contained type XIII collagen. Co-localisation of type XIII collagen with the focal adhesion markers was evident at any time between 0 and 90 minutes.

5.3 Cell surface expression of type XIII collagen changes adhesiveness of Sf9 insect cells (I)

The possible involvement of type XIII collagen in cell adhesion, as suggested by cellular localisation, was addressed by expressing it in Sf9 insect cells. Stable triple-helical human collagens, also type XIII collagen, can be produced in insect cells using a baculovirus system by co-expression of prolyl 4-hydroxylase α and β subunits with collagen(s) (Lamberg et al., 1996, Snellman et al., 2000b). Sf9 cells adhere poorly to the ECM in cell culture conditions, indicating minimum interference from other adhesion molecules. This makes them suitable as test cells. Sf9 cells were infected with viruses coding for type XIII collagen and prolyl 4-hydroxylase or prolyl 4-hydroxylase alone as a control. Non-permeabilised cells following co-infection of the two viruses showed a strong type XIII collagen cell surface staining, while only background staining was observed in the control cells. Cell adhesion assays demonstrated that the insect cells expressing type XIII collagen and prolyl 4-hydroxylase attached better on vitronectin, type I collagen and poly-D-lysine when compared to the control cells.

5.4 Localisation of type XIII collagen on tissue level (I)

5.4.1 Type XIII collagen localises to the adherens junctions in skeletal muscle and the intercalated disks in heart (I)

The cell culture results pointed to the possible role of type XIII collagen in cell adhesion. To explore this possibility further, various adhesive structures in tissues were studied. Skeletal muscle contains integrin-based structures specialised in cell–matrix adhesions. In

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human muscle, type XIII collagen was concentrated in the myotendinous junctions together with vinculin. The same localisation was observed in mouse skeletal muscle. In addition, type XIII collagen staining that coincided with vinculin was seen along the sarcolemma. This staining has been reported to be present in costameres (Pardo et al. 1983), suggesting that these structures also contain type XIII collagen. In the cross-sectioned samples there was a weak staining on the sarcolemma.

The small and large peripheral nerves within the endomycium were also stained. Here the type XIII collagen immunofluorescence staining was localised in the endoneurial sheet. The signal localisation was closely related but not totally identical to the staining of the type IV collagen, which also stained the perineurium. This suggests that type XIII collagen is expressed in the Schwann cells surrounding nerve fibres. Schwann cells are attached to the basement membranes by integrins, for instance by the α6β1 and the αV family receptors (Martin et al. 1996). Fluorescein-conjugated α-bungarotoxin staining, marking acetylcholine receptors, rarely overlapped with the nerve branches near the neuromuscular junctions which stained strongly with the type XIII collagen antibody. The lack of co-localisation suggests type XIII collagen staining in the junction area is probably derived from the Schwann cells that surround the nerve terminals although localisation at the junction is not excluded.

The intercalated discs in the heart are an analogue of myotendinous junctions, because they transmit mechanical forces. However, the junctional components in them are specialised in cell–cell adhesion and communication (Forbes & Sprelakis 1985, Severs 1990). Staining of the adult mouse heart with specific antibodies detected type XIII collagen in the intercalated discs and also in the lateral sarcolemma. At the latter site, the type XIII collagen antibody exhibited a striated staining pattern which also co-contained costameric vinculin. An attempt was made to identify the types of junctions in the intercalated discs that contain type XIII collagen. In these stainings type XIII collagen co-localised with the adherens-type junction marker vinculin, the cadherin-based junction marker α-catenin and the desmosome marker desmoplakin I/II. No decisive distinction could be made between the exact co-localisation with the various marker proteins by light microscopy. Therefore it is impossible to specifically associate type XIII collagen with any of the junction types mentioned above.

5.4.2 Type XIII collagen in other tissues (I)

Smooth muscle cells use dense bodies to attach to the ECM. These structures resemble focal adhesions of cultured cells by containing talin, vinculin and α-actinin. They can also be regarded as functional equivalents of the adherens junctions of striated muscle (Bagby 1986, Small 1995). When stained with the type XIII collagen antibody, small bright spots were seen in the smooth muscle layers of the intestine and larger blood vessels.

Structural and functional polarisation is a typical feature of epithelial cells. Also, their plasma membranes are divided into apical and basolateral compartments by junctional complexes. These junctions adhere the cells tightly to their neighbours, restrict the diffusion of the luminal contents into the surrounding tissue and allow communication and nutrient exchange between the cells. These complexes include cadherin-based

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adherens junctions, desmosomes and gap junctions. All of these junction types are present in intercalated discs. Epithelial cells also have tight junctions that appear exclusively in various epithelia. (Burgeson & Christiano 1997, Lampugnani & Dejana 1997). In the small intestine the type XIII collagen antibody stained the basal side of the epithelial cells. This signal was continuous, but uneven when compared to that of type IV collagen or laminin (not shown). Staining of foetal lung epithelia, testis and epididymis showed a similar basal localisation for type XIII collagen. These results suggest that type XIII collagen is not a ubiquitous component of adherens-type cell–cell junctions and desmosomes, but is mainly present at sites of cell–matrix adhesion.

5.5 Type XIII collagen is partly localised in membrane microdomains (II)

Light buoyant density and detergent insolubility at low temperatures are the main characteristics used to classify membrane rafts. Because of these features, raft-associated proteins can be found in the light density fractions in an isopycnic density floatation gradient (Claas et al. 2001, Rebres et al. 2001, Maile et al. 2002). Density gradient ultracentrifugation was used to study the association of type XIII collagen with membrane microdomains. The results showed that the membrane-bound type XIII collagen and furin of the proprotein convertases partly subfractionated into the lipid-enriched light density caveolin-positive membrane fractions under detergent-free conditions. This compartmentalisation was abolished by a strong non-ionic detergent TX-100, but was retained in a milder detergent CHAPS. Also the heavy density transferrin receptor CD-71-positive fractions contained type XIII collagen and furin. Caveolin-1 and CD71 were used as marker proteins for the lipid raft and non-raft domains, respectively (Claas et al. 2001, Maile et al. 2002, Wickström et al. 2003).

Sensitivity to cholesterol deprivation is another criterion defining the microdomain association of a given protein (Claas et al. 2001). The reduced distribution of type XIII collagen and furin in the light density fractions resulting from the depletion of plasma membrane cholesterol with the cholesterol-extracting agent methyl-β-cyclodextrin indicated the association of type XIII collagen and furin with the membrane microdomains. The segregation was restored if the membrane cholesterol was replenished with a methyl-β-cyclodextrin-cholesterol complex. It has been presumed that the lipid rafts are associated with actin-rich regions. The actin cytoskeleton itself or the associated proteins are proposed to stabilise rafts in the membrane. (Thomsen et al. 2002, Upla et al. 2004, Wickström et al. 2003). However, type XIII collagen and furin partitioned into the membrane microdomains even if the actin cytoskeleton was depolymerised with cytochalasin D.

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5.6 Analysis of type XIII collagen release from cells (II)

5.6.1 Type XIII collagen ectodomain shedding is sensitive to cholesterol deprivation (II)

The relationship between ectodomain shedding and the partial localisation of type XIII collagen in the cholesterol-enriched membrane microdomains was examined next. Microdomains were disrupted by manipulation of the cellular cholesterol level, and its effect on ectodomain shedding was determined by whole-cell shedding assay (Väisänen et al. 2004). Sequestration of cholesterol with β-cyclodextrins, aggregation of cholesterol to larger complexes by filipin, or inhibition of cholesterol biosynthesis with Lovastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, were used to lower the cholesterol content (Ilangumaran & Hoessli 1998, von Tresckow et al. 2004). Methyl-β-cyclodextrin is not associated with the plasma membrane, instead it removes cholesterol from the plasma membrane in a strictly cell surface-acting manner (Ilangumaran & Hoessli 1998). When used alone, it reduced the cholesterol in the HT-1080 cells by up to 35%, as also seen in the reduced intensity of filipin staining. This change induced a minor inhibition of ectodomain shedding without signs of cell toxicity. The effect of methyl-β-cyclodextrin was a specific one resulting from cholesterol depletion and not from an unspecific effect of the reagent, since the methyl-β-cyclodextrin-cholesterol complex did not reduce the cholesterol level or affect type XIII collagen ectodomain shedding. Filipin makes the cholesterol unavailable for raft formation and disrupts rafts by incorporating into the plasma membrane and aggregating with cholesterol to form larger complexes (Ilangumaran & Hoessli 1998). Filipin reduced ectodomain shedding by up to 50% in a dose-dependent manner, even though it did not markedly lower the cholesterol content of the HT-1080 cells. These two mechanistically dissimilar treatments caused the same eventual effect on ectodomain shedding by compromising the availability of membrane cholesterol for raft formation. A combination of methyl-β-cyclodextrin, Lovastatin and mevalonate were employed to achieve a more robust decrease in the cellular cholesterol level of HT-1080 cells. Mevalonate shunts the synthesis to a non-sterol pathway whereas Lovastatin inhibits the de novo biosynthesis of cholesterol (Keller & Simons 1998, Simons et al. 1998, Ehehalt et al. 2003). The cellular cholesterol level was reduced by up to 80% by using 4 μM Lovastatin and 0.25 mM mevalonate with increasing concentrations of methyl-β-cyclodextrin. This treatment also reduced type XIII collagen ectodomain shedding by up to about 75% in a dose-dependent manner. The cells with the lowest cholesterol level resulting from this treatment exhibited initial signs of reduced viability. Therefore, the experiment was repeated with a milder treatment to ascertain that the observed effect did not result from the compromised well-being of the cells. A clear reduction in the cholesterol level was achieved with 2 μM Lovastatin, 0.25 mM mevalonate and with reduced methyl-β-cyclodextrin concentrations. The effect on cholesterol level was also seen in a reduced filipin staining intensity. This time no signs of adverse cellular effects were noted and type XIII collagen ectodomain shedding was reduced.

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5.6.2 Extracellular Golgi-derived vesicles contain cleaved ectodomain (II)

Our earlier study on ectodomain shedding suggested that the shedding may occur both at the cell surface and intracellularly in the Golgi apparatus (Väisänen et al. 2004). Based on this, the cellular site(s) at which the methyl-β-cyclodextrin-induced cholesterol reduction affects the ectodomain shedding were studied. The membrane proteins at the cell surface were biotinylated, and then recovered by streptavidin pull-down (Arribas et al. 1996, Baldwin & Ostergaard 2002, Väisänen et al. 2004). A clear decrease in the amount of cell surface-labelled ectodomain was detected in the pull-down medium, indicating that the cholesterol depletion with methyl-β-cyclodextrin reduced type XIII collagen ectodomain shedding. Expectedly, PMA upregulated ectodomain shedding at the cell surface. Interestingly, the residual medium after the streptavidin pull-down contained unbiotinylated ectodomain. The amount of ectodomain in this subfraction had increased relative to the untreated or PMA-treated cultures, suggesting a causative role for cholesterol depletion by methyl-β-cyclodextrin. Lack of biotin indicated intracellular origin for this ectodomain fraction.

Ultracentrifugation of the culture media was used to explore the origin of the residual unbiotinylated ectodomain. All media contained material that sedimented at 100000x g. The size of type XIII collagen present in the sedimented material was found to be smaller in size than the full-length type XIII collagen, indicating that it was in cleaved form. A size difference of about 10-kDa between the full-length type XIII collagen and that in either the media or the sedimented material coincided well with earlier findings (Väisänen et al. 2004). It also agreed well with the consensus cleavage site for proprotein convertases at the stem of ectodomain being the cleavage site (Bennett et al 2000, Messageo et al. 2003, Väisänen et al. 2004). The sedimented material also contained furin. Vesicle secretion from the Golgi is known to be induced by methyl-β-cyclodextrin (Ilangumaran & Hoessli 1998, Claas et al. 2001, Gutwein et al. 2002). Therefore it was hypothesised that the pelleted material consisted of Golgi-derived vesicles, which was, in fact, ascertained by using several marker proteins. The material was found to be immunopositive for the Golgi marker GM130, but immunonegative for calnexin and Rab-5. The material also stained positive for γ-adaptin, which serves as another marker for the Golgi as well as for the transport vesicles. Calnexin and Rab-5 serve as markers of the endoplasmic reticulum and endosomal vesicles, respectively. The intracellular origin of the vesicles was verified by the cell surface biotinylation, in which the vesicle-derived ectodomain was found to be unbiotinylated, indicating intracellular origin.

If type XIII collagen is cleaved at the consensus cleavage site at the stem of the ectodomain, the liberation of ectodomain per se makes it more hydrophilic than the intact membrane-bound type XIII collagen. This results from the presence or absence of the transmembrane domain, creating a hydrophobicity difference between the uncleaved and released variants. The hydrophilicity of the vesicle-derived material was studied by using TX-114 phase separation assay. All the cleaved ectodomain was found to segregate in the aqueous phase with no intact type XIII collagen seen in the detergent-saturated phase. This confirmed the hydrophilic nature, i.e. the cleaved form, of the ectodomain found in the vesicles.

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5.6.3 Type XIII collagen found in the Golgi is of full length (II)

HT-1080 cell fractionation was used to analyze the site of intracellular cleavage of the type XIII collagen ectodomain. Full-length type XIII collagen was detected in the microsomal calnexin-positive fraction representing the rough endoplasmic reticulum and, although very faintly, in the Golgi membrane fraction. Also, density gradient sucrose ultracentrifugation was used to fractionate the cell constituents based on their buoyancy differences. Immunoblotting of the collected fractions showed that type XIII collagen was enriched in the same fractions as the Golgi marker GM130 but not in Rab-5-positive endosomes. The high-density calnexin-immunopositive endoplasmic reticulum fractions were devoid of type XIII collagen, most likely due to the low amounts. The trans-Golgi/transport vesicle marker γ-adaptin was also present in type XIII collagen containing fractions. The effect of the intracellular transport blockage on the putative intracellular ectodomain cleavage in HT-1080 cells was assessed by treating cells with brefeldin A. This substance inhibits anterograde vesicle transport from the Golgi to the plasma membrane by fusing the Golgi membranes to the endoplasmic reticulum, while leaving the trans-Golgi network unaffected (Gomez & Chrispeels 1993, Young et al. 1999, Bennett et al. 2000). Expectedly, the brefeldin A-treatment caused shifting of type XIII collagen and the Golgi marker protein GM130 to the heavy fractions, which also contained the bulk of the calnexin due to the Golgi-endoplasmic reticulum fusion. The distribution of the trans-Golgi/transport vesicle marker γ-adaptin was left unchanged. Despite the cessation of intracellular transport to the cell surface, only the full-length type XIII collagen was found in the Golgi fraction, as ascertained by size comparison with the variant in the cell lysate.

5.6.4 Inhibition of proprotein convertases removes ectodomain from the Golgi-derived vesicles (II)

Chloromethylketones inhibit proprotein convertases (Garten et al. 1994, Nakayama 1997). Earlier it has been shown that the treatment of cells with chloromethylketones results in a total lack of type XIII collagen ectodomain in the medium (Väisänen et al. 2004). Also here the chloromethylketone treatment totally abolished the presence of the cleaved ectodomain in the vesicles without, however, affecting vesicle formation. The cellular type XIII collagen expression also remained unaffected by the chloromethylketone treatment.

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5.7 Type XIII collagen in human cancers (III)

5.7.1 Type XIII collagen mRNA expression is upregulated in tumour stromal cells and in mesenchymal tumours (III)

Tissue microarray combined with ISH was used to explore the expression of type XIII collagen in epithelial and mesenchymal tumours. The analysis of the epithelial cancers encompassed colon adenocarcinoma, squamous cell carcinoma (SCC) of the cervix, urinary bladder carcinoma, endometrial adenocarcinoma and cystadenocarcinoma of the ovary. The specificity of the ISH probe was tested in Northern blotting, where the probe correctly detected a single band of expected size from total RNA of COS-1 cells, expressing full-length human type XIII collagen. In ISH, a progressive increase in type XIII collagen mRNA expression from normal to dysplastic to cancer stroma was detected in all of the epithelial cancers studied. Semi-quantitative and statistical analysis of the ISH (Bachmeier et al. 2000) revealed that the differences in the overall staining signal intensities between normal and cancer stromal expression of type XIII collagen mRNA in each TMA was statistically significant. The transition from pre-neoplastic dysplasia to malignancy induced a notable rise in stromal mRNA levels, whereas no marked differences were found between the cancer grades. There was a slightly enhanced type XIII collagen mRNA expression in the dysplastic and malignant epithelia relative to the normal epithelium, but the change was always weaker compared with that found in the stroma and was not statistically significant. TMA of the mesenchymal tumours containing fibrotic histiocytoma, dermatofibroma, fibrosarcoma, dermatofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma and chondrosarcoma showed a clear and statistically significant increase in the expression of type XIII collagen mRNA relative to the control tissue. The only exception in this group was dermatofibrosarcoma.

5.7.2 Type XIII collagen expression is upregulated in the invasive fronts of neoplastic foci and in the placenta (III)

The cancer cells in the invasive foci of epithelial cancers, as shown in SCC of the cervix, had a strong membrane-associated type XIII collagen protein expression. The IHC positivity coincided with type XIII collagen mRNA expression. Type XIII collagen expression was not only restricted to invasive fronts, since it was also evident in cancer cells deeper inside tumour tissue. As in the ISH, there was a clear local expression of type XIII collagen in the adjacent stromal cells surrounding the cancer foci. To positively identify the epithelial cancer cells in the tissue samples, cytokeratin staining was performed. Cytokeratin co-localised with the epithelial type XIII collagen ISH and IHC staining signals, indicating that the cells in the cancer foci were of epithelial origin. The presence and expression of type XIII collagen in physiologically invasive cells was investigated by ISH of the placenta. The staining results showed that type XIII collagen mRNA expression was upregulated in the invasive cytokeratin-positive cytotrophoblasts.

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5.7.3 Induction of type XIII collagen expression and phenotypic cellular transition of fibroblasts by cancer cell-derived factors (III)

Cancer cells produce soluble factors that affect stromal cells (Powell et al. 1999, Tomasek et al. 2002). An in vitro cell culture model system was set up to study the effects of cancer-derived factors on the expression of type XIII collagen. Primary fibroblasts were cultured in conditioned medium (CM) derived from either epithelial (HeLa) or mesenchymal (HT-1080) cancer cell cultures. In the CM cultures the cells eventually changed their morphology when compared to the untreated cells; the eventual cells were then named CM(HT-1080) and CM(HeLa), depending on the origin of the CM. The CM(HT-1080) cells acquired a less branched shape, whereas the CM(HeLa) cells became more rounded and flattened with distinctive membrane ruffling at the cell edges, when compared to the control cells. This phenotypic change was irreversible, since extended deprivation of the cancer-derived media (and hence the factors) did not lead to phenotypic reversion of the treated cells (data not shown). Based on Western blotting, there was also an induction of type XIII collagen expression in both the CM cell lines. This upregulation most likely originated at the transcriptional level because Northern blotting, normalised to β-actin, revealed elevated type XIII collagen mRNA levels in both CM cell lines. Type XIII collagen protein expressions in the various cells can be summarised as follows: non-malignant cells < the cultured cancer cells < CM cells. In other words, even the previously identified higher expression level in cultured cells, observed in HT-1080 cells, was markedly lower than that of both the CM cell lines. As expected, the high expression level of type XIII collagen in these cells resulted in rich liberation of ectodomain into the cell culture media.

The expression levels of several cell-matrix and cell-cell binding receptors as well as cytoskeletal and matrix proteins in the CM cell lines were analyzed and found to be altered when compared to the original ancestor cells. These changes included upregulation of α2 integrin as well as downregulation of fibronectin or E-cadherin, for example. Notably, the changes in the α-SMA and type XIII collagen levels were temporally congruent, indicating a transition to the myofibroblasts-like cells. The CM cells also showed a higher proliferation rate than their ancestor cells, concomitantly the expression of the proliferation marker PCNA was enhanced.

5.7.4 TGF-β1 upregulates COLXIII expression (III)

TGF-β1 is thought to be one of the most important factors in mediating the interactions between cancer and stroma (Akhurst & Derynck 2001). The effect of TGF-β1 and, as a comparison, PMA on the regulation of type XIII collagen mRNA expression, was assessed in primary fibroblasts. Serum-starved quiescent cells were treated with 1 or 5 ng/ml TGF-β1 or 100 ng/ml PMA for 24 hours. Based on the Northern blot analysis of mRNA normalised to β-actin, TGF-β1 induced type XIII collagen mRNA expression in a concentration-dependent manner up to about 1.8-fold, whereas PMA was only induced by about 1.3-fold. Blocking of TGF-β1 function with a specific antibody prevented the

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induction of the type XIII collagen expression and morphological changes, strongly suggesting that TGF-β1 plays a role in phenotypic transition during the CM treatment.

5.7.5 The soluble ectodomain of type XIII collagen reduces cell adhesion and spreading on vitronectin (III)

Since TMA and the CM experiments suggested that type XIII collagen is upregulated in the cancer stroma, the role of the soluble ectodomain was explored in cell culture. To model the effects of the soluble ectodomain on the matrix, vitronectin and fibronectin were immobilised on cover slips followed by a secondary coating with the purified recombinant ectodomain. No effect was seen on the fibronectin surface, whereas on vitronectin, the adhesion of primary fibroblasts, HT-1080 and HeLa cells was decreased by the presence of ectodomain. Spreading was also affected. HT-1080 cells plated on a double-coated vitronectin surface only showed signs of initial spreading after three hours, whereas on the control surface without ectodomain, majority of cells were in an advanced stage of spreading at the same point in time. The same effect was evident with the HeLa cells. Although these cells spread more slowly than HT-1080 cells, at 6.5 hours the difference between the single and double-coated vitronectin surfaces could be observed. After 24 hours morphological differences had vanished, suggesting a transient nature of the ectodomain influence.

5.8 Type XIII collagen in pathological cornea (IV)

5.8.1 Immunohistochemistry of normal and pathological cornea (IV)

The epithelial cells of normal human cornea express type XIII collagen, based on the immunohistochemical staining with the type XIII collagen NC4 domain antibody 777B. Diffuse expression was observed throughout the epithelium and in some cases the staining was concentrated in cell membranes. No clear differences were seen between the central and peripheral epithelium. The stromal keratocytes showed a faint immunopositivity for type XIII collagen throughout the cornea. The basement membrane of the corneal epithelium, Bowman’s membrane, Descemet’s membrane and the endothelial cells did not stain with type XIII collagen antibody.

Characteristic features of the keratoconus corneas were the irregular thinning of the epithelium, degeneration and ruptures of the Bowman’s membrane and distorted subepithelial stroma. The corneal epithelium and the stroma in the non-affected areas had a staining pattern comparable to the normal stroma. In the distinct spots where the Bowman’s membrane had been disrupted or the corneal epithelium was thinned, the epithelial expression of type XIII collagen was decreased or sometimes lacking altogether. However, the notable feature was that in these sites there was often an

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increased type XIII collagen-positive immunostaining of the stromal keratocytes located beneath the Bowman’s membrane.

In the case of corneal scar tissues, type XIII collagen expression was markedly induced in stromal cells. The staining intensity had a gradient pattern, the strongest staining being at the scar foci and the weakest in the unscarred adjacent areas. The epithelium of the scar tissue was, in most cases, irregular and thinner than in unscarred areas. The staining pattern for type XIII collagen varied distinctly since some epithelial cells did not exhibit any immunopositivity for type XIII collagen. In other parts of the scarred cornea, the epithelial immunostaining of type XIII collagen was comparable to the epithelium of unscared cornea. As a control, the antigen-blocked primary antibody did not stain cornea tissue at all.

5.8.2 In situ hybridisation of normal and pathological cornea (IV)

The same cases were subjected to ISH. The epithelial cells and to a lesser degree the stromal keratocytes in the normal cornea expressed type XIII collagen mRNA. No differences in the expression were detected between the central or peripheral epithelium and the stroma or between the upper and lower stromal compartments.

Type XIII collagen mRNA expression in keratoconus was located uniformly in the corneal epithelium. In areas which contained Bowman’s membrane ruptures and thinning of the epithelium, the expression of type XIII collagen was decreased. In the areas of subepithelial scarring of the upper stroma, a weaker expression in the stromal cells was found. Otherwise, type XIII collagen mRNA expression resembled that of the normal cornea.

The stromal cells of the wound scar tissue had an exceptionally high type XIII collagen mRNA expression. The dense scar tissue showed the most intense staining, while the non-scarred areas resembled the normal cornea. In the areas where the scar was dense with an intense type XIII collagen mRNA expression, almost no CD34 keratocyte marker immunoreactivity was seen. The non-scarred areas showed an almost normal CD34 immunostaining. In areas of the stromal scars with the highest type XIII collagen expression, a significant portion of the cells expressed the α-SMA antigen (myofibroblast marker). Epithelial mRNA expression showed variability in scarred corneas, with some cells showing a constantly weak level of expression. A sense probe, used as a negative control, did not show any positive unspecific expression.

6 Discussion

After the retrieval of the first cDNA clones encoding type XIII collagen, it was soon realised that this protein is not a traditional fibril-forming collagen but instead has a structure in which the collagenous domains are interspersed with non-collagenous interruptions (Tikka et al. 1988, Pihlajaniemi & Tamminen 1990). When the complete coding sequence of type XIII collagen cDNA was finally assembled, it also revealed a stretch of hydrophobic amino acids near the N-terminus of the protein suggesting an integral integration in the cell membrane. As immunofluorescent staining studies then showed type XIII collagen to be present as a membrane spanning protein on the plasma membranes of the HT-1080 cancer cells as well as in the adherens junctions of skin sections and cultured keratinocytes (Hägg et al.1998, Peltonen et al. 1999), type XIII collagen was deduced to belong to transmembrane collagens.

In this thesis investigation of type XIII collagen was continued by studying primary skin fibroblasts. It was shown that in fibroblasts, type XIII collagen is integrated into the cell membranes in the same manner as in HT-1080 cells. In fully spread fibroblasts type XIII collagen localised in the adhesive structures of the cell membrane. As this localisation coincided with the widely used focal adhesion marker proteins talin and vinculin, these structures were deduced to be focal adhesions, which are specialised integrin-containing cell surface structures mediating the attachment of cells to their surroundings (Wozniak et al. 2004). In addition to adhesive structures of stationary cells, type XIII collagen was also detected in the extreme periphery of the newly attaching and spreading cells and in the leading lamellae (lamellipodia) of migrating cells. These locations contain focal complexes which eventually mature into focal adhesions (Cukierman et al. 2002, Wozniak et al. 2004). The presence of type XIII collagen in focal complexes indicates that type XIII collagen emerges in these cell adhesion structures very early during their development. Type XIII collagen was also seen in more centrally located fibrillar structures, which resembled α5β1 integrin and tensin containing fibrillar adhesions (Cukierman et al. 2002, Wozniak et al. 2004). Fibrillar adhesions, in turn, are considered to develop from focal adhesions (Cukierman et al. 2002). Additionally, as the disassembly of cell adhesions by microinjection of C3 exoenzyme (Rho inhibitor) resulted in the removal of type XIII collagen from the disintegrating focal adhesions in the same pace as the other components, together these findings indicated that type XIII

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collagen is an integral structural component of the focal adhesions and is regulated in concert with the other focal adhesion components.

Localisation in focal adhesions suggests that the function of type XIII collagen may be related to cell adhesion. This model was supported by the Sf9 insect cell adhesion assay where the insect cells expressing type XIII collagen adhered to type I collagen, fibronectin and poly-D-lysine better than the control cells. However, despite the localisation findings and the observed enhancement of cell adhesion resulting from expression of type XIII collagen, the exact function of type XIII collagen in focal adhesions is not yet fully known. Because it is already present in early focal complexes, it may be involved in organising the assembly of these structures. The insect cell results also suggest that the membrane-associated type XIII collagen may bind directly to the matrix components as a receptor or indirectly via another cell surface receptor molecule. In fact, the recombinant type XIII collagen ectodomain has been shown to be able to bind to a number of matrix proteins, including fibronectin and vitronectin (Tu et al. 2002, Väisänen et al. 2005). Binding to poly-D-lysine, which is a negatively charged substance, may indicate that the positively charged C-terminus of the COL3 domain could mediate adhesion to anionic components of the matrix by electrostatic interactions.

Immunofluorescent stainings of tissues showed that type XIII collagen is present in myotendinous junctions and adherens junctions of striated muscle, in intercalated discs of the heart, and in various epithelium-basement membrane interfaces. At all these sites, cells attach to the ECM or to other cells via integrin or cadherin cell surface receptors. Based on this data, type XIII collagen appears to associate with integrin-containing adhesion structures but it is also possible that it may be involved in cadherin-mediated adhesion structures as well. For instance, intercalated discs contain cadherin-based adherens junctions, desmosomes, gap junctions and integrin-based junctions (van der Flier et al. 1995, Belkin et al. 1996, van der Flier et al. 1997). In this study we were not able to assign type XIII collagen specifically to any particular connection structure between cardiac cells. However, the analysis of the epidermal cell-cell and cell–matrix contacts suggests that type XIII collagen is probably not associated with desmosomes, while it may be found in the adherence junctions between keratinocytes (Peltonen et al., 1999). Data from transgenic animals seem to support a role in cell adhesion for type XIII collagen, since mutations in type XIII collagen protein result in phenotypes with impaired adhesion properties, for example dysfunctional association between the muscle cells and the basement membrane and defective adherens junctions of the heart (Kvist et al. 2001, Sund et al. 2001). The adhesive function of type XIII collagen is not unique among collagens. Type XVII collagen, another member of the transmembrane collagen group, is analogously found in hemidesmosomes, which anchor epithelial cells to the basement membrane (Borradori & Sonnenberg 1996).

The mechanism directing type XIII collagen to the adhesive structures or the focal adhesions is unknown. The speculative mechanisms include intra- and extracellular interactions or lateral interactions at the plasma membrane. In the case of type XVII collagen, the association with α6β4 integrin is crucial for the correct placement of type XVII collagen within hemidesmosomes (Borradori et al. 1997, Hopkinson et al. 1998). Based on this, it could be possible that type XIII collagen-integrin interactions could have a role in directing type XIII collagen to focal adhesions. Both proteins are known to emerge in focal adhesions very early during the maturation process (Cukierman et al.

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2002). Another possibility is that extracellular ligands might be involved in directing type XIII collagen to focal adhesions. However, analysis where cells were cultured on different matrix proteins does not support this hypothesis, as no relationship between the presence of type XIII collagen in focal adhesions and the matrix composition has been shown (Väisänen et al., unpublished data).

In addition to being an integral membrane protein, type XIII collagen ectodomain can be cleaved by proprotein convertases to produce a soluble extracellular matrix molecule (Peltonen et al. 1999, Snellman et al. 2000a, Väisänen et al. 2004). The shed ectodomain has been shown to have distinct effects on cell migration, adhesion, spreading and proliferation, as well as on the fibronectin matrix assembly (Väisänen et al. 2004, Väisänen et al. 2005). Recently, membrane microdomains, called rafts, have been implicated in the shedding of a number of membrane-associated proteins (Ehehalt et al. 2003, Matthews et al. 2003, Phong et al. 2003, Wakatsuki et al. 2004, Zimina et al. 2005). To study if this is the case with type XIII collagen, its presence in membrane microdomains was analyzed. To be regarded as a raft protein, the protein of interest must be present in light density ultracentrifugation fractions in the presence of a detergent and be sensitive to cholesterol depletion leading to raft disruption (Brown & London 2000, Simons & Toomre 2000, Claas et al. 2001, Maile et al. 2002). First, type XIII collagen and furin partly subfractionated in light density fractions that were CHAPS-resistant but TX-100 sensitive. Although resistance to TX-100 has traditionally been a criterion for raft association, cell membranes contain raft-like microdomains that do not tolerate TX-100 detergent treatment but are instead CHAPS-resistant (Claas et al. 2001). Secondly, the membrane microdomain association was supported by the fact that both proteins responded similarly to the biochemical manipulation of the cellular cholesterol level. Some raft proteins require the functional cytoskeleton to be correctly included in or excluded from the membrane rafts (Leitinger & Hogg 2002, Thomsen et al. 2002, Maxfield 2002, Upla et al. 2004). However, in the case of type XIII collagen and furin, the involvement of cytoskeleton was uninfluental. Disruption of membrane microdomains by lowering the cellular cholesterol level by methyl-β-cyclodextrin with or without lovastatin or by reducing its availability by filipin clearly decreased the efficiency of type XIII collagen ectodomain shedding, especially on the plasma membrane. This suggests a model in which the shedding of the ectodomain is at least partially dependent on its raft association on the plasma membrane.

Methyl-β-cyclodextrin sequesters cholesterol at the cell surface, but as a result of lipid transport may also affect the cholesterol content of intracellular membranes (Ilangumaran & Hoessli 1998, Ikonen 2001). It is known that furin mainly resides in the trans-Golgi network, but it is also present at the cell surface because of intracellular recycling (Seidah & Chrétien 1997, Thomas 2002). As both the plasma membrane and the Golgi contain lipid-enriched membrane microdomains (Brown & London 1998, Simons & Toomre 2000), it is possible that the shedding process of type XIII collagen ectodomain may, in fact, occur both intracelluraly in the trans-Golgi as well as at the cell surface. When this was further studied only full-length type XIII collagen was found to be present in the rough endoplasmic reticulum and Golgi fractions, even after brefeldin A-induced cessation of anterograde vesicular transport. However, the cell medium contained Golgi-derived GM130-positive vesicles which contained only the cleaved ectodomain. Inhibition of proprotein convertases by chloromethylketones blocked the entrance of the

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cleaved ectodomain into these vesicles, but did not hamper the release of the vesicles. This suggests that the cleavage of the ectodomain indeed also occurs intracellularly. Cleavage may be a gating event for the exit of type XIII collagen ectodomain, destined to be secreted, from the Golgi via transport vesicles. These findings, as well as our earlier observations of the interference of the Golgi function by brefeldin A or monensin decreasing ectodomain shedding (Väisänen et al. 2004), support our hypothesis of a secondary cleavage site in the Golgi apparatus. They do not yet, however, determine which of the cleavage sites may be physiologically more important.

Cholesterol depletion with methyl-β-cyclodextrin decreased type XIII collagen ectodomain shedding. However, at the same time it increased the amount of the intracellularly derived ectodomain in the cell culture medium. This is explained by the fact that methyl-β-cyclodextrin can induce vesicle release from the Golgi compartment of cultured cells (Ilangumaran & Hoessli 1998, Claas et al. 2001, Gutwein et al. 2002). Since vesicles were also present in the media of untreated cultures, vesicle formation can occur without exogenous stimulation. Vesicular shedding adds a new aspect to type XIII collagen ectodomain cleavage. The vesicle components have been suggested to have a role in the modulation of cancer tissue stroma (Dolo et al. 1998, Ginestra et al. 1998, Angelucci et al. 2000, Taraboletti et al. 2002). We have observed upregulated expression of type XIII collagen in the tumour stroma and in various cancer cell lines (original article III). The upregulated expression and the concomitant ectodomain shedding of type XIII collagen may influence the stromal composition and, consequently, the behaviour of cells. For instance, the metastatic capacity of the cancer cells could be increased since the soluble ectodomain has been shown to downplay adhesion-related cell functions (Väisänen et al. 2004). At present, however, the regulation and proportions of vesicle vs. cell surface ectodomain shedding are not known, and their importance related to the functions of the soluble or membrane-bound type XIII collagen are unclear.

Grave’s disease in which type XIII collagen autoantibodies have been detected (Mizokami et al. 2004), has so far been the only human disease to which type XIII collagen has been linked. To widen the scope of diseases in which type XIII collagen could be involved, several epithelial and mesenchymal cancers were screened using TMA methodology. Analysis with ISH revealed an enhanced type XIII collagen mRNA expression in the reactive stroma of epithelial cancers and throughout mesenchymal tumours. The induction was observed in the dysplastic stroma, which suggests that type XIII collagen is already induced at an early stage of tumour progression. Notably, invasive cells, both pathological and physiological, showed upregulated expression of type XIII collagen. It is known that the expression level of type XIII collagen in normal mature tissues is very low compared to that of embryonal tissues (Sund et al. 2001a). Therefore it seems that the expression of type XIII collagen in tumours reverts to an embryonal mode. Increased collagen expression, i.e. desmoplasia, is a well-known characteristic feature for cancers, and several different collagens, including other transmembrane collagens, have been described to be upregulated in cancer (Brown et al. 1999, Powell et al. 1999, Bode et al. 2000, Burns-Cox et al. 2001, Fisher et al. 2001, Theret et al. 2001, Banyard et al. 2003). The list of collagens upregulated in cancer-related desmoplasia now also includes type XIII collagen.

Strikingly, in many cases type XIII collagen mRNA expression formed a gradient, with the strongest expression next to the transformed foci. This suggests that cancer cells

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secrete paracrine factors which can induce type XIII collagen expression. Myofibroblasts are presumed to be the main cells responsible for the stromal desmoplasia (Faouzi et al. 1999, Petrov et al. 2002, Tuxhorn et al. 2002), and the irreversible phenotypic change of stromal fibroblasts to myofibroblasts is known to be induced by soluble factors secreted by cancer cells (Ronnov-Jessen & Petersen 1993, Powell et al. 1999, Valenti et al. 2001, Sung & Chung 2002). The hypothesis that the paracrine effect of cancer cells could change normal stromal cells into myofibroblasts with an induced type XIII collagen expression was tested in the cell culture experiment. The transformed cells exhibited an altered expression pattern of a number of matrix proteins, an enhanced proliferation rate, an increased type XIII collagen expression as well as increased expression of myofibroblast marker proteins vimentin and α-SMA (Eyden 2001, Tomasek et al. 2002). Despite the fact that the morphology of the cells did not exactly resemble the one classically defined for myofibroblasts, the cells showed myofibroblast-like characteristics. All in all, the greatly altered protein expression pattern as well as the different morphology of the cells, showed that the paracrine effects of cancer cells can drastically affect the neighbouring stromal cells and also stimulate type XIII collagen expression.

TGF-β1 has been suggested to play a central role in the transdifferentation of fibroblasts to myofibroblasts and the development of desmoplasia (Ignotz et al. 1986, Ignotz et al. 1987, Kähäri et al. 1991, Ronnov-Jessen & Petersen 1993, Frazer et al. 1994, Powell et al. 1999, Eyden 2001, Petrov et al. 2002, Tomasek et al. 2002, Tuxhorn et al. 2002). Based on this premise and the fact that the cancer cells used in this study secrete TGF-β1 (Taipale et al. 1992, Liu et al. 1996, Dunning et al. 2003) and the primary fibroblasts express its receptor (Verrecchia et al. 2001), TGF-β1 was considered a good candidate for being the paracrine factor inducing type XIII collagen expression. The cell culture experiment, with a function-blocking antibody against TGF-β1, showed convincingly that TGF-β1 is able to increase type XIII collagen expression in cancer stromal cells during the malignant transformation. Little has been known about type XIII collagen ectodomain shedding in normal vs. cancer tissues. However, the cell culture experiments showed that the upregulation of type XIII collagen synthesis in the stromal cells by cancer cell-derived factors simultaneously enhanced the release of the soluble ectodomain. This suggests that the ectodomain shedding in vivo is plausible. If the composition of the matrix is suitable, for instance with vitronectin accumulation detected in some cancers (Aaboe et al. 2003), the shedding of the ectodomain and its subsequent binding to matrices could influence the growth, progression and spreading of the cancer via reduced cell adhesion and spreading. For example invasive cells, which show type XIII collagen expression, could render their surrounding matrix unfavourable for cell adhesion, thereby promoting the dissemination of the cancer.

The cornea constitutes a well-structured tissue with epithelial and endothelial layers separated by a highly organised stroma. Type XIII collagen is expressed in various parts of the foetal eye including the cornea (Sandberg-Lall et al. 2000). In this thesis, type XIII collagen protein expression in normal cornea was shown to be restricted to corneal epithelial cells and stromal keratocytes. Taking into account the fact that it has been localised to cell-cell and cell-matrix contacts, type XIII collagen may be involved in the attachment of the corneal basal epithelial cells to the basement membrane and may participate in cell-cell communication. In keratoconus corneas, epithelial type XIII

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collagen was downregulated or was totally missing from areas where the Bowman’s membrane was disrupted and the corneal epithelium was thinned. If the epithelial changes were associated with a local subepithelial scarring, the scar formations sometimes showed an increased type XIII collagen expression in stromal keratocytes. The type XIII collagen expression changes in the epithelium may be a secondary effect due to the thinning of the epithelium and protrusion of the stroma. Matrix and basement membrane proteins have been shown to display markedly altered expressions in keratoconus cornea (Zhou et al. 1996, Kenney et al. 1997, Tuori et al. 1997). This probably represents reactive stromal changes in the upper stroma caused by tissue protrusion and accumulation of the matrix proteins. Based on the results, type XIII collagen seems to be involved in this process. Strong upregulation of type XIII collagen expression was seen in corneal scars. In these cases type XIII collagen was expressed in stromal cells, and the most intense reaction was localised in scarred foci. The surrounding non-scarred areas showed a less intense immunostaining reminiscent of the normal stroma. A significant portion of type XIII collagen expressing cells were myofibroblasts based on the α-SMA-positivity and a loss of the expression of the keratocyte marker CD34. The transdifferentation of keratocytes into myofibroblasts has been linked to the upregulation of focal and cell adhesion molecules combined with the downregulation of CD34. This process is likely to be promoted by TGF-β2 (Masur et al. 1999, Espana et al. 2004). The involvement of type XIII collagen in corneal wound healing, its expression in the myofibroblasts of the activated corneal stroma and the involvement of TGF-β are closely reminiscent of the findings in cancer.

Combined with the available knowledge concerning type XIII collagen, the results presented this thesis indicate firstly, that type XIII collagen is a transmembrane protein functionally involved in adhesive interactions; secondly, type XIII collagen release from cells can be considered as a regulated dual pathway process with the released ectodomain having implications on cell behaviour; thirdly, type XIII collagen expression is upregulated in situations where tissue stroma is activated.

7 Future perspectives

The localisation of type XIII collagen in focal adhesions is an important finding when considering the biology of this protein. The detailed mechanisms that govern the correct placement of this collagen in focal adhesions could be studied by expressing tagged type XIII collagen proteins in cultured mammalian cells. Various controlled deletions of type XIII collagen may provide information about the protein domains crucial for this process. These same deletion variants would also be useful when studying the lateral and intracellular interactions of type XIII collagen with other cellular components that may play a role in type XIII collagen function. In these respects analysis of cells derived from the various transgenic mouse lines would also be useful. Visualisation of the dynamics of tagged type XIII collagen in living cells, using time-lapse microscopy, could be used to provide information about the involvement of the type XIII collagen in cell migration and maturation of the focal adhesions.

The finding presented in this thesis concerning type XIII collagen localising in the membrane microdomains is a novel aspect with respect to its membrane localisation. The relationship between focal adhesions and lipid rafts is largely unresolved at the moment. The effects of manipulation of the cellular cholesterol levels could provide information not only about the involvement of rafts in type XIII collagen localisation in focal adhesions but also about the localisation mechanisms of focal adhesion components in general. The link between rafts and type XIII collagen shedding at the cell surface could also be important in the regulation of the turnover of the cell surface type XIII collagen during dissassembly of the adhesion complexes, for example during cell migration.

The involvement of Golgi apparatus in the ectodomain shedding provides a totally new perspective to the biology of type XIII collagen. It may be that intracellular shedding should be regarded as a secretion mechanism in order to distinguish it from cell-surface shedding. An important question that should be examined is if it is connected to particular cellular functions in physiological or pathological processes.

In this thesis it has been shown that type XIII collagen expression is upregulated in the activated stroma of various pathological situations, namely in cancer and some corneal diseases. It was shown that the ectodomain is capable of altering the biological characteristics of the stroma. The role type XIII collagen expression in the desmoplastic stroma could be studied by examining the behaviour (tumour growth, metastatic capacity)

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of various cancer cells in null and overexpressing type XIII collagen transgenic mice. Neoplastic cells derived from these same animals could be used to observe the role of type XIII collagen in invasive cells, for example. This line of study could be extended further, for instance by investigating if the ectodomain has any effect on angiogenesis. Additionally, the applicability of type XIII collagen ectodomain, if present in circulation, to serve as an early disease marker could be analyzed.

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