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D 1354
OULU 2016
UNIVERSITY OF OUL U P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLA ND
U N I V E R S I TAT I S
O U L U E N S I S
ACTA
A C TA
D 1354
ACTA
U N I V E R S I T AT I S O U L U E N S I S
Jyri Moilanen
University Lecturer Santeri Palviainen
Postdoctoral research fellow Sanna Taskila
Jyri Moilanen
Professor Esa Hohtola
FUNCTIONAL ANALYSIS
OF COLLAGEN XVII IN
EPITHELIAL CANCERS AND
A MOUSE MODEL
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Director Sinikka Eskelinen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-1168-8 (Paperback)
ISBN 978-952-62-1169-5 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE;
MEDICAL RESEARCH CENTER;
OULU UNIVERSITY HOSPITAL
D
MEDICA
ACTA UNIVERSITATIS OULUENSIS
D Medica 1354
JYRI MOILANEN
FUNCTIONAL ANALYSIS OF
COLLAGEN XVII IN EPITHELIAL
CANCERS AND A MOUSE MODEL
Academic Dissertation to be presented with the assent
of the Doctoral Training Committee of Health and
Biosciences of the University of Oulu for public defence
in Auditorium 3 of Oulu University Hospital, on 4 May
2016, at 12 noon
U N I VE R S I T Y O F O U L U , O U L U 2 0 1 6
Copyright © 2016
Acta Univ. Oul. D 1354, 2016
Supervised by
Professor Kaisa Tasanen-Määttä
Docent Tiina Hurskainen
Reviewed by
Professor Juha Peltonen
Professor Malin Sund
Opponent
Professor Jyrki Heino
ISBN 978-952-62-1168-8 (Paperback)
ISBN 978-952-62-1169-5 (PDF)
ISSN 0355-3221 (Printed)
ISSN 1796-2234 (Online)
Cover Design
Raimo Ahonen
JUVENES PRINT
TAMPERE 2016
Moilanen, Jyri, Functional analysis of collagen XVII in epithelial cancers and a
mouse model.
University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Medical
Research Center; Oulu University Hospital
Acta Univ. Oul. D 1354, 2016
University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Basement membranes (BM) underlie epithelia and endothelia and surround many tissues. In
cutaneous BM epithelial cells are attached to the stroma via multiprotein complexes called
hemidesmosomes (HD). Collagen XVII and integrin α6β4 are components of HD and they bind
to laminin 332, a component of anchoring filaments, extracellularly. The main interest of this
study is the function of collagen XVII and its interactions with these proteins.
What is known about the function of collagen XVII is mostly derived from its role as an
adhesive component in cutaneous HD. Here we demonstrate for the first time that collagen XVII
is expressed by podocytes in the human and murine glomerulus and that mutant mice lacking
collagen XVII in addition to small size, blisters and diffuse hair loss, also have deficient
glomerular development and a high mortality rate.
We also show for the first time at the protein level that collagen XVII is expressed, and
probably has a functional interaction with laminin 332, in normal colon epithelia. We demonstrate
that collagen XVII is expressed by the invasive cells of human colorectal carcinoma (CRC)
samples and its immunostaining is increased in metastasis in CRC. The higher proportion of
collagen XVII positive tumor cells correlates with decreased disease-free survival and cancerspecific survival times and we also suggest a functional interaction between collagen XVII and
laminin 332 in CRC.
Previous studies have suggested that collagen XVII participates in keratinocyte migration by
affecting the correlation of HD disassembly and assembly, its expression is increased in squamous
cell carcinoma (SCC) and it may have a role in cell adhesion and migration in SCC carcinogenesis.
Here we demonstrate upregulated collagen XVII, integrin β4 and laminin γ2 expression in actinic
keratosis, Bowen’s disease and SCC. The expression of collagen XVII was increased with a high
degree of variation, especially in samples taken from areas where SCC is particularly invasive. We
also demonstrate in the SCC-25 cell line that lack of collagen XVII or integrin β4 severely disrupts
the adhesion, migration and invasivity of these cells.
Taken together, in this study we show that collagen XVII is needed for normal glomerular
development, is expressed in normal colon epithelia and participates in CRC and SCC
carcinogenesis together with laminin 332 and integrin β4.
Keywords: basement membrane, collagen, colorectal carcinoma, glomerulus, squamous
cell carcinoma
Moilanen, Jyri, Kollageeni XVII toiminnan
syöpäsoluissa ja poistogeeninen hiirimalli.
analysointi
epiteliaalisissa
Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical Research
Center; Oulun yliopistollinen sairaala
Acta Univ. Oul. D 1354, 2016
Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Tyvikalvot sijaitsevat epiteelin ja endoteelin alla ja ympäröivät monia kudoksia. Ihon tyvikalvossa epiteelisoluja alla olevaan verinahkaan kiinnittää rakenne, jota kutsutaan hemidesmosomiksi
(HD). Kollageeni XVII ja integriin α6β4 ovat HD:n rakenneproteiineja. Ne kiinnittyvät solun
ulkopuolella laminiin 332 nimiseen proteiiniin, joka muodostaa ankkurifilamentit. Kollageeni
XVII ilmentyminen ja toiminta yhdessä näiden kahden proteiinin kanssa on tämän tutkimuksen
keskeisin kohde.
Valtaosa tutkimuksista, jotka käsittelevät kollageeni XVII:ää, koskevat sen toimintaa ihon
keratinosyyteissä. Tässä tutkimuksessa osoitimme ensi kertaa, että hiiren ja ihmisen munuaiskerästen podosyyttisolut ilmentävät kollageeni XVII. Geenimanipuloidut hiiret, joilta kollageeni
XVII oli poistettu, olivat pieniä, kehittivät rakkuloita ja karvattomuutta, niillä oli korkea kuolleisuus ja niiden munuaiskerästen kehitys oli häiriintynyt. Kollageeni XVII esiintymistä proteiinitasolla, sekä mahdollista toiminnallista yhteyttä laminiin 332:een, ei aiemmin ole osoitettu paksusuolen epiteelissä. Havaitsimme, että paksu- ja peräsuolen adenokarsinooman (CRC) invasiivinen solukko ilmentää kollageeni XVII:ää, kollageeni XVII esiintyminen on merkittävän voimakasta CRC:n metastasoinnin yhteydessä ja lisääntynyt kollageeni XVII esiintyminen lyhentää syöpävapaata aikaa ja heikentää syöpäspesifistä selviytymistä. Myös CRC:ssä kollageeni
XVII toiminta voi liittyä laminiini 332:een.
Aiempien tutkimusten mukaan kollageeni XVII osallistuu keratinosyyttien migratioon vaikuttamalla toimivien HD:ien määrään. Sen määrän on havaittu olevan korkeampi okasolusyövässä (SCC) ja sen on ehdotettu osallistuvan syöpäsolujen adheesioon ja migraatioon SCC:n
kehittyessä. Me osoitimme kohonneen kollageeni XVII, integriini β4 ja laminiini γ2 ilmenemisen aktiinisessa keratoosissa, Bowenin taudissa sekä SCC:ssä. Kollageeni XVII määrä oli korkea, mutta vaihteli paljon, sekä hiiren että ihmisen invasiivisilla SCC alueilla. Havaitsimme
myös SCC-25 solulinjalla, että kollageeni XVII tai integriini β4 puutos häiritsee vakavasti solujen adheesiota, migraatiota ja invaasiota.
Yhteenvetona tässä työssä osoitimme, että kollageeni XVII:ää tarvitaan munuaiskerästen
kehittymisessä, sitä esiintyy paksusuolen epiteelissä, ja että kollageeni XVII osallistuu CRC:n ja
SCC:n kehittymiseen yhdessä integriini β4:n ja laminiini 332:n kanssa.
Asiasanat: kollageeni, munuaiskeränen, okasolusyöpä, paksusuolen adenokarsinooma,
tyvikalvo
To my family
8
Acknowledgements
This study was carried out at the Department of Dermatology, Department of
Pathology, Clinical Research Center, Biocenter and Oulu Center for Cell Matrix
Research, University of Oulu from 2007 to 2016.
In particular, I owe my sincerest gratitude to my supervisor, the Head of the
Department of Dermatology, Professor Kaisa Tasanen-Määttä, MD, Ph.D, for all
the support, advice and patience you have given me during the years. It has been a
long journey and your thorough knowledge of this field of study has guided me
through the difficulties.
I wish to thank my second supervisor, Docent Tiina Hurskainen, Ph.D, who has
guided and supervised my work through the practical phases and assisted me in
each step to complete the research. You have also given me the much needed moral
support during the years.
I would like to express my deepest thanks to the former Head of the Department
of Dermatology, the chair of my follow-up group, Professor Aarne Oikarinen, MD,
Ph.D. You have always given your valuable time to provide me with the requested
information, comments and your support.
I wish to express my deepest thanks to the best lab technician there is. Anja
Mattila, without your vast expertise this project would not have been finished.
I also wish to appreciate the official pre-examinators of this dissertation,
Professor Malin Sund, MD, Ph.D, and Professor Juha Peltonen, MD, Ph.D. Your
insightful and thought-provoking comments have made a significant improvement
to this manuscript. I also thank Steve Smith, for his skilled linguistic editing of the
dissertation.
I thank all the other members of our research group, Laura Huilaja, MD, Ph.D,
Nina Kokkonen, Ph.D, Minna Kubin, MD, and Anna-Kaisa-Försti, MD, for your
intellectual support and ideas during the years. Your collegial support and advice
have given me a great deal of wisdom and strength during this project.
I also wish to thank all the collaborators that have participated in this project
and enabled its outcome. I especially would like to acknowledge: Docent Raija
Sormunen, Ph.D, Claus-Werner Franzke, PhD, Raija Soininen, PhD, Stefanie
Löffek, PhD, Docent Matti Nuutinen, MD, Ph.D, Professor Leena BrucknerTuderman MD, Ph.D, Docent Helena Autio-Harmainen, MD, Ph.D, Juha Väyrynen,
MD, PhD, Erkki Syväniemi, MD, Professor Markus Mäkinen, MD, Ph.D, Kai
Klintrup, MD, PhD, Ritva Heljäsvaara, Ph.D, Professor Jyrki Mäkelä, MD, Ph.D,
9
Sirpa Salo, PhD, Docent Aki Manninen, Ph.D, Professor Tuula Salo, DDS, Ph.D,
Pilvi Riihilä, MD, PhD and Professor Veli-Matti Kähäri, MD, Ph.D.
I would also like to express my gratitude to the whole staff in the Department
of Dermatology and Clinical Research Center for your understanding and kind
assistance whenever needed.
I would also wish to show my deepest gratitude to my parents, Irma and Leo,
for their love and support. You have taught me the ethics of hard work and therefore
given me the chance to succeed in life.
I cannot go without mentioning my beloved brothers Jani and Raine, and my
sister Tanja. If there has been anything I have needed help with, work-related or
not, I have always been able to count on your help and support.
My thanks are also owed to my friends and colleagues for your endless support
and advice. You have been there to help me through some very frustrating times.
You know who you are.
Finally, above all, I wish to thank my best friend and my wife, Elisa, and our
family. You have kept me on the right path and shown me what is truly important
in life.
16.3.2016
10
Jyri Moilanen
Abbreviations
BP
BP180
BP230
BM
cDNA
CRC
CSS
DNA
DFS
EB
ECM
FP
GFB
GFR
HD
IEM
IHC
KD
MAPK
MARCO
NC
RNA
SEM
SCC
SD
TEM
TMA
bullous pemphigoid antigen
bullous pemphigoid antigen 180
bullous pemphigoid antigen 230
basement membrane
complementary DNA
colorectal carcinoma
cancer-specific survival
deoxyribonucleic acid
disease-free survival
epidermolysis bullosa
extracellular matrix
foot process of podocyte
glomerular filtration barrier
glomerular filtration rate
hemidesmosome
immunoelectron microscopy
immunohistochemistry
knockdown
mitogen-activated kinase
macrophage receptor with collagenous structure
non-collagenous
ribonucleic acid
scanning electron microscopy
squamous cell carcinoma
slit diaphragm
transmission electron microscopy
tissue micro array
11
12
Original articles
This thesis is based on the following publications, which are referred throughout
the text by their Roman numerals:
I
Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Löffek S, Huilaja L,
Nuutinen M, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2012)
Transmembrane collagen XVII is a novel component of the glomerular filtration barrier.
Cell Tissue Res 348(3):579-88.
II Moilanen J, Kokkonen N, Löffek S, Väyrynen JP, Syväniemi E, Hurskainen T, Mäkinen
MJ, Klintrup K, Mäkelä J, Sormunen R, Bruckner-Tuderman L, Autio-Harmainen H,
Tasanen K (2015) Collagen XVII correlates with the invasion and metastasis of
colorectal cancer. Hum Pathol. 46(3):434-42.
III Moilanen J, Löffek S, Kokkonen N, Salo S, Väyrynen JP, Hurskainen T, Manninen A,
Riihilä P, Heljäsvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen MJ, Tasanen K
Collagen XVII and integrin β4 show similar expression in squamous cell carcinoma and
their knockdown suppresses migration and invasion only of the less aggressive
squamous cell carcinoma cells. Manuscript.
13
14
Contents
Abstract
Tiivistelmä
Acknowledgements
9
Abbreviations
11
Original articles
13
Contents
15
1 Introduction
17
2 Review of the literature
19
2.1 Basement membranes and hemidesmosomes ......................................... 19
2.1.1 Cutaneous basement membrane ................................................... 21
2.1.2 Glomerular basement membrane .................................................. 23
2.1.3 Colorectal basement membrane ................................................... 24
2.2 Collagen XVII ......................................................................................... 25
2.2.1 Introduction to collagens .............................................................. 25
2.2.2 Transmembrane collagens ............................................................ 26
2.2.3 Collagen XVII .............................................................................. 27
2.2.4 Localization and function of collagen XVII in the skin ............... 28
2.2.5 Collagen XVII in other tissues ..................................................... 29
2.2.6 Collagen XVII in blistering skin diseases .................................... 29
2.3 Collagen XVII, integrin α6β4 and laminin 332 in cell migration
and invasion ............................................................................................ 30
2.3.1 The role of collagen XVII in cell migration and invation ............ 30
2.3.2 Integrin α6β4 in cell migration and invasion ............................... 31
2.3.3 Laminin 332 and cell migration and invasion .............................. 33
3 Aims of this study
35
4 Materials and methods
37
4.1 Patients and tumor samples (II-III) ......................................................... 37
4.2 Generation of Col17a1-/- mice (I) ............................................................ 37
4.3 Cell culture (I-III).................................................................................... 38
4.4 RNA isolation, quantitative real time PCR, Western Blot (II-III) ........... 38
4.5 Immunohistochemistry and in-situ hybridisation (I-III) ......................... 38
4.6 Electron microscopical analysis (I-II) ..................................................... 40
4.6.1 Transmission electron microscopy ............................................... 40
4.6.2 Immunoelectron microscopy ........................................................ 40
4.6.3 Scanning electron microscopy ...................................................... 40
15
4.7 Determing glomerular volume fraction and slit diagram count (I) ......... 40
4.8 Laser capture microdissection (II)........................................................... 41
4.9 RNAi and overexpression of collagen XVII (II-III)................................ 41
4.10 Cell adhesion, migration and invasion analyses (II-III) .......................... 41
4.11 Statistical analysis of collagen XVII in CRC (II).................................... 42
5 Results
43
5.1 Collagen XVII expression and function in normal tissue ....................... 43
5.1.1 Collagen XVII is expressed in the human and murine
glomerulus and in human colon epithelia (I-II) ............................ 43
5.1.2 Lack of collagen XVII alters the phenotypes of mice and
the function of glomeruli (I) ......................................................... 44
5.2 Expression and function of collagen XVII, laminin γ2 and
integrin β4 in CRC and SCC ................................................................... 46
5.2.1 Collagen XVII and laminin γ2 are upregulated in CRC (II) ......... 46
5.2.2 Collagen XVII, laminin γ2 and integrin β4 are upregulated
in human and mouse SCC (III) ..................................................... 47
5.2.3 Collagen XVII expression is associated with TNM and
metastasis survival times in CRC patients (II) ............................. 48
5.2.4 Collagen XVII participates in adhesion, migration and
invasion (II-III) ............................................................................. 49
6 Discussion
51
6.1 Collagen XVII is a newly characterised component of the
glomerulus and affects glomerular development .................................... 51
6.2 Collagen XVII is expressed in the normal colon epithelium and
may interact with laminin 332 ................................................................. 52
6.3 Collagen XVII is expressed in CRC and its expression correlates
with tumor progression and metastasis in CRC ...................................... 53
6.4 Collagen XVII, integrin α6β4 and laminin 332 are upregulated in
SCC ......................................................................................................... 54
6.5 Collagen XVII and integrin α6β4 affect the adhesion, migration
and invasion of SCC-25 cells but has no effect on HSC-3 cells ............. 55
7 Conclusions
59
References
61
Original articles
71
16
1
Introduction
Proteins are the essential building blocks of body tissue. They are large molecules
consisting of one or more long chains of amino acid residues. The structural size of
proteins ranges from tens to thousands of residues (Rodwell & Kennelly 2003,
Kannan et al. 2012).
The functional range of proteins is wide. They participate in metabolic
reactions, DNA replication, transportation, response to stimuli and serve as crucial
structural components in tissues (Rodwell & Kennelly 2003). Besides the correct
amino acid sequence, a proteins needs to be folded into a specific three-dimensonal
arrangement to function correctly. In addition, correct function also requires
posttranslational modifications such as the addition of new chemical groups or the
removal of transiently needed peptide segments (Rodwell & Kennelly 2003).
At the cellular level individual cells are dependent on proteins. The attachment
of cells to the extracellular matrix or to other cells, cell migration (pathological or
physiological) and cell invasion are all dependent on the correct function and
structure of specific proteins (Margadant et al. 2008, Kashyap et al. 2011, Buchheit
et al. 2012).
Our study concentrates on the functions and interactions of three proteins:
collagen XVII, integrin α6β4 and laminin 332, in both normal and pathological
conditions. Collagen XVII and integrin α6β4 are both best known for being
components of multiprotein structures called the type I hemidesmosome (HD)
(Margadant et al. 2008). Laminin 332 is a major component of anchoring filaments
in the skin. Collagen XVII, integrin α6β4 and laminin 332 all interact with each
other and are well known for their role as adherence molecules, attaching epithelial
cells to the underlying basement membrane (BM) (Walko et al. 2015).
17
18
2
Review of the literature
2.1
Basement membranes and hemidesmosomes
BMs are thin, specialised extracellular matrices that surround many tissues and
which are capable of isolating a cell from, and connecting a cell to, its environment
in all metazoans. They provide mechanical stability to tissues and support
important functions, including differentiation, proliferation, migration, and
chemotaxis of cells during development.
The composition of BMs depends on the type of tissue in which they are
located. BMs are composed of diverse extracellular matrix (ECM) molecules, the
deposition and arrangement of which determines the ability of BMs to adapt to the
cell’s changing biological requirements. The complexity of BM composition
continues to be characterised in increasing detail with the discovery of previously
unknown components and isoforms. An impressive number of tissue- and sitespecific BMs are now described in the skin, kidney, colon and lung, among other
tissues (Yurchenco & Patton 2009, Kruegel & Miosge 2010, Breitkreutz et al. 2013).
In general BMs contain at least one member of laminin, type IV collagen,
nidogen, and perlecan proteins or their subtypes which determines their common
structure. Several laminins isoforms are found in BMs and they all consist of an α,
a β and a γ chain linked by disulfide bonds to form a cross-shaped structure. To
date, more than 15 isoforms with different combinations of the α1–5, β1–3 and γ1–
3 chains have been identified (Miyazaki 2006).
Tissue-specific functional diversity is determined by differential expression of
the BM’s component proteins. As the main structural components, laminin and
collagen IV form distinct networks which bond noncovalently with nidogen and
perlecan and thus form irregular polymers (Breitkreutz et al. 2013). Table 1
summarises the components of the BM, their receptors and localization sites
(Kramer 2005, Kruegel & Miosge 2010). Changes in the structure, localization or
the qualitative or quantitative composition of BM proteins are involved in the
development of different disease states (Kramer 2005, Kruegel & Miosge 2010).
BM is important in forming the epithelial barrier. In addition to the BM there
are other important structures that connect epithelial cells to each other and to the
underlying BM and mesenchyme. Adhesive epithelial cell-to-cell contacts contain
large multiprotein aggregates with structural and signaling functions. There are
three structurally prominent types of cell-to-cell junction: tight junction, adherens
19
junction and desmosome (Capaldo et al. 2014). The structures and functions of
these junctions are beyond the scope of this study and thus will not be discussed in
more detail.
The attachement of epithelial cells to the underlying BM is mediated via HDs.
HDs are classified as belonging to one of two types on the basis of their components
(Figure 1). Type I (classical) HDs are found in (pseudo-) stratified epithelia, for
example, in the skin and consist of integrin α6β4, plectin, tetraspanin CD151,
collagen XVII (also referred to as bullous pemphigoid antigen 180 or BP180) and
BP230 (Borradori & Sonnenberg 1999, Margadant et al. 2008). Type II HDs are
found in simple epithelia such as that of the intestine, and consist of integrin α6β4
and plectin (Fontao et al. 1999).
Table 1. Overwiev of BM components in specialized ECMs.
BM isoform
Interactive receptors/ligands
Site of expression
Laminin-111
Lam α1: α-dystroglycan
Development, ubiquitously in BMs, adult
Laminin-211
Lam α2: α7β1, α-dystroglycan
Skeletal muscle, schwann cells,
Laminin-221
-
Skeletal muscle, schwann cells,
Laminin-332
Lam α3: α3β1, α6β1, α6β4
Laminin-411
Lam α4: αvβ3
Vasculature
Laminin-421
-
Vasculature, neuromuscular junction
Laminin-511
Lam α5: Lutheran Bloodgroup
Development, vasculature, epithelium
articular cartilage
eye (limbal compartment)
neuromuscular junction
Skin, embryonic cartilage
glycoprotein, B-CAM, α3β1, α6β1,
α6,β4
Perlecan
Collagen IV
Interaction with and support of
Ubiquitously in BMs, articular cartilage,
diverse growth factors
growth plate
α1β1, α2β1, α3β1, α6β1, α10β1,
Development, ubiquitously in BMs, kidney,
α11β1, αvβ3, CD44, DDR-1
inner ear (cochlea), eye, testis, lung, skin,
esophagus, smooth muscle cells, synovia
(knee)
Nidogen-1
αvβ3, α3β1, Laminin γ1, collagen IV, Ubiquitously in BMs, limb development, rib
perlecan, fibronectin, collagen I,
anlagen, adult articular cartilage
collagen II
Nidogen-2
Laminin, collagen IV, perlecan,
Like nidogen-1, but more restricted in muscle,
fibronectin, collagen I, collagen II
neuromuscular junction and cartilage
Data adapted from Kruegel & Miosge 2010.
20
Fig. 1. Simplified diagram of the structure and components of type I and II
hemidesmosomes. The main components of type I HD are α6β4-integrin, plectin,
tetraspanin CD151 and collagen XVII. Type II HDs consist only of α6β4-integrin and
plectin (modified from Margadant et al. 2008).
2.1.1 Cutaneous basement membrane
Although cutaneous BM is not a major target of this study, understanding its
structure is important since the basic functions of the molecules of interest
(collagen XVII, laminin 332 and integrin β4) can be better understood via their
21
function in skin. The cutaneous BM zone is a complex multiprotein structure that
attaches the epidermal keratinocytes to the underlying dermis via type I HDs. The
type I HD proteins integrin α6β4 and collagen XVII bind extracellularly to laminin
332, a major component of anchoring filaments in the skin. The intracellular HD
stabilization occurs via their association with keratin intermediate filaments
through the two plakins, plectin and BP230, thus creating a stable anchoring
complex (Zillikens et al. 1999, Margadant et al. 2008, Guess & Quaranta 2009,
Rousselle & Beck 2013) (Figure 2).
The cutaneous BM also controlls cell traffic and the diffusion of bioactive
molecules in both directions and binds a variety of cytokines and growth factors,
serving as a reservoir for their controlled release (Breitkreutz et al. 2013).
The ultrastucture of cutaneous BM as observed by transmission electron
microscopy (TEM) can be divided into two segments: the lamina densa and lamina
lucida (Figure 2). The laminin and collagen IV polymers form the body of the
lamina densa below the lamina lucida. However, the lamina lucida is not detectable
in electron microscopy (EM) specimens fixed by cryopreservation, indicating that
the lamina lucida represents an artificial structure rather than the real topography
of BM (Breitkreutz et al. 2013).
Fig. 2. Schematic of the structure of the dermal-epidermal junction (modified from
McMillan et al. 2003, Masunaga 2006, Aumailley et al. 2006).
22
2.1.2 Glomerular basement membrane
The glomerulus is the principal dynamic filtering unit in the kidney. It consists of
endothelial cells and podocytes, separated by a glomerular basement membrane
(GBM), which is primarily comprised of collagen IV, laminins and
glycosaminoglycans, as well as supporting mesangial cells (Miner 2005). The
three-layered structure of the endothelium, GBM, and podocytes facilitates the flow
of plasma and small solutes and restricts the flow of large plasma proteins like
albumin. The presence of high levels of albumin in the urine is considered to be
indicative of a defect in at least one of the layers of the glomerular filtration barrier
(GFB) (Miner 2011, Miner 2012). The structure of the GFB is shown in Figure 3.
Mature podocytes extend into several large projections, which divide into
smaller foot processes (FP) that interdigitate with the FPs of adjacent podocytes
(Quaggin & Kreidberg 2008). The basal surfaces of the FPs of a pair of adjacent
podocytes adhere to the GBM. The contact of two FPs form a cell-cell junction
called the slit diaphragm (SD) (Michaud & Kennedy 2007, Quaggin & Kreidberg
2008). The SD is formed by a complex of plasma membrane proteins including
nephrin, podocin, Fat1, vascular endothelial cadherin, P-cadherin, Nck proteins and
Cd2-associated protein (Patrakka & Tryggvason 2010). The SD maintains spacing
between the FPs and permits the efficient flow of water and small solutes across
the filtration barrier. Podocyte FPs are anchored to the GBM via transmembrane
cell receptors, including α3β1 integrin, α- and β- dystroglycans, and tetraspanin
CD151 (Pozzi et al. 2008, Patrakka & Tryggvason 2010).
23
Fig. 3. The structure of the glomerular filtration barrier. Capillary loops are covered by
endothelial cells (red), glomerular basement membrane (green) and podocytes with
their FPs (blue). The glomerular basement membrane divides the glomerulus into two
compartments; the inner one containing the capillaries and the mesangial cells and the
outer one containing podocytes and the space into which the filtrate passess.
M = mesangial cells, C = capillary loop, BC = Bowman’s capsule, P = podocytes,
E = endothelial cells (modified from Quaggin & Kreidberg 2008).
2.1.3 Colorectal basement membrane
The BM of the colon adheres colonic epithelial cells to the underlying stroma
(Debruyne et al. 2006). The epithelium of the human colon consists of a single
sheet of columnar epithelial cells, which form finger-like invaginations into the
underlying connective tissue forming crypts, the most important functional units of
the intestine. There are millions of crypts in the intestine. The crypts are surrounded
by BM which separates the epithelial cells of the crypt from the mesenchymal cells
of the stroma. (Humphries & Wright 2008). The columnar epithelial cells are
attached to the colonic BM via type II HDs (Figure 1) (Fontao et al. 1999). The
structure of the intestinal wall including the BM zone is shown in Figure 4.
Colonocytes, mucus-secreting goblet cells, peptide hormone-secreting
endocrine cells and Paneth cells are four major terminally differentiated epithelial
cell types present in the colon. There is well founded evidence supporting the
concept that all these cells can originate from a single multipotential stem cell
through a number of committed progenitors (Cheng & Leblond 1974, Humphries
24
& Wright 2008). There is also strong evidence that these stem cells are located at
the base of the crypt. The interaction between the stem cells and underlying
mesenchymal cells of the myofibroblast lineage through the BM is considered
critical for the stem cell behaviour. It is thought that pericryptal myofibroblasts
control the stem cell habitus, cell migration and differentiation via multiple
signalling pathways, including Wnt and Notch signalling (Humphries & Wright
2008).
Fig. 4. The structure of intestinal wall (modified from www.patiencenter.com Colon and
Rectal Cancer Center).
2.2
Collagen XVII
2.2.1 Introduction to collagens
Members of the collagen family are the most abundant proteins in the extracellular
matrix. They are the major structural element of all connective tissues and are also
found in the interstitial tissue of virtually all parenchymal organs, where they
contribute to the stability of tissues and organs and maintain their structural
integrity. To date 28 types of collagen have been identified. They are composed of
three polypeptide chains, called α chains. Different α chains are numbered with
Arabic numerals (Ricard-Blum 2011). The three a chains can be identical
25
(homotrimer) or different (heterotrimer). Collagens are characterised by containing
domains with repetitions of the proline-rich tripeptide Gly-X-Y, which form
trimeric collagen triple helices. As well as triple-helical domains, collagens also
contain non triple-helical non-collagenous (NC) domains, which are used as
building blocks by other extracellular matrix proteins and are thus modular proteins
(Gelse et al. 2003, Ricard-Blum & Ruggiero 2005).
Collagens can be grouped based on their structure and supramolecular
organisation into fibril-forming collagens, fibril-associated collagens with
interrupted triple helices, network-forming collagens, anchoring fibrils, membrane
associated collagens with interrupted triple helices and others with unique functions
(Gelse et al. 2003, Jain et al. 2014).
Several diseases, such as Alport syndrome, Ehler’s Danlos syndrome and
epidermolysis bullosa (EB), arise from genetic defects that affect the molecular
structure of collagens. Thus it is crucial to understand their molecular structure,
biosynthesis, assembly and turnover (Gelse et al. 2003, Ricard-Blum & Ruggiero
2005).
2.2.2 Transmembrane collagens
The transmembrane, or membrane collagens and their distribution are summarized
in table 2. They include the homotrimeric collagens XIII, XVII, XXIII, XXV. This
subgroup also includes collagen-like membrane proteins that have, to date not been
classified as collagens, such as ectodysplasin, the macrophage receptor with
collagenous structure (MARCO) and macrophage scavenger receptors, (Tenner
1999).
Table 2. Membrane collagens.
Collagen type
Distribution
XIII
Endothelial cells, dermis, eye, heart
XVII
Widespread: Skin, kidney, brain, intestine
XXIII
Heart, retina
XXV
Brain, heart, testis
Data adapted from Jain et al. 2014.
Collagens XIII, XVII, XXIII and XXV are type II transmembrane proteins with the
N-terminus located inside the cell and with a single pass hydrophobic
transmembrane domain and several extracellular collagenous domains (Franzke et
26
al. 2005) (Figure 5). The folding of the triple helix proceeds from the N- to the Cterminus in the ectodomain of collagens XIII and XVII, in the opposite orientation
to that of the fibrillar collagens (Areida et al. 2001, Snellman et al. 2000).
Collagens XIII, XXIII and XXV, MARCO and ectodysplasin-A1 contain two
separate coiled-coil motifs, which are thought to function as independent
oligomerization domains (Latvanlehto et al. 2003, McAlinden et al. 2003,
Snellman et al. 2000). The NC4 C-terminal domain (20 amino acids) of collagens
XIII, XXIII and XXV is thought to be involved in functional interaction with the
cell surface or with extracellular matrix proteins (Banyard et al. 2003).
Membrane collagenous proteins function both as cell surface receptors and as
soluble extracellular molecules because their ectodomains are released from the
cell surface by shedding. Membrane collagens all have a triple-helical extracellular
domain. The length of the extracellular component varies between the collagens –
for example, it consists of three collagenous domains in domains in collagen XIII
(Pihlajaniemi & Rehn 1995), XXIII (Banyard et al. 2003) and XXV (Hashimoto et
al. 2002) and of 15 domains in collagen XVII (Franzke et al. 2003) (Figure 5).
Fig. 5. The structure of membrane collagens (modified form Ricard-Blum 2011).
2.2.3 Collagen XVII
Collagen XVII is the largest transmembrane collagen. It consists of three 180-kDa
α1 (XVII) chains, each with an intracellular N-terminal domain of 466 amino acids,
a short transmembrane stretch of 23 amino acids, and an extracellular C-terminal
ectodomain of 1008 amino acids (Franzke et al. 2004). Its rod-like structure is
flexible and triple-helical, with pronounced thermal stability (Schacke et al. 1998,
Tasanen et al. 2000, Areida et al. 2001). The ectodomain consists of 15 collagenous
27
subdomains (COL1–COL15) with typical collagenous Gly-X-Y repeat sequences
and 16 short non-collagenous sequences (NC1–NC16A) (Franzke et al. 2003). The
NC16A sequence is located between the plasma membrane and the COL15 domain
and can therefore be considered the extracellular linker domain. It is functionally
important because it is believed to play a role in both the shedding and triple-helical
folding of collagen XVII (McLaughlin & Bulleid 1998, Myllyharju & Kivirikko
2001, Franzke et al. 2002, Franzke et al. 2004, Nishie et al. 2010).
The ectodomain can be shed from the cell surface both in vitro and in vivo to
achieve a shorter collagenous triple-helical molecule (Franzke et al. 2002, Hirako
et al. 2003, Nishie et al. 2010). The cleavage occurs at different sites within the
juxtamembranous NC16A domain (Hirako et al. 2003, Nishie et al. 2010). Under
physiological conditions, ADAM9, -10, and -17 appear to be the major sheddases,
but involvement of neutrophil elastase and serine proteinases has been suggested
in pathological settings such as BP (Franzke et al. 2009, Hofmann et al. 2009, Lin
et al. 2012, Nishie et al. 2010). The regulation mechanisms and biological functions
of collagen XVII ectodomain shedding remain uncertain, but it plays an apparent
role in binding to laminin 332, migration and differentiation of keratinocytes
(Franzke et al. 2003, Tasanen et al. 2004, Franzke et al. 2005, Qiao et al. 2009,
Nishie et al. 2011, Van den Bergh et al. 2011, Loffek et al. 2014).
2.2.4 Localization and function of collagen XVII in the skin
Collagen XVII is expressed in the HDs of the epidermal basal keratinocytes. It is a
major transmembrane constituent of the epidermal anchoring complex. The
extracellular ligands of collagen XVII are α6 integrin and laminin 332 (Fig. 1), and
its intracellular ligands include β4 integrin, plectin and BP230 within the HD
plaque (Powell et al. 2005). It has an N-terminal globular head region that localizes
to the HD plaque and a C-terminal collagenous tail that projects into the basal
lamina. The structure and location of collagen XVII mean that it acts as a core
anchor protein, connecting the intracellular and extracellular HD proteins (Van den
Bergh et al. 2011). Also strong evidence of collagen XVII involvement in directed
keratinocyte migration have been demonstrated (Qiao et al. 2009, Loffek et al.
2014)
28
2.2.5 Collagen XVII in other tissues
Most of what is currently known about collagen XVII is derived from studies of
skin and keratinocytes. However, collagen XVII is also expressed in various other
tissues (Aho & Uitto 1999, Van den Bergh & Giudice 2003, Claudepierre et al.
2005, Seppanen et al. 2006, Huilaja et al. 2008) including:
–
–
–
–
–
–
–
–
–
buccal mucosa
upper oesophagus
small intestine
colon
urinary bladder
ocular cornea, conjunctiva and retina
heart
central nervous system
placenta and amniotic membrane
However, little is known of the function of collagen XVII in tissues other than the
skin.
2.2.6 Collagen XVII in blistering skin diseases
Bullous skin diseases caused by dysfunctional collagen XVII can be divided into
inherited and acquired types. In inherited diseases mutations in COL17A1 gene
lead to either the absence of collagen XVII or the expression of a structurally
altered protein. They are associated with diminished epidermal adhesion and with
skin blistering in response to minimal shearing forces in patients with junctional
EB, a genodermatosis with variable localized or generalized phenotype (McGrath
et al. 1995, Powell et al. 2005, Fine et al. 2014). Ultrastructural rudimentary HDs
and separation of the basal keratinocytes from the underlying BM can be detected.
Clinical characteristics include skin fragility and blisters, atrophic scarring, nail
dystrophy and loss, and diffuse alopecia. Deletion of the collagen XVII cytoplasmic
domain leads to a rare form of EB simplex, with mechanical fragility occurring
within the basal keratinocytes (Darling et al. 1997, Powell et al. 2005, Fine et al.
2014).
In acquired diseases autoantibodies are formed against collagen XVII (BP180).
Antibodies are found in patients with BP, pemphigoid gestationis, linear
immunoglobulin A disease, and mucous membrane pemphigoid, which are
29
clinically distinct subepidermal immunobullous diseases characterized by antibody
binding to components of the cutaneous basement membrane zone and the
development of cutaneous or mucosal blisters (Powell et al. 2005). It has been
shown with mouse models that collagen XVII is a major autoantigen in BP (Nishie
2014). BP is the most common subepidermal immunobullous disease and
autoreactivity to a 180-kDa epidermal antigen was first demonstrated in BP.
Historically autoimmunity to collagen XVII has been most studied in relation to
BP (Labib et al. 1986, Powell et al. 2005, Nishie 2014).
2.3
Collagen XVII, integrin α6β4 and laminin 332 in cell migration
and invasion
Although HDs are anchoring structures that provide stable adhesion they also
participate in the migration of epithelial cells. Components of the HDs undergo a
rapid turnover, which makes it possible for epithelial cells to change from a
stationary to a migratory state by changing the balance quickly to HD disassembly
(Kashyap & Rabinovitz 2012). HD disassembly is required for several
physiological processes, for example in cell division, differentiation and directed
migration in the wound healing process (Margadant et al. 2008, Tsuruta et al. 2011,
Loffek et al. 2014).
However, loss of attachment via HD disassembly is also a crucial criterion that
allows squamous cell carcinoma (SCC) cells to migrate and invade (Buchheit et al.
2012, Margadant et al. 2008). The roles of hemidesmosomal components and their
binding partners in SCC carcinogenesis have been studied widely, and the
importance of laminin 332 and α6β4 integrin in SCC cell migration and invasion is
well established (Beaulieu 2010, Chao et al. 1996, Giannelli & Antonaci 2000,
Guess & Quaranta 2009, Kligys et al. 2012, Miyazaki 2006). The role of collagen
XVII in SCC carcinogenesis is not as clearly characterized as those of its binding
partners, but its involvement in cancer cell migration, invasion and adhesion is
evident (Qiao et al. 2009, Nishie et al. 2011, Loffek et al. 2014).
2.3.1 The role of collagen XVII in cell migration and invation
It has been reported that collagen XVII siRNA knockdown affects keratinocyte
migration through the p38 mitogen-activated protein kinase (MAPK)-signaling
pathway. The MAPK pathway influences many cellular processes, including cell
migration via direct action on cytoskeletal components of migratory cells (Qiao et
30
al. 2009). A recent study also demonstrated a connection between collagen XVII
and the phospho-FAK/PI3K pathway in keratinocyte migration, which suggests
that collagen XVII plays a part in directed keratinocyte migration by dampening
integrin dependent PI3K activation and by stabilizing lamellipodia (Loffek et al.
2014). In collagen XVII-deficient keratinocytes, the genetic ablation of collagen
XVII leads to increased expression and increased phosphorylation of the integrin
β4 subunit and to phosphorylation of FAK, which enhance PI3K activity and induce
undirected cell migration via Rac1 activation. Phosphorylation of the collagen
XVII β4 domain is thought to inhibit the binding of the endodomain (Loffek et al.
2014). Also there has been speculation about the role of the cleaved ectodomain of
collagen XVII in adhesion and migration and there is evidence that the extodomain
may interact with laminin 332, thus independently participating in adhesion and
migration (Nishie et al. 2011). Aside from its function in migration, many studies
have been published on the participation of collagen XVII in invasion although the
information is mostly based on collagen XVII’s expression and localization.
Collagen XVII is abnormally distributed and up-regulated in solar keratosis,
Bowen’s disease, basal cell carcinomas and SCCs (Yamada et al. 1996, Parikka et
al. 2003, Parikka et al. 2006, Stelkovics et al. 2008). In SCC increased collagen
XVII expression is pronounced in areas of invasive growth (Parikka et al. 2003,
Parikka et al. 2006, Stelkovics et al. 2008, Yamada et al. 1996).
2.3.2 Integrin α6β4 in cell migration and invasion
Integrins are heterodimeric cell surface glycoproteins that are composed of α and
β subunits (Dydensborg et al. 2009, Maalouf et al. 2012). To date 24 αβ
heterodimeric members has been identified (Barczyk et al. 2010). They are
transmembrane receptors for extracellular matrix proteins and transmit signals
from extracellular matrix components to the cell interior (Dydensborg et al. 2009,
Maalouf et al. 2012). Integrins play roles in several normal cellular processes that
influence the development of tumors, including the regulation of proliferation and
apoptosis, cellular motility and invasion, cell surface localization of
metalloproteinases, and angiogenesis (Hood & Cheresh 2002). Integrin interaction
with a large range of scaffold proteins results in the activation of several signalling
molecules, such as Ras and PI3K, which in turn leads to activation of other
molecules including JNK (c-JUN N-Terminal Kinase), Jun, Erk and CyclinD
(Giancotti & Tarone 2003).
31
The α chain of α6β4 integrin is very similar to the α chains of other integrins
and consists of 1073 amino acids (120 kDa), however, the β4 subunit is atypical
compared with other integrins (Mercurio et al. 2001b, Yoon et al. 2006). The β4
subunit differs those of other integrins in three ways: 1) the cytoplasmic domain is
larger (over 1000 amino acid residues and 202 kDa in size); 2) it lacks some of the
conserved cysteines in the extracellular domain; and 3) it has a unique domain
organization (Mercurio et al. 2001a, Yoon et al. 2006, Kligys et al. 2012). In intact
skin α6β4 integrin mediates the interaction of basal keratinocytes with laminin-332
located at the interface of the epidermis and dermis (Kligys et al. 2012).
The participation of α6β4 integrin in migration and invasion has been studied
widely and the evidence that α6β4 integrin facilitates the formation of some
carcinomas as well as the migration, invasion, and survival of carcinoma cells is
strong (Chao et al. 1996, Mercurio et al. 2001b, Mercurio & Rabinovitz 2001,
Chung et al. 2002, Nikolopoulos et al. 2004, Yoon et al. 2006, Beaulieu 2010,
Kligys et al. 2012, Koivisto et al. 2014). As with collagen XVII, the expression of
α6β4 integrin is often upregulated in carcinoma cells (Kligys et al. 2012). The
mechanisms by which α6β4 integrin participates in migration and carcinogenesis
of SCC are not yet fully clear but there are some theories. In cell motility and
migration it has been suggested that α6β4 integrin could be the regulator of other
integrins in keratinocytes. Regarding migration and invasion a pivotal role for α6β4
integrin has been suggested. Firstly it promotes migration and invasion directly via
variety of signalling pathways (Shaw et al. 1997, O'Connor et al. 1998, Sehgal et
al. 2006, Kashyap et al. 2011, Kligys et al. 2012). These signaling pathways
include the Rac1 and cofilin pathways, regulating laminin 332 matrix assembly and
keratinocyte motility. Secondly, via phosphorylation of 4EBP1 and activation of
PI3K it determines the expression of α3β1 integrin which is shown to regulate
cellular velocity (Kligys et al. 2012). It has also been shown that α6β4 integrin is
required for tumorigenesis in a murine xenograft model of human SCC (Dajee et
al. 2003). It is believed that the right correlation of HD assembly/disassembly is
the key for both migration and invasion and it has been suggested that the reduction
of HDs in SCC could be due to β4 integrin serine phosphorylation (Kashyap et al.
2011, Kashyap & Rabinovitz 2012).
32
2.3.3 Laminin 332 and cell migration and invasion
Laminins are major cell adhesion substrates in epithelial BM. They have a crucial
role in the regulation of epithelial cell adhesion and also in normal cellular
functions such as proliferation, polarity and differentiation (Miyazaki 2006).
Laminin 332 consists of an α3 (200kDa), a β3 (140kDa) and a γ2 (140kDa)
chain from which the α3 and γ2 chains undergo further processing to smaller
species of 165/145 kDa and 105 kDa, respectively (Colognato & Yurchenco 2000).
Laminin 332 is unique in both structure and activity and functions as a major
adhesion component of anchoring filaments in the epidermal BM (Miyazaki 2006).
Laminin 332 is thought to be crucial for the migration and invasion of SCC
cells, although the exact mechanisms via which laminin 332 participates in
migration and invasion remain to be clarified (Miyazaki 2006, Hamasaki et al.
2011). Like α6β4 integrin, laminin 332 or its subunits are highly expressed in
various types of human cancers. In particular, the laminin γ2 chain is expressed in
tumor cells at the invasion front or in budding tumor cells in many types of human
cancers such as adenocarcinomas of the colon, breast, pancreas and lung, and
squamous cell carcinomas and melanomas (Ono et al. 1999, Pyke et al. 1995,
Sordat et al. 1998). Overexpression of the laminin γ2 chain by tumor cells, as well
as lowered or impaired expression of the laminin α3 and/or β3 chains, may
contribute to the loss of BM structures in invasive carcinomas. Another possible
mechanism is that the laminin γ2 chain monomer itself enhances tumor invasion.
Quaranta and his group reported that the proteolytic cleavage of the 150-kDa γ2
chain of rat laminin 332 to a 80-kDa form elevates the cell migration activity of the
laminin-332 (Giannelli et al. 1997, Koshikawa et al. 2000). Another study with
recombinant laminin 332 mutants which clearly showed that the cleavage of human
laminin γ2 chain to the 105-kDa isoform increases its cell motility activity but
decreases its cell adhesion activity (Ogawa et al. 2004). In addition, it has been
shown that cleavage of the γ2 or the β3 chain impairs the ability of laminin 332 to
deposit onto, or to be assembled into, the ECM (Gagnoux-Palacios et al. 2001,
Ogawa et al. 2004, Tsubota et al. 2005). The cleavage of the β3 chain leads to a
decrease in cell adhesion activity and complete loss of the type VII collagenbinding activity of laminin 332 (Miyazaki 2006). Cleavage of the α3 chains in the
human laminins 311 and 332 is reported to lead to an enhancement in both cell
adhesion and motility activities (Hirosaki et al. 2002, Tsubota et al. 2005). In
addition a soluble form of laminin 332 is able to stimulate cell migration by binding
to integrin α3β1 on the cell surface (Kariya & Miyazaki 2004). It has also been
33
shown that laminin 332, like α6β4 integrin, is required for tumorigenesis in a
murine xenograft model of human SCC (Dajee et al. 2003).
34
3
Aims of this study
The overall aim of this study was to broaden our knowledge concerning the
extracutaneous localization and function of collagen XVII. The specific aims were
to describe:
1.
2.
3.
4.
The localization and function of collagen XVII in the normal kidney
glomerulus.
Collagen XVII in normal colorectal epithelia.
The role of collagen XVII in the pathogenesis of CRC.
Function of collagen XVII and α6β4 integrin in SCC carcinogenesis.
35
36
4
Materials and methods
4.1
Patients and tumor samples (II-III)
The study included all patients who were operated on for CRC in Oulu University
Hospital between 2006 and 2010 (Kantola et al. 2012). The study complied with
the Declaration of Helsinki and the design was approved by the Ethical Committee
of Oulu University Hospital (58/2005, 184/2009). Tumor samples were classified
according to the TNM (tumor-node-metastasis) classification (Sobin & Wittekind
2002) and graded according to World Health Organization criteria (Hamilton et al.
2000)
Tissue collection for our SCC study was performed at the Department of
Pathology, Turku University Hospital (Kivisaari et al. 2008, Kivisaari et al. 2010,
Riihila et al. 2015). The use of archival tissue specimens and the collection of
normal skin and SCC tissues was approved by the Ethics Committee of the Hospital
District of Southwest Finland, Turku, Finland and was conducted according to the
Declaration of Helsinki.
Chemical skin carcinogenesis of wild type mice was performed using the
method described earlier (Brideau et al. 2007). The samples gathered from mice
were classified by a pathologist in a blinded manner on the basis of hematoxylin
and eosin (HE) stained sections. All animal experiments were approved by the
Animal Care and Use Committee of the University of Oulu and by the State
Provincial Office of Oulu.
4.2
Generation of Col17a1-/- mice (I)
The targeting vector contained 6.7 kb of genomic DNA with arms of 4.3 kb and 2.4
kb. Exon 18 and the surrounding intron sequences of the Col17a1 gene were
replaced with the neomycin resistance gene, driven by a phosphoglycerate kinase
promoter. Embryonic stem cell culture and the generation of chimeric mice were
performed by the Biocenter Oulu Transgenic Core Facility. Chimeric mice were
generated by blastocyst injection of embryonic stem cells carrying the targeted
mutation, and were mated with C57BL/6J OlaHsd females to produce a targeted
mouse line. F1 heterozygous mice were backcrossed for seven generations and then
intercrossed to generate Col17a1-/- mice.
37
4.3
Cell culture (I-III)
For this study we used multiple commercial cell lines including HaCaT cells
(Invitrogen, Paisley, UK), human oral squamous carcinoma cell lines SCC-15,
SCC-25 (ATCC cultures, Teddington, UK) and HSC-3 (JRCB 0623, Japan Health
Science Research Resources Bank, Osaka, Japan) cells, tumour derived primary
UT-SCC-105 cells (a generous gift from professor Reidar Grenman, University of
Turku, Finland) and the human colon adenocarcinoma cell line CaCo-2 (HTB-37,
ATCC). Cells were cultured according to the instructions provided by the
manufacturers. Primary keratinocytes from the skin of collagen XVII-deficient and
control mice were cultured in a defined serum-free keratinocyte medium
supplemented with 100 pm cholera toxin, 100 units/ml penicillin, 100 μg/ml
streptomycin, and 0.25 μg/ml amphotericin B (all from Invitrogen) on 6-well plates
coated with gelatin (2.5 mg/ml; Sigma-Aldrich). Cells were maintained at 37 °C,
5% CO2, and 95% humidity.
4.4
RNA isolation, quantitative real time PCR, Western Blot (II-III)
Total RNA was isolated from cells using QIAamp RNA Blood Mini Kit (Qiagen,
Crawley, UK) using the protocol provided by the manufacturer. cDNAs were
synthesized by standard methods (Thermo Fisher Scientific, Waltham, MA, USA).
mRNA levels were measured by quantitative RT-PCR analysis using SYBR Green
Supermix (Bio-Rad, Hercules, CA, USA) with iQ5 multicolour real-time PCR
equipment (Bio-Rad). The relative changes in collagen XVII, laminin γ2 and
integrin β4 mRNA expression were calculated by Livak’s 2-∆∆CT method (Livak &
Schmittgen 2001).
Western Blot was done using standard methods. Total protein concentration
was determined using the BioRAD DC protein Assay kit (Bio-Rad). Samples were
separated using SDS-PAGE and protein was visualized using the antibodies listed
in table 3. Images were taken with the Fusion SL imaging system (Vilber Lourmat,
Marne-la-Vallee Cedex, France).
4.5
Immunohistochemistry and in-situ hybridisation (I-III)
The antibodies used for immunohistochemical (IHC) stainings of human colon and
human and mouse kidney and SCC samples are listed in table 3. Immunoperoxidase
detection was performed using to the DAKOReal EnVision Detection System
38
(Dako Cytomation, Glostrup, Denmark). Kidney immunofluorescence (IF) staining
was viewed with an Olympus FluorView1000 confocal microscope using x60 oil
objectives and appropriate filters for the green and red channels (Alexa 488 and
Texas Red, Molecular Probes). The images were collected and saved using
Olympus software. Murine skin IF slides were mounted in VECTORSHIELD
Mounting Medium (Vector Laboratories, Peterborough, UK) containing DAPI and
recorded with Axiophot 2E microscope system (Carl Zeiss, Munchen, Germany).
Table 3. List of antibodies used in this study.
Antibody
Manufacturer / reference
Original article used in
Polyclonal NC14A1 (mouse)
(Franzke et al. 2009)
I, III
Polycolonal Endo-2 (mouse and
(Franzke et al. 2002)
human)
I-II
Polyclonal tubulin (mouse)
Abcam, Cambridge, MA, USA
I
Polyclonal NC16A (human)
(Schumann et al. 2000)
I-II
Polyclonal GAPDH (human)
Santa Cruz Biotechnology, Heidelberg,
Germany
I
Collagen XVII Ecto-4 (human)
(Huilaja et al. 2008)
I
CD-34 (mouse)
Novocastra, UK
I
Collagen IV (mouse)
Millipore, Billerica, MA, USA
I
Polyclonal integrin β4
Santa Cruz
III
Integrin α3 (mouse)
BD Biosciences, Franklin Lakes, NJ, USA
I
Integrin β1 (mouse)
Gift from Dr. Aki Manninen, Biocenter Oulu,
Oulu, Finland
I
Laminin α5 (mouse)
Santa Cruz
Nephrin (mouse)
(Putaala et al. 2001)
I
Integrin α6 (mouse)
Progen, Heidelberg, Germany
I
Laminin γ2
Santa Cruz
I-III
Monoclonal human cytokeratin
Dako, Glostrup, DK-2600, Denmark
III
I
A tissue microarray (TMA) was constructed as previously described (Vayrynen et
al. 2014). Bound antibodies were detected using the EnVisionTM system (Dako,
Copenhagen, Denmark); 3,3’-Diaminobenzidine was used as the chromogen and
hematoxylin as the counterstain. Histological images were captured with an
Olympus DP25 camera (Olympus, Center Valley, PA) attached to a Nikon Eclipse
E600 microscope (Nikon, Tokyo, Japan) using x20 and x10 objectives.
Immunoreactivity evaluation was done semi-quantitatively based on the proportion
of tumor cells found to be positive (0 to 100%).
39
Goat anti-mouse IgG-Alexa Fluor 488 and goat anti-rabbit IgG-Alexa Fluor
594 (Invitrogen, Paisley, UK) were used as secondary antibodies for IF studies of
frozen human colon tissue samples. Images were taken using an Olympus Fluor
View1000 confocal microscope and a x20 objective with appropriate filter settings.
4.6
Electron microscopical analysis (I-II)
4.6.1 Transmission electron microscopy
Standard methods were used for TEM. Thin sections were examined in a Philips
CM100 transmission electron microscope. Images were captured using a CCD
camera and analyzed with TCL-EM-Menu version 3 from Tietz Video and Image
Processing Systems GmbH (Gaunting, Germany).
4.6.2 Immunoelectron microscopy
Human kidney samples for immunoelectron microscopy (IEM) were collected
from diagnostic biopsies at the Department of Paediatrics, Oulu University
Hospital. All specimens were embedded in methylcellulose and examined using a
Philips CM100 transmission electron microscope (FEI Company, Eindhoven and
The Netherlands). Control samples were prepared by carrying out the labelling
procedure without the primary antibody. The Ethical Committee of Northern
Ostrobothnia Hospital Districts approved this element of the study, which was
performed according to the Declaration of Helsinki second revision (1983).
4.6.3 Scanning electron microscopy
Specimens were examined using a Zeiss Ultra Plus scanning electron microscope,
(Carl Zeiss MT- Nanotechnology System Division, Carl Zeiss NTS Gmbtt,
Oberkochen, Germany) with an accelerating voltage of 5 kV.
4.7
Determing glomerular volume fraction and slit diagram count
(I)
Glomerular volume fraction (VG) and glomerular surface density (SG) of the kidney
cortex were determined using a point counting method with a multipurpose test
40
grid of 100 points placed on a microscopic digital image on a monitor. Ten random
fields were counted for each case. Three Col17a1-/- mice and two control mice from
the same litter were analysed.
Three different sample pairs (wt/knock out) were chosen for counting slit
diaphragms. Statistical significance was calculated using the independent samples
t-test (SPSS, version 16).
4.8
Laser capture microdissection (II)
Two normal mucosal samples and five CRC samples were chosen for the
microdissection experiment. The PALM MicroLaser Systems (PALM
RoboSoftware 4.3 SP1, Carl Zeiss MicroImaging Gmbh, Jena, Germany) were
used to cut cancer cell islets according to manufacturers’ instructions. Total RNA
was isolated from microdissection samples using the QIAcube instrument
(QIAGEN Nordic, Sollentuna, Sweden) according to the manufacturer’s
instructions.
4.9
RNAi and overexpression of collagen XVII (II-III)
Collagen XVII knockdown (KD) by retrovirus-mediated RNAi for SCC-25 and
HSC-3 cells was generated as described in more detail previously (Schuck et al.
2004). β4 integrin knockdown was generated using the MISSION shRNA
lentivirus (Sigma, Munchen, Germany) according to the manufacturer’s protocols.
KD efficiency was evaluated by quantitative RT-PCR.
To create a stable CaCo-2 cell line overexpressing murine collagen XVII,
cDNA of full-length murine collagen XVII was cloned into the retroviral MSCVIRES-GFP, pMIG expression vector (Addgene, Cambridge, MA, USA). CaCo-2
cells transfected with an empty pMIG vector were used as a control. CaCo-2 cells
infected with virus particles were subsequently FACS-sorted for green fluorescent
protein to obtain stable cell lines.
4.10 Cell adhesion, migration and invasion analyses (II-III)
Cell adhesion assays were conducted using the electric cell–substrate impedance
sensing (ECIS) system (Applied Biophysics, Inc., Troy, NY, USA) according to the
manufacturer’s protocol. An elevated voltage pulse was applied with a frequency
of 40 kHz, amplitude of 5 V for 45 s.
41
The migration assay was performed using Ibidi Culture-Inserts (Ibidi,
Martinsfried, Germany) on glass-bottom 6 well plates (Mat Tek Corporation,
Ashland, MA, USA). The imaging was performed using Olympus Cell^P livecell/timelapse imaging system. Images were taken every 20 minutes until the
wound was closed in control cells.
Invasion analyses were done using an organotypic myoma model according to
method described earlier (Nurmenniemi et al. 2009). A MatrigelTM (BD
Biosciences, Bedford, MA) invasion analysis was done according to the
manufacturer’s protocol. Statistical significance was calculated by the independent
sample t-test using SPSS (version 22).
4.11 Statistical analysis of collagen XVII in CRC (II)
The statistical analyses for normally distributed continuous variables were
conducted using IBM SPSS Statistics 22.0 (IBM, Chicago, IL); values are
presented as mean (standard deviation), whereas other continuous variables are
presented as median (IQR). The statistical significance of the associations of
collagen XVII expression with clinicopathological factors was analyzed by the
Mann-Whitney U test (comparing two classes) or the Kruskal-Wallis test
(comparing three or more classes). A receiver operating characteristics (ROC)
analysis was used to determine an optimal cut-off score, with the shortest distance
to the coordinate (0,1), for the collagen XVII expression level to discriminate
survivors from non-survivors. A univariate survival analysis was conducted
according to the Kaplan-Meier method. Cox’s proportional hazards regression
model was used to analyze the independent prognostic significance of the
expression of collagen XVII. A two-tailed p value <0.05 was considered
statistically significant.
42
5
Results
5.1
Collagen XVII expression and function in normal tissue
5.1.1 Collagen XVII is expressed in the human and murine
glomerulus and in human colon epithelia (I-II)
Collagen XVII has not previously been studied in the kidney and only a little in the
colon. We wanted to confirm its expression in these tissues. Collagen XVII mRNA
expression was clearly detectable in the human glomerulus (Fig. 6A). In situ
hybridization showed clear expression of collagen XVII mRNA in podocytes,
endothelial cells and in the cells of the Bowman’s capsule. IHC of mouse kidney
samples showed similar localization of collagen XVII in the mouse glomerulus and
partial co-localization with nephrin in glomerular podocytes with double IF
staining. The ultrastructural localization of collagen XVII in podocytes was
investigated in normal human samples. IEM of the normal human glomerulus
showed gold particles within the pedicle and close to the cell membrane of the
pedicle soles in the lamina rara externa of the GBM (Fig. 6B). Some gold labelling
was also present in the vicinity of the slit pore between the neighbouring FPs.
The epithelial cells of the normal human colon mucosa showed collagen XVII
expression (Figure 6C). The basal cells in the anal squamous epithelium were
strongly positive for collagen XVII immunostaining. Collagen XVII was present in
the colon epithelial cells of the surface and in the crypt epithelium. Also IF staining
showed clear collagen XVII expression in normal colorectal epithelium and partial
colocalization with laminin γ2 was detected.
43
Fig. 6. Modified from original articles I-II. Collagen XVII is expressed in the human and
murine glomerulus and in human colon epithelia. IHC of mouse kidney samples showed
collagen XVII reaction in podocytes (arrows) with 40 x original magnification (A). IEM
demonstrated collagen XVII between two neighbouring foot processes and in the
glomerular basement membrane (B). IHC of colon epithelia showed clear positivity of
collagen XVII in the basal cells (arrow) in picture C. Scale bar is 100μm.
5.1.2 Lack of collagen XVII alters the phenotypes of mice and the
function of glomeruli (I)
The creation of a mouse line lacking collagen XVII enabled us to study further and
in more detailed the role and importance of collagen XVII in kidneys. These mutant
mice showed clear differences compared to normal wild type mice. Mutant mice
were smaller in size and suffered from high mortality (over 90%) within the first
weeks after birth. They developed blisters and detachment of epidermis especially
on the tail as well as both fore and hind paws. Diffuse, non-pigmented hair growth
and hair loss was also detected (Fig 7A).
The glomeruli of mutant mice appeared immature, densely packed and small
in size (Fig. 7B). Suprisingly, no major structural differences between the wild type
and mutant kidneys could be detected outside the glomerulus. Ultrastructural
analysis revealed abnormally shaped and hypertrophic podocytes and effacement
of podocyte FPs, but no major SD disruption (Fig. 7C upper pictures). In addition
endothelial cells were swollen, the capillary lumen was clogged in some places and
splitting of GBM was detected in some areas (Fig. 7C upper pictures). SEM showed
enlarged primary FPs and abnormal interdigitation of secondary pedicles
confirming the presence of disorganized podocytes (Fig. 7C lower pictures).
When analyzing functional differences in kidneys both glomerular volume
fraction (VG) and surface density (SG) were elevated in mutant mice at two weeks
44
of age, indicating that the glomerular content in mutant kidneys was increased
although the PAS staining showed no GBM thickening under light microscopy
resolution.
The impact of the absence of collagen XVII on the expression and organization
of other proteins known to be involved in GBM and FP integrity was studied by IF
stainings. No alterations in the expression and localization of collagen IV, integrins
α3, β1, laminin α5 chain or nephrin was observed compared to wild-type animals.
Fig. 7. Modified from original article I. Lack of collagen XVII affects both the structure
and function of mouce kidney and alters the fenotype of mutant mice. Mutant mice were
45
smaller in size and developed blisters and hair loss (A). Glomeruli of mutant mice were
small and immature compared to the control mice in HE staining (B). TEM (upper
pictures in C) showed abnormally shaped podocytes, foot process effacement and GBM
splitting and SEM (lower pictures in C) hypertrophic podocytes and disorganized foot
processes in mice lacking collagen XVII.
5.2
Expression and function of collagen XVII, laminin γ2 and
integrin β4 in CRC and SCC
5.2.1 Collagen XVII and laminin γ2 are upregulated in CRC (II)
Having verified the expression of collagen XVII in the normal colon, it was in our
interest to investigate whether upregulation of collagen XVII expression occurs in
CRC. 141 CRC patients were included in our study. Collagen XVII expression was
clearly detectable in most TMA patient samples. Thirty-one patients showed
negative or almost negative staining. In most positive cases the staining was both
membranous and intracellular, and more intensive than in the normal mucosa
(Figure 8A-B). Partial co-localization of collagen XVII and laminin γ2 was also
found in CRC similarly to the normal colon epithelia samples.
The localization of collagen XVII in colon epithelium and CRC was analyzed
further with immunoelectron microscopy, which revealed that the gold labeling of
collagen XVII was clearly more abundant in the cancer tissue than in the normal
colon epithelium. The localization was extracellular although some minor labelling
was also observed intracellularly (Figure 8C). In some cases concentrated
expression of collagen XVII was detected in the invasive tips of the malignant cells
(Figure 8C).
The expression of collagen XVII in CRC was quantitated by collecting normal
epithelial and colon tumor cells from patient samples by laser capture
microdissection. The collagen XVII mRNA levels of tumor samples were clearly
elevated compared to normal mucosa samples (Figure 8D).
46
Fig. 8. Modified from original article II. Collagen XVII is expressed in human CRC. In
CRC samples collagen XVII staining was strongly positive in invasive cells (arrows in
A-B), the scale bar is 100μm. In image C IEM of human CRC samples showed collagen
XVII expression intracellularly and in the extracellular matrix near basement membrane
(upper picture) and at the invasive tip of a malignant cell (lower picture). With laser
capture microdissection higher collagen XVII expression mRNA levels were
quantifically verified in CRC compared to normal epithelia (D).
5.2.2 Collagen XVII, laminin γ2 and integrin β4 are upregulated in
human and mouse SCC (III)
Due to the evident significance of collagen XVII, laminin 332 and integrin α6β4
in SCC carcinogenesis, we wanted to study their expression and localization in
precancerous, in-situ carcinoma and SCC samples compared to normal skin in a
more detailed fashion. In order to study the expression and localization of these
proteins multiple IHC stainings of human SCC samples were performed, which
showed strong expression of collagen XVII, laminin γ2 and integrin β4. Compared
to normal skin, where collagen XVII is positive in basal cells, SCC invading cells
showed membranous and cytoplasmic immunoreaction against collagen XVII. For
laminin γ2 cytoplasmic staining was limited to cells in and around the invasive
margin. A weaker immunoreaction was observed within areas of BM formation.
The staining pattern of integrin β4 was very similar to that of collagen XVII.
To investigate further, a quantitative IHC analysis of TMA samples of normal
skin, actinic keratosis, Bowen’s disease and SCC was conducted. Altogether 95
cases were included in the study (13 normal skin samples, 36 actinic keratosis
samples, 30 Bowen’s disease samples and 16 SCC samples). The results
demonstrated an increase of expression that was linear for laminin γ2 in normal to
SCC samples but not for collagen XVII or integrin β4. The highest expression
levels were found in Bowen’s disease samples, and were lower in aggressive SCC
samples. However, the positivity and intensity varied massively in Bowen’s disease
47
samples and especially in SCC samples with all three antibodies compared to
normal skin samples.
Several stainings of chemically induced mouse carcinoma samples were
performed to compare murine with human samples. The IHC of these samples
showed that the expression of collagen XVII and γ2 chain of laminin-332 were upregulated in the basal hyperplastic tissue of benign papillomas and in the individual
invasive cells of malignant SCCs similarly to the human samples. The signals for
both collagen XVII and laminin γ2 in the basement membrane zone were also much
stronger in all papilloma and carcinoma samples compared to normal skin samples.
To analyze whether the expression of collagen XVII, laminin γ2 and β4 integrin
was also increased in human SCC cell lines, we determined their relative expression
levels in UT-SCC-105, SCC-15, SCC-25, and HSC-3 cell lines by qRT-PCR.
Collagen XVII, laminin γ2 and β4 integrin were overexpressed in all SCC cells
compared to HaCaT keratinocytes. Collagen XVII expression was at its highest
(5,13-fold greater than in HaCaT keratinocytes) in SCC-15 cells and also elevated
in other SCC cell lines. Upregulated expression of laminin γ2 and β4 integrin was
also detected in all SCC cell lines. Moreover, an increase of collagen XVII
expression was found at the protein level.
5.2.3 Collagen XVII expression is associated with TNM and
metastasis survival times in CRC patients (II)
The upregulation of collagen XVII in CRC led to further investigation of the
correlation between clinicopathological variables in CRC patients and collagen
XVII. Analyses of the percentage of tumor cells with positive collagen XVII
staining relative to these variables revealed significant association between
increased collagen XVII expression and higher TNM stage. In particular, a higher
percentage of collagen XVII positive cells correlated with lymph node and distant
metastasis. In addition, an infiltrative growth pattern and tumor budding were
significantly associated with increased collagen XVII expression. No statistically
significant associations were found between collagen XVII staining and patient age,
gender, tumor location, WHO grade or preoperative treatments. According to the
Kaplan-Meier curve a high percentage of collagen XVII positive cancer cells
associates both with decreased disease-free survival (DFS) and cancer-specific
survival (CSS). Nevertheless, Cox regression analysis did not indicate an
independent prognostic value for collagen XVII expression.
48
5.2.4 Collagen XVII participates in adhesion, migration and invasion
(II-III)
To analyze the functional role of collagen XVII and integrin β4 in SCC, we created
KD cell lines to see how the absence of these proteins affects the adhesion,
migration and invasion of SCC cells. Both SCC-25 and HSC-3 cell lines were used
for collagen XVII KD but only SCC-25 for integrin β4 KD. The KD efficiency
varied from 78% (collagen XVII KD in SCC-25 cells) to 91% (β4 integrin KD in
SCC-25 cells). The adhesive capabilities of the cell lines were analyzed using the
ECIS analysis with either collagen XVII or integrin β4 SCC-25 KD cells. The
ability of integrin β4 SCC-25 KD cells to attach was decreased by 23% and the
ability of collagen XVII SCC-25 KD cells by 10% compared to the control cells.
KD did not affect HSC-3 cells.
The wounding assay revealed that both SCC-25 KD cell lines seemed to have
lost their ability to migrate. The KD cells were stationary and rotating; no migration
was detected. Similarly to the adhesion analysis, there was no difference in
migration between the HSC-3 control and KD cells.
The effect of KD on the ability to invade was studied using organotypic myoma
model. IHC of myoma samples using pancytokeratin antibody showed that SCC25 control cell lines invaded through the whole myoma section. SCC-25 cells
lacking either β4 integrin or collagen XVII did not invade to the myoma tissue at
all. However, the KD of collagen XVII did not affect the invasion of HSC-3 cells.
Since the expression of level of collagen XVII is normally very low in CaCo2 cells, the function of collagen XVII was studied creating CaCo-2 cells
overexpressing collagen XVII. The expression of cull-length murine collagen XVII
in the CaCo-2 cell line promoted migration of these cells in the MatrigelTM assay.
The overexpression of collagen XVII led to a 2.2-fold more effective migration in
CaCo-2-cells.
49
50
6
Discussion
6.1
Collagen XVII is a newly characterised component of the
glomerulus and affects glomerular development
Although previous studies have demonstrated collagen XVII expression in various
other tissues (Kondo et al. 1998, Aho & Uitto 1999, Van den Bergh & Giudice 2003,
Claudepierre et al. 2005, Seppanen et al. 2006) our study was the first to
demonstrate collagen XVII expression in the podocytes of the human glomerulus
and that collagen XVII is very likely needed for normal kidney development. Firstly,
the glomeruli of mutant mice lacking collagen XVII were immature and small in
size and the glomerular content of their kidneys was increased. Secondly, mutant
mice had podocyte FP effacement, which is the central cytopathological feature of
glomerular injury in the vast majority of human glomerulopathies and animal
models of glomerular diseases (D'Agati 2008, Patrakka & Tryggvason 2010). SDs
of the mutant mice were mostly normal, suggesting that collagen XVII is not an
actual component of SDs.
To understand the function of collagen XVII in the glomerulus at a molecular
level, it would be crucial to know which proteins it interacts with. However the
binding partners of collagen XVII in the glomerulus are not yet clear. In tissues
where collagen XVII functions as a component of type I HD the main intracellular
binding partners of collagen XVII are BP230, plectin and the integrin β4 subunit,
whereas the extracellular part of collagen XVII interacts with laminin 332 and the
integrin α6 subunit (Van den Bergh & Giudice 2003, Aumailley et al. 2006, Mihai
& Sitaru 2007, Nishie et al. 2011). Our hypothesis is that in the glomerulus,
collagen XVII binds components of FPs and/or the GBM, and disturbances in these
interactions result in the deficient glomerular development, podocyte effacement
and GBM splitting detected in mutant mice. Based on collagen XVII binding
partners in skin, integrins could be also be a rational possibility for collagen XVII
interaction in the kidney. Podocytes can express both α3β1- and β4-integrins
(Adler 1992, Kambham et al. 2000, Patrakka & Tryggvason 2010). Although the
exact localization and function of β4 integrin in the glomerular filtration unit is not
known, the cytoplasmic tail of β4 integrin might also be a ligand for collagen XVII
in podocytes. In keratinocytes collagen XVII also associates intracellularly with an
adherens junction protein, p120 catenin (Aho & Uitto 1999) and a microfilament
associated protein, α actinin-4 (Gonzalez et al. 2001). Both these proteins are
51
expressed in podocytes and could interact as intracellular binding partners for
collagen XVII (Michaud & Kennedy 2007). In contrast, the extracellular partners
of collagen XVII in the glomerulus are most probably different from those in the
dermo-epidermal junction, since laminin 332 is not expressed in healthy kidneys
and the integrin α6 subunit is expressed in the epithelial lining of healthy and
damaged tubules, but not in the glomerulus (Joly et al. 2006, Hahm et al. 2007).
Our finding that the expression and localization of both type IV collagen and
laminin α5 chain were unchanged in the GBM of mutant mice suggests that there
is no direct association between collagen XVII and these major GBM components.
The function of collagen XVII in podocytes is therefore still uncertain although our
findings suggest a possible role of collagen XVII in establishing cell-cell or cellGBM interactions of podocytes.
Interestingly, there is only one recent paper about the association between renal
lesions and BP (Hoorn et al. 2015) and no systematic analysis of kidney
involvement in this disease has yet been performed (Ross & Ahmed 1989, Plaisier
et al. 2002). Pemphigoid patients are treated with effective immunosuppressive
drugs for skin blistering, which may prevent the emergence of renal symptoms,
such as proteinuria. Based on data from the National EB Registry, renal failure may
occur in junctional EB, though mainly for secondary reasons (Fine et al. 2004,
Almaani & Mellerio 2010). Hereditary nephritis has been found in junctional EB
with pyloric atresia caused by mutations in the integrin β4 gene, although only a
minority of patients with integrin β4 mutations have renal iinvolvement (Kambham
et al. 2000, Dang et al. 2008, Almaani & Mellerio 2010). Integrin β4 null mice die
immediately after birth due to denuding of the skin (Dowling et al. 1996). The renal
consequences of integrin β4 knockout in mice have not been analysed. In the future,
the kidney function of the carriers of different type of collagen XVII mutations
must be carefully studied, since renal involvement in these cases has not yet been
analyzed in detail. However, it is also possible that patients with congenital or
autoimmune collagen XVII related skin diseases do not present any major renal
difficulties because human collagen XVII is more critical to cutaneous adhesion
than the integrity of the glomerular filtration barrier.
6.2
Collagen XVII is expressed in the normal colon epithelium and
may interact with laminin 332
We showed that collagen XVII is expressed in the colon epithelia but its role in
normal colonic epithelium is unclear. As does the kidney, the colon lacks type I
52
HDs meaning collagen XVII must have a different role in the colon than in the skin
(Fontao et al. 1999, Margadant et al. 2008). Our study demonstrated that the
expression of collagen XVII was distributed from the bottom of the crypt axis to
the tip of the normal surface colon epithelium, and thus resembled the localization
of the laminin γ2 chain and α6β4 integrin (Beaulieu 2010, Sordat et al. 1998). We
also demonstrated partial co-localization with laminin γ2. The co-localization of
collagen XVII and laminin 332 suggests a functional interaction between these
proteins in the colon epithelium.
Patients with junctional epidermolysis bullosa lacking collagen have been
found to have lower gastrointestinal tract complications such as constipation,
chronic diarrhea, anal fissures or rectal bleeding (Fine et al. 2008). These symptoms
may be explained by the fact that the epithelium of the colon is subject to milder
mechanical forces than the skin and the epithelial-mesenchymal interface of the
anorectal mucosa is very similar to that of the skin.
Based on these data it is evident that collagen XVII also has a role in colonic
epithelia. Since collagen XVII is not thought to be a component of intestinal type
II HD, its possible importance in attachment/detachment and migration could come
about via its interaction with laminin 332.
6.3
Collagen XVII is expressed in CRC and its expression
correlates with tumor progression and metastasis in CRC
The involvement of collagen XVII in CRC has not previously been described. In
our study we found out that the expression of collagen XVII in TMA patient
samples was both membranous and intracellular, and the staining was more
intensive than in normal mucosa. As in normal epithelia, partial co-localization of
collagen XVII and laminin γ2 was also detected in tumor tissue which together with
mainly extracellular localization could suggests a functional interaction between
these proteins in CRC. There were also similarities to the collagen XVII expression
in SCC since collagen XVII expression was more abundant in the tumor tissue than
in normal epithelia and there was a great deal of variation in the expression intensity
of collagen XVII within the tumor tissue (Parikka et al. 2003, Parikka et al. 2006,
Stelkovics et al. 2008, Yamada et al. 1996).
There are no previous data about the functional role of collagen XVII in CRC.
The results with TMA patient samples revealed significant correlation between
increased collagen XVII expression and advanced TNM stage and more aggressive
tumor behavior. Higher collagen XVII expression also correlates with tumor
53
budding and an infiltrative growth pattern. These are acknowledged histologic
features associated with poor outcome and are predictive of lymphovascular
invasion and lymph node involvement in CRC (Sagaert 2014). Interestingly, and
similarly to the laminin γ2 chain, increased collagen XVII immunostaining was
associated with both lymph node and distant metastasis in CRC (Shinto et al. 2005).
These similarities further strengthen our suggestion of collagen XVII and laminin
γ2 functional interaction. High expression of the laminin γ2 chain is regarded as an
independent prognostic factor in CRC (Guess & Quaranta 2009, Shinto et al. 2005).
However multivariate analysis revealed no independent significance of the
increased expression of collagen XVII, although collagen XVII positively
correlated with decreased DFS and CSS in univariate analyses. Similar to our
findings of the increased collagen XVII expression correlation to the metastasis,
another recent study had similar results in lung adenocarcinoma, where increased
collagen XVII expression had a significant correlation with brain metastasis
(Fabian et al. 2014). Our findings of increased invasion in collagen XVIIoverexpressing CaCo-2 cells strongly supports the suggestion that collagen XVII
may have an important role in CRC pathogenesis and indicates for the first time
that collagen XVII influences the migration and invasion of cancer cells derived
from non-stratified epithelia as well.
6.4
Collagen XVII, integrin α6β4 and laminin 332 are upregulated in
SCC
The upregulated expression of collagen XVII, laminin 332 and α6β4 integrin in
SCC has been reported previously (Hamasaki et al. 2011, Miyazaki 2006). Our
study was the first to quantitate and compare the expression of these binding
partners in actinic keratosis, Bowen’s disease and SCC tumours. Laminin γ2
expression increased linearly from normal skin through pre-malignant and
carcinoma in situ lesions to invasive SCC samples, which is in line with the findings
of Hamasaki and co-workers (2011) who found that the expression of laminin γ2
was higher in SCC than in Bowen’s disease (Hamasaki et al. 2011). The expression
patterns of collagen XVII and integrin β4 were alike but differed from that of
laminin γ2. In actinic keratosis and Bowen’s disease the expression of collagen
XVII and integrin β4 was up-regulated, but, surprisingly, their expression in
Bowen’s disease was higher than in SCC samples. Previous studies have
demonstrated higher expression for both collagen XVII and integrin β4 in SCC
compared to actinic keratosis or carcinoma in situ samples (Stelkovics et al. 2008,
54
Kashyap et al. 2011). However, the methods used in those studies differed from
ours. Stelkovics et al. used a different scoring system from ours and in the study by
Kashyap et al. immunostaining was performed with a phospho-specific antibody
(Stelkovics et al. 2008, Kashyap et al. 2011). It also has to be noted that as in CRC
samples, there was vast variation of the expression intensity within the tumor tissue,
both in Bowen’s disease and especially in SCC samples. We also observed that the
distribution pattern of laminin γ2 does not correspond with either collagen XVII or
integrin β4 in SCC samples which differs from what is seen in CRC. However, the
immunolocalization of collagen XVII and integrin β4 was very similar suggesting
that they interact with each other in SCC.
The elevated expression of collagen XVII and laminin γ2 in DMBA-TPA
induced cutaneous papillomas and carcinomas supports our findings in human
samples, and suggests that collagen XVII and laminin 332 are involved in both UVlight- and chemically- induced skin carcinogenesis. The expression of collagen
XVII, laminin γ2 and integrin β4 were also clearly elevated in all investigated SCC
cell lines which was to be expected based on the findings of the aforementioned
previous studies.
6.5
Collagen XVII and integrin α6β4 affect the adhesion, migration
and invasion of SCC-25 cells but has no effect on HSC-3 cells
As previously shown, the suppression of laminin γ2 by siRNA significantly inhibits
A431 SCC cell invasion (Hamasaki et al. 2011). This prompted us to investigate
the effect of collagen XVII and integrin β4 KD by virus-mediated RNAi on the
behavior of SCC cells. The deficiency of collagen XVII or integrin β4 markedly
reduced the adhesion of SCC-25 cells, athough it was to be expected as they both
are important components of type I HDs. Thus, the consequences of collagen XVII
or integrin β4 depletion on the adhesion of SCC-25 cells are comparable to those
on keratinocytes, indicating that they also act as adhesion molecules on the surface
of malignant cells (Qiao et al. 2009, Hamill et al. 2011, Loffek et al. 2014).
Previous observations of how the lack of collagen XVII changes keratinocyte
migration are controversial: Keratinocytes derived from human or murine genetic
models show increased, but undirected motility, whereas keratinocytes with viral
collagen XVII KD have reduced migratory ability (Qiao et al. 2009, Hamill et al.
2011, Loffek et al. 2014). We observed that collagen XVII KD SCC-25 cells
exhibited stationary rotation and markedly delayed migration in the scratch wound
healing assay. Thus the effect of viral collagen XVII KD is similar in keratinocytes
55
and in SCC-25 cells. The conflicting results of previous studies may have been due
to different compensatory mechanisms of genetic or viral systems (Loffek et al.
2014) or perhaps total or partial depletion of collagen XVII has a different impact
on the migration of epithelial cells. SCC-25 cells lacking integrin β4 behaved the
same way as collagen XVII KD SCC-25 cells.
The invasion of SCC cells with collagen XVII and integrin β4 KD was
analyzed using an organotypic assay, which is based on the use of human myoma
tissue and mimics the tumor microenvironment better than collagen organotypic
models (Nurmenniemi et al. 2009, Vered et al. 2015). The effect of collagen XVII
or integrin β4 depletion on the invasion of SCC-25 cells was very clear: Instead of
invading to the myoma tissue as the control SCC-25 cells did, KD SCC-25 cells
remained proliferating at the surface of the myoma disc.
However, the results with the HSC-3 cells lacking collagen XVII were quite
the opposite compared to the SCC-25 cells. KD did not have any effect on adhesion,
migration or invasion of the very aggressive HSC-3 cells. The fact that lack of
collagen XVII does not affect aggressive HSC-3 cells but disturbs severly functions
of less aggressive SCC-25 cells, combined with our findings with IHC stainings of
TMA tumor samples, suggests that collagen XVII is involved in the early
development of malignant transformation, but it is not required anymore in highly
invasive stages of SCC. Interestingly, even though there are many similarities in
collagen XVII expression in CRC and SCC as our results clearly demonstrate, the
association of collagen XVII expression with histopathological grades, tumor
invasion or metastases in SCC has not been addressed so far.
These functional results together with expression and localization results of
collagen XVII, integrin β4 and laminin γ2, suggest that increased expression of
collagen XVII may be involved in the migration and invasion of cancer cells, and
strengthens the already strong data regarding integrin β4 and collagen XVII
participation and interaction especially in the early stages of SCC carcinogenesis.
The mechanisms via which collagen XVII and α6β4 integrin as well as their
binding partner laminin 332 participate in migration and invasion have been studied
previously in multiple studies (Borradori & Sonnenberg 1999, Franzke et al. 2003,
Franzke et al. 2005, Walko et al. 2015). It seems that the understanding of the
phosphorylation of β4 integrin would clarify the mechanisms of the attachment,
migration and invasion of SCC cells, and the role of collagen XVII is linked to this
phosphorylation process. On one hand, the absence of collagen XVII favours the
phosphorylation of the β4 integrin intracellular domain which leads to enhanced
HD disassembly and therefore to the activation of migration and invasion. But on
56
the other hand, since migration and invasion are dependant on both the creation and
disassembly of adhesive components, the total loss of collagen XVII leads to
undirected migration and noninvasive SCC cells (Kashyap & Rabinovitz 2012,
Loffek et al. 2014). Only the presence of α6β4 integrin together with collagen
XVII/BP230 and the actin cytosleleton together with laminin 332 can lead to
directed cell migration (Tsuruta et al. 2011) which is in line with our findings.
Table 4 summarizes the known expression, distribution and function of
collagen XVII in normal human tissues and collagen XVII-related pathological
conditions. Based on the current knowledge it appears clear that collagen XVII has
functional similarities regardless of its expression site and its main role seems to be
connected to cell attachment/detachment and migration together with laminin 332
and integrin α6β4.
57
Table 4. Distribution and function of collagen XVII in human.
Site of expression Localization in tissue
Function in normal tissue
Related pathological
conditions
Skin
Basal keratinocytes, type I HD Attaches keratinocytes to the
BM and participates in
EB, BP, PG,LAD,
MMP, skin
keratinocyte migration (Powell carcinomas (Powell et
Buccal mucosa
Oral keratinocytes
et al. 2005)
al. 2005)
Attaches keratinocytes to the
Similar to those of the
BM similarly to the skin
skin (Ziober et al.
2001)
Colon/intestine
Kidney
Epithelial cells similarly to
Unclear, possibly functional
laminin 332
interaction with laminin 332
Podocyte FPs
Unclear, expressed by
CRC
N/A
podocytes and needed for
normal glomerular
development
Bladder
Epithelial cells
N/A
Amnionic
Syncytial trophoblasts and
Participation in the migration of N/A
membrane and
cytotrophoblasts
invasive trophoblasts (Huilaja
placenta
Nervous system
N/A
et al. 2008)
Soma and proximal axons of
Participation in neuronal
neurons
migration suggested
Müller cells
Functional interaction with
N/A
(Seppanen et al. 2006)
Cornea,
conjunctiva and
retina
Heart
N/A
laminin 332 suggested
(Claudepierre et al. 2005)
N/A
N/A, though similarly to the
N/A
skin attachment as a
component of type I HD
suggested (Kondo et al. 1998).
Abbreviations: HD: hemidesmosome; BM: basement membrane; EB: epidermolysis bullosa; PG;
pemphigoid gestationis; LAD: linear immunoglobulin A disease; MMP: mucous membrane pemphogoid;
CRC: colorectal carcinoma; FP: foot process.
58
7
Conclusions
This study expands the knowledge of the extracutaneous function of collagen XVII
and the role of collagen XVII and integrin α6β4 in epithelial cancers. It is tempting
to speculate whether in the future these findings will lead to the more careful
analyzing of the kidney function and potential defects in kidneys in patients with
collagen XVII deficiency. To speculate further it will be interesting to see whether
in the future controlling the amount of collagen XVII (soluble or membrane-bound)
could be used to control the migration and invasion of early stage SCC or CRC in
patients.
Regardless of the speculations, based on our results we were able to make
following conclusions:
1.
2.
3.
4.
5.
Collagen XVII is a newly characterised component of the glomerulus
localizing in the podocyte FPs.
Specific deletion of collagen XVII in a gene-targeted murine model results in
severe kidney abnormalities.
Collagen XVII is expressed in normal the colon epithelium and there may be a
functional interaction between collagen XVII and laminin 332 in CRC.
The level of collagen XVII expression strongly correlates with tumor
progression and metastasis in CRC.
Collagen XVII and integrin α6β4 have a crucial role in the adhesion, migration
and invasion of SCC cells and there is likely a functional interaction between
collagen XVII and integrin α6β4 in SCC.
59
60
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Original articles
IV Hurskainen T, Moilanen J, Sormunen R, Franzke CW, Soininen R, Löffek S, Huilaja L,
Nuutinen M, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen K (2012)
Transmembrane collagen XVII is a novel component of the glomerular filtration barrier.
Cell Tissue Res 348(3):579-88.
V Moilanen J, Kokkonen N, Löffek S, Väyrynen JP, Syväniemi E, Hurskainen T, Mäkinen
MJ, Klintrup K, Mäkelä J, Sormunen R, Bruckner-Tuderman L, Autio-Harmainen H,
Tasanen K (2015) Collagen XVII correlates with the invasion and metastasis of
colorectal cancer. Hum Pathol. 46(3):434-42.
VI Moilanen J, Löffek S, Kokkonen N, Salo S, Väyrynen JP, Hurskainen T, Manninen A,
Riihilä P, Heljäsvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen MJ, Tasanen K
Collagen XVII and integrin β4 show similar expression in squamous cell carcinoma and
their knockdown suppresses migration and invasion only of the less aggressive
squamous cell carcinoma cells. Manuscript.
Reprinted with the permissions from Springer Science and Elsevier Science.
Original publications are not included in the electronic version of the dissertation.
71
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Jyri Moilanen
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Jyri Moilanen
Professor Esa Hohtola
FUNCTIONAL ANALYSIS
OF COLLAGEN XVII IN
EPITHELIAL CANCERS AND
A MOUSE MODEL
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
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