Functional genomics of a three-dimensional epithelial cell culture and coeliac small intestinal mucosal biopsy samples

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Functional Genomics of a Three-Dimensional Epithelial Cell Culture and Coeliac Small Intestinal

Mucosal Biopsy Samples

ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine of the University of Tampere, for public discussion in the Auditorium of Finn-Medi 1,

Biokatu 6, Tampere, on June 13th, 2008, at 12 o’clock.

KATI JUUTI-UUSITALO

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Distribution Bookshop TAJU P.O. Box 617

33014 University of Tampere Finland

Cover design by Juha Siro

Acta Universitatis Tamperensis 1319 ISBN 978-951-44-7340-1 (print)

Tel. +358 3 3551 6055 Fax +358 3 3551 7685 taju@uta.fi

www.uta.fi/taju http://granum.uta.fi

Acta Electronica Universitatis Tamperensis 730 ISBN 978-951-44-7341-8 (pdf )

ACADEMIC DISSERTATION

University of Tampere, Institute of Medical Technology Tampere University Hospital, Department of Paediatrics

Tampere Graduate School in Biomedicine and Biotechnology (TGSBB) Finland

Supervised by

Professor Markku Mäki University of Tampere Professor Heikki Kainulainen University of Jyväskylä

Reviewed by

Professor Markku Heikinheimo University of Helsinki

Professor Erkki Savilahti University of Helsinki

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Meno:“ How will you look for it, Socrates, when you do not know at all what it is?

How will you aim to search for something you do not know at all?

If you should meet with it, how will you know that this is the thing that you did not know?”

Meno’s paradox, 80d (Plato 1997) "Miten sinä muka aiot tutkia sellaista, mistä et edes tiedä mitä se on?

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ABSTRACT

The small intestinal epithelium has a highly organised structure with well- differentiated secretory and absorptive properties. Epithelial cells undergo rapid turnover, and are therefore closely regulated to maintain homeostasis between proliferation, differentiation and apoptosis. The regulation of these processes affects transforming growth factor β (TGFβ) and wingless and receptor tyrosine kinase pathways. The T84 three-dimensional epithelial differentiation model, developed in the coeliac disease study group, is has been shown to mimic the epithelial cell differentiation of intestinal crypt-villus axis. The molecular events which synergistically regulate the cellular differentiation and development in T84 epithelial cells await for further studies.

In coeliac disease gluten induces an immunological reaction in genetically susceptible subjects resulting in villus atrophy and crypt hyperplasia. Though CD4+ specific T-cells, intraepithelial lymphocytes, infiltrating plasma cells and autoantibodies are known to affect the pathogenesis of coeliac disease, the mechanisms responsible for remodelling the small intestinal mucosa, leading to tissue injury, increased epithelial cell proliferation and failure in differentiation, have remained ambiguous.

The present study has three sections. The aim of the first section was to characterise genes whose transcription is affected in the differentiation of epithelial cells in three-dimensional epithelial cell differentiation model. In the second section the aim was to find gene transcripts which might have a role in coeliac disease pathogenesis. In the third section the genes which operate similarly in epithelial proliferation and differentiation in the epithelial cell differentiation model and in coeliac patient small intestinal mucosal biopsy samples were sought to by combining the data from a two previous sections.

All the gene expression studies were performed using a complementary strand of deoxyribonucleic acid (cDNA) filter microarray. The expression of some selected genes was also defined at protein level.

The first section revealed that epithelial cell differentiation altered the expression of 372 genes, and 47 of them were affected by both TGFβ1 and soluble factors such as TGFβ1 secreted from fibroblasts. The major change upon differentiation of epithelial cells was a diminishing expression of most the significantly affected

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In the second part the expression of 156 genes was found to be altered when untreated coeliac disease patients were compared to healthy controls, and 60 genes were affected when treated coeliac disease patients were compared to healthy controls. Altogether 98 genes evinced altered expression when treated and untreated coeliac disease patients were compared. The expression of several inflammatory mediators was increased in the untreated coeliac patient biopsies.

None of the genes on the filter of the coeliac predisposition regions (5q30-33, 2q31-33 and 15q11-13) were affected. There were eight genes which were similarly affected in both untreated and treated coeliac patients compared to healthy controls. In addition, there was only one gene, the distal-less homeobox 4 (DLX4), exhibiting increased gene expression in all comparisons.

In the third section, the cDNA microarray data originating from the epithelial differentiation model and from the small intestinal mucosal biopsy samples were combined, and 30 genes evinced similar expression. Removal of gluten from the diet induced reversed expression of 29 genes. Nine out of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Further characterisation by blotting and labelling revealed increased epidermal growth factor receptor (EGFR),β-catenin and Wiskott-Aldrich syndrome protein family member 1 (WAVE1) protein expression in the small intestinal mucosal epithelium of untreated coeliac disease patients compared to healthy controls and treated patients.

It can be concluded that in the three-dimensional epithelial cell differentiation model TGFβ1 appears to be a more potent differentiating agent than the fibroblast secreted soluble factors. TGFβ1 seems to modulate, either directly or indirectly, the transcription of members on the wingless and receptor tyrosine kinase signalling pathways such as EGFR and β-catenin. Many of the genes found to be affected in the coeliac disease patient small intestinal mucosal biopsies are directly or indirectly connected to inflammatory reaction or epithelial cell differentiation. Some of these genes might be of importance in the pathogenesis of coeliac disease. The results of the current study suggest that by combining gene expression data from the three-dimensional epithelial cell culture model with small intestinal mucosal biopsies it is possible to focus on the changes originating from epithelial cells. Furthermore, the results would suggest that the epidermal growth factor (EGF)-mediated signalling pathway is likely to be involved in the epithelial cell differentiation and coeliac disease pathogenesis.

These findings indicate that the epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.

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TIIVISTELMÄ

Ohutsuolen epiteeli muodostuu useista erityyppisistä soluista, jotka syntyvät kryptoissa olevista kantasoluista. Vaeltaessaan kohti villuksen kärkeä epiteelisolut erilaistuvat ja muuttuvat erittävistä epiteelisoluista absorptiivisiksi enterosyyteiksi. Ohutsuolen villuksen solut elävät vain noin viisi päivää. Solujen lyhyt elinkaari ja jatkuva uusien erilaistuneiden epiteelisolujen tarve asettavat tiukat vaatimukset jakaantumisen, erilaistumisen sekä apoptoosin säätelylle.

Solunsisäiset signalointireitit, kuten transformoiva kasvutekijä beta- (TGFβ), wingless- sekä reseptori tyrosiini kinaasi-reitti säätelevät solujen jakaantumista ja erilaistumista. Tampereen yliopiston Keliakiaryhmässä kehitetyn kolmiulotteisen soluviljelymallin epiteelisolujen erilaistumisen on osoitettu muistuttavan ohutsuolen krypta-villusakselilla tapahtuvaa solujen erilaistumista.

Signalointireittien toimintaa on kartoitettu monin eri tutkimuksin, mutta edelleenkään ei tiedetä tarkasti, kuinka nämä reitit toimivat erilaistuvissa T84 epiteelisoluviljelmässä.

Keliakiassa, geneettisen alttiuden omaavien henkilöiden ohutsuolen limakalvolla vehnän gliadiini, ja vastaavat ohran ja rukiin varastoproteiinit aiheuttavat tulehdusreaktion, jonka vaikutuksesta suolinukka lyhenee (villus atrofia) ja kryptat pidentyvät (krypta hyperplasia). Jakautuvien epiteelisolujen määrän on osoitettu olevan suurempi keliakiaiaa sairastavan kuin terveen verrokin suolessa.

Lisäksi keliakiaa sairastavan henkilön epiteelisolujen erilaistuminen on häiriintynyt, ja apoptoosi kiihtynyt. Vaikka tulehdussolujen tiedetään ottavan osaa keliakiassa ilmenevän suolivaurion syntyyn, silti limakalvovaurion syntymekanismi on edelleenkin epäselvä.

Tässä tutkimuksessa geenien ilmentymisessä tapahtuvia muutoksia tutkittiin komplementaarisella deoksiribonukleiniinihappo (cDNA) filtterimicroarray- tekniikan avulla. Muutamien valikoitujen geenien ilmentymistä tutkittiin myös proteiinitasolla.

Ensimmäisessä osassa geenien ilmentymistä tutkittiin suolen krypta-villusakselia mimikoivalla kolmiulotteisella soluviljelmällä. Tarkoituksena oli selvittää kuinka TGFβ:lla sekä fibroblastien liukoisilla tekijöillä erilaistettujen epiteelisolujen geenien ilmentyminen on vaikuttunut verrattuna erilaistumattomiin epiteelisoluihin. Toisessa osassa tutkittiin geenien ilmentymistä hoitamattomien

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selvittää, mitkä havaituista gluteenin aiheuttamista muutoksista geenien ilmentymisessä ja solujen toiminnassa johtuvat häiriöstä epiteelisolujen erilaistumisessa.

Kolmiulotteisessa soluviljelmässä kaikkiaan 372 geeniä muutti ilmentymistään solujen erilaistuessa, ja 47 näistä geeneistä oli muuttanut ilmentymistään samankaltaisesti sekä TGFβ:lla että fibroblastien liukoisilla tekijöillä erilaistuneissa soluissa. Tulokset osoittivat, että TGFβ:n aiheuttamat geenien ilmentymisen muutokset olivat suurempia kuin fibroblastien liukoisten tekijöiden aiheuttamat muutokset. TGFβ:lla erilaistaminen sai aikaan muutoksia myös wingless- ja reseptori tyrosiini kinaasi-signaalireittien jäsenten ilmentymisessä.

Potilasnäytteistä saadusta aineistosta löytyi 156 ilmentymistään muuttanutta geeniä, kun hoitamattomia keliakiapotilaita verrattiin terveisiin verrokkeihin ja 60 geeniä kun hoidettuja keliakiapotilaita verrattiin terveisiin verrokkeihin. Kun taas hoitamattomia keliakiapotilaita verrattiin hoidettuihin keliakiapotilaisiin oli 98 geeniä muuttanut ilmentymistään. Useiden tulehdusvälittäjäaineiden ilmentyminen oli lisääntynyt hoitamattomilla keliakiapotilailla kun näitä varrattiin hoidettuihin keliakiapotilaisiin ja terveisiin verrokkeihin. Yksikään keliakian geneettisen tautialttiuden alueille (5q30-33, 2q31-33 and 15q11-13) sijoittuvista geeneistä ei ollut merkittävästi muuttanut ilmentymistään.

Tutkimuksessa löytyi kahdeksan geeniä, joiden ilmentyminen oli samalla tavalla vaikuttunut, kun sekä hoitamattomia että hoidettuja keliakiapotilaita verrattiin terveisiin verrokkeihin. Lisäksi löytyi vain yksi geeni, distal-less homeobox 4 (DLX4), jonka ilmentyminen oli merkittävästi koholla kaikissa vertailtavissa ryhmissä.

Yhdistämällä biopsioista saadut tulokset soluviljelmästä saatuihin tuloksiin löydettiin 30 samansuuntaisesti muuttunutta geeniä. Gluteenittomalla dieetillä näistä 30:stä geenistä 29:n geenin ilmentyminen palautui. Yhdeksän geeniä näistä 30:stä on suorasti tai epäsuorasti reseptori tyrosiinikinaasin (RTK) säätelemällä reitillä. Lisätutkimukset osoittivat, että epidermaalisen kasvutekijäreseptorin, (EGFR), β-kateniinin ja Wiskott-Aldrich syndrome protein family member 1 (WAVE1) proteiinin ilmentyminen oli myös proteiinitasolla koholla, kun hoitamattomia keliakiapotilaita verrattiin terveisiin verrokkeihin.

TGFβ näyttäisi erilaistavan epiteelisoluja tehokkaammin kuin fibroblastien liukoiset tekijät, lisäksi TGFβ näyttäisivaikuttaa joko suorasti tai epäsuorasti muihin signalointireitteihin. Potilasnäytteiden tulokset viittasivat siihen, että monet keliakiapotilailla ilmentyvät geenit osallistuvat tulehdusreaktion syntyyn sekä vaikuttavat epiteelisolujen erilaistumiseen. Jotkut näistä geeneistä saattavat olla tärkeitä keliakian patogeneesissä. Vertaamalla soluviljelmästä saatuja tuloksia potilasaineistosta saatuihin tuloksiin, gluteeni näyttäisi aiheuttavan muutoksia epiteelisoluissa epidermaalisen kasvutekijän aloittamalla signalointireitillä. Tämä tutkimus osoitti, että kolmiulotteinen soluviljelymalli on toimiva työkalu ohutsuolen krypta-villus-akselilla tapahtuvien muutosten

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original publications, which will be referred to by their Roman numerals I-III:

I Juuti-Uusitalo KM, Kaukinen K, Mäki M, Tuimala J, Kainulainen H. (2006). Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model. BMC Genomics. Oct 31;7:279. (BMC Journals)

II Juuti-Uusitalo K, Mäki M, Kaukinen K, Collin P, Visakorpi T, Vihinen M, Kainulainen H. (2004). cDNA microarray analysis of gene expression in coeliac disease jejunal biopsy samples. J Autoimmun.

May;22(3):249-65. (Reprinted with permission of Elsevier Limited)

III Juuti-Uusitalo K, Mäki M, Kainulainen H, Isola J, Kaukinen K.

(2007). Gluten affects epithelial differentiation-associated genes in small intestinal mucosa of coeliac patients. Clin Exp Immunol. Nov;150(2):294-305.

(Reprinted with permission of Wiley-Blackwell) In addition, previously unpublished data are presented.

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ABBREVIATIONS

ADAMTS3 Disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 3

AJ Adherens junction

AJC Apical junctional complex

Akt V-akt murine thymoma viral oncogene homolog, a serine/threonine kinase APC Adenomatous polyposis coli

aPKC Atypical protein kinase C

AR Amphiregulin

ARNT2 Aryl-hydrocarbon receptor nuclear translocator 2 ATCC American type culture collection

BHMT2 Betaine-homocysteine methyltransferase 2

BSA Bovine serum albumin

BTC Betacellulin

Caco-2 Adenocarcinoma cell line

CASK/Lin2 Calcium/calmodulin-dependent serine protein kinase CD44 Cluster of differentiation, surface glycoprotein CD44 Cdc42 Cell division cycle 42 (GTP binding protein, 25kDa) cdk4 Cyclin-dependent kinase 4

cDNA Complementary strand of deoxyribonucleic acid CREB cAMP responsive element binding protein 1 CREBBP CREB binding protein

CRYAB Crystallin, alpha B CTNNB1 β-catenin gene D123 D123 gene product

DAB Diaminobenzidine

DC Dendritic cell

DGKD Diacylglycerol kinase, delta 130kD DIO Deiodinase, iodothyronine, type II DLX Distal-less homeobox

DPAGT1 Dolichyl-phosphate(UDP-N-acetylglucosamine)N-

acetylglucosaminephosphotransferase 1 (GlcNAc-1-P transferase) DRPLA Dentatorubral-pallidoluysian atrophy (atrophin-1)

ECM Extracellular matrix EGF Epidermal growth factor

EGFR Epidermal growth factor receptor, ErbB1

EGFR-RS Likely ortholog of mouse epidermal growth factor receptor, related sequence

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EPR Epiregulin

ErbB Epidermal growth factor receptor

Erk Extracellular signal-regulated kinase (Erk1 and -2)

EST Expressed sequence tag, a sequence known to be transcripbed to mRNA, having no known product

FGFR Fibroblast growth factor receptor FTS Fused toes homolog (mouse)

FXR2 Fragile X mental retardation, autosomal homolog 2 Fzd Frizzled, receptor in the wingless signalling pathway GAPDH Glyceraldehyde-3-phosphate dehydrogenase GM2 GM2 ganglioside activator protein

GO Gene ontology

GSK3β Glycogen-synthase kinase 3β

HB-EGF Heparin binding-EGF-like growth factor HEMBA1005314, Homo sapiens cDNA FLJ11723 fis HLA Human leucocyte antigen

IEL Intraepithelial lymphocyte

IFNγ Interferon gamma, a proinflammatory cytokine IGFR Insulin-like growth factor

IL Interleukin (for example IL-2,-4, -15, -18 etc) IL-10 Interleukin-10

IMR-90 Human mesenchymal cell line JAK Janus like-kinase

JAM Junctional adhesion molecule (JAM-A, JAM-B) KBRAS I-kappa-B-interacting Ras-like protein 2 KTR19 Keratin 19, cytokeratin 19

LEF Lymphoid enhancer

LOWESS Locally weighted linear regression LoVo Colorectal adenocarcinoma cell line

LRP LDL-receptor-related protein 5/6 or Arrow, a frizzled co-receptor LY6G5B Lymphocyte antigen 6 complex, locus G5B

MAG-1 Membrane-associated guanylate kinase with inverted domain structure 1 MAPK Mitogen-activated protein kinase

MgCl Magnesium chloride

MIC Non-classical MHC class I molecule MMP Matrix metalloproteinase

MRLP4 Mitochondrial ribosomal protein 4 mRNA Messenger ribonucleic acid

MS4A1 Membrane-spanning 4-domains, subfamily A, member 1 MTCP1 Mature T-cell proliferation 1

MUPP1 Multi-PDZ domain protein 1

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NLK Nemo-like kinase

PALS1 Protein associated with Lin seven 1 Par3 Partitioning defective 3

PATJ Protein associated with Lin seven 1 (PALS1)-associated tight junction protein PBS Phosphate buffered saline

PCR Polymerase chain reaction

PI3K Phosphatidyl-inositor triphosphate kinase PKB Phosphorylase kinase B, Akt1

PLC Phospholipase C (PLCγ)

PLCGI1 Phospholipase C, gamma 1 (formerly subtype 148)

PSM Proteasome complex protein (such as PSMA3 and PSMB6) PSMB Proteasome (prosome, macropain) subunit, beta type PTDSS1 Phosphatidylserine synthase 1

qRT-PCR Quantitative real-time PCR

Rac1 Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1)

Raf MAPK kinase kinase

RhoA Ras homolog gene family, member A

RNA Ribonucleic acid

ROK1 ATP-dependent RNA helicase RPS5 Ribosomal protein

R-Smad Receptor mothers against decapentaplegic homologs RTK Receptor tyrosine kinase

RT-PCR Reverse real-time PCR

SARA Smad anchor of receptor activation

SF Splicing factor (such as SFRS1 and SF3A1) SIAH1 Seven in absentia homologue 1

Smad Mothers against decapentaplegic homolog SOS Son of sevenless homolog 1

STAT Signal transducer and activator of transcription T84 Colon carcinoma cell line

TCF T-cell factor

Tcf-4 T-cell factor-4

TGFβ Transforming growth factorβ TGFα Transforming growth factorα

TGIF TGFB-induced factor homeobox 1, a TGFβ co-repressor TIEG Early growth response gene, Kruppel-like factor 10 TIMP Tissue inhibitor of metalloproteases

TNFα Tumour necrosis factor alpha, a proinflammatory cytokine tTG Tissue transglutaminase,

VRK Vaccinia-related kinase

WAVE1 Wiskott-Aldrich syndrome protein family member 1; verprolin homology domain-containing protein 1, WASF1

WNT Wingless-type MMTV integration site family, member

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INTRODUCTION

The small intestinal epithelium has variety of assignments, among them protection from pathogens, digestion and absorption of nutrients. These functions are reflected in the delicate differentiation of epithelial cells into several cell lineages (Rubin 2007). The rapid turnover of epithelial cells is strictly governed to maintain the homeostasis between proliferation, differentiation and apoptosis (Babyatsky and Podolsky 2003). The regulation of these processes is mediated by external signals from apical, basal and lateral sides of epithelial cells (Babyatsky and Podolsky 2003). Proliferation and differentiation is adjusted by the integrity of intracellular junctions, which induces polarisation of the epithelial cells (Madara and Anderson 2003). The polarisation and extracellular signals control signalling in the TGFβ, wingless and receptor tyrosine kinase pathways (Matter et al. 2005, Aijaz et al. 2006, Perez-Moreno and Fuchs 2006, Matter and Balda 2007).

In coeliac disease, in the small intestinal mucosa of genetically susceptible individuals the dietary wheat gluten and related prolamins from rye and barley induce an inflammatory reaction. The ingested gliadin activates both the innate and adaptive immune responses. The inflammation in turn induces mucosal lesion formation and morphological changes in the small intestinal mucosa, villous atrophy and crypt hyperplasia. Elimination of gluten from the diet results in the healing of the mucosa. However, the disease will relapse if gluten is reintroduced (Koning et al. 2005, Brandtzaeg 2006, Jabri and Sollid 2006, Kagnoff 2007).

In untreated coeliac disease epithelial cell junctional integrity and barrier function are affected (Laukoetter et al. 2006), and at least the TGFβ and wingless signalling pathways, and probably also the receptor tyrosine kinase signalling pathway, are also involved (Perry et al. 1999, Barone et al. 2007b, Benahmed et al. 2007). The epithelial cells in untreated coeliac small intestinal mucosa show decreased differentiation and increased apoptosis in the surface epithelium, and increased proliferation in the crypts (Diosdado et al. 2005). The mechanisms responsible for the remodelling of the small intestinal mucosa leading to this increased epithelial cell proliferation and failure in differentiation, have nonetheless remained unclear (Koning et al. 2005, Brandtzaeg 2006, Jabri and

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a three-dimensional epithelial cell culture model (Halttunen et al. 1996) and coeliac patient and healthy control small intestinal mucosal biopsy samples.

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REVIEW OF THE LITERATURE

Small intestinal crypt-villus axis

The small intestine is the main gastrointestinal compartment for the absorption of nutrients. The upper part of the small intestine is called the duodenum, which is followed by the jejunum and ileum, altogether from four to seven metres in length. The absorbing area of the small intestine is increased by circular folds, villi and microvilli. The folds being located in the submucosa of the duodenum and the jejunum. On top of these lie finger-like projections, called villi. These are covered by a single layer of columnar epithelial cells. The apical surface of these cells is covered by microvilli, which composes a brush border (Rubin 2003).

The absorption of dietary components from the gastrointestinal lumen occurs via the highly organised epithelium (Rubin 2003, Sancho et al. 2003, Sancho et al.

2004, Blanpain et al. 2007). All epithelial cells covering the crypts and the villi, are the descendants of epithelial stem cells (Figure 1). The stem cell is located four to five cells above the crypt base. At birth there are four to 16 multipotent stem cells in every crypt (Rubin 2003). Subsequently only one stem cell is left alive in every crypt (Babyatsky and Podolsky 2003). The stem cell forms sister progenitors by asymmetric division. These migrate bi-directionally up- and downwards on the crypt-villus axis. During upward migration cells differentiate towards three different lineages: enteroendocrine, absorptive and goblet cells.

Each of the villi is covered by linear stripes of epithelial cells originating from one to ten crypts surrounding each villi. The downwards migrating cells differentiate to Paneth cells, which reside in the base of the crypt. (Babyatsky and Podolsky 2003).

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Fig 1. One villus and one crypt(indicated by bracket) in the normal human small intestinal mucosa. The luminal side is covered with epithelial cells. In the saclike invaginations, in the crypts of Lieberk hn, stem cells proliferate continuously generating new sister progenitors which migrate bi-directionally. During their migration cells differentiate. The upward migrating cells differentiated into three cell types, the absorptive enterocytes and goblet cells depicted in the figure, and enteroendocrine cells visualised only with special labelling. The journey of the epithelial cells towards the villus tip takes three to five days, after which they are exfoliated to the lumen of the gut. The downward migrating cells differentiate into the Paneth cells.

These reside at the bottom of the crypts for around 20 days, until they are phagocytosed (Sancho et al. 2003). Epithelial cells are attached to the basement membrane.

Myofibroblasts lie beneath the basement membrane in the lamina propria.

At the onset, the descendants of the stem cells are undifferentiated crypt cells.

These intermediate progenitors of stem cells are still able to differentiate into several lineages. The precise mechanism of the determination of lineage commitment is not known, but it has been hypothesised to occur by consecutive binary decisions (Sancho et al. 2004). The undifferentiated epithelial cells diverge in several ways from the differentiated cells. Their cytoplasm is

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channels and Na+/K+ exchangers which can secrete sodium out from the cells in response to enterotoxins (Rubin 2003). As the cells migrate upwards and differentiate, the number of ribosomes decreases and the number of mitochondria and rough endoplasmic reticulum increases. During the migration towards villus tip the cell acquires the special features of the cell type it is committed to (Rubin 2003).

The cells committed to the absorptive enterocytes start to express cell specific proteins, immediately upon emerging above the crypt-villus junction (Rubin 2003). The higher they have migrated, the longer and more numerous microvilli they have on their apical side. The microvillus membrane of differentiated cells expresses enzymes such as alkaline phosphatase, receptors such as bile acid receptor, transporters such as the Na+-dependent glucose transporter and amino acid transporters, and peptidases. All these are needed for the digestion and absorption of nutrients (Rubin 2003).

The cells committed to the enteroendocrine line begin to produce peptides or amines, this affecting on bowel motility and behaviour of neighbouring epithelial cells. The exocrine enteroendocrine cells secrete these active peptides into the lumen of the gut. L cells produce glucagon-like peptide 1, D cell somatostatin, enterochromaffin cell serotonin, secretin cell secretin and cholecystokinin cell cholecystokinin. There are also endocrine cells oriented towards basal surface.

These have secretory granules located below the nucleus. They secrete peptides into the lamina propria. Enteroendocrine cells may also produce more than one product (Rubin 2003).

The cells committed to the goblet cell line form into the shape of cup. They mature earlier than absorptive enterocytes and are already active in the crypts.

Differentiated goblet cells have granules containing lubricating mucus, pS2 and spasmolysin and trefoil factor (Rubin 2003).

The journey of all three types of epithelial cells towards the villus tip takes three to five days. When they have reached the tip they are committed to regulated cell death, apoptosis. Their attachment to the basement membrane is loosened in a process called anoikis, and they are exfoliated to the lumen of the gut (Rubin 2003, Sancho et al. 2004). The fourth lineage, the Paneth cells, migrate downwards. They grow into pyramid-shaped cells which secrete lysozyme, cryptidins, defensis and other antibiotic proteins able to exert microbicidial activity (Rubin 2003). The Paneth cells live longest from the differentiated small intestinal epithelial cells, around 20 days at the bottom of the crypts, until they are phagocytosed (Sancho et al. 2003, Sancho et al. 2004, Blanpain et al. 2007).

Several gene expression products are used as markers to define cell type in the intestinal epithelium. The markers for intestinal epithelial stem cells are

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against decapentaplegic (Smad) 1, -5, and -8. The cell type specific marker for the goblet cells is mucin 2, for the enterocytes intestinal fatty acid binding protein 2, for absorptive enterocytes alkaline phosphatase and sucrose isomaltase; for the enteroendocrine cells the markers are secretin, serotonin and lysozyme, and for the Paneth cells cryptidins (Sancho et al. 2004).

Regulation of proliferation and differentiation of epithelial cells

The epithelium of the small intestine undergoes rapid turnover, and in the gastrointestinal mucosa particularly delicate regulatory mechanisms are required to maintain homeostasis. The external signals from all four sides of epithelial cells can have an effect on epithelial cell proliferation, differentiation and apoptosis (Babyatsky and Podolsky 2003).

The apical side of the epithelial cell facing towards the lumen of the gut is exposed to nutrients, bacteria and gastrointestinal proteins with growth- modulating properties (Babyatsky and Podolsky 2003). The small intestinal villus height and crypt depth ratio varies depending on the part of the world the subject lives in. The reason for differences in height has been proposed to be dissimilarity in the nutritional status. In underdeveloped countries the villi are short and broad whereas in developed countries they are long and slender (Babyatsky and Podolsky 2003, Rubin 2003). Glucose, galactose, methylglucose and sodium chloride (NaCl) have growth-inducing effects in the intestinal epithelium. All these substances are transported by the intestinal epithelial cells, but only glucose is metabolised. Therefore, the growth inducing stimuli generating slender villi are not attributable the amount of energy, but the amount of active transport of the intestinal epithelial cells (Babyatsky and Podolsky 2003). Several enteroendocrine cell products, for example the glucagon-like peptide 2, controls proliferation of the epithelial cells (Rubin 2003). The trefoil factor, secreted by the goblet cells, inhibits crypt epithelial cell apoptosis via activation of epidermal growth factor receptor (EGFR), phosphatidylinositol 3- phosphate (PI3K) and the Akt (a serine/threonine kinase) pathway. Several gastrointestinal proteins, among them epidermal growth factor (EGF) are produced by the salivary glands and the duodenal Brunner glands, can exert growth-modulating effects through interaction with the luminal mucosal surface (Babyatsky and Podolsky 2003).

The basal side of the epithelial cell is attached to the extracellular matrix which lies between epithelial and subepithelial myofibroblasts. The latter are also called mesenchymal cells. The extracellular matrix is formed from several components such as heparin sulphate proteoglycan, type IV collagen, laminin. These are required for the complete morphogenesis and differentiation of the epithelial cells (Birchmeier and Birchmeier 1993, Madara and Anderson 2003). Laminin-1 has a direct role in the differentiation of epithelial cells. It induces both caudal-

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mediate signals in epithelial-mesenchymal crosstalk (Birchmeier and Birchmeier 1993). The mesenchymal cells produce soluble factors such as TGFβ, hepatic growth factor/ scatter factor, neuregulin and keratinocyte growth factor. These interact with epithelial cells across the matrix and regulate their behaviour (Kedinger et al. 1998a).

The lateral side of epithelial cells attaches to the adjacent epithelial cells by an which has an important role in enabling the polarisation of the epithelial cells (Matter et al. 2005, Matter and Balda 2007).

Polarisation and differentiation of epithelial cells

Polarisation of the epithelial cells is the first requirement for cellular differentiation. It takes place in consequence of the cellular asymmetry between the apical and basolateral cell surface domains (Aijaz et al. 2006). The process requires a rigid cell shape, stable interaction to basement membrane and tight lateral adhesion between adjacent cells (Madara and Anderson 2003). The polarisation of the continuous sheet of epithelial cells enables formation of a selective barrier between the external side facing the lumen of the gut and the internal side facing towards lamina propria (Matter et al. 2005). Dysregulation of intestinal barrier integrity is associated with several diseases, among them coeliac disease and inflammatory bowel disease (Laukoetter et al. 2006). The loss of epithelial cell polarity and tissue morphology associated with epithelial cell hyperproliferation, is often the first step in the formation of adenomatous polyps, which precede intestinal cancer (Chung 2000).

The polarisation of the intestinal epithelial cells is mediated by the apical junctional complex, presented in Figure 2. This consists of tight and adherens junctions (Matter et al. 2005). The apical junctional complex forms a semi permeable diffusion barrier (Aijaz et al. 2006). The assembly of intracellular junctions starts with the formation of primordial adherens junctions by recruitment of E-cadherin, nectin, zonula occludens protein-1 (ZO-1) and junctional adhesion molecule (JAM) A (Aijaz et al. 2006). The junctional complex then matures and establishes distinct adherens and tight junctions (Aijaz et al. 2006). These junctions are important in controlling the formation and maintenance of cellular adhesion (Madara and Anderson 2003, Perez-Moreno and Fuchs 2006). They also regulate migration of cells within the epithelia, intracellular organisation, the transmission of information from out-to inside the cell, and the differentiation of the cells (Mege et al. 2006, Perez-Moreno and Fuchs 2006). The major component in the adherens junctions is E-cadherin. This is a single pass transmembrane protein assembled in the presence of calcium α-

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(Mege et al. 2006, Perez-Moreno and Fuchs 2006). The actin cytoskeleton has a crucial role in the regulation of the adherens junction assembly. The formation of these and organisation of the actin cytoskeleton are regulated by Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (Rac1), Cell division cycle 42 (Cdc42) and Ras homolog gene family, member A (RhoA) (Aijaz et al. 2006) (Perez-Moreno and Fuchs 2006).

The tight junction forms a dynamic paracellular diffusion barrier which allows passage only to certain solutes, not all. It thereby creates and maintains cellular polarity (Madara and Anderson 2003, Matter and Balda 2007). The tight junctions are composed of anastamosing strands of fibrils of transmembrane proteins which completely surround the apical region of the cell (Harhaj and Antonetti 2004). They are composed of the transmembrane, the scaffolding and the signalling proteins (Figure 2) (Harhaj and Antonetti 2004, Matter and Balda 2007). There are at least three main types of transmembrane proteins: occludin, claudins and JAM (Matter and Balda 2007). Occludin is a four-pass transmembrane protein having two extracellular loops and two intracellular domains. It interacts directly with ZO-1, -2 and -3, and indirectly with JAMs and the actin cytoskeleton via interaction with the ZO proteins (Matter and Balda 2007). The JAMs are single-pass transmembrane proteins (Matter and Balda 2007) which regulate junction assembly (Aijaz et al. 2006). The claudins (-1 -2, - 3 and -4) are four-pass transmembrane proteins having, similarly to occludin, two extracellular loops and two intracellular domains. They have an important role in the normal paracellular permeability of the magnesium and calcium ions and in determining barrier function (Aijaz et al. 2006, Matter and Balda 2007).

The claudin proteins interact with each other, with the ZO proteins and the protein associated with Lin seven 1 (PALS1) and the protein associated with the Lin seven 1-associated tight junction protein (PATJ) (Matter et al. 2005) (Aijaz et al. 2006). PATJ associates claudins with the Rac and to the atypical protein kinase C (aPKC) signalling network (Matter and Balda 2007). The adaptor proteins (also called scaffolding or peripheral membrane proteins) are the those which form a molecular bridge between adhesion proteins and cytoskeleton (Aijaz et al. 2006, Matter and Balda 2007). They include ZO-1,-2 and -3, PATJ, Pals1, partitioning defective (Par)-3 and -6, calcium/calmodulin-dependent serine protein kinase (CASK/Lin2), membrane-associated guanylate kinase with inverted domain structure 1 (MAG-1), multi-PDZ domain protein 1 (MUPP1) and partitioning defective 3 (Par3) (Aijaz et al. 2006, Matter and Balda 2007).

Several of these adaptor/scaffolding proteins interact with signalling proteins (Aijaz et al. 2006).

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Fig 2. Schematic presentation of the apical junctional complex in intestinal epithelial cells. The complex is composed of tight and adherens junctions. Colours code different types of proteins. In the tight junctional complex: light green indicates transmembrane proteins such as occludin (drawn with dotted line) or occludin (drawn with solid line), brown adaptor proteins such as ZO-1, light blue signalling proteins and red transcriptional regulators. In the adherens junctional complex: dark blue indicates transmembrane proteins, pink adaptor proteins. Directly

Adaptors

•ZO-1 occludin, claudins, ZO-2, cingulin,ZONAB, F-actin

•ZO-2 claudins, ZO-1, cingulin,CBP, AP-1, F-actin

•ZO-3 claudins, ZO-1, cingulin, F- actin, p120 catenin

•Cingulin JAM, ZO-1, -2, -3, myosin, F-actin

•Pals1 PATJ, PAR

•PATJ Pals, ZO-3

•Par3 JAM, Par6, aPKC, Cdc42

•MAGI-1 papillomavirus, adenovirus

•MAGI-2, -3 PTEN

•MUPP1 claudins, JAMs,

Transcriptional regulators

•twist

•snail

•β-catenin

Actin Transmembrane proteins

•Occludins ZO-1-, -2, -3, TGFβreceptor type II

•Claudins ZO-1-, -2, -3, MUPP1

•JAMs CASK, ZO-1, cingulin, PAR3

•CRB3 Pals1, Par6

Signalling molecules

•aPKC Occludin, Par3, Par6

•GEF-H1 Cingulin, RhoA

•CDK4 ZONAB

•Rab13 Protein kinase A Transcriptional regulators

• ZONAB CDK4, ZO-1

• Symplektin Hear shock factor 1

• AP-1 ZO-2

•Arp 2/3 α-catenin, actin

•Rho α-catenin

•Rac1 ARP 2/3, VASP Adaptors

•p120 Rac1, RhoA, cdc42

•β-catenin α-catenin

•α-catenin β-catenin, actin, ZO-1, afadin

Transmembrane proteins

•E-cadherin β-catenin,α-catenin p120, PI3-kinase, PLC Apical membrane

Brush border

Tight Junction

Claudin Occludin

JAMs

α-catenin β-catenin Actin

Lateral membrane

p120 Paracellular space

Adherens Junction

Adaptors

•ZO-1 occludin, claudins, ZO-2, cingulin,ZONAB, F-actin

•ZO-2 claudins, ZO-1, cingulin,CBP, AP-1, F-actin

•ZO-3 claudins, ZO-1, cingulin, F- actin, p120 catenin

•Cingulin JAM, ZO-1, -2, -3, myosin, F-actin

•Pals1 PATJ, PAR

•PATJ Pals, ZO-3

•Par3 JAM, Par6, aPKC, Cdc42

•MAGI-1 papillomavirus, adenovirus

•MAGI-2, -3 PTEN

•MUPP1 claudins, JAMs,

Transcriptional regulators

•twist

•snail

•β-catenin

Actin Transmembrane proteins

•Occludins ZO-1-, -2, -3, TGFβreceptor type II

•Claudins ZO-1-, -2, -3, MUPP1

•JAMs CASK, ZO-1, cingulin, PAR3

•CRB3 Pals1, Par6

•aPKC Occludin, Par3, Par6

•GEF-H1 Cingulin, RhoA

•CDK4 ZONAB

•Rab13 Protein kinase A Transcriptional regulators

• ZONAB CDK4, ZO-1

• Symplektin Hear shock factor 1

• AP-1 ZO-2

•Arp 2/3 α-catenin, actin

•Rho α-catenin

•Rac1 ARP 2/3, VASP Adaptors

•p120 Rac1, RhoA, cdc42

•β-catenin α-catenin

•α-catenin β-catenin, actin, ZO-1, afadin

Transmembrane proteins

•E-cadherin β-catenin,α-catenin p120, PI3-kinase, PLC Apical membrane

Brush border

Tight Junction

Claudin Occludin

JAMs

α-catenin β-catenin Actin

Actin

Lateral membrane

p120 Paracellular space

Adherens

Junction

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An important recently identified class are the dual localisation proteins. These shuttle between the junction and the nucleus, regulating both junctional assembly and gene expression. Currently known dual localisation proteins are β-catenin, the cdk4, ZO-1-associated nucleic acid-binding protein (ZONAB) and symplectin (Aijaz et al. 2006). The junctional adaptors can suppress proliferation by sequestering free dual localisation proteins to the junctions (Aijaz et al. 2006) Polarisation and the intracellular junctions contribute to regulation of proliferation and differentiation of epithelial cells. This takes place via effects on signalling cascades. Thus the assembly of the intracellular junctions both alters and maintains differentiation by controlling gene transcription (Madara and Anderson 2003, Matter et al. 2005, Aijaz et al. 2006, Perez-Moreno and Fuchs 2006, Matter and Balda 2007).

Signalling pathways involved in differentiation of epithelial cells

Intracellular signalling pathways within the intestinal epithelial cells are induced by external stimuli both from the apical side of the gut lumen and the basal side of the lamina propria (Kedinger et al. 1998a, Babyatsky and Podolsky 2003, Rubin 2003). They are also controlled by stimuli from the lateral sides of the cell from the apical junctional complex (Matter et al. 2005, Aijaz et al. 2006, Perez- Moreno and Fuchs 2006, Matter and Balda 2007). Especially the dual localisation proteins in the apical junctional complex are associated with the regulation of signalling cascades (Aijaz et al. 2006). The complex signalling networks of TGFβ, wingless and receptor tyrosine kinase regulate the proliferation and the differentiation of epithelial cells.

TGFβ signalling pathway

Ligands for the TGFβ receptor includes TGFβ, activin, and nodal and bone morphogenetic protein, which are secreted mediators controlling proliferation, differentiation, migration and apoptosis (Itoh and ten Dijke 2007). TGFβ is involved in the normal development and differentiation of the intestine (Sancho et al. 2004, Kapoor et al. 2007). The TGFβ1 ligand inhibits the growth of epithelial cells (Carethers and Boland 2003). In the small intestine the mRNA of TGFβ is found mainly in the crypts (Babyatsky and Podolsky 2003), which the TGFβ receptors and ligands are predominantly expressed in the differentiated compartment (Sancho et al. 2004). The expression of genes on the TGFβ signalling pathway is up-regulated in the differentiated cells inin vitro epithelial cell culture (Fleet et al. 2003). Furthermore, supplementation of TGFβ to the cell culture induces differentiation of epithelial cells (Halttunen et al. 1996).

Normally cells secrete TGFβ in latent form, to be activated by tissue transglutaminase (tTG) (Nunes et al. 1997). The TGFβ-signalling cascade, which

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receptor. Binding of the ligand to the type II TGFβ-receptor is a trigger inducing the dimeric assembly of two type-I and two type-II receptors (Massague 2000).

The type-II receptor phosphorylates type-I. These activated receptor complexes are internalised either by caveolin-coated or by clathrin-coated pits. From the former of these complexes cargo is targeted to degradation.

Clathrin-coated pits target cargo to endosomes, in which the signal is transmitted to the smad anchor of receptor activation (SARA), which recruits mothers against decapentaplegic homologs 2/3 (Smad 2/3). Smad 2/3 in turn complexes with Smad4, which accumulates in the nucleus to control transcription of target genes such as Id1 or c-myc (Itoh and ten Dijke 2007). The duration, intensity and the context in which the signal arrives determines the outcome of the pathway (Itoh and ten Dijke 2007). The outcome is fine-tuned by several specific co- activators and co-inhibitors, ubiquitination and phosphorylation of modulator proteins and selective sequestration of signalling proteins (Itoh and ten Dijke 2007).

Wingless signalling pathway

β-catenin is a dual localisation protein having an essential role in the cell adhesion when bound to E-cadherin, and also in regulating transcription in the wingless signalling pathway when bound to a member of T-cell factor-4 (Tcf-4) (Perez-Moreno and Fuchs 2006). The wingless signalling pathway is of major importance in intestinal epithelial homeostasis, regulating proliferation and renewal of epithelial stem cells (Reya and Clevers 2005, Kapoor et al. 2007).

The inhibition of wingless signalling by transgenic Dickkopf-1, the negative modulator of the wingless pathway, induces complete loss of crypts (Pinto et al.

2003, Kuhnert et al. 2004), whereas over-activity leads to adenomatous polyp formation and cancer (Sancho et al. 2004, Kapoor et al. 2007). In the normal small intestine β-catenin accumulates to the nucleus in the proliferating crypt epithelial cells, whereas in differentiated cells the nuclear label disappears and the β-catenin staining is concentrated in the junctions (Sancho et al. 2004). The expression of genes on the wingless signalling pathway is down-regulated in differentiated cells in in vitro epithelial cell differentiation models (Mariadason et al. 2002, Fleet et al. 2003).

The signalling in the wingless pathway, which is outlined in Figure 3, starts when the wingless-type MMTV integration site family, member (WNT) -1, -3A, -8, produced by the adjacent cells, binds on frizzled (Fzd) receptor and LDL- receptor-related protein 5/6 or Arrow (LRP) co-receptors. Especially WNT2b,- 3, 4, 5, 6,7 and Frizzled 4,6,7 have been shown to be expressed in the small

Functional genomics of a three-dimensional epithelial cell culture and coeliac small intestinal mucosal biopsy samples