Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in MolecularCell Biology presented at Uppsala University in 2002, from the Ludwig Institute forCancer Research

Abstract

Brodin, G. 2002 Smad7 in TGF-β Signalling. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine1135. 57pp. Uppsala. ISBN 91-554-5271-X.

Members of the transforming growth factor-β (TGF-β) superfamily of growth anddifferentiation factors regulate a vast array of biological functions in the adult, and areof great importance in governing cell fate determination and patterning in thedeveloping embryo. The TGF-β signal is propagated intracellularly by Smad proteinsresulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known todownregulate the TGF-β signal and thereby possibly modulating the biologicalresponse. This thesis describes a functional analysis of the inhibitory Smad7 from an invitro and in vivo perspective.

The prostate gland is dependent on androgens for its growth and differentiation.Androgen withdrawal can cause regression and apoptosis in normal and malignantprostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. Weinvestigated the expression levels of Smad proteins in the rat ventral prostate as well asin an androgen sensitive prostate tumor model (Dunning R3327 PAP) byimmunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, aswell as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 werealso increased in both normal and malignant prostate in response to castration.

Several studies have shown that Smad7 is upregulated in response to TGF-βstimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response.We investigated the molecular mechanism behind that response by studying thetranscriptional regulation of the Smad7 gene. We identified a palindromic Smad bindingelement (SBE) in the promoter. Point mutations introduced into the SBE abolishedtranscriptional activation via TGF-β. We also observed that mutating or deletingbinding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediatedtranscriptional induction as well as the basal promoter activity.

Gene ablation of Smad proteins has revealed specific physiological anddevelopmental roles. We analysed mice targeted on the Smad7 locus. The miceappeared viable and fertile with a slight reduction in litter size, suggesting a perinatalloss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no majordifference between wild type and mutant MEFs.

Greger Brodin, Ludwig Institute for Cancer Research, Biomedical Centre, Box 595, SE-751 24, Uppsala, Sweden

© Greger Brodin 2002

ISSN 0282-7476ISBN 91-554-5271-X

Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002

ToMy Parents

&Veronika

4

This thesis is based on the following papers, referred to in the text by their Romannumerals:

I. Brodin, G., ten Dijke, P., Funa, K., Heldin, C., and Landström, M. (1999)Increased Smad expression and activation are associated with apoptosis innormal and malignant prostate after castration. Cancer Research 59, 2731-2738

II. Brodin, G., Åhgren, A., ten Dijke, P., Heldin, C., and Heuchel, R. (2000)Efficient TGF-β induction of the Smad7 gene requires co-operation betweenAP-1, Sp1, and Smad proteins on the mouse Smad7 promoter. J. Biol. Chem.275, 29023-29030

III. Brodin, G., Cheng, A., Åhgren, A., Kulkarni, S., Pawson, T., Heldin, C.H.,and Heuchel, R. Targeting of the mouse Smad7 gene. Manuscript.

Reprints were made with permission from the publishers

We shall not cease from explorationAnd the end of all our exploringWill be to arrive where we startedAnd know the place for the first time.

T.S. Eliot

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TABLE OF CONTENTS

PageAbbreviations 6Introduction 7Transforming growth factor-β (TGF-β) 8

TGF-β superfamily of ligands 8TGF-β 9Activin / Inhibin 9BMP 9Other members 10Control of TGF-β production and activation 11TGF-β superfamily binding proteins 11

TGF-β superfamily receptors 12Type I and II receptors 12Type III/Endoglin receptors 13

TGF-β superfamily intracellular signalling 13The Smad family of signal transducers 13The Smad pathway 15Non-Smad pathways 17Cross-talk between Smads and the MAP kinase pathway 18Specificity in Smad signalling 19Genetic targeting 20

Smad7 23Smad7 signalling 24Smad7 in physiological and pathological conditions 25

Vasculogenesis 25Lung morphogenesis 27Apoptosis 27Fibrosis 28Immunological disorders 28Carcinogenesis 29

Present investigations 30Smad expression in prostatic carcinoma (paper I) 30Transcriptional regulation of the Smad7 gene (paper II) 31Targeting of the Smad7 gene (paper III) 32

Future perspectives 34Acknowledgements 35References 37

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ABBREVIATIONS

ActR activin receptorALK activin receptor-like kinaseAMH/MIS anti-Müllerian hormone/Müllerian inhibiting

substanceATF2 activating transcription factor 2AVM arteriovenous malformationsBAMBI BMP and Activin membrane-bound inhibitorBMP bone morphogenetic proteinCBFA/AML core-binding factor A/acute myeloneus leukaemiaEMSA electrophoretic mobility shift assaysFAST forkhead activin signal transducerGDFs growth and differentiation factorsGDNF glial cell line-derived neurotrophic factorHAT histone acetyltransferaseHDAC histone deacetylasesJNK Jun N-terminal kinase LAP latency-associated proteinLEF1/TCF lymphoid enhancer-binding factor 1/T-cell specific

factorLLCs large latent complexesLTBP latent TGF-β-binding proteinMEF mouse embryonic fibroblastsMH1/2 Mad homology ½SAD Smad activation domainSARA Smad anchor for receptor activation SBE Smad binding elementSki Sloan-Kettering Institute proto-oncogeneSLC small latent complexSMURF Smad ubiquitylation regulatory factorSnoN Ski-related novel gene NTβR TGF-β receptorTAK1 TGF-β-activated kinaseTFE3 transcription factor binding to immunoglobulin

heavy constant mu enhancer 3TGF-β transforming growth factor-β TGIF TG3-intercating factorVSMC vascular smooth muscle cellsXIAP X-chromosome-linked inhibitor of apoptosis protein

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INTRODUCTION

We are all wired. The term “we” is used in a rather generous way referring to ourselvesand all the multicellular organisms sharing life with us. The fact that prevents us allfrom being a shapeless pile of individual cells is biochemical communication. This isachieved through an elaborate signalling network relaying a continuous flow ofinformation within cells and between the cells and their environment. Pathological ordysfunctional conditions may arise when the regulation of this information is perturbed,which ultimately can affect the development of an organism or the maintenance ofphysiological functions in the adult organism. Cells exchange information by means ofdirect cell to cell interactions, cell contact with extracellular matrix components or byresponding to soluble factors such as hormones, neurotransmitters, cytokines, growthfactors or small ions. The extracellular signal is converted into an intracellular one bythe use of a specific class of proteins, the so-called receptors. The initiated signalcascade will affect cellular functions such as survival, division, death, movement ordifferentiation.

Transforming growth factor-β (TGF-β) belongs to a superfamily of cytokines that exerta multitude of cellular functions on the organism affecting such diverse things as tissuehomeostasis, cell division and multicellular patterning and development. These diverseeffects are mediated through the combination and activation of specific type I and typeII serine/threonine receptor kinases that transduce the signal from the cellular surface tothe nucleus via downstream effector proteins, called Smads. These proteins can bedivided into three subclasses, receptor activated (R-Smads), common mediator Smad(Smad4) and the inhibitory Smads (Smad6 and Smad7). The R-Smads (Smad1, 2, 3, 5and 8) become phosphorylated in their C-termini and thus activated by the type Ireceptors. They associate with the common mediator Smad4 and subsequentlytranslocate to the nucleus where they trigger transcriptional responses. The inhibitorySmads exert their effect as attenuators and modulators of the TGF-β signal. Thequestion is how different Smads can influence such a vast array of cellular effects andhow pathological conditions such as fibrosis, rheumatoid arthritis and carcinogenesis,can arise in response to a deregulation of this TGF-β signal.

The scope of this thesis was to investigate the functional role of the inhibitory Smad7from an in vivo and an in vitro perspective. This was achieved by assessing the level ofSmad expression in a rat prostate carcinoma model, by characterising the transcriptionalregulation of the Smad7 promoter and finally, by disrupting the Smad7 gene in mice, inorder to identify phenotypic alterations that would suggest different functional roles forSmad7.

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TRANSFORMING GROWTH FACTOR-β (TGF-β)

The basis for the discovery of TGF-β was a finding by de Larco and Todaro, where theyobserved that a pool of polypeptide growth factors released from mouse fibroblaststransformed with murine sarcoma virus, was able to transform cells in a soft agar assay(De Larco and Todaro, 1978). Using a similar approach on normal rat kidney fibroblastsit was demonstrated that the growth factor cocktail actually consisted of two distinctpolypetide growth factors, coined transforming growth factor (TGF)- α and -β (Anzanoet al., 1983; Roberts et al., 1981). Whereas TGF-α displayed mitogenic activity, it laterbecame clear that TGF-β served as a potent growth inhibitor in most other cell types(Roberts and Sporn, 1990), rather suggesting a role as an important suppressor oftumourigenesis.

TGF-β superfamily of ligands

30 40 50 60 80 100% Identity

MIS/AMHTGF-βActivinNodal

BMP-3

Vgr-2

GDF-1

GDF-5

BMP-7

BMP-2/4

Dpp

Figure 1. The ligands of the TGF-β superfamily. Dendrogram indicating the relativelevel of amino acid sequence similarity between members.

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TGF-β serves as the prototype for the large and still growing TGF-β superfamily,consisting of more than 30 members which include bone morphogenetic proteins(BMPs), activins, inhibins, anti-müllerian hormone (AMH) and growth anddifferentiation factors (GDFs) (see Figure 1) (reviewed in Massague, 1998).

TGF-β TGF-β exists in three highly similar isoforms termed TGF-β1 (Derynck et al., 1985),TGF-β2 (de Martin et al., 1987; Madisen et al., 1988), and TGF-β3 (Derynck et al.,1988; ten Dijke et al., 1988), all encoded by distinct genes. Besides forminghomodimers, it has been reported that heterodimers can form between TGF-β1 andTGF-β2, and between TGF-β2 and TGF-β3 (Cheifetz et al., 1987; Ogawa et al., 1992).The functions of TGF-β ligands include cell cycle arrest in epithelial andhaematopoietic cells and control of cell proliferation and differentiation in mesenchymalcells. They are also strong inducers of extracellular matrix production and are involvedin wound healing and immunosuppression, (Massague, 1990; Roberts and Sporn, 1990;Roberts and Sporn, 1993). The three TGF-β isoforms affect TGF-β signalling in a rathersimilar and redundant way in vitro, but display different in vivo expression patterns andfunctions (reviewed in (Roberts and Sporn, 1992). Gene analysis also revealed that eachTGF-β isoform is controlled by a unique and differently regulated promoter (reviewedin Roberts et al., 1991).

Activin/InhibinMembers of the activin subfamily can induce pituitary follicle-stimulating hormone(FSH) production. Other functions include erythroid cell differentiation and induction ofmesoderm as shown in Xenopous. The different members of the activins can formhomo- or hetero-dimers between different β-subunits: βA, βB, βC and βE (Gaddy-Kurten et al., 1995; Harland, 1994; Vale et al., 1990).

Inhibin consists of a distantly related α-subunit, which can form hetero-dimers with thedifferent activin β-subunits. Inhibin can act as a counterplayer of activin, and inhibitFSH production as well as other effects of activin (Gaddy-Kurten et al., 1995; Vale etal., 1990).

BMPBone morphogenetic proteins (BMPs) constitute the largest group within the TGF-βsuperfamily of growth and differentiation factors, with over 20 members. These can befurther subdivided into different subfamilies based on structural homology andphysiological effects.

Spencer et al. observed in 1982 that flies mutant for the Drosophila genedecaplentaplegic (dpp) displayed a variety of pattern deficiencies and structureduplications. They suggested that that the dpp gene complex influenced positional

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information within developing epidermal tissue (Spencer et al., 1982). In a search forfactors able to induce ectopical bone and cartilage formation, Wozney et al. identifiedtwo mammalian homologues of dpp, BMP2 and BMP4 (Wozney et al., 1988). BMP4and dpp was later shown to be able to functionally substitute for each other in flies andmammals, suggesting a high degree of conservation between structure and function(Padgett et al., 1993; Sampath et al., 1993).

The BMP2 subfamily consists of BMP2, BMP4 and DPP (a BMP2/4 homologue inDrosophila). This subfamily affects gastrulation, neurogenesis, and interdigitalapoptosis in mammalians. In Xenopus the BMPs influence patterning of the mesodermand in Drosophila they affect dorsalization and eye and wing development (Harland,1994; Hogan, 1996; Mehler et al., 1997). The second subfamily, BMP5, includesmembers like BMP5, 60A (a BMP5 homologue in Drosophila), BMP6/Vgr1,BMP7/OP1, and BMP8/OP2. Together with BMP2 and BMP4 this subfamily isinvolved in the development of almost every organ and plays many roles in neuronaldevelopment (Hogan, 1996; Mehler et al., 1997).

The BMP5 subfamily members BMP5 (Celeste et al., 1990), BMP7/OP1 (Celeste et al.,1990; Özkaynak et al., 1990), and BMP8/OP2 (Özkaynak et al., 1992), were allidentified as bone-inducing proteins. In a search for mammalian homologues for Vg1,previously discovered in Xenopus oocytes (Weeks and Melton, 1987), BMP6/Vgr1 wasidentified (Lyons et al., 1989). Wharton et al. isolated the Drosophila gene 60A whilesearching for homologues of TGF-β (Wharton et al., 1991).

Other membersGrowth and differentiation factors (GDFs) belonging to the GDF5 subfamily have beenreported to affect chondrogenesis in the developing limbs (Hogan, 1996; Kingsley,1994) while Vg1, belonging to the Vg1 subfamily, affects axial mesoderm induction infrog and fish (Kingsley, 1994).

Members of the BMP3 subfamily, such as BMP3/osteogenein and GDF10 have beenreported to influence osteogenic differentiation, endochondral bone formation andmonocyte chemotaxis (Cunningham et al., 1992).

Nodal, one of the intermediate members has been shown to be involved in axialmesoderm induction and left-right asymmetry (Beddington, 1996; Hogan, 1996). Otherintermediate members, i.e. Dorsalin and GDF8, are involved in regulation of celldifferentiation within the neural tube (Basler et al., 1993) and inhibition of skeletalmuscle growth (McPherron et al., 1997), respectively.

More distant members of the TGF-β superfamily include anti-Müllerian hormone(AMH) also called Müllerian inhibiting substance (MIS) and glial cell line-derivedneurotrophic factor (GDNF). AMH induces Müllerian duct regression, resulting in malegenital organs (Cate et al., 1986; Josso et al., 1993). GDNF is a factor that promotesdopaminergic neuron survival and differentiation (Lin et al., 1993), but also affects

11

kidney development (Massague, 1996). Surprisingly, GDNF has also been reported tosignal via the Ret tyrosine kinase receptor (Massague and Weis-Garcia, 1996).

Control of TGF-β production and activationTGF-βs are synthesised as inactive dimeric precursors that later become proteolyticallyprocessed in the Golgi apparatus by furin, a member of the convertase family ofendproteases (Dubois et al., 1995). Furin cleaves the precursor at RXXR sites, located112-114 amino acids from the C-terminal end resulting in a C-terminal TGF-β part andan N-terminal remnant called latency-associated protein (LAP) (Cui et al., 1998;Munger et al., 1997b). The N-terminal and the mature part of TGF-β, form a stillinactive, non-covalent complex called the small latent complex (SLC) which is muchmore stable than the bioactive form of TGF-β. The process continues in the Golgi by theformation of disulphide bonds between the LAP and latent TGF-β-binding protein(LTBP), resulting in large latent complexes (LLCs). LTBPs serve to enhance stabilityand secretion of the SLC complex, ensure correct folding of TGF-β, and target the latentTGF-β complex either to the cell surface for activation, or to the extracellular matrix ofdistinct cells and tissues for storage (reviewed in Munger et al., 1997b; Taipale andKeski-Oja, 1997). An additional function of the LTBPs might be to influence TGF-β toactivate integrin signalling. It has been shown that LTBP-1, -2, and -4 have RGDsequences which are integrin binding sites (Munger et al., 1998b; Saharinen et al.,1998). Indeed, it has been shown that large latent TGF-β complexes can associatedirectly to integrin αVβ1 at the cell surface (Munger et al., 1998b). In addition, it hasbeen shown that the epithelial specific integrin αVβ6 can bind the RGD-motif in LAP,suggesting a cytoskeleton-mediated activation of TGF-β (Munger et al., 1998a).

Final activation of TGF-β is physically controlled by the binding of LAP to mannose-6-phosphate receptors, and by proteases such as plasmin and cathepsin that cleave LAP(Munger et al., 1997a; Taipale and Keski-Oja, 1997). Thrombospondin-1 appears to beanother important activator of TGF-β in vivo. It induces a conformational change inLAP, which results in TGF-β activation (Crawford et al., 1998). Interestingly, TGF-βinduces PAI-1 (plasminogen activator inhibitor-1), suggesting some level of self-regulation of the activation process. Another activation candidate is matrixmetalloproteinase-9 (MMP-9) which can activate latent TGF-β2 and TGF-β3 in vitro(Yu and Stamenkovic, 2000).

TGF-β superfamily binding proteinsBioactive TGF-β superfamily members can associate with several extracellular proteinsthat can modify their activity. Extracellular matrix proteoglycans: decorin and biglycan,have been shown to inhibit TGF-β activity (Yamaguchi et al., 1990). In addition, a 60-kDa protein has been reported to inhibit the interaction between the receptor and TGF-βligands (Piek et al., 1997). Furthermore, it has been demonstrated that α2-macroglobulin acts as a clearance factor for circulating TGF-βs and activins in serum(Mather, 1996).

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BMPs are regulated by soluble factors that play important roles during embryonicdevelopment. By competing with BMPs for ligand-receptor interactions, noggin(Zimmerman et al., 1996), chordin (Piccolo et al., 1996), short gastrulation (Sog) aDrosophila homologue of chordin (Francois and Bier, 1995), Dan and gremlin (Hsu etal., 1998), modulate differentiation of mesoderm and ectoderm. Follistatin can alsoinhibit receptor-ligand interactions by interacting directly with activin (Nakamura et al.,1990), as well as with BMPs (Yamashita et al., 1995a). In addition, cerberus has beenidentified as an antagonist not only for BMP and activin, but also for nodal signalling(Hsu et al., 1998).

TGF-β superfamily receptors

Receptors for polypeptide growth factors are transmembrane proteins that are able totransduce the extracellular message across the plasma membrane into an intracellularsignal. The TGF-βs and related factors signal through a group of transmembrane proteinserine/threonine kinases known as the TGF-β receptor family.

Type I and II receptorsThe TGF-β receptor family is divided into two subfamilies, type I and type II receptors,based on their structural and functional characteristics. The vertebrate type I receptorsubfamily forms three subgroups based on similarities in kinase domains and signallingactivities. In mammals, one group includes TβR-I, ActR-IB, and ALK7, anotherincludes BMPR-IA and -IB, and a third group includes ALK1 and ALK2 (reviewed in(Massague, 1998)). As a result of being cloned simultaneously by different groups, mosttype I receptors received different names. Initially the neutral ALK (activin receptor-like kinase) nomenclature was used whereas a more descriptive name was designatedafter the identification of a physiological ligand. As a consequence, the TGF-β type Ireceptor initially identified as ALK-5 (Franzen et al., 1993) is now called TβR-I(Yamashita et al., 1994). On a similar note, the activin receptor type I previously knownas ALK-4 (ten Dijke et al., 1993) is now called ActR-IB (Carcamo et al., 1994), andALK-3 and ALK-6 are referred to as BMPR-IA and BMPR-IB, respectively (Koenig etal., 1994; Yamashita et al., 1995b). Type I receptors with no known ligands includemammalian ALK-7 (Rydén et al., 1996; Tsuchida et al., 1996) and the related XenopusXTrR-I (Mahony and Gurdon, 1995). TGF-β can bind to ALK-1 (Attisano et al., 1993)but more weakly than to TβR-I (ten Dijke et al., 1994a). ALK-1 has been reportedmediate a TGF-β response (Attisano et al., 1993). ALK-2 is also known as ActR-I sinceit can bind to activin and mediate certain activin responses (Attisano et al., 1993;Yamashita et al., 1995a). ActR-I is a bit promiscuous since it also can binds BMP2 andBMP4 (Liu et al., 1995; ten Dijke et al., 1994b). In addition, the ActR-I mousehomologue can when overexpressed bind TGF-β (Ebner et al., 1993; ten Dijke et al.,1994a). It has also been proposed that ALK-2/ActR-I can function as an MIS/AMHtype I receptor (He et al., 1993).

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The type II receptor subfamily includes in vertebrates TβR-II, BMPR-II, and AMHR,which bind selectively to TGF-β (Lin et al., 1992), BMPs (Liu et al., 1995; Nohno et al.,1995; Rosenzweig et al., 1995) and MIS, respectively (Baarends et al., 1994; diClemente et al., 1994). ActR-II and -IIB can together with the BMP type I receptorsbind BMP2, BMP4 and BMP7 as well as GDF5 (Hoodless et al., 1996; Nishitoh et al.,1996; Yamashita et al., 1995a). In addition, ActR-II and -IIB can also associate withactivins, either alone or in concert with activin type I receptors (Attisano et al., 1992;Mathews and Vale, 1991; Mathews et al., 1992).

The type I receptor contains a unique region, known as the GS-domain (Wrana et al.,1994). It has been shown that ligand-induced phosphorylation of TβR-I by the type IIreceptor takes place on serines and threonines in a TTSGSGSG sequence in the GS-domain. This is important for receptor activation and signalling (Souchelnytskyi et al.,1996; Wieser et al., 1995; Wrana et al., 1994).

Type III/Endoglin receptorsLigand crosslinking experiments have identified an additional class of accessoryreceptors, the type III receptors. These consists of two related proteins, betaglycan andendoglin (Gougos and Letarte, 1990; Wang et al., 1991). Since these receptors lackintrinsic signalling activity they are believed to regulate the TGF-β access to thesignalling receptors. Betaglycan has been reported to bind to all three TGF-β ligandswith high affinity (Cheifetz and Massagué, 1991; Segarini et al., 1989), and to facilitateligand binding to the type II receptor (Lopez-Casillas et al., 1993; Wang et al., 1991). Incontrast, endoglin has been shown to bind to TGF-β1 and -β3, but not to TGF-β2(Cheifetz et al., 1992).

TGF-β superfamily intracellular signalling

The signal initiated by the TGF-β superfamily ligands is transduced by type I and typeII serine/threonine kinase receptors into the intracellular space. The ligands bind type IIreceptor, forming a heterodimeric complex which can recruit and activate the type Ireceptor by phoshorylating serine and threonine residues located primarily in the GS-domain (Souchelnytskyi et al., 1996; Wrana et al., 1994). Ligands such as TGF-β1,TGF-β3, and activins have been shown to associate to the receptors through sequentialbinding (Massague, 1998). In contrast, TGF-β2 (Rodriguez et al., 1995), as well asBMP2 and BMP7, have affinity for both type I and II receptors, and associate with thereceptor complex through co-operative binding (Massague, 1998). The activatedreceptor can recruit downstream signalling molecules, known as Smads (figure 2).

The Smad family of signal transducersIn a genetic screen looking for enhancers of a weak decapentaplegic (dpp) maternalphenotype in Drosophila, a new gene mad (mothers against dpp) was isolated (Rafteryet al., 1995; Sekelsky et al., 1995). This was followed by the discovery of three Mad-

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homologues: sma-2, sma-3 and sma-4 in C. Elegans (Savage et al., 1996). Mutations ofthese sma genes resembled the small body sized phenotype observed in the Daf4mutants (type II serine/threonine receptor) of C. Elegans. The vertebrate homologues ofthe mad- and sma-genes are called smads. The proteins derived from these genes can bedivided into three different subclasses (figure 2), i) receptor activated Smads (R-Smads), ii) common mediator Smads (Co-Smads), and iii) inhibitory Smads (I-Smads)depending on their diverse roles in signalling. The N-terminal and C-terminal regions ofthe R-Smads and the Co-Smads display a great deal of homology and have beendesignated the Mad homology 1 (MH1) and Mad homology 2 (MH2) domainsrespectively. A proline-rich linker domain exists between the MH1 and MH2 domain.The R-Smads, but not the Co- or I-Smads, contain a C-terminal SSXS motif whichbecomes phosphorylated upon receptor interaction (reviewed in Heldin et al., 1997).

ActR-IIAActR-IIB

BMPR-IIActR-IIAActR-IIB

TGF-β Activin BMP

TβRII

TβRI/ALK-5 ALK-1

?

Smad1Smad5Smad8

ALK-4/ActR-IB

Smad2Smad3

ALK-2/ActR-IALK-3/BMPR-IAALK-6/BPMR-IB

Smad2Smad3

Smad1Smad5Smad8

Smad4 Smad4 Smad4 Smad4

Smad6Smad7

Smad6Smad7

Smad6Smad7

Smad6Smad7

TGF-β superfamily

Type-II R

Type-I R

R-Smads

Co-Smads

InhibitorySmads

Biologicalresponses

Inhibition of mitogenicityInduction of extracellular matrix

Induction of dorsal mesoderm, erythroid differentiation,and FSH-release

Induction of ventral mesoderm,cartilage and bone,and apoptosis

Figure 2. TGF-β superfamily members, their signalling molecules and some biologicalresponses. Adapted from Heldin et al., 1997.

The R-Smads Smad2 and Smad3 mediate signals from TGF-β and activin ligandsthrough the TβR-I/Alk-5 and ActR-IB receptors, respectively (see Figure 2) (Eppert etal., 1996; Macias-Silva et al., 1996; Zhang et al., 1996). BMP signalling is mediatedthrough R-Smads 1, 5 and 8 which become phosphorylated and activated by the ActR-I,BMPR-IA or BMPR-IB receptors (Thomsen, 1996). The determinant of specificity

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between the R-Smads and their interaction with either TGF-β/activin or BMP receptors,is the L3 loop region within the MH2 domain (Lo et al., 1998). However, some reportssuggest that Smad1, 5, and 8 might be promiscous towards the TGF-β receptors as well(Lux et al., 1999; Macias-Silva et al., 1998; Oh et al., 2000). The Co-Smad, Smad4(also known as DPC4, deleted in pancreatic carcinomas), appears to play a critical rolein both BMP- and TGF-β/activin-mediated pathways.

The affinity of Smad4 for R-Smads can be increased through phosphorylation of the C-terminal part of R-Smads leading to the formation of a complex (Souchelnytskyi et al.,1997). Moreover, Smad4 has a unique Smad activation domain (SAD) in the linkerregion, which governs transcriptional activation via the co-activator p300 (deCaestecker et al., 2000). One Co-Smad has been identified in mammals, but there mightbe others. Two Co-Smads have been found in Xenopus laevis (Howell et al., 1999;Masuyama et al., 1999).

The Smad pathwayUpon receptor activation Smad molecules can be recruited to the receptor complex(figure 3). It has been shown that microtubules play an important role in the guiding ofSmads to the plasma membrane (Dong et al., 2000), where a FYVE-domain protein,Smad anchor for receptor activation (SARA), presents the R-Smads to the receptor(Tsukazaki et al., 1998). The FYVE-domain is a protein structure that in other proteinshas been associated with anchoring to endosomes (Tsukazaki et al., 1998). This hasraised the possibility that the receptor needs to be internalised before it can bind theSARA-sequestered Smad proteins. Besides restraining the movement of Smads it seemsas if SARA also masks a region of Smad2 that otherwise mediates nuclear import (Xu etal., 2000). While receptor-mediated phosphorylation and activation of R-Smadsincreases the affinity for Smad4 it decreases the affinity for SARA at the same time (Xuet al., 2000).

The R-Smads are presented to the receptor complex and become activated byphosphorylation on the C-terminal SSXS motif (Heldin et al., 1997). The L45 loop, adistinct region in the type I receptor containing two β strands (number 4 and 5) flankingthe kinase domain, has been reported to determine the association of Smad molecules tothe receptor (Feng and Derynck, 1997). To underscore the importance of the L45 loopin determination of specificity, it has been shown that swapping of the L45 loop ofTβR-I and BMPR-IB also alters Smad1 and Smad2 recognition, subsequently leadingto switched transcriptional response (Chen et al., 1998; Persson et al., 1998). Afterreceptor activation, the R-Smads oligomerise with the Co-Smad, forming a heteromericcomplex that is translocated to the nucleus where it regulates transcription of targetgenes (Heldin et al., 1997). Smad3 and Smad4 have been shown to interact directly withspecific DNA sequences via their MH1 domain (Dennler et al., 1998; Vindevoghel etal., 1998; Yingling et al., 1997). In order to fully activate transcription of the targetpromoters, the Smad complexes must recruit additional factors, like the transcriptionfactor components AP-1 (Liberati et al., 1999), DNA-binding adaptors like FAST-1(Chen et al., 1996), or co-activators such as CBP/p300 (Feng et al., 1998; Janknecht et

16

al., 1998; Nishihara et al., 1998; Shen et al., 1998; Topper et al., 1998). Thistransactivating role of the Smad proteins has been ascribed to the MH2 domain(reviewed by Massagué and Wotton, 2000).

R-SMAD

Co-SMAD

Nucleus

Ras

Erk

SNIP1

COREPRESSORS

TGIFSki/SnoN TGF-β

CO-ACTIVATORSP300CBP

SIGNALLINGRECEPTORS

ACCESSORY RECEPTORS

III

III

LIGANDS

DNA-BINDING COFACTORS

BetaglycanEndoglinCrypto

LEF1/TCFCBFA1CBFA3

JUN

β-cateninSMAD1SMAD2

JNK

BMPTGF-βTNF-αIFN-γEGF

SMAD1SMAD2NFκBSTAT

Co-SMAD

R-SMAD

CO

FAC

TO

R

BAMBISMURF1

SMAD7SMAD6

WntBMPTGF-βTNF-α

TGF-β superfamily

Figure 3. Schematic overview of the transforming growth factor-β (TGF-β) signallingpathway. Adapted from Massagué et al., 2000.

To date, two I-Smads have been identified in mammals, Smad6 and Smad7 (Imamura etal., 1997; Nakao et al., 1997; Topper et al., 1997). They have been characterised asinhibitors of TGF-β/activin and BMP signalling and have been proposed to function innegative feed-back loops, since their expression is induced by TGF-β/activin and BMP-members (Christian and Nakayama, 1999). It has been shown that I-Smads can interactstably with the type I receptor and block further activation of R-Smads (Imamura et al.,1997; Nakao et al., 1997; Souchelnytskyi et al., 1998). For Smad6 an additionalmechanism has been suggested, where Smad6 competes with Smad4 for binding toSmad1, thereby preventing the formation of a functional heteromeric Smad1/Smad4complex (Hata et al., 1998). Smad7 is considered as a general inhibitor of TGF-βsuperfamily-induced responses, whereas Smad6 is thought to preferentially block BMP-mediated signalling (Itoh et al., 1998), although this is controversial (Imamura et al.,1997).

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Self-regulation of the TGF-β pathway through negative feedback has been reported tooccur through other mechanisms than antagonistic I-Smads. BMP has been shown toexert a negative feedback control via the protein BAMBI (BMP and Activin membrane-bound inhibitor) which is a truncated and kinase-deficient type I receptor that interfereswith activation by binding to the BMP receptors (Onichtchouk et al., 1999).

Smads are disposed from the cytoplasm and the nucleus by ubiquitination andproteasomal degradation. The cytoplasmic Smad1 and Smad2 become targeted byproteolysis via the ubiquitin-ligase proteins Smurf1 and Smurf2, respectively (Lin et al.,2000; Zhu et al., 1999). The nuclear Smad2 on the other hand, is degraded via theUbcH5 family of ubiquitin-conjugating proteins (Lo and Massagué, 1999).

Non-Smad pathwaysTo this date, only Smads are recognised as TGF-β receptor substrates and signaltransducers. However, numerous reports indicate that TGF-β and BMP are also able totransduce signals via the mitogen-activated protein kinase (MAPK) pathway (figure 4)(reviewed by Massagué and Wotton, 2000) TGF-β has been reported to activate the JunN-terminal kinase (JNK) via MKK4 (MAPK kinase 4) in a rapid fashion in afibrosarcoma cell line (Hocevar et al., 1999). In addition, p38 seems to be activated byTGF-β in lung and kidney epithelial cell lines via MKK3 (Hanafusa et al., 1999) leadingto activation of the nuclear target ATF2 (activating transcription factor 2) (Sano et al.,1999). The biochemical link between the TGF-β receptor and the MKKs is to this datestill unknown. One candidate could be TAK1 (TGF-β activated kinase), a member ofthe MAPK kinase kinase (MAPKKK) family, implicated in activation of p38 viaMKK3/MKK6 (Moriguchi et al., 1996) and activation of JNKs via MKK4 (Shirakabe etal., 1997). JNK signalling by TAK1 is also reported during Drosophila development(Takatsu et al., 2000). Other factors such as TAB1 and XIAP might be located furtherupstream in the signalling pathway affecting TAK1.

TAB1 is one of the proposed activators of TAK1 (Shibuya et al., 1996). Also, sinceinjection of TAB1- and TAK1-mRNA into the dorsal marginal zone of Xenopusinduced ventral mesoderm, TAB1 and TAK1 are suggested to play a functional role inBMP signalling during early Xenopus development (Shibuya et al., 1998).

XIAP, a positive regulator of BMP signalling, has been proposed as a link between theBMP receptors and TAB1-TAK1. XIAP can associate not only with TAB1, but alsowith BMP-receptors in mammalian cells. Furthermore injection of XIAP mRNA intodorsal blastomeres enhanced the ventralisation of Xenopus embryos in a TAB1-TAK1dependent manner (Yamaguchi et al., 1999).

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GRB2

IIIPlasma membrane

TGF-β

TGF-β receptor

Growth factor

Growth factorreceptor

P P

SOS

Nuclear membrane

Ubiquitinationand

degradation

R-SMAD

Co-SMAD

R-SMAD

CO

FAC

TO

R

Fos Jun AFT2

TGIF

DNA

P

MKK4 MKK3

JNK p38

RAS MEK

P

XIAP

TAK1

Cytokinereceptor

Cytokine

ERK

Figure 4. Cross-talk between Smads and the MAP kinase pathway. Adapted fromMassagué et al., 2000.

Cross-talk between Smads and the MAP kinase pathwayThe Erk-activated Ras pathway has been reported to modify the TGF-β signallingpathways at different levels (figure 4). It has been shown that a hyperactive Raspathway can downregulate TGF-β receptors in H-Ras transformed rat intestinalepithelial cells (Zhao and Buick, 1995). Moreover, it has been reported that oncogenicRas can inhibit BMP and TGF-β signalling negatively by decreasing the accumulationof R-Smads in the nucleus. It was observed that EGF and HGF could phosphorylateSmad1, Smad2 and Smad3 on MAPK and Erk consensus sites located in the linkerregion, which led to a retention of R-Smads in the cytoplasm and reducedtranscriptional activity (Kretzschmar et al., 1997a; Kretzschmar et al., 1999;Kretzschmar et al., 1997b). When mutations were introduced into the Erkphosphorylation sites of Smad3, it resulted in a Ras-resistant form that could rescue thegrowth inhibitory response of TGF-β in Ras-transformed cells. Moreover, EGF induceda less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3, ascompared to oncogenic H-Ras, which might explain the silencing of anti-mitogenicTGF-β functions by hyperactive Ras in cancer cells (Kretzschmar et al., 1999).

The Erk/MAP kinase pathway has also been implicated in regulation of Smad mediatedtranscription at other levels than Smad retention. It has been reported that the

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transcriptional co-repressor TGIF becomes upregulated in response to Erk signalling(Lo et al., 2001).

Specificity in Smad signallingHow can specificity in Smad signal transduction be achieved? It has been shown thatthe different branches of TGF-β/activin and BMP signalling can access distinct sets oftarget genes, via their respective down-stream modulators such as Smad1 and Smad 2,(Chen et al., 1998). An example of the opposing and somewhat complementary rolesbetween Smad1- and Smad2-dependent pathways, is demonstrated in Xenopous laevisembryogenesis. Smad2 can, in response to Nodal related factors (Xnr-1, Xnr-2 andVg1), activate genes responsible for induction of dorsal mesoderm. In contrast Smad1,in response to BMP4, activates a different set of genes that affect induction of ventralmesoderm and suppression of neural fates. Ectopic expression of Smad1 or Smad2induces of ventral and dorsal structures, respectively (Baker and Harland, 1996; Graff etal., 1996). In a similar complementary fashion, BMP and Nodal dictate the specificationof left-right symmetry in the developing vertebrate embryo (Rodriguez Esteban et al.,1999; Saijoh et al., 2000; Yokouchi et al., 1999).

As transcription factors, R- and Co-Smad complexes are able to recognise and interactwith distinct DNA sequences, e.g. CAGAC, GTCTAGAC, and other GC richsequences. This interaction is rather weak which suggests that the Smads need theassistance of other proteins for correct binding (Shi et al., 1998). Since the pentamericCAGAC DNA-binding motif statistically occurs rather frequently in the genome, a highaffinity of the Smads to their recognition sequence would result in a rather general andunspecific association with DNA. Other factors are therefore required for increasedspecificity and target selection such as co-factors (figure 3). The co-factors arestructurally diverse proteins that share the ability to associate with Smad molecules anda neighbouring DNA sequence (reviewed (Massagué and Wotton, 2000)). Cell typespecific responses could be explained by the fact that certain co-factor combinations areexpressed in distinct cells or tissues (Chen et al., 1997; Hata et al., 2000).

One group of co-factors can be described as adaptors for the DNA-Smad interaction. Toachieve DNA binding Smad1 has been shown to bind to OAZ (olf-associated zincfinger) (Hata et al., 2000), while Smad2 binds FAST (forkhead activin signaltransducer) (Chen et al., 1996) and Mixer (Germain et al., 2000). The adaptor proteinsOAZ, FAST, and Mixer lack intrinsic transcriptional activity. The recognition betweenthe Smad molecule and the adaptor protein requires a bulging alpha-helix 2 region ofthe MH2 domain in the Smad proteins and a Smad-interaction domain, which isconserved in FAST and Mixer but not in OAZ (Chen et al., 1998; Germain et al., 2000;Hata et al., 2000).

Transcription factors are another group of proteins that can form functional complexeswith Smad molecules (figure 3). Examples are JunB (Zhang et al., 1998), TFE3(transcription factor binding to immunoglobulin heavy constant mu enhancer 3) (Hua etal., 1999), core-binding factor A/acute myeloneus leukaemia (CBFA/AML) proteins

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(Hanai et al., 1999; Pardali et al., 2000; Tsuji et al., 1998) and lymphoid enhancer-binding factor 1/T-cell specific factor (LEF1/TCF) (Labbe et al., 2000; Nishita et al.,2000). LEF1/TCF is the mediator of WNT/β-catenin signalling and is able to co-operatewith Smads in the activation of Xtwn (Xenopous twin) in response to Nodal relatedsignals (Nishita et al., 2000).

Smads are able to recruit transcriptional repressors as well as activators (figure 3).Smads can associate with repressors such as TG3-interacting factor (TGIF) (Wotton etal., 1999), Sloan-Kettering Institute proto-oncogene (Ski) (Luo et al., 1999) and Ski-related novel gene N (SnoN) (Sun et al., 1999). The repressors can bind histonedeacetylases (HDAC) which are generally implicated in chromatin condensation andtranscriptional silencing. The HDAC binding would counteract the effect of histoneacetyltransferase (HAT) activity associated with the co-activators (figure 3) CBP andp300, and transcriptional activation (Massagué and Wotton, 2000). The relative levelsof repressor versus co-activator could determine the final outcome of transcription. Forexample, it has been reported that the repressor TGIF can associate with Smad2 andSmad3 in competition with the co-activator p300 (Wotton et al., 1999).

Genetic targetingThe functional importance of TGF-β superfamily members in embryonic patterning andtissue homeostasis has been assessed by gene ablation experiments in mice (Table 1-3).They revealed a plethora of effects that range from defective vasculogenesis,inflammation, autoimmunity, and skeletal abnormalities to early malformationsaffecting egg cylinder formation, gastrulation and mesoderm formation. Interestingly,Smad3 deficient mice developed metastatic colorectal cancer at 4-6 months of age(Table 3) (Zhu et al., 1998), highlighting the potential role of TGF-β as a potent tumoursuppressor. This is a complex issue, since other groups have reported phenotypicalvariations for the same targeted genes, perhaps revealing the importance of geneticstrain background in determining the phenotypic outcome of specific null mutants.

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Table 1. Major defects in TGF-β superfamily ligand-deficient mice. Adapted fromGoumans and Mummery, 2000.

Targeted gene Phenotype ReferencesTGF-β1

TGF-β2

TGF-β3

- Defective yolk sac vasculogenesis andhaematopoiesis. Embryonic lethal (E9.5-11.5)

- Inflammation and autoimmunity

- Cardiac, lung, craniofacial, limb, spinalcolumn, eye, inner ear, urogenitaldefects. Perinatal lethality. - Cleft palate, delayed lung maturation.Perinatal lethality.

(Dickson et al., 1995)

(Shull et al., 1992)(Kulkarni et al., 1993)(Letterio and Roberts,1996)(Sanford et al., 1997)

(Proetzel et al., 1995)(Kaartinen et al., 1995)

BMP-2

BMP-4

BMP-7

- Embryonic lethal (E7.5-10.5)- Failure of proamniotic canal to close,heart malformation.- Embryonic lethal (E7.5-9.5)- 1. Arrest at egg cylinder stage, lack ofmesoderm.- 2. Develop until early somite stage w.disorganised/truncated posteriorstructures and reduced extra-embryonicmesoderm.- Defects in primordial germ cells andallantois formation.- Skull, eye and kidney defects.- Perinatal lethality

(Zhang and Bradley,1996)

(Winnier et al., 1995)

(Lawson et al., 1999)

(Dudley et al., 1995)(Luo et al., 1995)

Activin βA

Activin βB

Activin βA/βB

- Cleft pallet, lack of whiskers andincisors.- Failure of eyelid fusion. Females showimpaired reproductive ability.- No additional defects.

(Matzuk, 1995) (Schrewe et al., 1994)(Vassalli et al., 1994)(Matzuk, 1995)

Nodal - Embryonic lethal (E7.5). Failure ingastrulation and primitive streakformation

(Conlon et al., 1994)

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Table 2. Major defects in TGF-β superfamily receptor-deficient mice. Adapted fromGoumans and Mummery, 2000.

Targeted gene Phenotype ReferencesALK-1

ALK-2

ALK-3

ALK-4

ALK-5

ALK-6

- Embryonic lethal (E10.5-11.5).- Defects in angiogenesis and vascularsmooth muscle cell differentiation.- Embryonic lethal (E7.5-9.5)- Failure in primitive streak elongation,delayed mesoderm formation andmalformed visceral endoderm. - Embryonic lethal (E7.5-9.5)- Fail to form mesoderm, reducedproliferation of the epiblast.- Embryonic lethal (E7.5-9.5)- Defect in epiblast and extraembryonicectoderm organisation and gastrulation.- Embryonic lethal around E 10.5. - Severe defects in vascular developmentof placenta and yolk sac.- Intact haematopoietic potential ofprecursors.- Defects in endothelial cell proliferation,migration and fibronectin production.- Defects in digit formation and fore- andhindlimb development

(Oh et al., 2000)

(Gu et al., 1999)

(Mishina et al., 1995)

(Gu et al., 1998)

(Larsson et al., 2001)

(Yi et al., 2000)(Baur et al., 2000)

TβRII

ActR-IIA

ActR-IIB

ActR-IIA/ActR-IIb

- Embryonic lethal (E10)- Defective yolk sac vasculogenesis.- Skeletal and facial abnormalities inpercentage of mice.- Perinatal lethality- Cardiac defects associated with defectsin left-right asymmetry- Homeotic transformation of theskeleton - Embryonic lethal- Defect in primitive streak formationand gastrulation.

(Oshima et al., 1996)

(Matzuk, 1995)

(Oh and Li, 1997)

(Song et al., 1999)

Endoglin -Embryonic lethal (E10.5-11.5). -Defective yolk sac vasculogenesis,embryonic angiogenesis and vascularsmooth muscle cell development. - Cardiac malformations.

(Arthur et al., 1999)(Li et al., 1999)

(Bourdeau et al., 1999)

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Table 3. Major defects in Smad-deficient mice. Adapted from Goumans and Mummery,2000.

Targeted gene Phenotype ReferencesSmad1

Smad2

Smad3

Smad4

Smad5

Smad6

-Embryonic lethal (E9.5) - Failure in establishing chorion-allantoiccirculation. - Embryonic lethal (E7.5-8.5)- Failure in egg cylinder elongation,gastrulation and mesoderm formation.- Metastatic colorectal cancer (4-6months of age)- Impaired immunity and chronic infection.

- Accelerated wound healing.- Embryonic lethal (E7.5-8.5)- Growth retardation, no mesodermformation, abnormal visceral endoderm.- Embryonic lethal (E9.5-10.5)- Defect in angiogenesis, left/rightasymmetry, craniofascial abnormalitiesand induced mesenchymal apoptosis.- Cardiovascular abnormalities.- Defect in endocardial cushiontransformation

(Lechleider et al.,2001)

(Weinstein et al., 1998)(Nomura and Li, 1998)(Waldrip et al., 1998)(Zhu et al., 1998)

(Datto et al., 1999)(Yang et al., 1999b)

(Ashcroft et al., 1999)(Yang et al., 1998)(Sirard et al., 1998)

(Chang et al.,1999),(Chang et al.,2000)(Yang et al., 1999a)(Galvin et al., 2000)

SMAD7

Two research groups, using two different experimental approaches, discovered Smad7.In a screening of Smad homologues in mouse EST and human cDNA libraries, Nakao etal. identified a protein of 426 amino acids and termed it Smad7 (Nakao et al., 1997).Topper et al. using a differential display approach identified Smad6 and Smad7 as novelgenes upregulated in response to laminar vascular flow-stress in human endothelial cells(Topper et al., 1997).

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Smad7 signalling

Smad7 has been proposed to function as an antagonist of TGF-β signalling. Nakao et al.observed that Smad7 could block responses initiated by TGF-β1 in Mv1Lu- and HaCaTcells. They also demonstrated that injection of Smad7 mRNA in a Xenopous embryoblocked the activin/TGF-β signalling. Moreover, ectopic expression of Smad7 resultedin a failure to form head and tail structures and showed a negative effect on theformation of mesodermal derivatives such as muscle and notochord (Nakao et al.,1997). This mimicked the effect of a dominant negative form of the activin receptor(Hemmati-Brivanlou and Melton, 1992), or dominant negative Smad4 (Lagna et al.,1996). The expression of Brachyury, a mesodermal marker gene, was alsodownregulated by Smad7 injection in the blastomere of two-cell Xenopus embryos(Nakao et al., 1997). Taken together, these data underscored the possible function ofSmad7 as a possible attenuator of TGF-β/activin- mediated signalling, both in vivo andin vitro.

Based on the observations that TGF-β mediated phosphorylation of Smad2 and Smad3is blocked in Smad7-transfected cells and that overexpressed Smad7 associates with theTGF-β type I receptor in COS cells, a molecular mechanism was proposed stipulatingthat the inhibitory effect resides in the ability of Smad7 to compete with R-Smads forthe type I receptor (Hayashi et al., 1997; Nakao et al., 1997).

An additional Smad7 function was proposed by Kavsak et al. where Smad7 can targetthe TGF-β receptor for proteasomal and lysosomal degradation, through the interactionwith an ubiquitin ligase, Smurf2. In addition, they also observed that the associationbetween Smurf2 and Smad7 induced a translocation of Smurf2 from nucleus to theactivated TGF-β receptor. This Smurf2/Smad7-complex formation and TGF-β receptorturnover could be further enhanced by interferon-γ (IFN-γ) (Kavsak et al., 2000).

On a similar note, Ebisawa et al. observed that Smurf1, an E3 ubiquitin ligase for BMP-specific Smads, also could bind Smad7 and thereby induce TGF-β receptor degradation(Ebisawa et al., 2001). Moreover, it has been demonstrated that STRAP, a WD40 repeatprotein could associate with both TβR-I and TβR-II (Datta et al., 1998). This stabilisedthe interaction between Smad7 and the activated receptor, thereby assisting in Smad7inhibition of TGF-β signalling (Datta et al., 1998; Datta and Moses, 2000).

Based on observations that Smad7 mRNA expression could be induced by TGF-βstimulation, Smad7 has been proposed to take part in a negative feedback loopdownregulating the TGF-β signal (Nakao et al., 1997). Additional pathways have beenshown to upregulate Smad7 transcription. Bitzer et al. reported that the mRNAexpression of Smad7 was upregulated via the NF-κB/RelA pathway by the pro-inflammatory molecules tumour necrosis factor-α and interleukin 1β (Bitzer et al.,2000). In addition, it was demonstrated that IFN-γ also could upregulate Smad7transcription via Jak1/Stat1 (Ulloa et al., 1999).

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I-Smads have conserved C-terminal Mad homology 2 (MH2) domains, whereas theamino acid sequences of their N-terminal regions (N-domain) are highly divergent, notonly between the Smad families but also between the I-Smads of different species.(Christian and Nakayama, 1999; Nakayama et al., 2000). Both the N- and C-terminalMH2-domains are important to obtain full activity of Smad6 and Smad7 in Xenopus(Nakayama et al., 2001). The N-domain and MH2-domain can bind to each other, whichfacilitates the interaction between Smad7 and TGF-β receptors and thereby enhancesthe inhibitory effect of Smad7/MH2-complex (Hanyu et al., 2001).

Smad7 has been suggested to exert other functional roles distinct from the antagonisticeffect in receptor-mediated Smad activation. It has been reported that overexpressedSmad7 is predominantly located to the nucleus in the absence of ligand and becomestranslocated to the cytoplasm in response to TGF-β stimulation (Itoh et al., 1998).Moreover, Pulaski et al., observed that phosphorylation of Smad7 at serine residue 249could affect Smad7-dependent transcriptional activation on an SV40 minimal promoter(using the GAL4 DNA binding domain fused to Smad7) (Pulaski et al., 2001). Inaddition, it has been proposed that I-Smads can act as transcriptional co-repressors byrecruiting histone deacetylases (HDACs) (Bai and Cao, 2002; Bai et al., 2000).

Smad7 in physiological and pathological conditions

VasculogenesisThe developing vascular system of the early embryo originates from a small populationof mesodermal endothelial precursor cells. These cells join to form a capillary plexusthat is gradually remodelled by the development of distinct arterial-venous parts and therecruitment of mural cells such as capillary-associated pericytes and vascular smoothmuscle cells (VSMC). These structures constitute the basis for future angiogenesisusing existing vessels to form new ones (Daniel and Abrahamson, 2000; Hungerfordand Little, 1999; Risau, 1997; Yancopoulos et al., 2000). A multitude of TGF-βsuperfamily ligands, receptors and signalling molecules have been implicated in thedevelopment of the vascular system (Gatherer et al., 1990; Pelton et al., 1990; Pelton etal., 1989; Roberts and Sporn, 1992; Schmid et al., 1991). Several in vitro studiessuggest that TGF-β can regulate the growth of endothelial cells as well as influencetheir migration and fusion into capillary tubes. In addition, TGF-β can also affect vessellumen size (Gajdusek et al., 1993; Madri et al., 1988; Merwin et al., 1990; Pepper,1997; Pepper et al., 1990; Pepper et al., 1993; Roberts and Sporn, 1989). However, thebehaviour of the endothelial cell in response to TGF-β stimulation, varies greatlybetween experimental set ups, making functional assessments difficult (Daniel andAbrahamson, 2000; Klagsbrun and D'Amore, 1991). A different approach used toinvestigate the function of individual genes in vasculogenesis in vivo is gene targeting.Such studies have revealed that activin receptor-like kinase 1 (ALK1) a TGF-β/activinbinding receptor, endoglin (Eng) a TGF-β superfamily co-receptor, and Smad5 areexamples of proteins necessary for normal angiogenesis. Null mutants lacking either ofthese three gene functions display fragile, haemorrhagic and dilated vessels. In addition,

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extracellular matrix remodelling enzymes have abnormal expression levels and therecruitment of vascular smooth muscle cells (VSMC) to the vascular endothelium isimpaired (Chang et al., 1999; Li et al., 1999; Oh et al., 2000; Pepper, 1997; Urness etal., 2000; Yang et al., 1999b). Moreover, ALK1 null mutant mice display anarteriovenous malformation phenotype, characterised by the development of shuntsbetween the arterial and venous circulatory systems (Urness et al., 2000). Interestingly,these phenotypes resemble hereditary haemorrhagic telangiectasia, a humanpathological condition associated to haploinsufficiency of ALK1 or Eng (Shovlin andLetarte, 1999).

What is the role of TGF-β signalling in the developing vascular system? Recent modelssuggest that the primary function is to promote VSMC recruitment and differentiationwhile inhibiting endothelial proliferation through VSMC-endothelial interactions.However, non-TGF-β superfamily (e.g. angiopoeitins, PDGFs) signalling mutants alsoexhibit vessel dilations, fragility and haemorrhage associated with disturbed VSMC-endothelial interactions (Puri et al., 1995; Sato et al., 1995; Suri et al., 1996; Vikkula etal., 1996). It is therefore not clear to what extent loss of VSMCs, vessel dilation andreduced vessel integrity is a direct consequence of impaired TGF-β signalling or if it isa secondary effect caused by other disturbed TGF-β dependent functions (Folkman andD'Amore, 1996; Li et al., 1999; Oh et al., 2000).

Smad6 and Smad7 were originally identified as genes induced in vascular endotheliumin response to steady laminar shear stress, a physiologic biomechanical stimulus. Thissystem mimicking the effect of blood flowing past the endothelial cells, suggested animportant role in the maintenance of the vascular endothelium in the adult organism.But could these Smads also play a role in the developing endothelium? Zwijsen et al.investigated the expression of Smad7 mRNA in early mouse development by RT-PCR.They found that Smad7 was indeed upregulated in the developing vascular system ofthe mouse embryo, especially in endothelial cells of larger blood vessels, possibly as aresult of a larger blood flow. In addition, they observed a very early Smad 7 expressionduring preimplantation and gastrulation. Furthermore, overexpression of Smad7 in themouse zygote inhibited development at the 2-cell stage (Zwijsen et al., 2000).

In an approach that resembled the inhibition of TGF-β signals modulating angiogenesisand vasculogenesis, Vargesson et al. virally misexpressed Smad7 in the developingchick limb and head. They found that the larger vessels became dilated and frequentlydeveloped arteriovenous shunts similar to arteriovenous malformations (AVM).Expression of constitutively active BMP receptor could counteract the effect of Smad7overexpression, suggesting that a BMP-like pathway in contrast to TGF-β signallingwas the target of Smad7 inhibition. Moreover, Smad7 overexpressing vessels were nothaemorrhagic and had a normal structure. In addition, the dilation of vessels wasindependent of VSMCs and the recruitment of VSMCs was not affected. Takentogether, these findings suggest that the TGF-β pathway regulates vessel calibre and isnecessary for correct vessel connectivity in this experimental setting. They also indicatethat vessel dilation not necessarily must lead to vessel rupture and haemorrhages.Furthermore, the findings suggest that a VSMC coat is not a prerequisite for vessel

27

maintenance and that TGF-β signalling is not involved in VSMC recruitment anddifferentiation (Vargesson and Laufer, 2001). Another study suggests that Smad7overexpression can attenuate the growth inhibitory effects of TGF-β on cultured ratsmooth muscle cells (Kato et al., 2001).

Lung morphogenesisTGF-β is involved in the negative regulation of lung growth and development duringearly lung organogenesis (Warburton and Lee, 1999). It had been shown that receptorregulated Smad2 and Smad3 were required for the TGF-β mediated inhibition ofembryonic lung branching morphogenesis and epithelial cell differentiation (Zhao et al.,1998). The role of Smad7 in lung morphogenesis was investigated by introducingSmad7 antisense oligonucleotides in cellular embryonic lung explants. There it wasdemonstrated that TGF-β mediated inhibition of lung branching was significantlyincreased in cells with abrogated Smad7 gene expression. In addition, it was observedby immunohistochemistry that Smad7 together with Smad2 and Smad3 co-localised indistal bronchial epithelial cells (Zhao et al., 2000a). A different approach to assess therole of Smad7 in lung morphogenesis, used adenoviral Smad7 overexpression. Theobservation was that Smad7, but not Smad6, could abolish TGF-β mediated branchinginhibition. In addition, Smad7 also inhibited TGF-β induced down regulation ofsurfactant protein C, a bronchial epithelial differentiation marker (Zhao et al., 2000b).

ApoptosisThe idea that Smad7 works as a modulator and attenuator of TGF-β mediated signallinghas gained acceptance over time. The fact that Smad7 can inhibit TGF-β signals alsosuggested that Smad7 might inhibit apoptosis initiated by TGF-β. Indeed, studies inmouse B- and T-cells, supported this theory and suggested that Smad7 could act as aninhibitor of activin-induced growth arrest and apoptosis (Ishisaki et al., 1998).Moreover, studies in WEHI 231 B-lymphocytes further corroborated this notion,showing that Smad7 could protect these cells from TGF-β induced apoptosis (Patil etal., 2000). Other reports suggested that the picture is much more complicated. Somestudies indicated that Smad7 by itself could work as an inducer of apoptosis. Transgenicmice overexpressing TGF-β1 under the control of the albumin promoter displayedincreased apoptosis associated with a depletion of podocytes in progressiveglomerulosclerosis (Schiffer et al., 2001). TGF-β1 and Smad7 both seemed able toinduce apoptosis, but through different pathways. The TGF-β mediated effect requiredthe activation of p38 MAP kinase and caspase-3, which was not required for Smad7-mediated apoptosis. In contrast to TGF-β, Smad7 was able to inhibit the nucleartranslocation and transcriptional activity of the cell survival-promoting factor, NF-kB(Schiffer et al., 2001). Additional evidence in prostatic carcinoma cells (PC-3U)suggested that Smad7 antisense RNA could inhibit TGF-β induced apoptosis(Landström et al., 2000). Moreover, overexpressing Smad7 alone using an induciblepromoter also resulted in apoptosis, implicating Smad7 itself as an effector of apoptosis(Landström et al., 2000).

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What signalling pathway could serve as the transducer of this Smad7 mediatedapoptosis? One candidate is the c-Jun N-terminal kinase (JNK) cascade. Mazars et al.demonstrated that expression of Smad7 caused a strong and sustained activation ofJNK. The use of a dominant-interfering mutant of mitogen-activated protein kinasekinase 4 (MKK4) completely abolished the Smad7-induced activation of JNK.Furthermore, expression of the mutant MKK4 also blocked the ability of Smad7 topromote cell death (Mazars et al., 2001).

FibrosisTGF-β is a potent regulator of extracellular matrix (ECM) by inducing proteins such ascollagen, fibronectin but also PAI-1 and TIMP. It is believed that a sustainedoverproduction of TGF-β caused by repeated chemical and or biological injury canresult in pathological amounts of ECM being collected in the tissue leading to afunctional deterioration (Border and Noble, 1994). TGF-β has been implicated in manyfibrotic disorders such as idiopathic pulmonary fibrosis (Broekelmann et al., 1991),autoimmune lung diseases (Deguchi, 1992), and bleomycin-induced lung fibrosis inanimal models (Khalil et al., 1989; Khalil et al., 1993; Westergren-Thorsson et al.,1993; Zhang et al., 1995). TGF-β was found to be necessary for the bleomycin-inducedtissue fibrosis in rodents (Giri et al., 1993). Based on these findings, Nakao et al.introduced Smad7 and Smad6 by adenoviral transient gene transfer into bleomycintreated mice. They observed that Smad7, but not Smad6, was able to reduce expressionof type I precollagen mRNA and abolish the morphological fibrotic responses intransgenic mice. In addition, the phospho-Smad2 immunoreactivity was reduced inbleomycin treated Smad7 transgenic mice, suggesting inhibition of the TGF-β/Smadpathway (Nakao et al., 1999).

Immunological disordersTGF-β is a powerful modulator of immune responses. It affects the differentiation,proliferation and state of activation of all immune cells. The dysregulation of TGF-βsignalling has been implicated in immune abnormalities related to autoimmunity,opportunistic infections and fibrotic complications (Letterio and Roberts, 1998). Studiesin murine models clearly underscore the connection between disrupted TGF-βsignalling and inflammatory disease. Deletion of the TGF-β1 gene in mice results insystemic inflammation and early death (Shull et al., 1992). In addition, overexpressionof a dominant negative TGF-β type II receptor gives rise to CD4+ T-cell hyperactivityand autoimmunity (Gorelik and Flavell, 2000). On a similar note, targeted disruption ofthe Smad3 gene resulted in inflammation of mucosal surfaces (Yang et al., 1999c).Given the strong immunosuppressive role of TGF-β1, what would the effect be if aTGF-β signalling inhibitor like Smad7 was overexpressed in immune cells? Nakao et al.studied the effect of Smad7 overexpression in peripheral T-cells in transgenic mice. Themutant T-cells were no longer growth inhibited by TGF-β but no overt phenotype wasobserved in the unchallenged transgenic mice. However, when subjected to antigen-

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induced airway inflammation the transgenic mice displayed an increased airwayreactivity and inflammation (Nakao et al., 2000). Despite the fact that this was anexample of a highly artificial system it can still give us some idea how Smad7 couldinfluence TGF-β signalling in vivo. Interestingly, a human disorder exists where theexpression levels of Smad7 are increased. Monteleone et al. (2001) demonstrated thatthe Smad7 expression was highly upregulated in the colon mucosa and in T-cellspurified from the colonic mucosa in patients with inflammatory bowel disease (IBD)and Crohns disease. The diseased tissue displayed reduced immunoreactivity forphospho-Smad3, indicating a reduced level of TGF-β signalling and responsiveness,despite an increased level of TGF-β ligand. When subjecting cells derived from IBDpatient tissue to antisense Smad7 oligonucleotides, they showed a reduction in Smad7protein expression. More importantly, silencing of Smad7 not only restored the abilityto respond to exogenous TGF-β, but lead also to reduced mRNA levels of the pro-inflammatory cytokines IFN-γ and TNF-α, the major counterplayers of TGF-β in theregulation of the inflammation status. This resulted in a TGF-β mediated inhibition ofIFN-γ and TNF-α production and suppression of inflammation (Monteleone et al.,2001).

CarcinogenesisHuman Smad7 has been mapped to the chromosomal region 18q21, between the Mad-Related-2 (MADR2) and Deleted in Pancreatic Cancer-4 (DPC4) locus. MADR2 andDPC4 encode the Smad2 and Smad4 proteins, respectively (Boulay et al., 2001; Röijeret al., 1998). Deletions in the 18q21 region are the most common genetic variationsobserved in colorectal carcinoma. An analysis of the gene copy number from colorectaltumour biopsies revealed that Smad2 or Smad4 were more often deleted than Smad7.On the contrary, Smad7 appeared to be more often amplified than the other genes(Boulay et al., 2001). The most common genetic alteration of Smad genes in the tumoursamples were simultaneous deletions of Smad2 and Smad4 while Smad7 was ofteneither retained by normal diploidy or amplified (Boulay et al., 2001).

Mutational analyses performed in other tumour types such as hepatocellular carcinoma,ovarian cancers and pancreatic cancer made it seem unlikely that deletion or duplicationof the Smad7 gene would influence cancer progression (Jonson et al., 1999; Wang et al.,2000; Kawate et al., 2001). Kleeff et al. (1999) however, claims that Smad7 canenhance tumourigenicity in pancreatic cancer. They observed increased Smad7 mRNAlevels in human pancreatic cancer. When they transfected COLO-357 human pancreaticcancer cells with Smad7, the cells lost their growth inhibitory response to TGF-β1. Inaddition these transfected cells showed increased anchorage-independent growth andaccelerated growth in nude mice (Kleeff et al., 1999).

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PRESENT INVESTIGATIONS

Smad expression in normal and malignant prostate (paper I)

The prostate gland is dependent on androgens for its growth and differentiation. It hasbeen observed that androgen ablation (i.e. castration) leads to regression and apoptosis(Kerr et al., 1972) in the rat ventral prostate (Kyprianou and Isaacs, 1988) as well as inPC-82 human prostate cancer (Kyprianou and Isaacs, 1988). Additional studies haveimplicated TGF-β as an inducer of apoptosis in the normal prostate as well as inmalignant prostatic carcinoma epithelial cells (Hsing et al., 1996; Kyprianou and Isaacs,1989; Landström et al., 1994; Rajah et al., 1997). Moreover, castration and, inparticular, castration in combination with estrogen treatment in a rat prostaticadenocarcinoma model caused an increased protein expression of TGF-β1, as well as ofTGF-β receptors (Landström et al., 1996). Taken together, these observations suggesteda role for TGF-β in the induction of programmed cell death in the normal and androgen-sensitive malignant prostate. However, the molecular mechanism behind such a TGF-βmediated apoptosis remained poorly understood.

We wanted to investigate a possible functional role for Smad proteins, the transducersof the TGF-β signal, in the normal and malignant prostate after castration. To addressthis issue, we examined the immunoreactivity of receptor-activated Smads (1, 2, 3 and5), the common mediator Smad4 and inhibitory Smads (6 and 7), in the rat ventralprostate and in an androgen and estrogen sensitive prostate tumour model (DunningR3327 PAP) (Isaacs, 1987). In parallel, we performed TUNEL-staining to assess thelevel and localisation of apoptotic cells within the two prostate models, which allowedus to correlate the Smad expression profile with the presence of apoptotic cells.

We observed increased expression levels of the Smads involved in TGF-β signaltransduction (i.e. Smad2, Smad3 and Smad4) in the epithelial cells of the normalprostate after castration, as well as in the Dunning tumour cells. Furthermore, in prostateepithelial cells after castration, we demonstrated an increased activation of Smad2 asdetected by antisera specific for phosphorylated Smad2 (Chen et al., 1998; Persson etal., 1998; Piek et al., 1999). These elevated levels of phosphorylated Smad2 were alsoobserved in the Dunning tumour after castration, but were less prominent. Interestingly,the expression of Smad2 was very low in the Dunning tumour cells prior to castration,in contrast to the rat ventral prostate, where the Smad2 levels were elevated beforecastration and remained high after androgen ablation. Smad3 immunostaining was alsoelevated in response to castration, both in normal and to a lesser degree in malignantprostate. Interestingly, in the rat ventral prostate Smad4 expression first increased aftercastration, only to become sharply reduced at later time points. The expression levels ofinhibitory Smad6 and Smad7 were low in the Dunning tumour when compared withnormal prostate, but increased significantly after treatment.

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There was a significant increase of the number of apoptotic cells in the normal andmalignant prostate in response to castration. Estrogen treatment of rats transplanted withthe Dunning tumours had an additive effect on castration-induced apoptosis.Interestingly, we were able to correlate areas of apoptotic cells with increased Smadprotein expression.

In summary, we observed elevated protein expression levels for Smad2, Smad3, andSmad4, increased activation of Smad2, as well as elevated protein expression levels ofinhibitory Smads in normal prostatic epithelial cells and, to some extent also in themalignant prostatic epithelial cells, after castration. These observations lend credence tothe notion that there is a correlation between the TGF-β/Smad pathway and apoptosis invivo. Deregulated expression or inactivation of components in this pathway mayinterfere with TGF-β induced apoptosis and create a favourable milieu for prostatictumour development.

Transcriptional regulation of the Smad7 gene (paper II)

Smad7 has been proposed to be an inhibitor of TGF-β signalling (Hayashi et al., 1997;Nakao et al., 1997). Previous studies have shown that Smad7 is upregulated in responseto TGF-β stimuli (Afrakhte et al., 1998; Nakao et al., 1997), suggesting a possible rolein a negative feedback loop modulating the TGF-β signal. We wanted to investigate themolecular mechanism underlying Smad7 activation, by studying the transcriptionalregulation of the mouse Smad7 gene promoter by TGF-β.

To obtain the mouse Smad7 promoter, we isolated several overlapping λ-phage clonesspanning the Smad7 genomic region. Using the promoterless pGL3 luciferase reportersystem we found that a 3 kb (kilo base pair) Xho1 fragment conferred TGF-β inducibleluciferase activity in HepG2 cells. Sequential deletions of the 3 kb promoter fragmentrevealed the presence of a TGF-β-responsive region containing a palindromicGTCTAGAC motif. Point mutations introduced into the palindromic sequenceabolished TGF-β mediated transcriptional activation. This palindromic element wasinitially identified by Zawel and co-workers (Zawel et al., 1998) in a artificial PCR-based screen in search for optimal Smad binding motifs. Indeed we could show byelectrophoretic mobility shift assays (EMSA) that Smad2, Smad3, and Smad4 were partof a complex associating with the palindromic Smad binding element (SBE).

In addition we observed the presence of consensus binding sequences for AP-1 and Sp1,which we showed subsequently to bind c-Jun and c-Fos, and Sp1, respectively, in vitro,in electrophoretic mobility shift assays (EMSA). Deletion or mutation of either AP-1 orSp1 binding motifs led to a dramatic decrease in promoter activity while retaining someTGF-β inducibility. In contrast, the major consequence of deletion or point mutation ofthe SBE site, was the complete loss of TGF-β inducibility. These findings suggested theimportance of co-operation between Smads and general transcription factors, such asAP-1 and Sp1.

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What could explain the ability of Sp1 and AP-1 to support the TGF-β response?Previous studies had shown that Smad3 and Smad4 could physically interact with c-Jun,JunB, JunD, and c-Fos and mediate transcription on the 12-O-tetradecanoyl-13-acetate-responsive gene promoter element (Liberati et al., 1999; Zhang et al., 1998). Otherstudies performed on a number of different TGF-β responsive promoters revealed thatSp1 sites could serve as major TGF-β responsive promoter elements (Datto et al., 1995;Greenwel et al., 1997; Li et al., 1995; Li et al., 1998; Moustakas and Kardassis, 1998).Moreover, it had been shown that c-Jun could superactivate Sp1 on the p21Waf/Cip1promoter (Kardassis et al., 1999). Our findings, however, suggested that thegeneral transcription factors for AP-1 and Sp1 had to collaborate together with theSmads in order to orchestrate the basal and TGF-β inducible transcriptional activity ofthe Smad7 promoter.

Targeting of the Smad7 gene (paper III)

The family of Smad proteins represents the major intracellular transducer of TGF-ßsignalling. Gene ablation of the different Smad proteins has revealed specificphysiological and developmental roles (see Table 1-3). In an effort to elucidate thefunctional relevance of Smad7 in vivo, we targeted the Smad7 locus.

We used a targeting vector where the coding region of exon-I including the translationalstart site, was replaced by a neomycin phosphotransferase (neo) selection cassette. Themorula aggregation method was used to generate mutant mice from targeted embryonicstem (ES) cells. The Smad7 homozygous mutant (-/-) mice were viable and fertile. Allmajor organs of the targeted mice such as brain, heart, kidneys, lung, liver, spleen andthymus as well as the larger and smaller intestines, were examined by autopsy andrevealed no consistent differences compared to wild type animals. Also the skeleton wasstudied with no consistent macroscopic morphological variations observed. However,some of the homozygous Smad7 (-/-) pups were significantly smaller than their wildtype (wt) and heterozygous (+/-) siblings. Genotyping of new-borns and mouse embryosof different ages indicated a certain amount of postnatal lethality of -/- mice within thefirst 1-4 weeks of life.

We observed by Northern blot and RT-PCR analyses that there was still TGF-ßinducible Smad7 mRNA expression in embryonic fibroblasts derived from wt or -/-E12.5 mouse embryos. As expected, the mutant mRNA also contained the neosequence, but lacked the coding sequence for the Smad7 exon-I. Interestingly, we foundthe mRNA for Smad6 to be highly upregulated in unstimulated mutant cells. However,we were unable to unambiguously identify Smad7 protein in mouse embryonicfibroblasts (MEFs) or mouse tissues, independent of the genotype.

Since Smad7 had been proposed as a negative modulator of TGF-ß signalling weexamined a number of classical TGF-β responses using MEFs. Assays for plasminogenactivator inhibitor-1 (PAI-1), a well-studied TGF-ß target, showed no differencebetween wt and -/- MEFs, and neither did assays for mitogenicity. In order to test a

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”pure” Smad-dependent cellular response, we transfected a CAGA12-luciferase reporterconstruct into wt and -/- MEFs. Stimulation of these cells with TGF-ß resulted in similarlevels of reporter vector activation in wt and -/- MEFs. This finding was consistent withthe observation that we were unable to detect any significant differences in thephosphorylation pattern of Smad2 upon TGF-ß stimulation between the two cell types.

Taken together, we did not find any indication for a significantly changed TGF-ßresponse in fibroblasts from mutant mouse embryos, despite the effect of the Smad7gene targeting on the size and survival of Smad7 homozygous new-born mice. Apossible explanation could be that Smad6, the other known negative regulator of TGF-βsignalling, was able to compensate for the loss of Smad7. Since we were unable toidentify endogenous Smad7 protein, we can not exclude that a truncated form of Smad7was still produced by the mutant organism, resulting in a hypomorph. In addition, thereis also the possibility that Smad7 is not involved in the modulation of TGF-β signallingin cells of fibroblast origin.

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FUTURE PERSPECTIVES

A major emphasis for future work will lie in the improvement of tools for theidentification of endogenous Smad7 protein in order to verify if the targeting hasresulted in a true knockout or a hypomorph. In the case there is residual protein, it willlack the N-domain, due to the targeting strategy. Very little is known about the N-domain of Smad7 and most of known Smad7-functions regarding negative regulation ofTGF-β signalling reside in the MH2-domain. However, there are observations that bothdomains are necessary for proper function (Hanyu et al., 2001; Nakayama et al., 2001).

The fact that some homozygous animals do survive creates the option to investigatemore about the function of Smad7 by challenging the animals in specific ways.

The back crossing of the Smad7 mutation into different inbred mouse strains will addfurther important information to our picture of Smad7 and TGF-β signalling.

To investigate the biological function of Smad7, many research groups have ectopicallyoverexpressed Smad7 from very strong promoters, resulting in presumablyunphysiological protein levels. We therefore believe that Smad7 gene ablation will beinstrumental in dissecting the physiological role of Smad7 in vivo.

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ACKNOWLEDGEMENTS

The work presented in this thesis was carried out at the Ludwig Institute for CancerResearch in Uppsala. I have had the privilege to interact with so many wonderful peoplethat not only have made the scientific hurdles so much easier to negotiate, but have alsoprovided me with so many joyful memories to be savoured in the future…I would like to express my sincere gratitude to the following people:

Rainer Heuchel, my excellent supervisor…for possessing a scientific enthusiasm andoptimism (certainly temporal optimism…) of a global and highly contagious nature. Butmost of all, for having a great HEART…

Aive Åhgren, my wonderful college, co-worker and “MOM”… for being the centre ofgravity around which the more disorganised celestial bodies of the Gene TargetingGroup revolve…

Maréne Landström, my excellent second supervisor… for your endless support andencouragement…and for long discussions about things that REALLY matters…

Carl-Henrik Heldin, for allowing me to explore the realms of science… for hisoutstanding scientific passion and knowledge…not to mention his ability to digestpainstaking piles of “less than perfect” PhD student scribble…and still make sense of it.

Akira Ishisaki and Takanori Murayama, my crazy Japanese buddies from the Clinlab days… For broadening my point of view and being great friends…

Keiko Funa, my first supervisor… for giving me an excellent introduction to therealities of science.

Olle Nylander, my Studirektor från Biomedicinarlinjen… for your support andenthusiasm ….och vad hände egentligen med Kalle och den där fästingen ?

Michael Cross, for his unquestionable wit and reassuring words: “Greger…..you knowit makes sense…”

All my fellow PhD students through the years, Tobias, Anders, Johan, Simon, Olli-Pekka, Ninna, Sofia, Ann-Sofi, Urban, Peter, Maria, Roya, Catrin, Daniel,Kristian, Jonas, Inga, Kaisa, Camilla, Jan, Katerina and many more, for sharingand verbally expressing “The Ludwig Spleen”, and explaining why words like“motgång föder motgång”, “delad bitterhet är dubbel bitterhet” samt “inget gott sominte har något ont med sig..” actually helps you through the day…

All ye fantastic Lab Technicians, Aino, Anita, Jill, Lotti and Gudrun… for makingthe day at the bench quite enjoyable

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Ulf “Velocipedofilen” Hellman…for his magic greasy touch and everything else…

Ulla and Christer …for long discussions on different topics…mostly economy

Lasse, CPU-Uffe, Ingegärd, Eva H, Inger, and Gullbritt…for making life in thescience-lane run a lot smoother…

Lucie and Annegret…for soulfood and lengthy chats…

People who have broadened my scientific horizon and much, much more…such asLars, Johan E, Valeria, Ester, Peter tD, Aris, Marie-José, Serhiy, Pontus, Ivan, DrKoza, Norbert and Arne…MILLE GRACIE!!

Johan Sandin…my old “Biomed-sambo”…for great laughs and vocal partyperformances…

Eva “Pling” Grönroos, my former fellow commuter and the better half of the dynamicsong writer duo “Reclaim the bad lyrics..”, for some really “artistic” experiences..

Bengt “Becke” Ejdesjö, for his ability to violate natural laws in order to deliver or fixANYTHING … and for making people question the linear concept of time… You arethe best!

My buddies, Ola, Lars-Erik, Gustav and Kaj…who, after all these years, are stilltalking to me, the Eremitus Upsaliensis….

The Toronto crowd, Tony, Berri, Nicki and Venus …for making my stay “overthere”highly enjoyable and educational… Thanks a bunch!!!

Pernilla Grehag, for allowing me to share some really beautiful moments…

My parents, Inger and Lars, for your endless support and love…

Veronika, mitt allt, min snutta…för allt…Älskar Dig.

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