1360 NATURE MEDICINE • VOLUME 8 • NUMBER 12 • DECEMBER 2002

NEWS & VIEWS

Colorectal cancer, the third most com-mon cancer in the United States,

can be thought of as a disease of thelong-lived stem-cell populations. In theintestine, stem cells reside nearthe bottom of flask-like invagi-nations, called crypts.Normally, cells differentiate andmove in a continuous wave upthe crypt (Fig. 1) to be shed aftera total lifetime of about sevendays1,2. Cancer can arise throughloss of the strict control overproliferation and migrationevents in these structures1,2. In arecent issue of Cell, Clevers andcolleagues uncover some of thekey molecular events that holdcrypt cells in their place3,4. Intwo studies, they implicate theT-cell factor–β-catenin pathwayin regulating both differentia-tion and positional location ofcells in intestinal crypts.

T-cell factor (TCF) is a DNA-binding protein and transcrip-tion factor that can be activatedupon binding to β-catenin.Overexpression of β-catenin andtranslocation to the nucleus oc-curs in colorectal cancer, whereit binds TCF and stimulates cellproliferation. In normal cells,

the adenomatous poliposis coli (APC)protein competes with TCF to bind β-catenin and suppress proliferation. Lossof APC is the primary event in colorec-

tal cancer (hence its reputationas the ‘gatekeeper’ of tumor de-velopment). It is lost in thehuman syndrome FAP (familialadenomatous poliposis coli) inwhich individuals develop nu-merous intestinal adenomas rel-atively early in life, whichrapidly progress to carcinomas.

In many systems, the TCF–β-catenin pathway is regulated byextracellular Wnt protein,which binds to a membrane re-ceptor, called ‘frizzled’, andstimulates formation of the βcatenin–TCF complex5. Cleversand colleagues ask what hap-pens after the complex is acti-vated in mammalian crypt cells.

The authors undertook DNAmicroarray analysis in humancolon adenocarcinoma cell lineswith inducible dominant-nega-tive TCF mutations, which, byinhibiting TCF–β-catenin com-plex formation, induced G1 ar-rest. They found 120 genes withat least a two-fold drop in ex-pression, and 115 with increased

Crowd control in the cryptCancer can ensue when cells do not know who they are or where they are. In the intestine, a single regulator seems

to take care of both of these issues of identity.

CATHERINE BOOTH, GERARD BRADY &

CHRISTOPHER S. POTTEN

Ephrin

Eph B2

Eph B3

Migrationwith increasingvelocity

Boundarymyc–p21

Stem cellregion

Proliferativeregion

Differentiatedregion

Crypt-villusjunction

p21)( CMyc,

p21)( c-Myc,TCF/β-catenin complex

Free TCF

S S

Paneth cellsStem cells

Fig. 1 The intestinal crypt. Illustrated are protein expressionpatterns of TCF–β-catenin target genes EphB/Ephrin and c-MYC/p21. Also shown are the proliferative regions, including thestem-cell locations.

produced in Escherichia coli. It is thereforepossible that β-defensin 2 is essentially de-livering LPS to TLR4. This mechanismwould be similar to the function of theLPS-binding protein (LBP). LBP is a serumprotein that binds to LPS monomers withhigh affinity and transfers them intoTLR4 complexed with CD14, another pro-tein involved in LPS recognition by TLR4(ref. 3). In the absence of LBP, muchhigher concentrations of LPS are neededto trigger TLR4. Because β-defensin 2seems to signal through the same receptoras LPS (TLR4), TLR4 knockout cells couldnot be used to discriminate between theeffect of β defensin 2 and that of LPSbound to the bacterially produced recom-binant protein. A better approach to dis-tinguish between the two possible modesof action of β-defensin 2 would have beento produce the peptide in baculovirus oreven in mammalian cells. If the β-de-

fensin 2 produced in a eukaryotic expres-sion system activates TLR4, it wouldimply that it functions as a ligand in itsown right. Lack of activity would suggestthat β-defensin might function as an LPScarrier that, like LBP, allows activation ofTLR4 by minute amounts of LPS (ref. 3)(Fig. 1).

Regardless of the mechanism, this newfinding reveals a novel function of de-fensins and may have interesting practi-cal applications for preventive andtherapeutic vaccines. Because of itschemoattractive abilities, β-defensin 2,when linked to an appropriate antigen,can help recruit dendritic cells to presenta specific protein to T cells. Moreover, itcould be used as a safer adjuvant thatmay trigger TLR4 without the need fortoxic doses of LPS. In this way, β-defensin2 could be used to activate the essentialreceptors of the innate immune system

and prime the adaptive response againsta specific target.

1. Janeway, C.A. Jr. & Medzhitov, R. Innate immunerecognition. Annu. Rev. Immunol. 20, 197–216(2002).

2. Biragyn, A. et al. Toll-like receptor 4-dependent acti-vation of dendritic cells by β-defensin 2. Science 298,1025–1029 (2002).

3. Ulevitch, R.J. & Tobias, P.S. Receptor-dependentmechanisms of cell stimulation by bacterial endo-toxin. Annu. Rev. Immunol. 13, 437–457 (1995).

4. Raj, P.A. & Dentino, A.R. Current status of defensinsand their role in innate and adaptive immunity. FEMSMicrobiol. Lett. 206, 9–18 (2002).

5. Lehrer, R.I. & Ganz, T. Defensins of vertebrate ani-mals. Curr. Opin. Immunol. 14, 96–102 (2002).

6. Biragyn, A. et al. Mediators of innate immunity thattarget immature, but not mature, dendritic cells in-duce antitumor immunity when genetically fusedwith nonimmunogenic tumor antigens. J. Immunol.167, 6644–6653 (2001).

1Section of Immunobiology, Yale University2Howard Hughes Medical InstituteNew Haven, Connecticut, USAEmail: ruslan@biomed.med.yale.edu

Deb

bie

Mai

zels

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

NATURE MEDICINE • VOLUME 8 • NUMBER 12 • DECEMBER 2002 1361

NEWS & VIEWS

expression. Genes activated by TCF–β-catenin were expressed mainly in prolif-erating cells and repressed genes wereexpressed in differentiated cells. Amongfive genes known to control cell prolif-eration, only the transcription factor c-MYC was capable of overriding the G1growth arrest induced by dominant-negative TCF. The authors then showedthat c-MYC was able to repress thegrowth inhibitor p21CIP/WAF1 (probablyindirectly, by inhibiting the transcrip-tion factor cMIZ-1). The authors con-cluded that the formation of aβ-catenin–TCF complex in the nucleusresults in the upregulation of c-MYCand the inhibition of p21, thereby al-lowing cells to proliferate.

While the first study revealed part ofthe mechanism of β-catenin–TCF ac-tion, the second begins to show how itcan control cell location. This studystarts with an observation from the ini-tial DNA microarray analysis. It seemedthat TCF–β-catenin could upregulatetwo receptors associated with the con-trol of cell migration (EphB2 andEphB3) and downregulate their ligand(Ephrin B). This family of tyrosine ki-nase receptors are known to control cy-toskeletal remodeling during cellmigration6. Both receptors and ligandsin this system are membrane-anchored,suggesting that a two-way signalingprocess could occur, with the ligandsignaling the host cell. Intriguingly,there are hints that Eph family of regu-lators might contribute to tumorigene-sis. EphB4 is upregulated in someadenomas7 and EphB2 and the tyrisinekinase Abl can reciprocally phosphory-late each other8. Interestingly, Abl alsofunctions in the β-catenin signalingpathway.

Immunohistochemical analysis re-vealed that EphB2 and B3 were re-stricted to the crypt, with EphB3occurring only near the base where thedifferentiated Paneth cell populationresides. Ephrins were expressed gener-ally in the more mature upper cryptand villus cells. Notably, co-expressionof EphB2/Ephrins occurred at thecrypt–villus junction, which roughlymarks the end of the proliferative zoneand beginning of the differentiationzone. Using EphB2/EphB3 transgenicanimals the authors show that bothEphB2 and EphB3 help to control theorderly maturation or migration path-ways in normal animals. Specificallythey found that EphB3 normally re-

stricts Paneth cells (thought to fightmicrobes), to a location at the cryptbase. The data indicate that theEphB2/3-Ephrin interaction restrictsthe intermingling of proliferating anddifferentiated cells and possibly con-trols migratory direction.

Since the proliferation data are diffi-cult to interpret, the details of the inter-mingling remain to be thoroughlyevaluated. Further positional and cellturnover analysis are required, ideallyaccompanied by lineage tracking. Theseanalyses could address whether migrat-ing Paneth cells are dividing and assesscell-cycle times in both the stem cellsand their daughters. They also could ad-dress whether EphB controls stem-celllocation and asymmetric-division po-tential. Consistent with this possibility,lymphoid-enhancement Factor-1 (LEF-1) (a transcription factor related to TCF)is involved in regulating crypt stem-cellnumber in neonatal life, suggesting thatit may direct stem-cell divisions9.Notably, Ephrins interact with PAR-3, aregulator of asymmetric cell division inC. elegans10. Since PAR-3 is expressed inmammalian tissue it may have a similarrole in the crypt.

β-catenin, in addition to its functionin the nucleus as a binding protein forTCF, is known to act at the cell surfacein adhesion. Indeeed, it is expressed atthe cell surface throughout the crypt(Fig. 2) where it interacts with α-cateninand E cadherin to reduce crypt-cell mi-gration rate in mice11. Perhaps theEphBs promote attachment, along withβ-catenin/E-cadherin, and the Ephrinspromote detachment and cell migra-tion. As the cells move upwards, theEphrin effect begins to dominate andthe cells migrate more quickly, consis-tent with the known acceleration ofcells as they move upwards towards thelumen. However, this is undoubtedly anoversimplification—other cell-adhesionmolecules have expression gradients inthe crypt and villus, which will con-tribute to the sorting and migrationprocess.

If, as indicated in these studies,TCF–β-catenin directly targets only afew genes in the crypt, future anti-can-cer drug screen would be highly simpli-fied. In reality, the story is likely to bemore complicated. However, new stud-ies can now begin to investigate howthe processes described by Clevers andcolleagues may malfunction in humandisease.

1. Potten, C.S. & Loeffler, M. Stem cells: Attributes,cycles, spirals, pitfalls and uncertainties. Lessonsfor and from the crypt. Development 110,1001–1020 (1990).

2. Potten, C.S., Booth, C., Pritchard, D.M. The in-testinal epithelial stem cell: The mucosal gover-nor. Intl. J. Exp. Pathol. 78, 219–243 (1997).

3. Battle, E. et al. β-Catenin and TCF mediate by cellpositioning in the intestinal epithelium by control-ling the expression of EphB/EphrinB. Cell 111,251–263 (2002).

4. Wetering van de, M. et al. The β-catenin/TCF-4complex imposes a crypt progenitor phenotypeon colorectal cancer cells. Cell 111, 241–250(2002).

5. Bienz, M., and Clevers, H. Linking colorectal can-cer to Wnt signaling. Cell 103, 311–320 (2000).

6. Cowan, C.A. & Henkemeyer.M. Ephrins in reverse,park and drive. Trends Cell Biol. 12, 339–346(2002).

7. Stephenson, S.A., Slomak, S., Douglas, E.L.,Hewett, P.J. & Hardingham, J.E. Receptor proteintyrosine kinase EphB4 is up-regulated in coloncancer. BMC Mol. Biol. 2. 1–9 (2001).

8. Yu, H.H., Zisch, A.H., Dodelet, V.C. & Pasquale,E.B. Multiple signaling interactions of Abl and Argkinases with the EphB2 receptor. Oncogene 20,3995–4006 (2001).

9. Wong, M.H., Huelsken, J., Birchmeier W. &Gordon J.I. Selection of multipotent stem cellsduring morphogenesis of small intestinal crypts ofLieberkuhn is perturbed by stimulation of Lef-1/β-catenin signaling. J. Biol. Chem. 18, 15843–50(2002).

10. Etemad-Moghadam, B. et al. Asymmetrically dis-tributed PAR-3 protein contributes to cell polarityand spindle alignment in early C. elegans em-bryos. Cell 83, 743–752 (1995).

11. Wong, M.H., Rubinfield, B. & Gordon, J.I. Effectsof forced expression of an N-terminal truncated β-catenin on mouse intestinal epithelial homeosta-sis. J. Cell. Bio. 141, 765–777 (1998).

EpiStem Ltd.Manchester, UKEmail: c.booth@epistem.co.uk

Fig. 2 β-catenin in crypts (cross section ininset.) β-catenin is found at the cell surface andin the nuclei at the base (arrow).Controversially, nuclear localization has notbeen reported before in crypts, despite exten-sive investigation. Cells higher up lack nuclearβ-catenin (blue arrow).

Rep

rinte

d w

ith p

erm

issi

on fr

om E

lsev

ier

Scie

nce

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine