WNT and β-catenin signalling diseases and therapies

WNTs are secreted cysteine-rich glycoproteins that act as short-range ligands to locally activate receptor-mediated signalling pathways.Their functions have been the subject of investigation for more than 20 years. Our present knowledge has come from genetic studies in Drosophila mela noga ster,mouse,zebrafish and Caenorhabditis elegans,as well as biochemical and cell biological studies using Xenopus laevis,sea urchin, chicken embryos and mammalian cultured cells1,2.One conclusion from this work is that WNTs seem to acti-vate more than one type of signalling pathway3.This is perhaps to be expected given that there are 19 members of the WNT protein family in mammals alone,which are expressed in spatially restricted and dynamic pat-terns in embryos and in adults.The best understood pathway activates the nuclear functions ofβ-catenin, leading to changes in gene expression that influence cell proliferation and survival,as well as cell fate.By contrast, some WNTs seem to activate β-catenin-independent pathways,generally leading to changes in cell movement and behaviour.Despite considerable progress in investigating the mechanisms of WNT signalling pathways and their roles in development,it is only recently that studies have emerged that implicate WNT signalling in diseases and clinical conditions other than cancer.In an effort to provide an initial overview of these new studies,our main goal in the present review is to emphasize the unexpected diversity in medical conditions that involve altered WNT signalling (TABLE 1).Another goal is to highlight recent evidence that altering WNT signalling might be therapeutic in various clinical contexts and in the development of stem-cell-based therapies.Finally,given the emerging involvement of WNT signalling in myriad disease processes,we hope that readers who might wish to initiate their own WNT projects will find useful the brief summary of WNT online and experimental resources that we have included.WNT and β-catenin signalling pathwaysβ-catenin-dependent WNT signalling.The best under-stood WNT signalling pathway — the WNT/β-catenin pathway (FIG.1)— has been characterized by a combina-tion of genetics and biochemistry.A core set of proteins and an ever-growing list of proteins that modify their function have been identified in this way,as thoroughly reviewed elsewhere4,5.In the absence of WNT ligands,β-catenin is recruited into a ‘destruction complex’that contains adenomatous polyposis coli (APC) and AXIN, which facilitate the phosphorylation ofβ-catenin by casein kinase 1 (CK1) and then glycogen synthase kinase 3 (GSK3).This leads to the ubiquitylation and proteasomal degradation ofβ-catenin.In the nucleus,WNT AND β-CATENIN SIGNALLING:DISEASES AND THERAPIESRandall T.Moon,Aimee D.Kohn,Giancarlo V.De Ferrari and Ajamete KaykasAbstract | WNT signalling has been studied primarily in developing embryos, in which cellsrespond to WNT s in a context-dependent manner through changes in survival and proliferation,cell fate and movement. But WNT s also have important functions in adults, and aberrant signallingby WNT pathways is linked to a range of diseases, most notably cancer. What is the full rangeof diseases that involve WNT pathways? Can inhibition of WNT signalling form the basis of aneffective therapy for some cancers? Could activation of WNT signalling provide new therapiesfor other clinical conditions? Finally, on the basis of recent experiments, might WNT s normallyparticipate in self-renewal, proliferation or differentiation of stem cells? If so, altering WNTsignalling might be beneficial to the use of stem cells for therapeutic means.Howard Hughes MedicalInstitute,Department ofPharmacology,and theCenter for DevelopmentalBiology,University ofWashington School ofMedicine,Seattle,Washington 98195,USA.Correspondence to R.T.M.e-mail:rtmoon@doi:10.1038/nrg1427tumour-suppressor roles,in other cancers such as melanoma there is evidence that WNT5A and WNT11 might act through the WNT/calcium path-way to promote increased cell motility and invasive-ness,paving the way for future studies on the role of WNT signalling in metastasis 22,23.WNT1 and the neurodevelopmental hypothesis of schiz-ophrenia.Most WNTs and FZ receptors are expressed during development of the central nervous system,and many are expressed in the adult brain,indicating that future reports might reveal the roles of WNT signalling in the adult brain.It has been proposed that increased expression of WNT1might lead to altered cell adhesion,synaptic rearrangement and plasticity in the brains of people with schizophrenia 24,with the caveat that elevated expression of WNT ligand does not ensure elevated WNT signalling,given the diversity of WNT pathway inhibitors.However,consistent with the possible involve-ment of WNT signalling in schizophrenia,it has recently been reported that SNPs in FZ3 are associated with susceptibility to schizophrenia 25,whereas others have suggested that GSK3 activity is altered in schizophrenia 26.Similarly,deletion of Dsh1/Dvl1 in mice produces behavioural defects 27,further linking the WNT pathway to the modulation of brain activity.It remains unclear,but worthy of future studies,whether WNT signalling is actually altered in individuals with psychiatric condi-tions and whether this is mechanistically linked to the symptoms.sFRP3 and osteoarthritis.Osteoarthritis is a degenerative disease of the joints,characterized by cartilage deteriora-tion.A recent study 28has found a SNP that is associated with osteoarthritis in females.This polymorphism is found in secreted Frizzled-related protein 3 (sFRP3),which is a secreted antagonist of WNTs.The polymor-phism reduces the ability of sFRP3 to antagonize WNT signalling,indicating that WNT signalling might be elevated in osteoarthritis.Altered function of Frizzled and LRP5/6Loss-of-function mutations in FZ4 and LRP5 are linked to FEVR.Considerable evidence points to a role for WNT signalling in vascular development,including the identification of vascular endothelial growth factor (VEGF ) as a direct target gene of β-catenin 29.Familial exudative vitreoretinopathy (FEVR ) is an inherited disorder of the retinal vasculature in which peripheral capillaries fail to grow,resulting in compen-satory neovascularization,leakage and bleeding,scar-ring and ultimately blindness.Although genetically heterogeneous,positional cloning of FEVR loci has revealed that loss-of-function mutations in the putative WNT receptor FZ4,and the co-receptor LRP5,underlie many cases of FEVR 30–33.Consistent with some FZ4mutations acting in a dominant manner,one mutation encodes a truncated protein that acts in a dominant–negative manner to oligomerize with wild-type FZ receptors,and to trap them in the endoplasmic reticu-lum 34.Nonetheless,many questions remain.Forabnormal testicular vasculature and their testos-terone synthesis is inhibited.Through the analysis of cells cultured in vitro ,the authors found that WNT4interferes with the functional synergy between β-catenin and steroidogenic factor 1,and reduces recruitment of β-catenin to the steroidogenic acute regulatory protein promoter.Although the mecha-nisms remain unclear,the authors do not discount the possibility that WNT4 antagonism of β-catenin func-tion might be a result of WNT4 acting through the WNT/calcium pathway.In summary,the authors suggest that WNT4 acts as an anti-male factor by interfering with β-catenin functions.WNT4 and renal development and disease.WNT4 is also linked to organ formation and disease.In the mouse,Wnt4and Wnt11are involved in kidney devel-opment,as revealed by analysis of knockout mice 15.Although several additional WNTs might function in the kidney,a key role for Wnt4in RENAL TUBULE formation is clear.Terada et al.16used a rat model of acute renal failure in which renal arteries were occluded to show that Wnt4RNA and protein are significantly elevated in tubules within hours after induced ISCHAEMIA .So,the recovery from injury might involve WNT-dependent processes that in some respects mirror normal develop-mental processes and pathways.Similarly,ligation of a single URETER results in tubular damage followed by a repair process in which WNT4 expression is elevated,which in turn activates expression of C-type natriuretic peptide that regulates fluid and electrolyte homeostasis and inhibits FIBROSIS 17.Finally,WNT and β-catenin signalling might be involved in polycystic kidney disease (PKD ).The poly-cystic kidney disease 1 (PKD1) gene,which is often mutated in PKD,is a direct target of β-catenin/TCF 18.Interestingly,the level of PKD1 expression might be important in PKD,as both elevation and reduction in expression have been linked to the disease.Consistent with these data,elevated expression of Wnt4has been reported in polycystic kidneys in mice,and transgenic mice that overexpress active forms of β-catenin develop PKD 18.WNT5a — A tumour-suppressor gene and a modula-tor of metastasis.Several lines of evidence support the idea that Wnt5a might be a tumour-suppressor gene.For example,loss of Wnt5a in C57MG mammary epithelial cells promotes a phenotypic transformation that resembles activation of β-catenin signalling 19.In a uroepithelial cell line,a chromosomal deficiency that removes Wnt5a results in a transformation that can be blocked by overexpression of Wnt5a 20.Mice that are hemizygous for Wnt5a develop myeloid leukaemia and B-cell lymphomas,and WNT5A is deleted or its expression is reduced in most patients with compara-ble cancers 21.Although the pathway that is activated by WNT5A has not been clearly identified in most of these studies,the data of Liang et al.21support a role for WNT5A as a tumour suppressor that works through WNT/calcium signalling.Independent ofRENAL TUBULESThe kidney contains many units known as nephrons,which contain (renal) tubules that are involved in filtering the blood.ISCHAEMIAReduced flow of oxygenated blood to organs in response to blockage in an artery.URETERA tube leading from the kidney to the bladder.FIBROSISFormation of fibrous tissue during a repair process.mutations in the amino terminal phosphorylation sites of β-catenin,and loss-of-function mutations in AXIN,also activate β-catenin signalling and are linked to can-cer 43–45.Interestingly,in non-small-cell lung cancer,DSH/DVL genes are overexpressed,and small interfer-ing RNAs (siRNA S) that reduce their expression inhibit growth 47.Finally,β-catenin signalling might,in some circumstances,be required for the maintenance of the transformed state 48–50,indicating that therapies that tar-get this pathway might be successful.Intriguingly,despite the observation that muta-tions in the β-catenin pathway generally activate the pathway downstream of WNT,and usually at the level of the destruction complex,recent evidence indicates that epigenetic inactivation of sFRP genes occurs early in colorectal cancer,and restoring sFRP expression attenuates activation of the β-catenin pathway 44,51.These data indicate continuing roles for WNT ligands in some cancers,even those in which downstream components are mutated.AXIN2,familial tooth agenesis and colon cancer.Effects of mutant AXIN2/Conductin,a component of the β-catenin degradation complex that is related to AXIN,confirm that activated β-catenin signalling is linked to defects in development and to cancer.Specifically,WNT signalling genes are normally expressed during tooth dev-elopment,and Lef1knockout mice have tooth defects 52;consequently,tooth defects might be expected in humans with mutations in the WNT pathway.Indeed,a Finnish family with severe tooth agenesis and with a his-tory of colorectal cancer was studied recently;positional cloning identified mutations in AXIN253.This study further supports the conclusion that β-catenin sig-nalling has normal roles in several organs,including the gut and teeth,and that mutations in components of this pathway can have phenotypic consequences in several unrelated structures.Mutations linked to tuberous sclerosis activate β-catenin signalling.Even mutations in genes that modulate β-catenin signalling without any apparent link to WNT ligands affect cell proliferation.Mutations in the tuber-ous sclerosis complex (TSC) genes Tsc1or Tsc2,which encode novel proteins that associate to form a complex,are linked to the disease tuberous sclerosis ,in which tumour-like lesions form in several organs.Tsc2muta-tions in rats lead to elevated β-catenin levels 54,raising the questions of whether β-catenin target genes are acti-vated in this disease,and whether wild-type TSC nor-mally participates in regulating β-catenin levels.Consistent with this possibility,wild-type Tsc1and wild-type but not mutant Tsc2,if co-expressed in cultured cells,reduce β-catenin levels and reduce activation of a β-catenin-responsive luciferase reporter 54.Moreover,the TSC complex co-immunoprecipitates with AXIN and GSK3,which are involved in degrading β-catenin.So,components of the TSC might normally promote degradation of β-catenin,and mutations that interfere with this process might promote this tumour-like dis-ease.Whether β-catenin signalling is activated inexample,it is unclear whether FZ4 signalling is reduced in FEVR patients,as the data would predict.Neither is it clear why the disease develops relatively late,nor are there data on when and where FZ4 signalling is required to prevent the onset of the disease.Activating mutations in LRP5 are linked to high bone mass.WNTs are involved in regulating bone forma-tion 35.Several insightful studies have recently pro-vided compelling genetic evidence establishing that,in humans and mice,activating mutations in LRP5result in a high bone mass phenotype.One project began when an 18-year-old female Nebraska high-school student,following a motor accident and result-ing radiographs,was found to have unusually dense bones.The insightful referral of this patient to the Creighton Osteoporosis Research Center,and genetic analysis of her family,led to the discovery that an amino-acid change in the extracellular domain of LRP5 was linked to this high bone mass pheno-type 36–38and resulted in weak activation of the β-catenin pathway 39.Intriguingly,the carriers have no apparent increased cancer risk,probably because weak activation of the pathway at the level of LRP5 would still induce expression of two negative-feedback components,AXIN2/Conductin and βTrCP (β-transducin-repeat-containing protein),which would downregulate β-catenin signalling and therefore protect against can-cer.It is not unreasonable to expect that therapeutics that weakly activate LRP5 function might serve as treatments for osteoporosis.Loss-of-function mutations in LRP5 are linked to low bone mass and eye defects.Just as gain-of-function mutations in LRP5promote bone mass,loss of func-tion in LRP5in humans and mice results in the low bone mass observed in osteoporosis–pseudoglioma syn-drome (OPPG )40.Interestingly,and consistent with the FEVR studies mentioned above,this syndrome also affects eye development.In agreement with a role for WNT signalling in promoting bone formation,deletion of the WNT antagonist sFRP1 enhances bone forma-tion 41,and target genes of β-catenin signalling have been implicated in bone formation 42.Altered function of cytoplasmic componentsActivation of β-catenin signalling and cancer.The WNT/β-catenin pathway contains many repressors,indicating that it is imperative for this pathway to be tightly regulated.Supporting this idea,any mutations that strongly and constitutively activate the β-catenin pathway are probably involved in the initiation and pro-gression of cancer 43–45.The involvement of β-catenin sig-nalling in oncogenesis conforms with the role of the pathway as a “master switch that controls proliferation versus differentiation”46.The best studied pathway muta-tions are the inherited and sporadic mutations in the tumour suppressor APC,which reduce the degradation of β-catenin,causing an increase in β-catenin levels and activation of target genes such as the oncogenes cyclin D1 (CCND1) and c-MYC 46.Similarly,gain-of-functionsiRNASmall interfering RNAs,which target (in a sequence-specific manner) endogenous RNAs for degradation,thereby reducing the function of a gene.Stem cells.Stem cells are pluripotent cells that both self-renew and give rise to specialized cell types.WNT sig-nalling has been implicated in the maintenance and self-renewal of various pluripotent stem cells and prog-enitor cells 55,57,98–110.For example,stem cells of the gut require WNT activation for self-renewal,as demon-strated by the loss of INTESTINAL CRYPT progenitor cells dur-ing fetal development in Tcf4-deficient mice 46,111.WNT signalling in gut progenitor cells remains essential in adults as injection of an adenovirus expressing Dkk1,a soluble WNT inhibitor,into adult mice inhibits intestinal proliferation 112.This result raises the possibility of using WNT activators to regenerate gut epithelium as adjuvant therapy in inflammatory bowel disease 112.There is also evidence that in other contexts WNT signalling can provide an instructive signal that changes the fate of stem cells 104.Resolving the mechanisms by which WNT signals promote self-renewal versus differ-entiation will be important for controlling stem cells for therapeutic uses.In haematopoietic cells,overexpression of activated β-catenin results in stem-cell expansion whereas ectopic expression of inhibitors of WNT signalling reduces in vitro haematopoietic stem-cell growth and in vivo reconstitution 107.In vivo ,INTRAPERITONEAL injection of WNT5A-conditioned media into mice engrafted with human haematopoietic progenitor cells increases recon-stitution compared with controls and is also associated with a greater proportion of phenotypically primitive haematopoietic progenitor cells,indicating that WNT signalling promotes in vivo stem-cell expansion,engraftment or survival 109.These studies point towards potential therapeutic applications of WNT activation in stem-cell transplantation,in which WNT stimulation might facilitate haematopoietic recovery and umbilical cord blood expansion.In human embryonic stem cells (HESCs),activation of the WNT/β-catenin pathway by 6-bromoindirubin-3′-oxime (BIO),a pharmacological inhibitor of GSK3,maintains the undifferentiated phenotype 102.Under-standing the role of WNT signalling in determining the capacity to proliferate and differentiate is a step towards tissue engineering and transplantation that will require large-scale expansion of undifferentiated HESCs fol-lowed by differentiation into specific tissue types.As the potential therapeutic use of stem cells has already been demonstrated in animal models for Parkinson disease 113and diabetes mellitus 114,it is reasonable to predict that modulating WNT pathways in stem cells will ultimately have an impact on stem-cell therapies.ResourcesGiven the emerging involvement of WNT signalling in many disease processes,researchers who were previously unfamiliar with the WNT field might wish to initiate their own WNT studies.This prospect is made less daunting by the availability of several useful resources.The primary online resource for the WNT field is the Wnt Gene Homepage (see online links box),which is operated by R.Nusse of Stanford University.It includes comparisons of protein and gene sequences,models ofneurotransmitter system and the only risk factor that has so far been identified for AD,Apolipoprotein E (APOE).Interestingly,recent data indicate that APP processing or A βneurotoxicity might be modulated by WNT signalling components 76–78or compounds that mimic its activity,such as the GSK3 inhibitor,lithium 79,80.Collectively,avail-able data continue to point to a role for diminished WNT/β-catenin signalling in AD.Cardiovascular disease.Both WNT/β-catenin and the WNT/calcium pathways are involved in cardiogene-sis 81–83and in diseased hearts 84.For example,after induc-tion of MYOCARDIAL INFARCTION in Dsh1/Dvl1-null mice,75% of them develop an infarct rupture compared with less than 10% of controls.There is also evidence that WNT signalling might be involved in neovasculariza-tion in the infarct area 84.Moreover,overexpression of sFRP1,a WNT inhibitor,reduces infarct size and improves cardiac function in a mouse model 85.Finally,in cardiac hypertrophy,FZ2 is upregulated in the rat,whereas other data point to roles for GSK3 (REF .84).Therapeutic modulation of WNT pathwaysInhibitors of WNT/β-catenin signalling are under con-sideration or development,initially for treatment of cancers — a promising avenue given that WNT/β-catenin signalling seems to be involved in cancer pro-gression,and not just initiation 48–50.Candidate approaches include small-molecule inhibitors that block interaction of β-catenin with TCF 86or CREB-binding protein (CBP)87,as well as siRNAs 45and the therapeutic use of antibodies against WNTs 88,89.Other potential therapeutic inhibitors of β-catenin signalling include agents that have no obvious link to the β-catenin pathway,such as extracellular calcium 90,non-steroidal anti-inflammatory drugs,including exisulind,sulindac and aspirin 91–95,and the tyrosine-kinase inhibitor STI571/Gleevac 96.Conversely,activa-tors of β-catenin signalling will probably be useful in treating osteoporosis and AD,and might include activa-tors of LRP5 as well as inhibitors of GSK3.A summary of candidate therapeutic targets,focusing on those discussed in this review,is shown in FIG.3.Regeneration.Although clinical interest in organ regeneration has a long history,molecular under-standing of the underlying mechanisms is in its early inception.For example,it is well known that liver size can be restored after injury or loss.A study of chicken liver morphogenesis 97showed that hepatocyte precur-sors are localized to specific areas in the periphery of the developing liver where β-catenin and WNT3A are also enriched.Although localized overexpression of a stabilized form of β-catenin caused an increase in liver size with a greater distribution of growth zones,over-expression of WNT inhibitors decreased the size,thereby implicating WNT activation in liver growth and development and,by extension,liver regen-eration 97.Whether WNTs or pathway-activating small molecules will facilitate tissue and organ regeneration in the clinic remains to be tested.MYOCARDIAL INFARCTIONCommonly known as a heart attack,in which there is reduced blood flow to the heart,leading to reduced oxygen supply.INTESTINAL CRYPTAn involution in the intestine in which stem cells give rise to differentiated cells.INTRAPERITONEALAdministered or withdrawn from within the abdominal cavity.1. Wodarz, A. & Nusse, R. Mechanisms of Wnt signaling indevelopment. Annu. Rev. Cell Dev. Biol.14, 59–88 (1998). 2. Huelsken, J. & Birchmeier, W. New aspects of Wnt signalingpathways in higher vertebrates. Curr. Opin. Genet. Dev.11,547–553 (2001).3. Veeman, M. T., Axelrod, J. D. & Moon, R. T. A secondcanon. Functions and mechanisms of β-catenin-independent Wnt signaling. Dev. Cell5, 367–377 (2003). 4. T olwinski, N. S. & Wieschaus, E. Rethinking WNT signaling.Trends Genet.20, 177–181 (2004).5. Nelson, W. J. & Nusse, R. Convergence of Wnt, β-catenin,and cadherin pathways. Science303, 1483–1487 (2004). 6. He, X., Semenov, M., T amai, K. & Zeng, X. LDL receptor-related proteins 5 and 6 in Wnt/β-catenin signaling: arrowspoint the way. Development131, 1663–1677 (2004).7. T an,C.et al. Inhibition of integrin linked kinase (ILK) suppresses β-catenin-Lef/T cf-dependent transcription andexpression of the E-cadherin repressor, snail, in APC–/–human colon carcinoma cells. Oncogene20, 133–140(2001).8. Levina, E., Oren, M. & Ben-Ze’ev, A. Downregulation ofβ-catenin by p53 involves changes in the rate of β-cateninphosphorylation and Axin dynamics. Oncogene23,4444–4453 (2004).9. Doble, B. W. & Woodgett, J. R. GSK-3: tricks of the trade fora multi-tasking kinase. J. Cell Sci.116, 1175–1186 (2003).10. Moon, R. T., Bowerman, B., Boutros, M. & Perrimon, N. Thepromise and perils of Wnt signaling through β-catenin.Science296, 1644–1646 (2002).11. Westfall, T. A. et al. Wnt-5/pipetail functions in vertebrateaxis formation as a negative regulator of Wnt/β-cateninactivity. J. Cell Biol.162, 889–898 (2003).12. T opol, L. et al. Wnt-5a inhibits the canonical Wnt pathway bypromoting GSK-3-independent β-catenin degradation.J. Cell Biol.162, 899–908 (2003).13. Niemann, S. et al. Homozygous WNT3 mutation causestetra-amelia in a large consanguineous family. Am. J. Hum.Genet.74, 558–563 (2004).14. Jordan, B. K., Shen, J. H., Olaso, R., Ingraham, H. A. &Vilain, E. Wnt4 overexpression disrupts normal testicularvasculature and inhibits testosterone synthesis byrepressing steroidogenic factor 1/β-catenin synergy. Proc.Natl Acad. Sci. USA100, 10866–10871 (2003).15. Perantoni, A. O. Renal development: perspectives on aWnt-dependent process. Semin. Cell Dev. Biol.14,201–208 (2003).16. Terada, Y. et al. Expression and function of thedevelopmental gene Wnt-4during experimental acuterenal failure in rats. J. Am. Soc. Nephrol.14, 1223–1233(2003).17. Surendran, K. & Simon, T. C. CNP gene expression isactivated by Wnt signaling and correlates with Wnt4expression during renal injury. Am. J. Physiol. Renal Physiol.284, F653–F562 (2003).18. Rodova, M., Islam, M. R., Maser, R. L. & Calvet, J. P. Thepolycystic kidney disease-1 promoter is a target of theβ-catenin/T-cell factor pathway. J. Biol. Chem.277,29577–29583 (2002).19. Olson, D. J. & Gibo, D. M. Antisense wnt-5a mimicswnt-1-mediated C57MG mammary epithelial celltransformation. Exp. Cell Res.241, 134–141 (1998).20. Olson, D. J., Gibo, D. M., Saggers, G., Debinski, W. &Kumar, R. Reversion of uroepithelial cell tumorigenesis bythe ectopic expression of human wnt-5a. Cell GrowthDiffer.8, 417–423 (1997).21. Liang, H. et al. Wnt5a inhibits B cell proliferation andfunctions as a tumor suppressor in hematopoietic tissue.Cancer Cell4, 349–360 (2003).Strong evidence that WNT/calcium signalling is atumour-suppressor pathway.22. Weeraratna, A. T. et al. Wnt5a signaling directly affects cellmotility and invasion of metastatic melanoma. Cancer Cell1,279–288 (2002).Solid evidence that WNT/calcium signalling isinvolved in metastasis.23. Ouko, L., Ziegler, T. R., Gu, L. H., Eisenberg, L. M. &Yang, V. W. Wnt11 signaling promotes proliferation,transformation and migration of IEC6 intestinal epithelialcells. J. Biol. Chem. 279, 26707–26715(2004).24. Miyaoka, T., Seno, H. & Ishino, H. Increased expression ofWnt-1 in schizophrenic brains. Schizophr. Res.38, 1–6(1999).25. Katsu, T. et al. The human frizzled-3 (FZD3) gene onchromosome 8p21, a receptor gene for Wnt ligands,is associated with the susceptibility to schizophrenia.Neurosci. Lett.353, 53–56 (2003).WNT signalling,cartoons of protein interactions,lists of target genes and announcements of upcoming WNT meetings.It also includes references and methods for purifying WNTs,and for monitoring and inhibiting WNT signalling.The Signal Transduction Knowledge Environment (see online links box),operated by Science magazine,has a detailed model of WNT/β-catenin signalling on its Connections Map link (see online links box),which complements the data on the Wnt Gene Homepage by providing a general description of all WNT/β-catenin pathway components and links to publications. Animated movies are also provided.In addition to these online resources,TABLE 2pro-vides a summary of some of the reagents that are useful in various applications.Although incomplete,the list is offered in the hope that it will reduce some of the perceived barriers for initiating research with WNTs. Future directionsAberrant regulation of WNT signalling is involved in sev-eral diseases and syndromes,probably through modulat-ing the normal processes that are affected by WNT signals — notably,cell proliferation,survival,fate and behaviour. It is abundantly clear that mutations that lead to constitu-tive activation of the WNT/β-catenin pathway will gener-ally have deleterious consequences in humans,which is no surprise given the impressive number of antagonists that normally work to dampen the pathway,and the normal transient and local activation of WNT path-ways in embryos.It is also apparent that WNT5A, probably acting through the WNT/calcium pathway, can act as a tumour suppressor,although the mecha-nisms are unclear.So,multiple WNT signalling pathways are important in human diseases.Several unresolved issues that continue to vex us are offered here in the hope that this will stimulate their resolution.First,the mechanisms by which cells integrate their transcriptional and non-transcriptional respon-ses to WNT signals — such as changes in prolifera-tion,survival,fate or behaviour — are largely unknown.Resolving the mechanisms that control the cell’s responses to WNTs might be important for deter-mining whether stem cells can be developed for a wide range of therapies.In addition,it is possible to imag-ine that small-molecule modulators that switch a cell’s response to a WNT/β-catenin signal from pro-liferation to differentiation might be considered in cancer treatments.Second,WNT signalling does not occur in isola-tion from other signalling pathways,and there is con-siderable evidence to show that WNT signalling can cooperate in unexpected ways with other pathways. The functional interaction of WNT signalling with other pathways probably accounts for the observa-tion that WNT/β-catenin signalling regulates distinct sets of genes in different cells.The mechanisms by which this combinatorial signalling occurs,and whether it is functionally significant in the diseases and potential therapies discussed here,are largely unknown.Finally,can the insights gained by hundreds of investigators who have studied WNT signalling over the past 20 years actually be translated into tangible therapies for some of the diseases and conditions dis-cussed here? There are tantalizing positive hints —notably,the development of small molecules that antag-onize β-catenin function in the nucleus — that might lead to future therapies for treating proliferative diseases such as cancer.Another positive finding is that WNT sig-nalling is important in the normal biology of embryonic stem cells,raising the hope that modulating WNT sig-nalling in stem cells or in the local environments into which they are transplanted will eventually contribute to cell-based therapies.。