wnt bone formation

Molecular and Cellular Endocrinology 310 (2009) 52–62Contents lists available at ScienceDirectMolecular and CellularEndocrinologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /m ceReviewIs Wnt signalling the final common pathway leading to bone formation?Frances Milat a ,b ,c ,∗,Kong Wah Ng a ,baSt Vincent’s Institute,9Princes St,Fitzroy,Victoria 3065,AustraliabDepartment of Endocrinology and Diabetes,St Vincent’s Health,Fitzroy,Victoria 3065,Australia cPrince Henry’s Institute,PO Box 5152,Clayton,Victoria 3168,Australiaa r t i c l e i n f o Article history:Received 30April 2009Received in revised form 3June 2009Accepted 4June 2009Keywords:Bone formation Wnt LRPOsteoblasts PTH BMPsa b s t r a c tSince the discovery of the link between mutations in the LRP5gene and human bone mass,considerable progress has been made in our understanding of Wnt signalling and bone formation.The connection between canonical Wnt signalling and bone formation is convincing,and there is evidence of interaction between the Wnt signalling pathway and key growth factors,transcriptional factors and systemic hor-mones.More recently,the role of the non-canonical pathway in bone metabolism has also started to be explored as well as potential bone–gut interactions.This review focuses on the role of the Wnt pathway in osteoblast differentiation as well as the interplay between Wnt signalling and other pathways involved in bone formation.© 2009 Elsevier Ireland Ltd. All rights reserved.Contents 1.Introduction..........................................................................................................................................522.The osteoblast........................................................................................................................................533.The Wnt/␤-catenin signalling pathway .............................................................................................................533.1.Overview......................................................................................................................................534.The Wnt proteins and bone biology .................................................................................................................545.The Wnt receptors and bone biology................................................................................................................556.Antagonists of the Wnt signalling pathway .........................................................................................................566.1.Dickkopfs (Dkks)..............................................................................................................................566.2.Secreted frizzled related proteins (sFRPs)....................................................................................................566.2.1.Wnt inhibitory factor (Wif-1).......................................................................................................577.SOST,Wnt/␤-catenin signalling and bone formation................................................................................................578.Interaction between Wnt/␤-catenin signalling and key growth factors,transcriptional factors and systemic hormones.........................578.1.Bone morphogenetic proteins (BMPs)........................................................................................................578.2.Parathyroid hormone (PTH)..................................................................................................................589.Conclusions ..........................................................................................................................................59Acknowledgements..................................................................................................................................59References ...........................................................................................................................................591.IntroductionBone is a specialised connective tissue with an intricate system of metabolically active cells and extracellular matrix that are vital to the performance of its structural,mechanical and metabolic roles.∗Corresponding author at:Prince Henry’s Institute,PO Box 5152,Clayton,Victoria 3168,Australia.Tel.:+61395944372;fax:+61395946125.E-mail address:at@ (at).Osteoblasts and osteoclasts are continually involved in the regen-eration,remodelling and repair of a vast network of bone matrix.Our understanding of the regulation of bone formation continues to evolve,with genetic and molecular studies identifying key local and systemic factors involved in bone remodelling (Zaidi,2007).In 2001,new insights into the control of bone mass came from discoveries related to the gene for low-density lipoprotein receptor-related protein 5(LRP5).LRP5,LRP6and Frizzled (Fzd)function as co-receptors for Wnt proteins,and the LRP and Fzd receptor com-plex provide signal transduction for the canonical Wnt signalling0303-7207/$–see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.mce.2009.06.002at,K.W.Ng/Molecular and Cellular Endocrinology310 (2009) 52–6253pathway.Loss of function mutations in the LRP5gene were shown to be associated with low bone mass in the osteoporosis pseudoglioma syndrome(Gong et al.,2001),whereas a single amino-acid substitu-tion(G171V)in the same gene was associated with a high bone mass state(Boyden et al.,2002;Little et al.,2002).Later,six other amino-acid substitutions were also shown to result in the high bone mass phenotype(Van Wesenbeeck et al.,2003;Ai et al.,2005).Animal studies largely confirmed what was seen in human disease,with LRP5−/−mice demonstrating decreased osteoblast proliferation and low bone mass(Kato et al.,2002).In contrast,mice over-expressing the human G171V LRP5mutation associated with high bone mass had a phenotype with increased bone mass similar to that described in the original kindred(Babij et al.,2003).Since the original publication connecting Wnt signalling to bone formation less than a decade ago,there has been a stream of research aimed at elucidating the complex roles that Wnt proteins, endogenous Wnt inhibitors and various signalling pathways play in the regulation of bone mass.This review will focus on the role of the Wnt pathway(and its components)in the regulation of osteoblast differentiation and bone formation as well as the interplay between Wnt signalling and other pathways involved in bone formation. 2.The osteoblastThe osteoblast is derived from multipotential mesenchymal stem cells,which also give rise to bone marrow stromal cells, chondrocytes,muscle cells and adipocytes under the influence of local growth factors,hormonal regulators and transcriptional fac-tors(Aubin et al.,2006).Bone formation results from a series of events that involves the differentiation of mesenchymal precursor cells into osteoblast precursors(osteoprogenitor,pre-osteoblast), the maturation of osteoblasts and the formation and the miner-alisation of matrix(Martin et al.,2008).Under light microscopy, osteoblasts appear as plump cells approximately20–30␮m in diameter lined up on the surface of unmineralised osteoid.The osteoblast has an eccentric round nucleus,a strongly basophilic cytoplasm and a prominent Golgi apparatus(Doty and Schofield, 1976).The plasma membrane of the osteoblast is rich in alkaline phosphatase and this is a useful marker for the detection of mature osteoblasts in culture.Osteoblasts have receptors for parathyroid hormone,prostaglandins,oestrogen,vitamin D3,several adhesion molecules and cytokines reflecting the key factors involved in local and systemic regulation of osteoblast function.Osteoblasts express receptor activator of NF␬B ligand(RANKL)on its cell surface,which interacts with its cognate receptor RANK(receptor activator of NF␬B)on osteoclast precursors to promote osteoclast differentia-tion.Osteoblasts also secrete osteoprotegerin(OPG),a decoy RANK receptor,which inhibits osteoclast formation(Suda et al.,1999). Towards the end of the matrix secretory period,the osteoblast loses its synthetic capacity and either becomes a lining cell or becomes trapped behind an advancing calcification front and is embedded in bone as an osteocyte.Lining cells appear elongated andflattened under light microscopy,and cover cancellous and endocortical bone surfaces.Lining cells are also known as resting osteoblasts,surface osteoblasts,inactive osteoblasts andflattened mesenchymal cells(Martin et al.,2008).In comparison,the osteo-cyte is the most abundant cell type in bone,with as many as10times the number of osteocytes as osteoblasts.They have a stellate mor-phology and reside in lacunae in mineralised matrix(Parfitt,1977). The osteocytes in their lacunae communicate with each other and other cells via extensions of their plasma membrane that run along canaliculi.They have long been thought to be mechano-sensors in bone that communicate with osteoblasts and osteoblast pro-genitors through the canalicular network,and targeted ablation of osteocytes has been shown to induce osteoporosis with defective mechanotransduction(Tatsumi et al.,2007).Osteocytes are also responsive to changes in systemic factors as shown by increased osteocyte apoptosis with glucocorticoid excess or oestrogen with-drawal(Tomkinson et al.,1997;Weinstein et al.,1998,2000).In recent years however,the complex role of the osteocyte in the con-trol of bone mass has been appreciated through the discovery of the protein product of the SOST(SclerOSTeosis)gene,sclerostin.Scle-rostin is produced exclusively by osteocytes and its critical role in the inhibition of bone formation is evident in the sclerosing bone dysplasias caused by inactivating mutations in SOST(Balemans et al.,2001;Brunkow et al.,2001).Targeted deletion of the SOST gene in mice results in increased bone formation and bone strength(Li et al.,2008);conversely transgenic mice over-expressing SOST have low bone mass(Loots et al.,2005).The action of sclerostin is to antagonise LRP5/6-mediated Wnt signalling and the complex inter-play between sclerostin,hormonal and transcriptional regulators of bone formation will be examined in the latter part of this review.3.The Wnt/␤-catenin signalling pathway3.1.OverviewSince the discovery of thefirst Wnt gene27years ago(Nusse and Varmus,1982)research into the area of Wnt signalling has expanded to encompass areas as diverse as embryogenesis,oncoge-nesis,haematology,stem cell research and now bone biology.Wnt proteins are critical signalling molecules in developmental biology, with roles in early axis specification(Huelsken et al.,2000),brain patterning(Heisenberg et al.,2001),intestinal development(Batlle et al.,2002)and limb development(Hartmann and Tabin,2000, 2001).Wnt proteins play a vital role in adult tissue maintenance, with aberrations in Wnt signalling leading to disease.Diverse con-ditions such as familial adenomatous polyposis(Kinzler et al.,1991; Nishisho et al.,1991),familial exudative vitreoretinopathy with retinal angiogenesis(Robitaille et al.,2002;Niemann et al.,2004), osteoporosis pseudoglioma syndrome(Gong et al.,2001)and high bone mass disorders(Boyden et al.,2002;Little et al.,2002)are all caused by mutations in genes encoding Wnt signal transducing proteins.It is through understanding of the role of Wnt signalling in bone formation that new therapeutic targets may be sought to relieve the burden of osteoporosis and other diseases affecting the skeleton.Wnts are secreted glycoproteins that activate at least three receptor-mediated signalling pathways to regulate cellular activ-ities.Wnt family members are defined by sequence homology to Drosophila wingless(wg)and the murine int-1proto-oncogene(hence its name).To date,nineteen Wnt genes have been identified in the mouse and human genomes (/rnusse/wntwindow.htlm).Historically, Wnt proteins were grouped into two classes,canonical and non-canonical,based on their activity in in vivo assays or cell lines. Canonical Wnts(including Wnt1,Wnt3A,Wnt8,Wnt10b)stabilise ␤-catenin and induce transcription of TCF/LEF target genes.In con-trast,non-canonical Wnts(including Wnt4,Wnt5a and Wnt11) activate Wnt signalling independent of␤-catenin transcriptional function.Non-canonical Wnt pathways are diverse and include Wnt/planar cell polarity signalling,the Wnt–cGMP/Ca2+pathway and a protein kinase A pathway(Semenov et al.,2007).More recently,however,it has been proposed that Wnt proteins them-selves are not canonical or non-canonical,but that distinct sets of receptors determine the signalling pathway initiated by the Wnt proteins(van Amerongen et al.,2008).The Wnt–␤-catenin signalling pathway is the most extensively studied pathway with direct relevance to basic bone biology.The critical function of the Wnt pathway is to activate␤-catenin-54 at,K.W.Ng /Molecular and Cellular Endocrinology310 (2009) 52–62Fig.1.(A)Wnt signalling pathway in the inactive state,showing a ligand Wnt inhibited by a decoy sFRP,the co-receptor,LRP5/6,bound by either inhibitory protein,sclerostin or Dkk,and GSK-3␤in the active state,resulting in proteosomal degradation of ␤-catenin.(B)Active Wnt signalling,with LRP5/6engaging in receptor complex after Wnt binding,disruption of GSK-3␤inhibitory complex,stabilisation of ␤-catenin and its translocation to nucleus where it activates transcription.With kind permission from Springer Science +Business Media:Osteoporosis International,Regulatory pathways revealing new approaches to the development of anabolic drugs for osteoporosis,19(8),2008,pp.1125–38,Martin TJ,Sims NA,Ng KW,Fig.2.dependent transcription (Fig.1).In the absence of Wnt receptor activation,␤-catenin is phosphorylated by a complex that con-tains the scaffolding protein axin,adenomatous polyposis coli protein (APC),glycogen synthase kinase-3␤(GSK3)and casein kinase I (Krause et al.,2008).The kinases phosphorylate ␤-catenin,allowing it to be recognised by the E3ubiquitin ligase ␤-TrCP and targetted for proteosomal degradation (Aberle et al.,1997).␤-Catenin mutants that lack the phosphorylation sites necessary for degradation cannot transmit Wnt signals and acti-vate Wnt target genes (Munemitsu et al.,1996).Cytoplasmic ␤-catenin levels are normally kept low through this continu-ous degradation.In contrast,in the presence of Wnt ligands,this degradation pathway is inhibited and ␤-catenin accumu-lates in the cytoplasm and nucleus.Nuclear ␤-catenin can then interact with transcription factors such as lymphoid enhancer-binding factor 1/T cell-specific transcription factor (TCF/LEF)to affect transcription through the recruitment of various cofactors.A large number of Wnt target genes have been identified to date (/∼rnusse/pathways/targets.html ).The precise mechanism by which Wnt binding to its receptors triggers downstream signalling events is subject to debate and is part of ongoing research (Fuerer et al.,2008).The binding of Wnt to the receptors Fzd and LRP5or LRP6(LRP5/6)(Pinson et al.,2000;Tamai et al.,2000;Wehrli et al.,2000;He et al.,2008)mediates Wnt signalling at the plasma membrane.While the mechanisms by which this receptor complex initiates signalling remains to be understood,Wnt-induced phosphorylation of LRP6is critical.The phosphorylation of the PPPSP motif of LRP6by GSK3,which primes the phosphorylation of xS in the PPPSPxS motif by casein kinase I is thought to provide the docking site for axin within the LRP6intra-cellular domain (Tamai et al.,2004;Davidson et al.,2005;Zeng et al.,2005;Nusse,2008).This process also involves the cytoplasmic scaf-folding protein dishevelled (Dsh/Dvl),and recent work has shown that Wnt induces LRP6signalosomes and promotes dishevelled-dependent LRP6phosphorylation (Bilic et al.,2007).Fzd,Dvl and axin were all shown to be required for LRP6phosphorylation by GSK3(Zeng et al.,2008).Axin is a critical protein in the ␤-catenin destruction complex and acts as a negative regulator of Wnt signalling (Nusse,2008).The docking of Axin to the LRP cytoplasmic tail facilitates the dis-sociation of the Axin/APC/GSK3destruction complex,leading to ␤-catenin stabilisation and subsequent nuclear translocation.Once in the nucleus,␤-catenin interacts with LEF/TCF transcrip-tion factors to initiate transcriptional activation of target genes (Behrens et al.,1996).In the absence of Wnt signalling,TCF forms a complex with Groucho (Cavallo et al.,1998)and acts as a repressor of Wnt target genes.Interactions between Groucho and the histone deacetylase Rpd3mediate the repression (Chen et al.,1999).␤-Catenin displaces Groucho from LEF/TCF (Daniels and Weis,2005)and this is thought to be a mechanism for the switch from transcrip-tional repression to activation.There are other negative regulators of ␤-catenin including “inhibitor of ␤-catenin and TCF”(ICAT)and Chibby,a nuclear antagonist that binds to the C-terminus of ␤-catenin (Takemaru et al.,2003).Much of the molecular complexity of gene transcription in response to Wnt signalling remains to be elucidated.4.The Wnt proteins and bone biologyWnts are secreted glycoproteins with 350–400amino acids and 23–24residues of conserved cysteines (Uusitalo et al.,1999),many of which are likely to participate in intramolecular disulfide bonds.The structures of Wnt molecules remain elusive,as they are hydrophobic,highly insoluble and until recently,have been difficult to purify.The first isolation of an active Wnt molecule,Wnt3a (Willert et al.,2003),demonstrated palmitoylation on a conserved cysteine (C77),which explained the insoluble nature of Wnts.There is in vitro evidence that osteoblasts produce Wnt pro-teins.In mouse bone marrow stromal osteoblast cultures,a marked increase in expression of Wnt7b was noted after induction of differ-entiation (Zhang et al.,2004)and Wnt7b induced a 3-fold increase in LEF-1reporter activity.Wnt1,Wnt4and Wnt14were detected in calvaria and primary osteoblasts (Kato et al.,2002)and Wnt1,Wnt4,at,K.W.Ng/Molecular and Cellular Endocrinology310 (2009) 52–6255Wnt5a and Wnt7a were detected in two of four osteosarcoma cell lines(Hoang et al.,2004).Some Wnt proteins have been shown to have an effect on bone physiology.Wnt10b−/−mice have decreased trabecular bone and serum osteocalcin(Bennett et al.,2005).FABP4-Wnt10b mice that express the Wnt10b transgene in marrow have increased bone mass and strength and the elevated bone mass appeared to confer pro-tection against ageing and ovariectomy.The expression of Wnt10b in mesenchymal progenitors induced the expression of Runx2and osterix and stimulated osteoblastogenesis(Bennett et al.,2005). Wnt10b inhibited the adipogenic transcription factors C/EBP␣and PPAR␥and blocked adipogenesis.Wnt1,Wnt2and Wnt3a have been shown to induce the expres-sion of alkaline phosphatase in mesenchymal cell lines(Gong et al.,2001;Rawadi et al.,2003).Over-expression of␤-catenin-dependent Wnt1,Wnt2and Wnt3a resulted in an increase in alkaline phosphatase activity,whereas over-expression of the␤-catenin independent Wnt4and Wnt5a had no effect.Though Wnt3a induced alkaline phosphatase gene expression in mesenchymal cells,Runx2/Cbfa1,osteocalcin and collagen type1gene expression were not affected.Furthermore,the induction of alkaline phos-phatase by Wnt3a was independent of BMP2and sonic hedgehog (SHH),but inhibition of Wnt signalling disrupted the ability of BMP2 and SHH to induce alkaline phosphatase.Wnt3a had also been shown to inhibit adipocyte differentiation,with decreased expres-sion of PPAR␥in mesenchymal C3H10T1/2cells over-expressing Wnt3a.In a study of Wnt3a+/−male mice,a decrease in bone mass was seen(Takada et al.,2007),highlighting the role of the canon-ical Wnt cascade in osteoblastogenesis(Gong et al.,2001;Glass et al.,2005).However,adipogenesis seemed unaffected in Wnt3a+/−mice.Wnt5a has been shown to induce osteoblastogenesis through the attenuation of PPAR␥-induced adipogenesis in bone mar-row mesenchymal stem cells(Takada et al.,2007).Wnt5a activated the non-canonical Wnt signalling cascade through CaMKII–TAK1–TAB2–NLK and did not activate the TCF/LEF canoni-cal signalling.Furthermore,the same group showed that Wnt5a+/−mice had a reduction in bone mass with a reduced number of osteoblasts,whereas an increased number of adipocytes were seen in the bone marrow.Non-canonical Wnt signalling repressed PPAR␥function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase(Takada et al.,2007).Both the canonical and non-canonical Wnt signalling pathways play a role in osteoblastogenesis.5.The Wnt receptors and bone biologyThefirst identified Wnt receptors were members of the Friz-zled family of receptors(Wodarz and Nusse,1998).Several groups subsequently showed that members of the low-density lipopro-tein receptor family were essential co-receptors for Wnt signalling (Pinson et al.,2000;Tamai et al.,2000;Wehrli et al.,2000).There are currently10members of the Fzd gene family and Fzd receptors can bind Wnt proteins with a Kd of1.6nM(He,2003).Structurally, the Fzd receptor has an extracellular Wnt-binding domain,a seven transmembrane segment and an intracellular C-terminal tail.Wnts interact with Fzd through a cysteine-rich domain(CRD)on the extracellular surface(Hsieh et al.,1999;Wu and Nusse,2002).LDL receptor-related proteins have been classically known for their role in lipoprotein metabolism and atheroma formation but LDL receptor-related proteins are critical in the Wnt signalling pathway.The importance of thefly ortholog of LRP,arrow,as a co-receptor in the initiation of Wg signalling was demonstrated9years ago(Wehrli et al.,2000).LRP5and LRP6are71%homologous.At the amino terminus of the extracellular domain of LRP5and6,four ␤-propeller motifs alternate with four epidermal growth factor-like repeats.This creates binding sites for extracellular ligands(He et al.,2004;Williams and Insogna,2009).The cytoplasmic tail is anchored into the plasma membrane by a single pass transmem-brane segment.The cytoplasmic tail contains the series of highly conserved PPPS/TP motifs,which,as discussed earlier,are phos-phorylated following Wnt activation.The transport of LRP from the endoplasmic reticulum to the cell membrane requires a molecu-lar chaperone known as mesoderm development(Mesd)in mice (Hsieh et al.,2003).Molecular chaperones promote the folding of lipoprotein receptors in the endoplasmic reticulum.The effect of Mesd deficiency in the mouse is severe,with abnormal devel-opment of anterior–posterior polarity and impaired mesodermal differentiation(Hsieh et al.,2003).In humans,two kindreds with a familial high bone mass phenotype were found to have a Gly171-to-Val mutation in LRP5(Boyden et al.,2002;Little et al.,2002). This point mutation was predicted to disturb the structure of the first six-bladed␤-propeller module in LRP5.The G171V mutation inhibits the ability of Dkk1and potentially other proteins to bind to LRP5and inhibit Wnt signalling.In addition,the G171V mutation was shown to disrupt the interaction of LRP5with Mesd(Zhang et al.,2004),resulting in fewer LRP5molecules on the cell surface.Mice with disruption of LRP5in all cells,similar to patients with OPPG,have a low bone mass phenotype and persistent embryonic eye vascularisation(Kato et al.,2002).The low bone mass became evident in the postnatal period and was secondary to reduced osteoblast proliferation.Cbfa1expression was normal in the Lrp5−/−mice,revealing that the phenotypic bone changes were a Cbfa1 independent phenomenon.Interestingly,deletion of␤-catenin in differentiated osteoblasts in mice did not result in the phenotypic and molecular abnormalities in bone cells seen with the inactiva-tion of LRP5.This mutation primarily affected bone resorption,with an increase in osteoclasts secondary to a decrease in OPG(Glass et al.,2005).This demonstrates the complexity of Wnt signalling in bone formation,with␤-catenin controlling osteoclast differentia-tion by regulating OPG expression in differentiated osteoblasts.In contrast,conditional deletion of␤-catenin in limb and head mes-enchyme in mouse embryos resulted in osteoblast precursors that were blocked in differentiation with subsequent development into chondrocytes instead of osteoblasts(Day et al.,2005;Hill et al., 2005).Recently,Yadav et al.(2008)demonstrated that LRP5inhibited tryptophan hydroxylase(Tph1)expression,a rate-limiting enzyme in the gut-derived serotonin biosynthetic pathway,inhibiting sero-tonin synthesis in the duodenum.Serotonin had previously been implicated in the regulation of bone mass(Warden et al.,2005) and gut-specific deletion of LRP5was shown to result in low bone mass phenotype seen in LRP5null mice(Yadav et al.,2008).In contrast,osteoblast-specific deletion of LRP5did not cause a sim-ilar osteoblast defect.The authors pointed out that the low bone phenotype of LRP5null mice appeared postnatally,with normal bone formation during development,and furthermore that the osteoblast proliferation defect in LRP5−/−mice postnatally was not cell autonomous.The evidence for an important role for Wnt sig-nalling in bone formation remains compelling.It is possible that LRP6,rather than LRP5,is the critical co-receptor for Wnt signalling in bone.Though Yadav et al.(2008)did not exclude the possibility that LRP5may regulate osteoblast differentiation in bone postna-tally,their work provides convincing evidence from mouse genetics for an important role of LRP5in inhibiting serotonin production,and has demonstrated for thefirst time the role of complex gut–bone interactions in the regulation of bone mass.Likely consistent with this,the LRP6loss of function bone phenotype is much more severe than the LRP5loss of function phenotype,reflecting the critical role of LRP6during embryogenesis.Although LRP6−/−mice die at birth (Pinson et al.,2000),LRP6heterozygous mice display reduced bone56 at,K.W.Ng/Molecular and Cellular Endocrinology310 (2009) 52–62mass(Holmen et al.,2004).In2007,a mis-sense mutation in LRP6 that resulted in impaired Wnt signalling was reported in a family with autosomal dominant early coronary artery disease,metabolic risk factors and osteoporosis(Mani et al.,2007).Wnt proteins are not the only known ligands for the Fzd–LRP5/6 receptor complex.There are secreted proteins that antagonise the receptor directly.Other antagonists interfere with Wnt proteins themselves.6.Antagonists of the Wnt signalling pathwayThere are several known classes of proteins that antagonise vertebrate Wnts.Dickkopfs(Dkk)proteins and Wise compete for the LRP5/6receptor to prevent canonical signalling.In con-trast,secreted frizzled related proteins(sFRPs),Cerberus and Wnt Inhibitory Factor1(WIF-1)bind to directly to Wnts.Studies of Cerberus and Wise are largely limited to the Xenopus,so we will concentrate on Dkks,sFRPs and Wif-1and their contribution to Wnt signalling in bone.Furthermore,the product of the SOST gene,scle-rostin,will also be reviewed as an important inhibitor of canonical Wnt signalling.6.1.Dickkopfs(Dkks)The Dkk family has four members—Dkk1–Dkk4.Dkk gene expression is both temporally and spatially regulated throughout development,and there are elevated expression patterns in tissues that mediate epithelial-mesenchymal transformation(Monaghan et al.,1999),including the heart,limbs and bone.Dkk1is the most extensively studied DKK family member.In embryos,Dkk1is involved in head development(Glinka et al.,1997). Dkk−/−mice lack head structures anterior to the otic vesicle as well as anterior neural structures including the telencephalon,dien-cephalon and parts of the midbrain(Mukhopadhyay et al.,2001). Xenopus embryos over-expressing Dkk1develop enlarged heads and shortened tails(Glinka et al.,1997).However,Dkk1also has an important role in bone biology.Dkk1mRNA has been isolated from osteocytes(Zhang et al.,2004)and osteosarcoma cell lines (Gregory et al.,2003).Dkk1over-expression in transgenic mice resulted in severe osteopenia with a49%reduction in the number of osteoblasts(Li et al.,2006).In contrast,mice engineered to lack Dkk1led to an increase bone formation and bone mass(Morvan et al.,2006).Gene expression profiling in myeloma patients with and without bone lesions demonstrated that an elevated expression of Dkk1by plasma cells correlated with bone disease(Tian et al., 2003).Treatment of C2C12cells with recombinant Dkk1and patient sera containing high Dkk1levels inhibited BMP2/Wnt-induced ALP production.More recently,the treatment of myeloma-bearing SCID-rab mice with anti-Dkk1antibody decreased osteolytic bone lesions,suggesting a potential role for Dkk1as a therapeutic target in myeloma bone disease(Yaccoby et al.,2007).Dkk1binds to LRP6with high affinity(Bafico et al.,2001;Mao et al.,2001;Semenov et al.,2001)as well as to the Kremens(Mao et al.,2002),another class of transmembrane protein.There are two Kremen proteins—Kremen1and Kremen2.Kremen2,Dkk1and LRP6form a complex that disrupts Wnt/LRP signalling by promoting removal of the Wnt receptor from the plasma membrane by endo-cytosis(Mao et al.,2002).It is postulated that the internalisation of LRP through Kremen and Dkk1may inhibit recruitment of axin to the plasma membrane,blocking the stabilisation of␤-catenin (Kawano and Kypta,2003).Like Dkk1,Dkk4can cooperate with Kremen2to inhibit Wnt/␤-catenin through binding LRP6.In comparison,Dkk3cannot bind to LRP6and has no effect on Wnt signalling(Krupnik et al.,1999; Mao and Niehrs,2003).Dkk2functions as both an agonist and an antagonist of LRP6,depending upon the presence of Kremen2.In humanfibroblasts transfected with Dkk2,Wnt signalling is acti-vated.In the presence of a co-transfection with Dkk2and Kremen2, Wnt signalling is inhibited.The interaction with Kremen and LRP6 is mediated through the second cysteine-rich domain of Dkks (Mao and Niehrs,2003).Interestingly,mice lacking Dkk2were osteopenic(Li et al.,2005).Further work needs to be performed with animal models to address the role of Dkk2in bone(Rawadi, 2008).6.2.Secreted frizzled related proteins(sFRPs)Five sFRPs(sFRP1–sFRP5)bind directly to Wnt proteins to antag-onise Wnt signalling.Based on sequence homology,sFRP1,sFRP2 and sFRP5are related,whereas sFRP3and sFRP4form another dis-tinct group.The sFRPs contain a cysteine-rich domain(CRD)that is homologous to the CRD in the Fzd receptor;however,sFRPs lack a transmembrane domain and are secreted as extracellular proteins. sFRPs have a role in increasing apoptosis,and are down regulated in malignant disease(Suzuki et al.,2002)and up-regulated in some degenerative diseases(James et al.,2000).sFRP1,sFRP3and sFRP4 have demonstrated roles in skeletogenesis and bone formation.Human sFRP1encodes a314amino acid,35kDa pro-tein and is mapped to chromosome8p12(/cgi-bin/genecards).sFRP1has been identified as a regulator of osteoblast and osteocyte survival,with effects on trabecular bone mass.Expression of sFRP1increases with advanc-ing osteoblast differentiation and peak expression of sFRP1occurs at the pre-osteocyte stage(Bodine and Komm,2006).Deletion of the sFRP1gene in mice enhanced trabecular bone accrual in adult mice with increased trabecular bone mineral density,volume and bone apposition rates between the ages of13–52weeks(Bodine et al.,2004).sFRP1−/−animals had normal serum osteocalcin,type1 collagen C-terminal telopeptides(CTX),serum calcium and alkaline phosphatase levels.Deletion of sFRP1did not affect cortical bone or non-skeletal organs.Mechanistically,deletion of sFRP1led to a decrease in osteoblast and osteocyte apoptosis with a resultant increase in osteocyte num-ber in vivo.In vitro work from sFRP1−/−bone marrow isolates demonstrated inhibition of osteoblast lineage cell apoptosis and increased osteoclastogenesis(Bodine et al.,2004).In vitro assays on osteoclast formation have also shown that sFRP1is an inhibitor of osteoclast formation(Hausler et al.,2004).Deletion of sFRP1also affects chondrogenesis and endochondral bone formation.Gaur et al.(2006)showed that sFRP1is highly expressed in cartilaginous tissues of the developing mouse and histological analysis of the long bones from4-week-old mice demonstrated that loss of sFRP1 led to shortened columnar zones and increased calcification of the growth plates and primary spongiosa.sFRP3is also referred to as FrzB(Frizzled motif associated with bone development)and wasfirst identified from purified protein extracts of bovine articular cartilage(Hoang et al.,1998).sFRP3 is located on human chromosome2q32and encodes a36.3kDa protein.In the human,it is primarily expressed in the cartilagi-nous core of long bones during embryonic development and in the appendicular skeleton(6–13weeks).At13weeks of gestation,tran-scripts were present in early chondroblasts of the tarsal bones of the foot,the carpal bones of the hands and the epiphysis of long bones.sFRP3is highly expressed in placenta and heart,but can also be found in the brain,skeletal muscle,kidney and pancreas (/cgi-bin/genecards).sFRP3is a important modulator of chondrocyte maturation and function. Virally driven misexpression of sFRP3in chick limbs resulted in shortening of skeletal elements,joint fusion and delayed chondro-cyte maturation(Enomoto-Iwamoto et al.,2002).。