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β-Catenin in the race to fracture repair: in it to WntDavid Silkstone, Helen Hong and Benjamin A Alman*

INTRODUCTIONFracture repair is a regenerative process that involves tightly coordinated biological events that partially recapitulate normal bone develop-ment. Approximately one-third of all individuals will fracture a bone during their lifetime, and in most cases the reparative process results in successful fracture union.1 For 5% of fractures, however, there is a failure of normal healing, which results in non-union. In such cases, exten-sive surgery is required to achieve complete bone healing.2 A pharmacologic approach that enhances fracture repair would avoid the need for surgery and improve patient outcome. Understanding the signaling pathways that direct fracture repair could provide insight into possible therapeutic targets and thereby help to achieve this aim. One crucial signaling pathway involved in both embryonic bone development and fracture repair is the canonical Wnt/β-catenin signaling pathway. In the remainder of this Review we discuss the processes involved in normal bone development and fracture repair before considering the role of the Wnt/β-catenin signaling pathway—a pathway that is crucial for both embryonic bone development and fracture repair. The Wnt/β-catenin signaling pathway as a therapeutic target is also discussed.

NORMAL BONE DEVELOPMENTThe embryonic skeleton develops through two different ossification processes. Intramembranous ossification, which forms the flat bones of the skull, involves the condensation and direct differentiation of mesenchymal stem cells into osteoblasts, producing bone without the need for an intermediate cartilaginous template.3 By contrast, endochondral ossification is the forma-tion of bone from chondrocytes and osteoblasts through a cartilaginous model; this type of ossification is responsible for the formation of long bones.4

Endochondral ossification is initiated when pluripotent mesenchymal stem cells (PMSCs) undergo condensation and differentiation into

The Wnt/β-catenin pathway regulates multiple biological events during embryonic development, including bone formation. Fracture repair recapitulates some of the processes of normal bone development, such as the formation of bone from a cartilaginous template, and many cell-signaling pathways that underlie bone development are activated during the repair process. The Wnt/β-catenin signaling pathway is activated during fracture repair, and dysregulation of this pathway alters the normal bone-healing response. In early pluripotent mesenchymal stem cells, Wnt/β-catenin signaling needs to be precisely regulated to facilitate the differentiation of osteoblasts; by contrast, β-catenin is not needed for chondrocyte differentiation. Once mesenchymal stem cells are committed to the osteoblast lineage, activation of Wnt/β-catenin signaling enhances bone formation. This activity suggests that the Wnt/β-catenin pathway is a therapeutic target during bone repair. Indeed, treatments that activate Wnt/β-catenin signaling, such as lithium, increase bone density and also enhance healing.

Keywords chondrocytes, embryonic bone development, fracture repair, osteoblasts, wnt/β-catenin

D Silkstone and H Hong are graduate students and BA Alman is the AJ Latner Chair of Orthopaedic Surgery at the University of Toronto, Toronto, ON, Canada.

Correspondence*Program in Developmental and Stem Cell Biology, Hospital for Sick Children and University of Toronto, Toronto, Ontario M5G 1X8, Canada benjamin.alman@sickkids.ca

Received 4 February 2008 Accepted 8 May 2008 Published online 17 June 2008

www.nature.com/clinicalpracticedoi:10.1038/ncprheum0838

REVIEw CRITERIAPubMed and Ovid were searched in January 2008 for full papers, published in English-language journals, using the following keywords alone or in combination: “embryonic bone development”, “endochondral ossification”, “Wnt/β-catenin”, “fracture repair” and “molecular mechanisms”. Articles were selected from original research papers and reviews, without a time restriction. Articles from the authors’ own collections were also considered for inclusion. The reference list was updated in March 2008.

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chondrocytes, which secrete matrix compounds, including type II collagen.5 The matrix becomes a cartilaginous framework from which the bone will adopt its shape and orientation: the shaft and ends of the longitudinal bone become the diaphysis and epiphyses, respectively, both of which are encompassed by the perichondrium. As the collagen matrix lengthens towards the epiphyses, central chondrocytes hypertrophy, whereas perichondral cells differentiate into osteoblasts to form a surrounding layer of bone called the bone collar.6 Hypertrophic chondro-cytes secrete factors that initiate angiogenesis into the cartilage, and then eventually undergo apoptotic cell death.7 Invading osteoblasts replace the chondrocytes and convert mineral-ized cartilage matrix into bone. Elongation of the long bone occurs at the growth plate, an area that is occupied by chondrocytes, and persists until skeletal maturity.

Various transcription factors and signaling molecules involved in bone development and homeostasis have been identified. These factors work in a coordinated manner in various cell types. Bone morphogenic protein (BMP) signaling has an important role throughout bone formation, particularly in mesenchymal stem cell condensation and chondrocyte proliferation.8,9 Indian hedgehog signaling controls chondro-cyte differentiation and proliferation, as well as osteoblast differentiation, and is negatively regu-lated by parathyroid-hormone-related protein signaling.9,10 Many other factors, such as fibro-blast growth factor, also regulate chondrocyte and osteoblast proliferation and differentiation.11,12

FRACTURE REPAIRFracture repair is a dynamic process that involves the coordination of cell proliferation and differentiation, apoptosis, angiogenesis and remodeling.13–16 Cellular events activated during bone development, as well as the underlying mechanisms that direct these processes, are also activated during this reparative process. Fracture repair can occur through either primary (direct) or secondary (indirect) healing. In a manner similar to intramembranous ossification, primary healing entails direct repair via bridging of the fracture by osteoblasts, a process that occurs in the absence of chondrocytes. Secondary healing is the more commonly observed mechanism of fracture healing, and more closely resem-bles endochondral bone development, but with minor, yet important, differences.15

Inflammation is a physiological response that is present at the start of both primary and secondary bone healing, but is not present during embryonic bone development.17 Immediately after an injury occurs, hematoma formation, platelet degranulation and inflamma-tion are initiated. During this initial phase of healing, recruited inflammatory cells, damaged local cells and the extracellular matrix release growth factors and cytokines that are thought to be important for the proliferation and differ-entiation of PMSCs.18,19 PMSCs differentiate into osteochondral progenitor cells, which can transform into either mature chondrocytes or osteoblasts.19–22

Fracture repair can occur through both intramembranous and endochondral ossifica-tion processes. During intramembranous ossification,15 cells from the bone marrow, as well as local osteochondral progenitor cells, differentiate into an osteoblastic phenotype and contribute to the formation of the ‘hard callus’ observed during intramembranous ossification. In regions of endochondral ossification, PMSCs undergo chondrogenesis and give rise to hyper-trophic chondrocytes, which are responsible for the resorption of cartilage and mineraliza-tion of the ‘soft callus’, as observed in fetal bone development.15 At the end of the endochondral ossification process, a substantial amount of woven bone is extensively remodeled and eventually replaced by lamellar structures.

THE wNT/β-CATENIN SIGNALING PATHwAYWnt signaling is crucial for normal embryogenesis and has a role in various regenerative processes, including bone repair.23 Several intracellular pathways are induced on activation of the Wnt receptor, such as the β-catenin-dependent canon-ical pathway and the β-catenin-independent noncanonical pathways (planar cell polarity pathway and Ca2+-mediated pathway).23 The canonical Wnt/β-catenin signaling pathway (Figure 1) is the most studied of these pathways, and has been shown to have a dominant role in bone development and repair.24

The canonical Wnt/β-catenin signaling pathway is initiated by the interaction of Wnt ligands with their cognate receptor complex. The receptor complex consists of a seven-transmembrane- domain receptor of the Frizzled family of receptors, and an LDL-related protein 5/6 (LRP5/6) co-receptor.23 There are numerous Wnt ligands: some activate the canonical or noncanonical

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signaling pathways, and some even inhibit Wnt signaling. In the absence of appropriate Wnt proteins, the cytoplasmic phosphoprotein Dishevelled is not phosphorylated and is, there-fore, unable to inhibit the multiprotein complex. Two scaffolding proteins within this complex—adenomatous polyposis coli (APC) and Axin—then bind cytoplasmic β-catenin, which becomes phosphorylated by serine/threonine kinases, casein kinase 1γ (CK1γ) and glycogen synthase kinase 3β (GSK3β).23 As a result, β-catenin is targeted for ubiquitination and degradation by proteasomal machinery.23 In the presence of appropriate Wnt proteins, Dishevelled becomes phosphorylated, which sequesters the action of Axin and ultimately inhibits the kinase activity of the multiprotein complex.25 β-Catenin accumulates in the cyto-plasm and translocates into the nucleus, where it interacts with members of the T-cell factor/lymphocyte elongation factor (TCF/LEF) family to activate the transcription of several target genes.23 TCF/LEF transcription factors are archi-tectural factors, and as such the target genes vary between cell types depending on the presence of other transcription factors.26

wNT/β-CATENIN SIGNALING IN ENDOCHONDRAL BONE FORMATIONData from mouse studies show that Wnt/ β-catenin signaling has an essential role throughout endochondral bone develop-ment.27–29 During early bone development, β-catenin is not necessary for chondrocyte differentiation. Wnt molecules such as Wnt1, Wnt3a, Wnt4, Wnt7a and Wnt11 can inhibit the differentiation of PMSCs into chondrocytes through the regulation of downstream cellular pathways.5,30–33 Once osteochondral progen-itor cells differentiate into chondrocytes, Wnt molecules have opposing effects on chondrocyte maturation: overexpression of Wnt5a or Wnt5b delays maturation of hypertrophic chondro-cytes, whereas overexpression of Wnt4 and Wnt8 expedites their maturation.30,32,34,35

In contrast to its role in chondrocyte differ-entiation, β-catenin is required for osteoblast differentiation. Progenitor cells in mice that lack β-catenin develop into chondrocytes, and the mice have decreased bone density; furthermore, inhibition of Wnt signaling results in abnormal bone development.36–41 Later in development, Wnt signaling is important for maintaining bone

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Figure 1 The Wnt/β-catenin signaling pathway. (A) In the absence of an activating Wnt ligand, cytoplasmic protein Dsh remains unphosphorylated. This in turn allows a multiprotein complex, which includes the scaffolding proteins APC and Axin, CK1γ and GSK3β, to degrade β-catenin. Axin binds and phosphorylates β-catenin through the kinases CK1γ and GSK3β. Phosphorylated β-catenin is then targeted for ubiquitin-mediated degradation. As a result, Wnt target gene expression is repressed by transcriptional inhibitors. (B) In the presence of Wnt ligands, Frizzled and LRP5/6 inhibit the multiprotein complex through phosphorylated Dsh. As a result, β-catenin is stabilized in the cytoplasm and is able to translocate into the nucleus. β-Catenin can then bind to TCF/LEF transcription factors, thereby regulating expression of Wnt target genes. Abbreviations: APC, adenomatous polyposis coli; CK1γ, casein kinase 1γ; Dsh, Dishevelled; GSK3β, glycogen synthase kinase 3β; LRP5/6, LDL-related protein 5/6; TCF/LEF, T-cell factor/lymphocyte elongation factor.

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density over time. As previously demonstrated, mutations in the Lrp5 gene inhibit β-catenin activity, resulting in decreased bone density and decreased osteoblast differentiation; by contrast, mutations that activate β-catenin can lead to elevated bone density.27,40,41

During BMP-induced osteogenesis, β-catenin-mediated TCF-dependent trans-cription is activated by Wnt ligands.40 β-catenin is required downstream of BMP2 signaling for osteoblast mineralization during this process.41–44 The function of Wnt/β-catenin signaling in chondrogenesis and osteogenesis, and its inter-action with other signaling pathways, such as Indian hedgehog and BMP, underlies its crucial role in endochondral bone development.

wNT/β-CATENIN SIGNALING IN FRACTURE REPAIRThe molecular mechanisms and signaling pathways that regulate fracture repair are not as well understood as those that regulate bone development. Gene profiling studies in animals have found that Wnt4, Wnt5a, Wnt5b, Wnt10b, Wnt11, Wnt13, and β-catenin are expressed during fracture repair, which suggests that Wnt/β-catenin signaling is activated.41,45 In addition, β-catenin level has been shown to be elevated during human fracture repair.46

In our laboratory, the observation of fracture healing in TCF-reporter mice revealed that levels of β-catenin expression increased throughout the fracture healing process.46 However, β-catenin-mediated TCF-dependent trans-cription only occurred after PMSCs displayed either a chondrocytic or osteoblastic phenotype, and by 2 weeks after the fracture, this trans-cription was observed only in osteoblasts. Once the remodeling phase began, TCF activity was decreased and osteoblasts residing in the peri-osteum and distant from the fracture repair site showed only residual activity.46

THE IMPLICATIONS OF DYSREGULATION OF wNT/β-CATENIN SIGNALING IN FRACTURE REPAIRSeveral groups have investigated the effects of altered Wnt/β-catenin signaling on fracture repair.42,44–47 In our study, mice were generated to have conditional β-catenin null or stabilized alleles that could be activated by Cre recombinase (Cre).46 In mice transfected with an adenovirus expressing Cre, conditional null alleles were activated in all cells during fracture repair. In the

same study, mice with conditional null alleles were crossed with transgenic mice with Cre expression driven by regulatory elements that were activated only in osteoblasts. In the mice with null alleles in all cells, there was no evidence of bone bridging at the fracture site, and histo-logical evaluation indicated that the fracture site consisted of undifferentiated mesenchyme-like tissue. When inactivated in committed osteo-blasts only, chondrocytes were still present at the fracture site, but osteoblasts did not form.

Hyperactivation of β-catenin during normal bone development causes an increase in bone density,42 so mice with stabilized alleles would be expected to have an increased amount of bone present at the fracture site. In our study, however, bone did not completely bridge the fracture gap when the stabilized allele was activated by adeno-virus-transfected Cre.46 By contrast, there was an increased amount of bone found at the frac-ture site when stabilized β-catenin alleles were activated only in committed osteoblasts under control of the type I collagen–Cre system.46

Once cells are committed to the osteoblast phenotype, β-catenin will positively regulate the amount of bone that forms at the fracture site.46 Furthermore, treatment with DKK-1 (Dickkopf 1) has been shown to inhibit Wnt/β-catenin activa-tion during fracture repair and result in a lack of runx2 expression (essential for osteoblastic differentiation) and bone mineralization at the fracture site, supporting the idea that Wnt ligands activate β-catenin.44,46

These results support an essential role for Wnt/β-catenin in fracture repair (Figure 2). Precursor cells lacking β-catenin are unable to differentiate into osteoblasts. Overexpression of β-catenin will also inhibit normal differentiation, but in cells already committed to the osteoblast lineage, it will increase the quantity of bone at the fracture site. This finding is supported by in vitro studies that demonstrate the importance of precise Wnt signaling during PMSC differentiation.42,47

THERAPIES MODULATING wNT/β-CATENIN SIGNALING IN FRACTURE REPAIRCurrently, there are no pharmacologic agents available that have been shown to improve bone repair; however, biologic agents that can be directly applied at the fracture site are avail-able. One such clinically approved biologic agent is BMP.38,48–51 Similar to the role of endogenous BMP in fracture repair, pharmacologic BMP induces heterotopic bone formation through

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β-catenin activation.40 As a consequence, BMP biologics might require β-catenin for success. In addition, clinically approved parathyroid hormone also activates Wnt signaling to enhance fracture repair.43

The ideal treatment for delayed bone healing would be a pharmacologic agent that did not require the addition of invasive procedures such as surgery or implantation of a biologic agent. Activation of β-catenin signaling in cells that have already committed to the osteoblast lineage would be one such method of encouraging fracture healing.β-Catenin can be pharmacologically activated

through the inhibition of GSK3β by agents such as lithium.46 Lithium is approved as a treat-ment for manic-depressive states and bipolar illnesses. In mice that experienced acceler-ated osteoporosis as a result of dysfunctional osteoblastogenesis, lithium treatment led to a significant improvement in bone mass.52–54

In our laboratory, we administered lithium treatment to mice either before or several days after generating a fracture.46 When treatment was initiated before the fracture, healing was inhibited. By contrast, when lithium treatment was started after the fracture was generated, bone formation was enhanced. This is consistent with our hypothesis that the β-catenin level needs to be precisely regulated to allow differentiation of cells to osteoblast precursors.46 Thus, the administration of lithium, starting several days after a fracture has occurred, has the potential to enhance repair in a clinical setting. As such, lithium treatment could potentially be used to improve healing in cases of slow union or non-union, avoiding the need for more-invasive treatments.55 In addition, a similar therapeutic approach could be applied to other situa-tions in which enhanced bone repair would be beneficial, such as in healing after a total joint replacement or in improving the rate of fusion after spinal surgery. Lithium can have adverse effects, however, including vomiting, weight gain, decreased thyroid function and impaired memory, although these effects are associated with long-term use and high dosages.

THERAPEUTIC INTERVENTION FOR OTHER DISORDERS OF OSSIFICATIONSeveral diseases are associated with excessive bone formation, or heterotopic ossification. This condition can occur after injury near a joint or after extensive muscle damage. Current

treatments include surgery to remove excess bone or radiation therapy to prevent its occurrence. Data from a model of BMP-induced heterotopic ossification show that β-catenin is required for BMP to induce such bone formation.40 This concept is supported by work involving tumor-necrosis-factor-induced rheumatoid arthritic joints, in which it was found that inhibition of DKK-1 leads to ectopic bone formation.56 It is possible, therefore, that inhibition of β-catenin could be used to treat heterotopic ossification. At present, there are no agents available that inhibit β-catenin, but this is an area of intensive ongoing research. As such agents are identified,

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Figure 2 The role of β-catenin in different cell types during fracture repair. During the initial phase of fracture repair, a hematoma forms at the fracture site. The tissue damage, hematoma and the inflammatory response liberate growth factors and cytokines, allowing leukocytes, monocytes, macrophages and pluripotent mesenchymal stem cells to reach the fracture site. In the cartilage and bone formation phase, pluripotent mesenchymal stem cells differentiate into osteochondral progenitor cells and then into chondrocytes and osteoblasts. At this stage, β-catenin levels need to be precisely regulated for differentiation to osteochondral progenitor cells. The cartilage that forms provides a template that is eventually replaced with bone. In the absence of β-catenin, osteochondral progenitor cells are committed to the chondrocyte lineage. Once cells become committed to the osteoblast lineage, β-catenin levels positively regulate bone formation. In the later phases of repair, there is extensive remodeling until the damaged skeletal element regains its original shape and size.

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they might be applicable in a clinical setting to treat this condition, avoiding the need for more-invasive therapies.

CONCLUSIONSWnt/β-catenin signaling is crucial for bone development, fracture repair, and the main-tenance of bone density. This signaling pathway mediates the differentiation of osteochondral progenitor cells during fracture repair, and pharmacologic agents that target this pathway could be developed into effective therapies to improve bone density and enhance bone repair. Given the central role of Wnt/β-catenin signaling in bone development and fracture repair, it is an enticing therapeutic target for disorders of bone repair.

KEY POINTS■ Wnt/β-catenin signaling has a major role in

embryonic bone development by controlling the differentiation of pluripotent mesenchymal stem cells into osteoblasts

■ Activation of the Wnt/β-catenin pathway during embryonic bone development increases bone formation

■ Wnt/β-catenin signaling is activated during fracture repair

■ Removal of β-catenin prevents osteochondral progenitor cells from differentiating into osteoblasts

■ Activation of β-catenin by pharmacologic agents such as lithium can enhance fracture repair and improve patient outcome

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Competing interestsThe authors declared no competing interests.

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