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A Wnt canon orchestrating osteoblastogenesis
Review
TRENDS in Cell Biology
Vol.16 No.3 March 2006
A Wnt canon orchestrating osteoblastogenesis Christine Hartmann Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria
Several transcription factors have been identified that control the differentiation of osteoblasts; however, relatively little is known about the signaling pathways involved in regulating the differentiation process. Recently, the canonical Wnt–b-catenin pathway has been implicated in osteoblastogenesis. This review focuses on the role of the canonical Wnt–b-catenin pathway during embryonic development, where it is required for the differentiation of osteoblasts from a precusor that is shared with the chondrocyte lineage and the requirement of this pathway during postnatal life in bone homeostasis. The recent findings covered in this review are major advances in our understanding of skeletal development and promise new therapeutic avenues for tissue engineering and treatment of osteoporosis.
Formation of the vertebrate skeleton The skeleton is composed of two types of tissue, cartilage and bone, which are formed by chondrocytes and osteoblasts, respectively. Bones are formed during embryonic development by two different processes – intramembranous and endochondral ossification. Some skull bones and the lateral halves of the clavicles are formed by intramembranous ossification, whereby boneforming cells, the osteoblasts, differentiate from within a connective tissue sheet of condensed mesenchymal cells and start to secrete osteoid (see Glossary), which later becomes mineralized and reorganized into ‘compact’ bone (Figure 1a). However, most bones are formed by endochondral ossification: mesenchymal condensations differentiate into chondrocytes, forming a cartilaginous template prefiguring the future skeletal element. Chondrocytes in the center of the cartilage element stop proliferating, become prehypertrophic and mature into hypertrophic chondrocytes, which are characterized by an increase in size, vacuolization and secretion of a distinct extracellular matrix. Mesenchymal cells surrounding the cartilage element flatten and form the perichondrium – a condensed multilayered tissue (Figure 1b). Perichondrial cells that are adjacent to the middle region of the cartilage template receive a signal, the protein Indian hedgehog (Ihh), from prehypertrophic chondrocytes, which induces the osteogenic program in these cells, coupling the process of chondrocyte matuCorresponding author: Hartmann, C. ([email protected]). Available online 7 February 2006
ration to the onset of osteoblast differentiation. Thus, during endochondral ossification the first osteoblasts develop within a condensed mesenchymal tissue, similar to the process of intramembranous ossification; however, in contrast to osteoblasts in the skull, these osteoblasts require the Ihh gene product as an inductive signal from the chondrocytes. It is therefore likely that chondrocytes and osteoblasts have a common mesenchymal skeletal precursor [1,2]. The bone formed by either intramembranous or endochondral ossification is remodeled by the osteoclasts, which are bone resorbing cells of hematopoietic origin. Differentiation of osteoclasts is controlled in part by factors secreted by osteoblasts. Here, we focus on the recent discoveries showing important roles for the canonical Wnt pathway (Box 1) in osteoblast lineage determination during embryonic development. We also discuss its postnatal role in regulating osteoblast proliferation, maturation and activity (Table 1).
Glossary Allele: alternative form of any given genomic locus on a chromosome; can be an alternative form of a gene, but can also refer to intergenic regions. Conditional allele: allele of a gene, in which the genomic region is modified in a way that an essential exon is flanked by loxP sites, which functionally behaves like a wild-type allele. Craniosynostosis: premature fusion of sutures. Cre: Cre recombinase, type 1 topoisomerase from P1 bacteriophage catalyzing site-specific recombination between distinct sequences referred to as loxP sites. Endochondral ossification: replacement of a cartilage template by bone. Intramembranous ossification: formation of bone from condensed fibrous cells. Lacunae: small spaces within compact bone. Osteo-chondroprogenitor: postulated progenitor cells, which are still bipotential and can differentiate into an osteoblast or chondrocyte. Osteocyte: a cell type with mechanosensory properties found within bone lacunae, which is derived from osteoblasts that became entrapped in the matrix during bone formation. Osteoid: non-calcified, fibrillar, extracellular matrix secreted by the osteoblast. Osteopenia: bone loss. Osteopetrosis: increase in bone density. Osteoporosis pseudoglioma (OPPG): Autosomal recessive inherited disorder, showing abnormalities of the eye (iris, lens or vitreous body) and low bone mass. Perichondrium: non-osteogenic fibrous connective tissue surrounding the skeletal elements. Periosteum: subpopulation of fibrous connective tissue surrounding the skeletal elements formed by endochondral ossification, in which the first osteoblasts differentiate. Suture: fibrous joint between the skull bones. Wnt: name based on the founding members of the family: Wingless (Wg) in Drosophila and int-1, a viral-insertional mutation causing mammary tumors in mice.
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(a) Osteocyte Osteoclast
Osteoblast
Compact bone (mineralized)
Osteoid Condensed mesenchyme Blood vessel
Loose mesenchyme
(b)
Chondrocytes
Loose mesenchyme
P e r i o s t e u m
Perichondrium
HTC Osteoid Osteoblasts
HTC Osteoclast Trabecular bone Osteocyte
Condensed mesenchyme Chondrocytes
Bone collar (compact bone) TRENDS in Cell Biology
Figure 1. Formation of the skeleton through intramembranous (a) and endochondral (b) ossification. (a) Intramembranous ossification process starts with osteoblast differentiation from within mesenchymal condensation. Osteoblasts first produce a fibrillar, non-mineralized matrix (osteoid), which becomes organized into compact bone. Osteoblasts entrapped in the bone matrix differentiate into osteocytes. Osteoclasts are bone resorbing cells, which are of hematopoietic origin (b) Endochondral ossification starts with the formation of condensed mesenchyme, in which chondrocytes develop forming a cartilaginous element prefiguring the future skeletal element. Mesenchymal cells that surround the element form the perichondrium, which consists of flattened densely packed mesenchymal cells. In the region of the perichondrium adjacent to the hypertrophic chondrocytes (HTC), the first osteoblast precursors differentiate after receiving a signal from prehypertrophic and hypertrophic chondrocytes. This region is also referred to as the periosteum. Osteoblasts secrete a non-mineralized, fibrillar extracellular matrix, the osteoid, which eventually becomes organized into mineralized compact bone. Bone collar and trabecular bone consist of compact bone. During the process of bone deposition, osteoblasts become entrapped in the bone matrix and differentiate into osteocytes and bone matrix is resorbed by osteoclasts. Osteoclasts are required for the production of the hollow architecture of vertebrate bones and their activity is important for the release of minerals such as calcium and phosphate.
b-catenin is a major player in osteoblast lineage differentiation Given that chondrocytes and osteoblasts share a common progenitor, which signals and signaling pathways control the differentiation into the two lineages? Transcription factors that are essential for the two lineages have been identified recently. Sox9, together with its targets Sox5 and Sox6, is required for the chondrogenic lineage [3,4], and Cbfa1/Runx2, Osterix, Atf4 and others are required for the osteoblast lineage [5,6] (Figure 2); however, little is known about the signaling pathways involved in lineage decision. The canonical Wnt pathway is an important mediator in regulating cell proliferation and differentiation and is conserved from flies to humans [7]. Immunohistochemical and reporter analyses hinted at a possible involvement of this pathway in osteoblastogenesis: b-catenin, an essential component of the pathway is found in the nuclei of osteoblast precursor cells in the periosteum [8,9]. Reporter mouse strains for active canonical Wnt signaling (TOPGAL mice), revealed activity in the www.sciencedirect.com
perichondrium and osteoblasts [8,10,11] at sites of endochondral and intramembranous bone formation. Recent experiments examining the conditional inactivation of b-catenin in skeletal progenitors and using different Cre lines revealed that b-catenin activity is essential for the differentiation of mature osteoblasts and, consequently, for bone formation in endrochondral bones (the long bones of the limbs) and membranous bones (in the skull) [8,9,12]. Chondrogenesis in the limb was not impaired by loss of b-catenin activity in the early limb mesenchyme [9], and chondrocytes continued to express the osteogenic factor Ihh. However, chondrocyte maturation was delayed [3]. Interestingly, the perichondrial and periosteal cells continued to express early markers of the osteoblast lineage, such as Alkaline phosphatase, Collagen 1a1 and Runx2 [8,9,12]. Therefore, lack of b-catenin does not impair the differentiation into the early osteoblastic precursors. However, perichondrial and periosteal cells failed to express the osteoblast commitment factor, Osterix, and acquired a chondrogenic fate [8,9]. Cre-mediated deletion in the head mesenchyme
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Box 1. The canonical Wnt/b-catenin pathway The canonical Wnt signaling pathway (Figure I) is activated by binding of a WNT ligand (19 WNT ligands are encoded in both the human and the mouse genome) to a receptor complex comprising a member of the Frizzled protein family, which encodes a serpentine receptor, and a member of the low density lipoprotein receptor related protein family LRP5/6. The ligand–receptor interaction is transmitted intracellularly through Dishevelled (Dsh) proteins, which leads to the inhibition of a multiprotein complex containing the proteins Axin, Adenomatous Polyposis Coli (Apc), and glycogen synthase kinase-3b (Gsk3). In the absence of a suitable ligand, this complex is involved in facilitating the phosphorylation of b-catenin, which leads to its degradation by the ubiquitin–proteasome pathway. Thus, ligand–receptor interactions ultimately lead to an increase in cytoplasmic b-catenin levels and translocation of bcatenin into the nucleus, where it acts as a co-activator in a complex with a member of the Tcf/Lef1 transcription factor family [7]. There are various extracellular secreted inhibitor molecules that can potentially modulate the signaling strength by binding either to WNTs (such as Wif and Sfrps) or the co-receptors of the LRP5/6 family (such as Dkk, Wise/Sost) [7,32,33]. Sfrps encode secreted decoy receptor-like molecules and are thought to inhibit Wnt signaling by binding to the ligands competing with their binding to Frizzled proteins. Dkks are thought to inhibit Wnt signaling by binding to Lrp5 or Lrp6 and another high-affinity receptor Kremen (1 or 2), which leads to rapid internalization of the ternary complex through endocytosis, thereby removing the Lrp co-receptor from the plasma membrane [58]. b-catenin is an obligatory and the only nonredundant component of the canonical Wnt pathway.
Sfrp Wnt Frizzled Wnt
Kremen
LRP Dkk
Dsh
Axin Gsk APC β-cat
β-cat TCF/Lef
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further demonstrated that b-catenin is a universal factor required for osteoblast development. Similar to the long bones, the osteoblastic progenitors differentiated in the absence of b-catenin into chondrocytes [8,9]. These findings were substantiated by in vitro deletion of b-catenin activity in dissociated calvarial cells. These cells would usually differentiate into mature osteoblasts in culture but in the absence of b-catenin 20–30% differentiated into chondrocytes [8,9]. It is likely that b-catenin activity is required in a bipotential precursor of the osteoblast lineage, the so-called osteo-chondroprogenitor, and b-catenin suppresses its chondrogenic potential. Chondrogenesis could be the default differentiation state of these precursors. Mice without Osterix also lack mature osteoblasts and cells within the periosteum of their long bones differentiate into chondrocytes, similar to that seen in b-catenin mutants. However, no ectopic chondrocytes are found in the skull of these Osterix-deficient mice [13]. It is not yet known whether Osterix is directly regulated by canonical Wnt signaling or if loss of Osterix affects b-catenin stability. On the basis of recent studies, it has been proposed that b-catenin concentrations have to be elevated to enable differentiation into osteoblasts and the upregulation of the commitment factor Osterix. By contrast, for differentiation into the chondrocyte lineage, b-catenin levels must be low (Figure 2) [8,9,12]. Various Wnt genes, such as Wnt1, Wnt4, Wnt5a, Wnt9a/14 and Wnt7b, are expressed in either osteoblast precursors or adjacent tissues during embryonic development, and Wnt3a and Wnt10b are expressed in bone marrow [12,14,15]. Most of these Wnt genes have been genetically inactivated in mice; however, none of the mutant mice has a reported defect in early osteoblastogenesis. Only Wnt10b mutants display a postnatal decrease in bone mass [16]. Conversely, retroviral overexpression of Wnt4 in chick and transgenic mice that express Wnt9a/14 under the control of the Col2a1 promoter had enhanced maturation of chondrocytes, shown by the premature appearance of hypertrophic chondrocytes and enhanced maturation of osteoblasts, which also resulted in a thickening of the bone collar [8,17], although this could be an indirect effect [18]. Thus, the endogenous ligand(s) required for the canonical Wnt and b-catenin pathway activity that enable osteoblast lineage differentiation from mesenchymal cells have yet to be identified. Investigation of the role of Wnt genes using deletion studies could be difficult, as some of the candidate Wnt ligands are expressed in overlapping or adjacent regions and might function redundantly during osteoblastogenesis. Therefore, it will be necessary to generate double or even triple knockout mice to uncover potential roles of Wnt proteins in osteoblast differentiation. It also remains an open question as to which of the Wnt receptors of the Frizzled family are involved in this process. Postnatal requirement of the canonical Wnt pathway for bone homeostasis In postnatal and adult life, three cell types are involved in regulating bone mass by the continuous removal and replacement of bone (remodeling and homeostasis): osteoblasts that produce bone matrix; osteocytes (which
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Table 1. Skeletal defects caused by human mutations, gene knockouts and mouse models with regard to components of the canonical Wnt pathway Human gene AXIN2 LRP5
Protein function Part of destruction complex Co-receptor
SOST Gene b-catenin
Wnt inhibitor Protein function Cell adhesion and transcriptional co-activator
Apc Axin2 Lef1
Lrp5
Part of destruction complex (adenomatosis polyposis coli) Part of destruction complex Transcription factor
Sfrp1 Tcf1 Wnt10b
Co-receptor of the canonical Wnt pathway Co-receptor of the canonical Wnt pathway Wnt antagonist Transcription factor Ligand
Wnt9a/14
Ligand
Lrp6
Phenotype Tooth agenesis Osteoporosis-pseudoglioma syndrome (OPPG) Osteopetrosis, increased bone mass Sclerostosis, increased bone density Allele Skeletal phenotype Conditional lof a Delayed chondrocyte maturation Col2a1–Cre Fusions of joints Conditional lof Loss of osteoblasts Dermo1-Cre, Prx1–Cre Conditional lof Osteopenia a1(I)Col-Cre, Oc–Cre Conditional gofb Osteopetrosis a1(I)Col–Cre Conditional lof Oc–Cre Osteopetrosis Null Col2a1 transgenic overexpression of constitutively active form of Lef1 Null Transgenic hLRP5G171V Heterozygous Hypomorph Null Null Null Transgenic FABP–Wnt10b Transgenic Col2a1–Wnt9a Transgenic Col2a1–Wnt9a
a
Craniosynostosis Pleiotrophic effects: disorganized growth plate, inhibition of chondrocyte and osteoblast maturation. Osteopenia High bone mass Enhances osteopenic phenotype of Lrp5K/K Low bone mass Increased bone mass Low bone mass Osteopenia Increased bone mass Accelerated maturation of chondrocytes and osteoblasts Joint formation
Refs [59] [60] [25,61,62] [63,64] Refs [3] [65] [8,9,12] [10,38] [10] [38] [37] [66]
[14,23,28] [24] [28] [29] [35] [10] [16] [8] [65]
Loss of function; bGain of function.
are osteoblasts entrapped within lacunae) [19]; and osteoclasts that resorb bone matrix. High bone mass can result from having either more (or more active) osteoblasts or fewer (or less active) osteoclasts. To balance bone production and resorption in healthy individuals, osteoblasts secrete factors that regulate the differentiation of osteoclasts [20] and osteocytes secrete factors regulating the activity of osteoblasts [21]. Osteoblast maturation and activity is regulated by the canonical Wnt pathway Mutations that map to the human LRP5 gene, which encodes a co-receptor of the canonical Wnt pathway (Box1), are responsible for different abnormal bone phenotypes, including osteoporosis pseudoglioma (OPPG) and high bone mass, indicating that canonical Wnt signaling has a role in regulating bone mass [22]. This has sparked the generation of various mouse models, which have confirmed the importance of this signaling pathway in bone homeostasis, primarily in the osteoblast lineage. Lrp5 knockout mice show a reduction in bone mass, although onset and severity of the phenotype differs between the independently generated knockout strains. Kato and colleagues reported an OPPG-like phenotype in young mice and a defect in osteoblast proliferation and maturation [14], whereas Fujino et al. reported a late onset and sexspecific reduced bone thickness, as well as a role for Lrp5 in cholesterol and glucose metabolism [23]. The reasons for the phenotypic differences could be intrinsic to the different knockout alleles or because of differences in the genetic background. www.sciencedirect.com
Similar to humans carrying the LRP5G171V mutation, transgenic mice that express a mutant form of human LRP5G171V in osteoblasts have increased bone mineral density. This phenotype is due to elevated numbers of active osteoblasts, which seem to be protected from apoptosis [24]. Interestingly, this is a missense mutation, which decreases the interaction of Lrp5 with the Wnt antagonist dickkopf1 (Dkk1), thereby diminishing its inhibitory effect on endogenous Wnt signaling [25] (Box 1). Recent studies suggest that this is a general mechanism for all Lrp5 mutations causing a high bone mass phenotype [26]. In the case of the LRP5G171V mutant protein, it is controversial whether this mutant protein is reaching the cell surface [26,27]. Lrp6, which is homologous to Lrp5, also functions as a Wnt coreceptor and has been implicated in bone mass accrual [28,29]. However, its exact role in osteogenesis and bone homeostasis has yet to be clarified. This will require the generation of a conditional allele, as the loss of Lrp6 causes embryonic lethality [30]. Lrp5 and Lrp6 function as receptors for other types of molecule, such as sclerostin (Sost) and connective tissue growth factor (CTGF) [31–33]. Interestingly, Sost is produced by osteocytes and inhibits proliferation and maturation, but stimulates apoptosis, of osteoblasts [34]. Mutations reducing Sost function in humans cause overgrowth of bone tissue (sclerosteosis; Table 1). Sclerosteosis shares many similarities with the high bone mass diseases caused by the LRP5G171V mutation. The Lrp5 and Sost loss-of-function mutations are complementary, whereas in osteoblasts loss of b-catenin, a downstream activator of the canonical Wnt pathway, results in different
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(a)
β-catenin
Osterix
Runx2 Osteo-chondroprogenitor Mesenchymal stem cell
β-catenin
Commited osteoprogenitor
Skeletal precursor Sox9
β-catenin
Chondroblast Sox9 Chondrocyte
Sox5 Sox6
(b) Terminal Proliferation Matrix maturation differention Axin2 Lrp5 β-catenin ATF Committed osteoprogenitor
Pre-osteoblast
Mature osteoblast
Osteocyte
Wnt10b OPG
Osteoclast precursor
Osteoclast
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Figure 2. Role of canonical Wnt–b-catenin signaling during lineage differentiation and maturation of osteoblasts. (a) b-catenin negatively regulates the differentiation of mesenchymal cells into a common skeletal precursor [7]. Skeletal precursor cells downregulate b-catenin and upregulate Sox9 and, subsequently, Sox5 and Sox6, causing the precursor cells to differentiate into chondroblasts and chondrocytes, whereas if the precursors upregulate Runx2 and elevate b-catenin levels they differentiate into osteoblast precursors. High levels of b-catenin are necessary to suppress the chondrogenic potential of uncommitted progenitors, such as the proposed osteo-chondroprogenitor. Osterix is required for the final commitment of progenitors to osteoblasts. (b) Wnt10b and Lrp5 signaling are required for the expansion of committed osteoprogenitors. This proliferative effect is attenuated by Axin2, at least in neural crest-derived osteoblasts. b-catenin activity and Tcf1 are required to inhibit osteoclastogenesis through the regulation of Opg.
phenotypic changes (discussed below). Therefore, Lrp5 and Lrp6 could have signaling activities that are independent from the canonical Wnt pathway. However, results from several other mouse models support the notion that activated Wnt signaling leads to a postnatal increase in bone mass, such as transgenic mice expressing Wnt10b in adipose tissue and bone marrow under the control of the Fabp4 promoter [16] and mice lacking the Wnt antagonist Sfrp1 [35]. It has also been proposed that Sfrp1 is a negative regulator of osteoclastogenesis [36], which is surprising, given that osteoclasts mediate bone resorption, this would be expected to lead to a reduction of bone mass rather than the increase reported in the Sfrp1 mutant mice. Mice deficient for Axin2, which encodes a protein that is part of multiprotein complex that facilitates b-catenin phosphorylation and destruction (Box 1), exhibit craniosynostosis. Loss of this negative regulator of b-catenin leads to increased proliferation of osteoblast progenitors in the metopic skull sutures and accelerated maturation and mineralization of osteoblasts in vitro [37]. www.sciencedirect.com
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Non-cell autonomous regulation of osteoclastogenesis by the canonical Wnt signaling pathway Recently, a novel role for canonical Wnt signaling in postnatal bone homeostasis has been discovered by inactivating b-catenin function in more mature osteoblasts using a Col1a1– and an Osteocalcin (OC)–Cre line [10,38]. Mice deficient in b-catenin develop osteopenia. By contrast, activation of b-catenin function in osteoblasts using the Col1a1– and the OC–Cre line in combination with a conditional b-catenin gain-of-function allele [39] and a conditional APC allele, respectively, resulted in increased bone mass [10,38]. Surprisingly, both the loss-offunction and the gain-of-function phenotypes were cause by changes in bone resorption rather than in bone formation, as would have been expected on the basis of the Lrp5 mutant phenotype, which affects osteoblast proliferation and maturation but not osteoclast differentiation. The altered bone resorption was caused by deregulation of Osteoprotegerin (Opg), a major inhibitor of osteoclast differentiation. Using a multipotent mesenchymal cell line, Opg expression was found to be upregulated by canonical Wnt signaling in an in vitro screen for Wnt-regulated genes [40]. Opg is also a direct target gene of the b-catenin–TCF complex in osteoblasts and Tcf1 is probably the relevant transcription factor required for Opg regulation; nevertheless, a possible role for Tcf4 cannot be excluded [10,40]. However, there are some discrepancies between the in vivo studies; Holmen et al. [38] reported that b-catenin deficient mice had increased levels of Rankl and showed a decrease in osteoblast numbers four weeks after birth. Furthermore, mice lacking Apc in osteoblasts (which would result in increased b-catenin levels because Apc is part of the complex involved in destabilizing b-catenin through phosphorylation) resulted in a complete loss of osteoblasts at two weeks after birth [38]. None of these phenotypes was reported by Glass et al. [10]. Nevertheless, these studies [10,38] demonstrate the importance of the canonical Wnt and b-catenin signaling in postnatal bone homeostasis, regulating osteoclastogenesis non-cell autonomously through Opg. Interestingly, Opg was also identified as a potential b-catenin target in two studies unrelated to osteoblastogenesis [40,41]. Although loss of the Wnt antagonist Sfrp1 leads to an increase in bone mass [35], mice lacking the Wnt antagonist Dkk2 are osteopenic [42]. Dkk2K/K mice have a complex bone phenotype that is associated with a defect in terminal maturation of osteoblasts and matrix mineralization and, in addition, they show an increase in osteoclasts. The effect on osteoclasts is non-cell-autonomous and is associated with higher expression of two osteoclastic differentiation factors, Rankl and M-Csf, but no changes in the levels of Opg have been observed [42]. The analysis of this mouse is puzzling because Dkk2 has been described as an inhibitor of Wnt signaling. Dkk proteins interact with Lrp5 and Lrp6 and with a receptor of the Kremen family (Krm1, Krm2). Krm2 has been shown to alter the activity of Dkk2, changing it from an agonist of Wnt signaling to an antagonist [43]. Therefore, it would be interesting to see whether Krm2 is expressed in osteoblasts. Nevertheless, it seems unlikely that the
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phenotype is only due to a decrease in Wnt signaling. The analysis suggests that Dkk2 has additional functions that are independent from antagonizing (or agonizing) Wnt signaling during osteogenesis, as was recently shown for Dkk1 in cardiogenesis [44]. Crosstalk between different signaling pathways How is the Wnt canonical pathway integrated with other pathways that have important roles in osteoblastogenesis? In vivo and in vitro analysis by Hu et al. [12] showed that signaling by a member of the hedgehog family, Ihh, (for further information on the hedgehog signaling pathway, see Ref. [45]) might be functioning upstream and in concert with the canonical Wnt–b-catenin pathway during osteoblastogenesis in long bones. However, Ihh is not required for the development of osteoblasts in the membranous bones of the skull [46]. Therefore, other signals might also be cooperating with the canonical Wnt– b-catenin pathway. One of these signals could be a member of the bone morphogenetic proteins (Bmps). Signaling by Bmp2 activates b-catenin signaling [47], possibly by inducing the expression of canonical Wnts [48] (for further information on the Bmp signaling pathway, see Ref. [49]). There is also evidence that Bmps, similarly to hedgehog proteins, require the canonical Wnt–b-catenin pathway to induce osteoblastic differentiation of the multipotent mesenchymal C3H10T1/2 cell line [48]. However, this point is controversial, as the report by Winkler et al. [50] provides evidence that Wnt proteins induce Bmps and that Bmp antagonists, such as Noggin and a soluble Bmp-receptor molecule, can block the activity of Wnt3a on C3H10T1/2 cells. This indicates that Bmp activity is required downstream of a Wnt stimulus [50]. This is further supported by the observation that cytosolic b-catenin levels are reduced in the bone marrow cells of transgenic mice that overexpress the Bmp antagonist gremlin [51]. Furthermore, a downstream target gene of Wnt signaling, Wisp1, is expressed in osteoblasts and potentiates Bmp2-induced osteoblastic differentiation [52]. Together, these studies suggest that Bmp and canonical Wnt signaling cooperate and coregulate each other, thereby promoting osteoblast differentiation. Fgf signaling, however, seems to inhibit canonical Wnt signaling indirectly through upregulation of the transcription factor Sox2 [53]. Most of the work on interactions between different signaling pathways has been performed in vitro using multipotent mesenchymal or osteoblastic cell lines. The analysis in these studies was often limited to assaying alkaline phosphatase induction and/or the mineralization potential of the cultures; two assays that are not absolutely specific for osteoblast differentiation. The assays will have to be refined to unravel cooperative activities during proliferation or maturation of osteoblasts. In some cases, similar experiments using the same cell lines have led to different results, which could be due to slight differences in reagents or culture conditions; however, this makes it difficult to integrate these in vitro results into a network. The integration of the different signaling pathways into a comprehensive network, www.sciencedirect.com
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leading to a better understanding of their collective functions and interactions in osteoblastogenesis, is a major future challenge. Conclusions Recent studies show that canonical Wnt signaling has multiple roles in osteoblastogenesis: it is essential for osteoblast lineage differentiation early in development and, postnatally, is involved in the regulation of various aspects of bone homeostasis, such as osteoblast proliferation and maturation. In addition, the pathway is involved in attenuating osteoclastogenesis through transcriptional regulation of Opg, an inhibitory factor of osteoclast differentiation that is produced and secreted by osteoblasts. Recent studies have also revealed that the signaling outcome is fine-tuned by several extracellular modulators of the canonical Wnt pathway, probably through intracellular interaction with other signaling pathways. The discovery that Wnt signaling has a part in bone homeostasis has initiated new therapeutic approaches, for example, testing lithium and other GSK3 inhibitors as potential drugs to treat osteoporosis. A recent case study reported a dose-dependent decreased fracture risk for spine and wrist in patients treated with high doses of lithium, whereas those treated with low doses showed an increased risk of fracture [54]. However, the risk for hip fractures, which are associated with the highest morbidity of osteoporotic patients, was not improved by lithium. These reagents are not absolutely specific for the canonical Wnt pathway and could therefore have side effects. Lithium, which is commonly used as an antidepressant in patients with bipolar disorder (manic depressive illness) has been shown to influence brain activity in healthy individuals [55]. The role of modulators of canonical Wnt signaling has also been investigated in stem cell biology [56,57]. Manipulation of b-catenin levels in mesenchymal progenitor cells might be a way to direct cell differentiation along the chondrocyte or osteoblast lineage and would open new avenues for tissue engineering. Despite the many questions (Box 2), it has become clear from in vivo and in vitro studies that the canonical Wnt-signaling pathway has essential roles during embryonic and postnatal osteoblastogenesis. Box 2. Outstanding questions † Which Wnt signals orchestrate the different events, such as controlling lineage differentiation during embryonic development and osteoblast proliferation, maturation and activity during postnatal life? † Are specific transcriptional cofactors recruited to the b-catenin–TCF complex to achieve different functions? † Which Frizzled receptors are involved? † What are the binding preferences of Wnts to the various Frizzled receptors, and is this modulated by the presence of Lrp5 or Lrp6? Does this result in different signaling outputs? †Are there signaling activities of the Lrp5 and Lrp6 receptors that are independent of the canonical Wnt pathway and that could be stimulated by the binding of Sost, CTGF or other ligands? † How are the different signaling pathways interconnected and integrated to coordinate osteoblastogenesis?
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References 1 Karsenty, G. and Wagner, E.F. (2002) Reaching a genetic and molecular understanding of skeletal development. Dev. Cell 2, 389–406 2 Zelzer, E. and Olsen, B.R. (2003) The genetic basis for skeletal diseases. Nature 423, 343–348 3 Akiyama, H. et al. (2004) Interactions between Sox9 and b-catenin control chondrocyte differentiation. Genes Dev. 18, 1072–1087 4 Lefebvre, V. et al. (2001) L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Osteoarthritis Cartilage 9(Suppl A), S69–S75 5 Nakashima, K. and de Crombrugghe, B. (2003) Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet. 19, 458–466 6 Kobayashi, T. and Kronenberg, H. (2005) Minireview: transcriptional regulation in development of bone. Endocrinology 146, 1012–1017 7 Logan, C.Y. and Nusse, R. (2004) The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 8 Day, T.F. et al. (2005) Wnt/b-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739–750 9 Hill, T.P. et al. (2005) Canonical Wnt/b-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev. Cell 8, 727–738 10 Glass, D.A., 2nd. et al. (2005) Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev. Cell 8, 751–764 11 Hens, J.R. et al. (2005) TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro. J. Bone Miner. Res. 20, 1103–1113 12 Hu, H. et al. (2005) Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 132, 49–60 13 Nakashima, K. et al. (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 14 Kato, M. et al. (2002) Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J. Cell Biol. 157, 303–314 15 Reya, T. et al. (2000) Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity 13, 15–24 16 Bennett, C.N. et al. (2005) Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc. Natl. Acad. Sci. U. S. A. 102, 3324–3329 17 Hartmann, C. and Tabin, C.J. (2000) Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development 127, 3141–3159 18 Kitagaki, J. et al. (2003) Activation of b-catenin-LEF/TCF signal pathway in chondrocytes stimulates ectopic endochondral ossification. Osteoarthritis Cartilage 11, 36–43 19 Knothe Tate, M.L. et al. (2004) The osteocyte. Int. J. Biochem. Cell Biol. 36, 1–8 20 Wagner, E.F. and Karsenty, G. (2001) Genetic control of skeletal development. Curr. Opin. Genet. Dev. 11, 527–532 21 Martin, R.B. (2000) Toward a unifying theory of bone remodeling. Bone 26, 1–6 22 Johnson, M.L. et al. (2004) LRP5 and Wnt signaling: a union made for bone. J. Bone Miner. Res. 19, 1749–1757 23 Fujino, T. et al. (2003) Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion. Proc. Natl. Acad. Sci. U. S. A. 100, 229–234 24 Babij, P. et al. (2003) High bone mass in mice expressing a mutant LRP5 gene. J. Bone Miner. Res. 18, 960–974 25 Boyden, L.M. et al. (2002) High bone density due to a mutation in LDL-receptor-related protein 5. N. Engl. J. Med. 346, 1513–1521 26 Ai, M. et al. (2005) Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling. Mol. Cell. Biol. 25, 4946–4955 27 Zhang, Y. et al. (2004) The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd. Mol. Cell. Biol. 24, 4677–4684 www.sciencedirect.com
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28 Holmen, S.L. et al. (2004) Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. J. Bone Miner. Res. 19, 2033–2040 29 Kokubu, C. et al. (2004) Skeletal defects in ringelschwanz mutant mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis. Development 131, 5469–5480 30 Pinson, K.I. et al. (2000) An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407, 535–538 31 Mercurio, S. et al. (2004) Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex. Development 131, 2137–2147 32 Li, X. et al. (2005) Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J. Biol. Chem. 280, 19883–19887 33 Semenov, M. et al. (2005) SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J. Biol. Chem. 280, 26770–26775 34 van Bezooijen, R.L. et al. (2004) Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J. Exp. Med. 199, 805–814 35 Bodine, P.V. et al. (2004) The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice. Mol. Endocrinol. 18, 1222–1237 36 Hausler, K.D. et al. (2004) Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation. J. Bone Miner. Res. 19, 1873–1881 37 Yu, H.M. et al. (2005) The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development 132, 1995–2005 38 Holmen, S.L. et al. (2005) Essential role of b-catenin in postnatal bone acquisition. J. Biol. Chem. 280, 21162–21168 39 Harada, N. et al. (1999) Intestinal polyposis in mice with a dominant stable mutation of the b-catenin gene. EMBO J. 18, 5931–5942 40 Jackson, A. et al. (2005) Gene array analysis of Wnt-regulated genes in C3H10T1/2 cells. Bone 36, 585–598 41 Willert, J. et al. (2002) A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Dev. Biol. 2, 8 42 Li, X. et al. (2005) Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation. Nat. Genet. 37, 945–952 43 Wu, W. et al. (2000) Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/b-catenin signalling. Curr. Biol. 10, 1611–1614 44 Pandur, P. et al. (2002) Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418, 636–641 45 Lum, L. and Beachy, P.A. (2004) The Hedgehog response network: sensors, switches, and routers. Science 304, 1755–1759 46 St-Jacques, B. et al. (1999) Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072–2086 47 Bain, G. et al. (2003) Activated b-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction. Biochem. Biophys. Res. Commun. 301, 84–91 48 Rawadi, G. et al. (2003) BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J. Bone Miner. Res. 18, 1842–1853 49 Nohe, A. et al. (2004) Signal transduction of bone morphogenetic protein receptors. Cell. Signal. 16, 291–299 50 Winkler, D.G. et al. (2005) Sclerostin inhibition of Wnt-3a-induced C3H10T1/2 cell differentiation is indirect and mediated by bone morphogenetic proteins. J. Biol. Chem. 280, 2498–2502 51 Gazzerro, E. et al. (2005) Skeletal overexpression of gremlin impairs bone formation and causes osteopenia. Endocrinology 146, 655–665 52 French, D.M. et al. (2004) WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair. Am. J. Pathol. 165, 855–867 53 Mansukhani, A. et al. (2005) Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation. J. Cell Biol. 168, 1065–1076 54 Vestergaard, P. et al. Reduced relative risk of fractures among users of lithium. Calcif. Tissue Int. (in press) 55 Bell, E.C. et al. (2005) Lithium and valproate attenuate dextroamphetamine-induced changes in brain activation. Hum. Psychopharmacol. 20, 87–96
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56 Gregory, C.A. et al. (2005) How wnt signaling affects bone repair by mesenchymal stem cells from the bone marrow. Ann. N. Y. Acad. Sci. 1049, 97–106 57 Kleber, M. and Sommer, L. (2004) Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell Biol. 16, 681–687 58 Mao, B. et al. (2002) Kremen proteins are Dickkopf receptors that regulate Wnt/b-catenin signalling. Nature 417, 664–667 59 Lammi, L. et al. (2004) Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am. J. Hum. Genet. 74, 1043–1050 60 Gong, Y. et al. (2001) LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107, 513–523 61 Little, R.D. et al. (2002) A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am. J. Hum. Genet. 70, 11–19
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62 Van Wesenbeeck, L. et al. (2003) Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am. J. Hum. Genet. 72, 763–771 63 Balemans, W. et al. (2001) Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum. Mol. Genet. 10, 537–543 64 Brunkow, M.E. et al. (2001) Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am. J. Hum. Genet. 68, 577–589 65 Guo, X. et al. (2004) Wnt/b-catenin signaling is sufficient and necessary for synovial joint formation. Genes Dev. 18, 2404–2417 66 Tamamura, Y. et al. (2005) Developmental regulation of Wnt/b-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification. J. Biol. Chem. 280, 19185–19195
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A Wnt canon orchestrating osteoblastogenesis Christine Hartmann Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria
Several transcription factors have been identified that control the differentiation of osteoblasts; however, relatively little is known about the signaling pathways involved in regulating the differentiation process. Recently, the canonical Wnt–b-catenin pathway has been implicated in osteoblastogenesis. This review focuses on the role of the canonical Wnt–b-catenin pathway during embryonic development, where it is required for the differentiation of osteoblasts from a precusor that is shared with the chondrocyte lineage and the requirement of this pathway during postnatal life in bone homeostasis. The recent findings covered in this review are major advances in our understanding of skeletal development and promise new therapeutic avenues for tissue engineering and treatment of osteoporosis.
Formation of the vertebrate skeleton The skeleton is composed of two types of tissue, cartilage and bone, which are formed by chondrocytes and osteoblasts, respectively. Bones are formed during embryonic development by two different processes – intramembranous and endochondral ossification. Some skull bones and the lateral halves of the clavicles are formed by intramembranous ossification, whereby boneforming cells, the osteoblasts, differentiate from within a connective tissue sheet of condensed mesenchymal cells and start to secrete osteoid (see Glossary), which later becomes mineralized and reorganized into ‘compact’ bone (Figure 1a). However, most bones are formed by endochondral ossification: mesenchymal condensations differentiate into chondrocytes, forming a cartilaginous template prefiguring the future skeletal element. Chondrocytes in the center of the cartilage element stop proliferating, become prehypertrophic and mature into hypertrophic chondrocytes, which are characterized by an increase in size, vacuolization and secretion of a distinct extracellular matrix. Mesenchymal cells surrounding the cartilage element flatten and form the perichondrium – a condensed multilayered tissue (Figure 1b). Perichondrial cells that are adjacent to the middle region of the cartilage template receive a signal, the protein Indian hedgehog (Ihh), from prehypertrophic chondrocytes, which induces the osteogenic program in these cells, coupling the process of chondrocyte matuCorresponding author: Hartmann, C. ([email protected]). Available online 7 February 2006
ration to the onset of osteoblast differentiation. Thus, during endochondral ossification the first osteoblasts develop within a condensed mesenchymal tissue, similar to the process of intramembranous ossification; however, in contrast to osteoblasts in the skull, these osteoblasts require the Ihh gene product as an inductive signal from the chondrocytes. It is therefore likely that chondrocytes and osteoblasts have a common mesenchymal skeletal precursor [1,2]. The bone formed by either intramembranous or endochondral ossification is remodeled by the osteoclasts, which are bone resorbing cells of hematopoietic origin. Differentiation of osteoclasts is controlled in part by factors secreted by osteoblasts. Here, we focus on the recent discoveries showing important roles for the canonical Wnt pathway (Box 1) in osteoblast lineage determination during embryonic development. We also discuss its postnatal role in regulating osteoblast proliferation, maturation and activity (Table 1).
Glossary Allele: alternative form of any given genomic locus on a chromosome; can be an alternative form of a gene, but can also refer to intergenic regions. Conditional allele: allele of a gene, in which the genomic region is modified in a way that an essential exon is flanked by loxP sites, which functionally behaves like a wild-type allele. Craniosynostosis: premature fusion of sutures. Cre: Cre recombinase, type 1 topoisomerase from P1 bacteriophage catalyzing site-specific recombination between distinct sequences referred to as loxP sites. Endochondral ossification: replacement of a cartilage template by bone. Intramembranous ossification: formation of bone from condensed fibrous cells. Lacunae: small spaces within compact bone. Osteo-chondroprogenitor: postulated progenitor cells, which are still bipotential and can differentiate into an osteoblast or chondrocyte. Osteocyte: a cell type with mechanosensory properties found within bone lacunae, which is derived from osteoblasts that became entrapped in the matrix during bone formation. Osteoid: non-calcified, fibrillar, extracellular matrix secreted by the osteoblast. Osteopenia: bone loss. Osteopetrosis: increase in bone density. Osteoporosis pseudoglioma (OPPG): Autosomal recessive inherited disorder, showing abnormalities of the eye (iris, lens or vitreous body) and low bone mass. Perichondrium: non-osteogenic fibrous connective tissue surrounding the skeletal elements. Periosteum: subpopulation of fibrous connective tissue surrounding the skeletal elements formed by endochondral ossification, in which the first osteoblasts differentiate. Suture: fibrous joint between the skull bones. Wnt: name based on the founding members of the family: Wingless (Wg) in Drosophila and int-1, a viral-insertional mutation causing mammary tumors in mice.
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(a) Osteocyte Osteoclast
Osteoblast
Compact bone (mineralized)
Osteoid Condensed mesenchyme Blood vessel
Loose mesenchyme
(b)
Chondrocytes
Loose mesenchyme
P e r i o s t e u m
Perichondrium
HTC Osteoid Osteoblasts
HTC Osteoclast Trabecular bone Osteocyte
Condensed mesenchyme Chondrocytes
Bone collar (compact bone) TRENDS in Cell Biology
Figure 1. Formation of the skeleton through intramembranous (a) and endochondral (b) ossification. (a) Intramembranous ossification process starts with osteoblast differentiation from within mesenchymal condensation. Osteoblasts first produce a fibrillar, non-mineralized matrix (osteoid), which becomes organized into compact bone. Osteoblasts entrapped in the bone matrix differentiate into osteocytes. Osteoclasts are bone resorbing cells, which are of hematopoietic origin (b) Endochondral ossification starts with the formation of condensed mesenchyme, in which chondrocytes develop forming a cartilaginous element prefiguring the future skeletal element. Mesenchymal cells that surround the element form the perichondrium, which consists of flattened densely packed mesenchymal cells. In the region of the perichondrium adjacent to the hypertrophic chondrocytes (HTC), the first osteoblast precursors differentiate after receiving a signal from prehypertrophic and hypertrophic chondrocytes. This region is also referred to as the periosteum. Osteoblasts secrete a non-mineralized, fibrillar extracellular matrix, the osteoid, which eventually becomes organized into mineralized compact bone. Bone collar and trabecular bone consist of compact bone. During the process of bone deposition, osteoblasts become entrapped in the bone matrix and differentiate into osteocytes and bone matrix is resorbed by osteoclasts. Osteoclasts are required for the production of the hollow architecture of vertebrate bones and their activity is important for the release of minerals such as calcium and phosphate.
b-catenin is a major player in osteoblast lineage differentiation Given that chondrocytes and osteoblasts share a common progenitor, which signals and signaling pathways control the differentiation into the two lineages? Transcription factors that are essential for the two lineages have been identified recently. Sox9, together with its targets Sox5 and Sox6, is required for the chondrogenic lineage [3,4], and Cbfa1/Runx2, Osterix, Atf4 and others are required for the osteoblast lineage [5,6] (Figure 2); however, little is known about the signaling pathways involved in lineage decision. The canonical Wnt pathway is an important mediator in regulating cell proliferation and differentiation and is conserved from flies to humans [7]. Immunohistochemical and reporter analyses hinted at a possible involvement of this pathway in osteoblastogenesis: b-catenin, an essential component of the pathway is found in the nuclei of osteoblast precursor cells in the periosteum [8,9]. Reporter mouse strains for active canonical Wnt signaling (TOPGAL mice), revealed activity in the www.sciencedirect.com
perichondrium and osteoblasts [8,10,11] at sites of endochondral and intramembranous bone formation. Recent experiments examining the conditional inactivation of b-catenin in skeletal progenitors and using different Cre lines revealed that b-catenin activity is essential for the differentiation of mature osteoblasts and, consequently, for bone formation in endrochondral bones (the long bones of the limbs) and membranous bones (in the skull) [8,9,12]. Chondrogenesis in the limb was not impaired by loss of b-catenin activity in the early limb mesenchyme [9], and chondrocytes continued to express the osteogenic factor Ihh. However, chondrocyte maturation was delayed [3]. Interestingly, the perichondrial and periosteal cells continued to express early markers of the osteoblast lineage, such as Alkaline phosphatase, Collagen 1a1 and Runx2 [8,9,12]. Therefore, lack of b-catenin does not impair the differentiation into the early osteoblastic precursors. However, perichondrial and periosteal cells failed to express the osteoblast commitment factor, Osterix, and acquired a chondrogenic fate [8,9]. Cre-mediated deletion in the head mesenchyme
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Box 1. The canonical Wnt/b-catenin pathway The canonical Wnt signaling pathway (Figure I) is activated by binding of a WNT ligand (19 WNT ligands are encoded in both the human and the mouse genome) to a receptor complex comprising a member of the Frizzled protein family, which encodes a serpentine receptor, and a member of the low density lipoprotein receptor related protein family LRP5/6. The ligand–receptor interaction is transmitted intracellularly through Dishevelled (Dsh) proteins, which leads to the inhibition of a multiprotein complex containing the proteins Axin, Adenomatous Polyposis Coli (Apc), and glycogen synthase kinase-3b (Gsk3). In the absence of a suitable ligand, this complex is involved in facilitating the phosphorylation of b-catenin, which leads to its degradation by the ubiquitin–proteasome pathway. Thus, ligand–receptor interactions ultimately lead to an increase in cytoplasmic b-catenin levels and translocation of bcatenin into the nucleus, where it acts as a co-activator in a complex with a member of the Tcf/Lef1 transcription factor family [7]. There are various extracellular secreted inhibitor molecules that can potentially modulate the signaling strength by binding either to WNTs (such as Wif and Sfrps) or the co-receptors of the LRP5/6 family (such as Dkk, Wise/Sost) [7,32,33]. Sfrps encode secreted decoy receptor-like molecules and are thought to inhibit Wnt signaling by binding to the ligands competing with their binding to Frizzled proteins. Dkks are thought to inhibit Wnt signaling by binding to Lrp5 or Lrp6 and another high-affinity receptor Kremen (1 or 2), which leads to rapid internalization of the ternary complex through endocytosis, thereby removing the Lrp co-receptor from the plasma membrane [58]. b-catenin is an obligatory and the only nonredundant component of the canonical Wnt pathway.
Sfrp Wnt Frizzled Wnt
Kremen
LRP Dkk
Dsh
Axin Gsk APC β-cat
β-cat TCF/Lef
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further demonstrated that b-catenin is a universal factor required for osteoblast development. Similar to the long bones, the osteoblastic progenitors differentiated in the absence of b-catenin into chondrocytes [8,9]. These findings were substantiated by in vitro deletion of b-catenin activity in dissociated calvarial cells. These cells would usually differentiate into mature osteoblasts in culture but in the absence of b-catenin 20–30% differentiated into chondrocytes [8,9]. It is likely that b-catenin activity is required in a bipotential precursor of the osteoblast lineage, the so-called osteo-chondroprogenitor, and b-catenin suppresses its chondrogenic potential. Chondrogenesis could be the default differentiation state of these precursors. Mice without Osterix also lack mature osteoblasts and cells within the periosteum of their long bones differentiate into chondrocytes, similar to that seen in b-catenin mutants. However, no ectopic chondrocytes are found in the skull of these Osterix-deficient mice [13]. It is not yet known whether Osterix is directly regulated by canonical Wnt signaling or if loss of Osterix affects b-catenin stability. On the basis of recent studies, it has been proposed that b-catenin concentrations have to be elevated to enable differentiation into osteoblasts and the upregulation of the commitment factor Osterix. By contrast, for differentiation into the chondrocyte lineage, b-catenin levels must be low (Figure 2) [8,9,12]. Various Wnt genes, such as Wnt1, Wnt4, Wnt5a, Wnt9a/14 and Wnt7b, are expressed in either osteoblast precursors or adjacent tissues during embryonic development, and Wnt3a and Wnt10b are expressed in bone marrow [12,14,15]. Most of these Wnt genes have been genetically inactivated in mice; however, none of the mutant mice has a reported defect in early osteoblastogenesis. Only Wnt10b mutants display a postnatal decrease in bone mass [16]. Conversely, retroviral overexpression of Wnt4 in chick and transgenic mice that express Wnt9a/14 under the control of the Col2a1 promoter had enhanced maturation of chondrocytes, shown by the premature appearance of hypertrophic chondrocytes and enhanced maturation of osteoblasts, which also resulted in a thickening of the bone collar [8,17], although this could be an indirect effect [18]. Thus, the endogenous ligand(s) required for the canonical Wnt and b-catenin pathway activity that enable osteoblast lineage differentiation from mesenchymal cells have yet to be identified. Investigation of the role of Wnt genes using deletion studies could be difficult, as some of the candidate Wnt ligands are expressed in overlapping or adjacent regions and might function redundantly during osteoblastogenesis. Therefore, it will be necessary to generate double or even triple knockout mice to uncover potential roles of Wnt proteins in osteoblast differentiation. It also remains an open question as to which of the Wnt receptors of the Frizzled family are involved in this process. Postnatal requirement of the canonical Wnt pathway for bone homeostasis In postnatal and adult life, three cell types are involved in regulating bone mass by the continuous removal and replacement of bone (remodeling and homeostasis): osteoblasts that produce bone matrix; osteocytes (which
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Table 1. Skeletal defects caused by human mutations, gene knockouts and mouse models with regard to components of the canonical Wnt pathway Human gene AXIN2 LRP5
Protein function Part of destruction complex Co-receptor
SOST Gene b-catenin
Wnt inhibitor Protein function Cell adhesion and transcriptional co-activator
Apc Axin2 Lef1
Lrp5
Part of destruction complex (adenomatosis polyposis coli) Part of destruction complex Transcription factor
Sfrp1 Tcf1 Wnt10b
Co-receptor of the canonical Wnt pathway Co-receptor of the canonical Wnt pathway Wnt antagonist Transcription factor Ligand
Wnt9a/14
Ligand
Lrp6
Phenotype Tooth agenesis Osteoporosis-pseudoglioma syndrome (OPPG) Osteopetrosis, increased bone mass Sclerostosis, increased bone density Allele Skeletal phenotype Conditional lof a Delayed chondrocyte maturation Col2a1–Cre Fusions of joints Conditional lof Loss of osteoblasts Dermo1-Cre, Prx1–Cre Conditional lof Osteopenia a1(I)Col-Cre, Oc–Cre Conditional gofb Osteopetrosis a1(I)Col–Cre Conditional lof Oc–Cre Osteopetrosis Null Col2a1 transgenic overexpression of constitutively active form of Lef1 Null Transgenic hLRP5G171V Heterozygous Hypomorph Null Null Null Transgenic FABP–Wnt10b Transgenic Col2a1–Wnt9a Transgenic Col2a1–Wnt9a
a
Craniosynostosis Pleiotrophic effects: disorganized growth plate, inhibition of chondrocyte and osteoblast maturation. Osteopenia High bone mass Enhances osteopenic phenotype of Lrp5K/K Low bone mass Increased bone mass Low bone mass Osteopenia Increased bone mass Accelerated maturation of chondrocytes and osteoblasts Joint formation
Refs [59] [60] [25,61,62] [63,64] Refs [3] [65] [8,9,12] [10,38] [10] [38] [37] [66]
[14,23,28] [24] [28] [29] [35] [10] [16] [8] [65]
Loss of function; bGain of function.
are osteoblasts entrapped within lacunae) [19]; and osteoclasts that resorb bone matrix. High bone mass can result from having either more (or more active) osteoblasts or fewer (or less active) osteoclasts. To balance bone production and resorption in healthy individuals, osteoblasts secrete factors that regulate the differentiation of osteoclasts [20] and osteocytes secrete factors regulating the activity of osteoblasts [21]. Osteoblast maturation and activity is regulated by the canonical Wnt pathway Mutations that map to the human LRP5 gene, which encodes a co-receptor of the canonical Wnt pathway (Box1), are responsible for different abnormal bone phenotypes, including osteoporosis pseudoglioma (OPPG) and high bone mass, indicating that canonical Wnt signaling has a role in regulating bone mass [22]. This has sparked the generation of various mouse models, which have confirmed the importance of this signaling pathway in bone homeostasis, primarily in the osteoblast lineage. Lrp5 knockout mice show a reduction in bone mass, although onset and severity of the phenotype differs between the independently generated knockout strains. Kato and colleagues reported an OPPG-like phenotype in young mice and a defect in osteoblast proliferation and maturation [14], whereas Fujino et al. reported a late onset and sexspecific reduced bone thickness, as well as a role for Lrp5 in cholesterol and glucose metabolism [23]. The reasons for the phenotypic differences could be intrinsic to the different knockout alleles or because of differences in the genetic background. www.sciencedirect.com
Similar to humans carrying the LRP5G171V mutation, transgenic mice that express a mutant form of human LRP5G171V in osteoblasts have increased bone mineral density. This phenotype is due to elevated numbers of active osteoblasts, which seem to be protected from apoptosis [24]. Interestingly, this is a missense mutation, which decreases the interaction of Lrp5 with the Wnt antagonist dickkopf1 (Dkk1), thereby diminishing its inhibitory effect on endogenous Wnt signaling [25] (Box 1). Recent studies suggest that this is a general mechanism for all Lrp5 mutations causing a high bone mass phenotype [26]. In the case of the LRP5G171V mutant protein, it is controversial whether this mutant protein is reaching the cell surface [26,27]. Lrp6, which is homologous to Lrp5, also functions as a Wnt coreceptor and has been implicated in bone mass accrual [28,29]. However, its exact role in osteogenesis and bone homeostasis has yet to be clarified. This will require the generation of a conditional allele, as the loss of Lrp6 causes embryonic lethality [30]. Lrp5 and Lrp6 function as receptors for other types of molecule, such as sclerostin (Sost) and connective tissue growth factor (CTGF) [31–33]. Interestingly, Sost is produced by osteocytes and inhibits proliferation and maturation, but stimulates apoptosis, of osteoblasts [34]. Mutations reducing Sost function in humans cause overgrowth of bone tissue (sclerosteosis; Table 1). Sclerosteosis shares many similarities with the high bone mass diseases caused by the LRP5G171V mutation. The Lrp5 and Sost loss-of-function mutations are complementary, whereas in osteoblasts loss of b-catenin, a downstream activator of the canonical Wnt pathway, results in different
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(a)
β-catenin
Osterix
Runx2 Osteo-chondroprogenitor Mesenchymal stem cell
β-catenin
Commited osteoprogenitor
Skeletal precursor Sox9
β-catenin
Chondroblast Sox9 Chondrocyte
Sox5 Sox6
(b) Terminal Proliferation Matrix maturation differention Axin2 Lrp5 β-catenin ATF Committed osteoprogenitor
Pre-osteoblast
Mature osteoblast
Osteocyte
Wnt10b OPG
Osteoclast precursor
Osteoclast
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Figure 2. Role of canonical Wnt–b-catenin signaling during lineage differentiation and maturation of osteoblasts. (a) b-catenin negatively regulates the differentiation of mesenchymal cells into a common skeletal precursor [7]. Skeletal precursor cells downregulate b-catenin and upregulate Sox9 and, subsequently, Sox5 and Sox6, causing the precursor cells to differentiate into chondroblasts and chondrocytes, whereas if the precursors upregulate Runx2 and elevate b-catenin levels they differentiate into osteoblast precursors. High levels of b-catenin are necessary to suppress the chondrogenic potential of uncommitted progenitors, such as the proposed osteo-chondroprogenitor. Osterix is required for the final commitment of progenitors to osteoblasts. (b) Wnt10b and Lrp5 signaling are required for the expansion of committed osteoprogenitors. This proliferative effect is attenuated by Axin2, at least in neural crest-derived osteoblasts. b-catenin activity and Tcf1 are required to inhibit osteoclastogenesis through the regulation of Opg.
phenotypic changes (discussed below). Therefore, Lrp5 and Lrp6 could have signaling activities that are independent from the canonical Wnt pathway. However, results from several other mouse models support the notion that activated Wnt signaling leads to a postnatal increase in bone mass, such as transgenic mice expressing Wnt10b in adipose tissue and bone marrow under the control of the Fabp4 promoter [16] and mice lacking the Wnt antagonist Sfrp1 [35]. It has also been proposed that Sfrp1 is a negative regulator of osteoclastogenesis [36], which is surprising, given that osteoclasts mediate bone resorption, this would be expected to lead to a reduction of bone mass rather than the increase reported in the Sfrp1 mutant mice. Mice deficient for Axin2, which encodes a protein that is part of multiprotein complex that facilitates b-catenin phosphorylation and destruction (Box 1), exhibit craniosynostosis. Loss of this negative regulator of b-catenin leads to increased proliferation of osteoblast progenitors in the metopic skull sutures and accelerated maturation and mineralization of osteoblasts in vitro [37]. www.sciencedirect.com
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Non-cell autonomous regulation of osteoclastogenesis by the canonical Wnt signaling pathway Recently, a novel role for canonical Wnt signaling in postnatal bone homeostasis has been discovered by inactivating b-catenin function in more mature osteoblasts using a Col1a1– and an Osteocalcin (OC)–Cre line [10,38]. Mice deficient in b-catenin develop osteopenia. By contrast, activation of b-catenin function in osteoblasts using the Col1a1– and the OC–Cre line in combination with a conditional b-catenin gain-of-function allele [39] and a conditional APC allele, respectively, resulted in increased bone mass [10,38]. Surprisingly, both the loss-offunction and the gain-of-function phenotypes were cause by changes in bone resorption rather than in bone formation, as would have been expected on the basis of the Lrp5 mutant phenotype, which affects osteoblast proliferation and maturation but not osteoclast differentiation. The altered bone resorption was caused by deregulation of Osteoprotegerin (Opg), a major inhibitor of osteoclast differentiation. Using a multipotent mesenchymal cell line, Opg expression was found to be upregulated by canonical Wnt signaling in an in vitro screen for Wnt-regulated genes [40]. Opg is also a direct target gene of the b-catenin–TCF complex in osteoblasts and Tcf1 is probably the relevant transcription factor required for Opg regulation; nevertheless, a possible role for Tcf4 cannot be excluded [10,40]. However, there are some discrepancies between the in vivo studies; Holmen et al. [38] reported that b-catenin deficient mice had increased levels of Rankl and showed a decrease in osteoblast numbers four weeks after birth. Furthermore, mice lacking Apc in osteoblasts (which would result in increased b-catenin levels because Apc is part of the complex involved in destabilizing b-catenin through phosphorylation) resulted in a complete loss of osteoblasts at two weeks after birth [38]. None of these phenotypes was reported by Glass et al. [10]. Nevertheless, these studies [10,38] demonstrate the importance of the canonical Wnt and b-catenin signaling in postnatal bone homeostasis, regulating osteoclastogenesis non-cell autonomously through Opg. Interestingly, Opg was also identified as a potential b-catenin target in two studies unrelated to osteoblastogenesis [40,41]. Although loss of the Wnt antagonist Sfrp1 leads to an increase in bone mass [35], mice lacking the Wnt antagonist Dkk2 are osteopenic [42]. Dkk2K/K mice have a complex bone phenotype that is associated with a defect in terminal maturation of osteoblasts and matrix mineralization and, in addition, they show an increase in osteoclasts. The effect on osteoclasts is non-cell-autonomous and is associated with higher expression of two osteoclastic differentiation factors, Rankl and M-Csf, but no changes in the levels of Opg have been observed [42]. The analysis of this mouse is puzzling because Dkk2 has been described as an inhibitor of Wnt signaling. Dkk proteins interact with Lrp5 and Lrp6 and with a receptor of the Kremen family (Krm1, Krm2). Krm2 has been shown to alter the activity of Dkk2, changing it from an agonist of Wnt signaling to an antagonist [43]. Therefore, it would be interesting to see whether Krm2 is expressed in osteoblasts. Nevertheless, it seems unlikely that the
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phenotype is only due to a decrease in Wnt signaling. The analysis suggests that Dkk2 has additional functions that are independent from antagonizing (or agonizing) Wnt signaling during osteogenesis, as was recently shown for Dkk1 in cardiogenesis [44]. Crosstalk between different signaling pathways How is the Wnt canonical pathway integrated with other pathways that have important roles in osteoblastogenesis? In vivo and in vitro analysis by Hu et al. [12] showed that signaling by a member of the hedgehog family, Ihh, (for further information on the hedgehog signaling pathway, see Ref. [45]) might be functioning upstream and in concert with the canonical Wnt–b-catenin pathway during osteoblastogenesis in long bones. However, Ihh is not required for the development of osteoblasts in the membranous bones of the skull [46]. Therefore, other signals might also be cooperating with the canonical Wnt– b-catenin pathway. One of these signals could be a member of the bone morphogenetic proteins (Bmps). Signaling by Bmp2 activates b-catenin signaling [47], possibly by inducing the expression of canonical Wnts [48] (for further information on the Bmp signaling pathway, see Ref. [49]). There is also evidence that Bmps, similarly to hedgehog proteins, require the canonical Wnt–b-catenin pathway to induce osteoblastic differentiation of the multipotent mesenchymal C3H10T1/2 cell line [48]. However, this point is controversial, as the report by Winkler et al. [50] provides evidence that Wnt proteins induce Bmps and that Bmp antagonists, such as Noggin and a soluble Bmp-receptor molecule, can block the activity of Wnt3a on C3H10T1/2 cells. This indicates that Bmp activity is required downstream of a Wnt stimulus [50]. This is further supported by the observation that cytosolic b-catenin levels are reduced in the bone marrow cells of transgenic mice that overexpress the Bmp antagonist gremlin [51]. Furthermore, a downstream target gene of Wnt signaling, Wisp1, is expressed in osteoblasts and potentiates Bmp2-induced osteoblastic differentiation [52]. Together, these studies suggest that Bmp and canonical Wnt signaling cooperate and coregulate each other, thereby promoting osteoblast differentiation. Fgf signaling, however, seems to inhibit canonical Wnt signaling indirectly through upregulation of the transcription factor Sox2 [53]. Most of the work on interactions between different signaling pathways has been performed in vitro using multipotent mesenchymal or osteoblastic cell lines. The analysis in these studies was often limited to assaying alkaline phosphatase induction and/or the mineralization potential of the cultures; two assays that are not absolutely specific for osteoblast differentiation. The assays will have to be refined to unravel cooperative activities during proliferation or maturation of osteoblasts. In some cases, similar experiments using the same cell lines have led to different results, which could be due to slight differences in reagents or culture conditions; however, this makes it difficult to integrate these in vitro results into a network. The integration of the different signaling pathways into a comprehensive network, www.sciencedirect.com
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leading to a better understanding of their collective functions and interactions in osteoblastogenesis, is a major future challenge. Conclusions Recent studies show that canonical Wnt signaling has multiple roles in osteoblastogenesis: it is essential for osteoblast lineage differentiation early in development and, postnatally, is involved in the regulation of various aspects of bone homeostasis, such as osteoblast proliferation and maturation. In addition, the pathway is involved in attenuating osteoclastogenesis through transcriptional regulation of Opg, an inhibitory factor of osteoclast differentiation that is produced and secreted by osteoblasts. Recent studies have also revealed that the signaling outcome is fine-tuned by several extracellular modulators of the canonical Wnt pathway, probably through intracellular interaction with other signaling pathways. The discovery that Wnt signaling has a part in bone homeostasis has initiated new therapeutic approaches, for example, testing lithium and other GSK3 inhibitors as potential drugs to treat osteoporosis. A recent case study reported a dose-dependent decreased fracture risk for spine and wrist in patients treated with high doses of lithium, whereas those treated with low doses showed an increased risk of fracture [54]. However, the risk for hip fractures, which are associated with the highest morbidity of osteoporotic patients, was not improved by lithium. These reagents are not absolutely specific for the canonical Wnt pathway and could therefore have side effects. Lithium, which is commonly used as an antidepressant in patients with bipolar disorder (manic depressive illness) has been shown to influence brain activity in healthy individuals [55]. The role of modulators of canonical Wnt signaling has also been investigated in stem cell biology [56,57]. Manipulation of b-catenin levels in mesenchymal progenitor cells might be a way to direct cell differentiation along the chondrocyte or osteoblast lineage and would open new avenues for tissue engineering. Despite the many questions (Box 2), it has become clear from in vivo and in vitro studies that the canonical Wnt-signaling pathway has essential roles during embryonic and postnatal osteoblastogenesis. Box 2. Outstanding questions † Which Wnt signals orchestrate the different events, such as controlling lineage differentiation during embryonic development and osteoblast proliferation, maturation and activity during postnatal life? † Are specific transcriptional cofactors recruited to the b-catenin–TCF complex to achieve different functions? † Which Frizzled receptors are involved? † What are the binding preferences of Wnts to the various Frizzled receptors, and is this modulated by the presence of Lrp5 or Lrp6? Does this result in different signaling outputs? †Are there signaling activities of the Lrp5 and Lrp6 receptors that are independent of the canonical Wnt pathway and that could be stimulated by the binding of Sost, CTGF or other ligands? † How are the different signaling pathways interconnected and integrated to coordinate osteoblastogenesis?
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