Elsevier

Gene

Volume 341, 27 October 2004, Pages 19-39
Gene

Review
Wnt signaling in osteoblasts and bone diseases

https://doi.org/10.1016/j.gene.2004.06.044Get rights and content

Abstract

Recent revelations that the canonical Wnt signaling pathway promotes postnatal bone accrual are major advances in our understanding of skeletal biology and bring tremendous promise for new therapeutic treatments for osteoporosis and other diseases of altered bone mass. Wnts are soluble glycoproteins that engage receptor complexes composed of Lrp5/6 and Frizzled proteins. A subgroup of Wnts induces a cascade of intracellular events that stabilize β-catenin, facilitating its transport to nuclei where it binds Lef1/Tcf transcription factors and alters gene expression to promote osteoblast expansion and function. Natural extracellular Wnt antagonists, Dickkopfs and secreted frizzled-related proteins, impair osteoblast function and block bone formation. In several genetic disorders of altered skeletal mass, mutations in LRP5 create gain-of-function or loss-of-function receptors that are resistant to normal regulatory mechanisms and cause higher or lower bone density, respectively. In this review, we summarize the available molecular, cellular, and genetic data that demonstrate how Lrp5 and other components of the Wnt signaling pathway influence osteoblast proliferation, function, and survival. We also discuss regulatory mechanisms discovered in developmental and tumor models that may provide insights into novel therapies for bone diseases.

Introduction

Wnts are secreted, lipid-modified glycoproteins that activate cell surface receptor-mediated signal transduction pathways to regulate a variety of cellular activities, including cell fate determination, proliferation, migration, polarity, and gene expression (Moon et al., 2002). Many Wnts are essential during embryogenesis, but Wnts are also active in regenerating adult tissues such as lymphoid tissues, colon, skin, hair follicles, and bone (Bienz and Clevers, 2000, Alonso and Fuchs, 2003, Staal and Clevers, 2003). As is typical for important developmental molecules, genes encoding Wnt signaling pathway components are altered by genetic and epigenetic mutations in human cancers and neurodegenerative diseases (Caricasole et al., 2003, Giles et al., 2003). In addition, mutations in four genes encoding Wnt signal transducing proteins are now linked to five inherited human disorders: familial adenomatous polyposis (APC) (Kinzler et al., 1991, Nishisho et al., 1991), familial exudative vitreoretinopathy with retinal angiogenesis (FZD4 and LRP5) (Robitaille et al., 2002, Toomes et al., 2004), tetra-amelia (WNT3) (Niemann et al., 2004), osteoporosis pseudoglioma syndrome (LRP5) (Gong et al., 2001), and high bone mass disorders (LRP5) (Boyden et al., 2002, Little et al., 2002). The discoveries that LRP5, a Wnt receptor, is differentially mutated in the latter two conditions initiated a flurry of experiments aimed at understanding how Wnts affect bone accrual and at developing new therapeutic strategies to regulate bone density and treat millions of people with osteoporosis. Wnts clearly have important roles in regulating many aspects of skeletal development, from limb formation to chondrogenesis and osteoblast maturation. In this review, we will focus most of our attention on osseous cells. To learn more about Wnt signaling in limb buds and cartilage, we refer you to reviews by Tuan (2003) and Yang (2003), respectively. For additional and updated information about the Wnt signaling pathways, we encourage you to visit these websites: The Wnt Gene Homepage (http://www.stanford.edu/~rnusse/wntwindow.html) and the Signal Transduction Knowledge Environment (http://www.stke.sciencemag.org/).

Wnts activate at least three distinct intracellular signaling cascades: the Wnt/β-catenin pathway, the Wnt/Ca+2 pathway, or the Wnt/planar polarity pathway. The Wnt/β-catenin pathway is commonly referred to as the canonical pathway. It promotes cell fate determination, proliferation, and survival by increasing β-catenin levels and altering gene expression through Lef/Tcf transcription factors (Behrens et al., 1996). The Wnt/Ca+2 pathway stimulates heterotrimeric G proteins, increases intracellular calcium levels, decreases cyclic GMP levels, and activates protein kinase C to induce NF-AT and other transcription factors (Wang and Malbon, 2003). The Wnt/planar polarity pathway activates Rho/Rac GTPases and Jun N-terminal kinase to modulate cytoskeletal organization and gene expression (Habas et al., 2003). Distinct Wnt ligands probably act through specific Frizzled (Fzd) receptors to initiate each pathway (Nusse et al., 2000, Rulifson et al., 2000). The available data from osteoblast models concern activation of the Wnt/β-catenin pathway; thus, this cascade will be the focus of our attention in this review.

The canonical Wnt signaling pathway affects cellular functions by regulating β-catenin levels and subcellular localization. In the absence of Wnts, β-catenin levels are kept at a steady state. Any β-catenin molecules that are not bridging cadherins to the actin cytoskeleton or participating in other activities are ubiquitinated and degraded by the 26S proteosome (Fig. 1) (Aberle et al., 1997). A multiprotein complex containing kinases (casein kinase (CK) 1 and glycogen synthase kinase (GSK) 3β) and scaffolding proteins (Axin, APC (adenomatous polyposis coli) and Disheveled (Dsh)) mediate the degradation of excess β-catenin by phosphorylating specific amino terminal residues and creating docking sites for F-box protein/E2 ligase complexes (Siegfried et al., 1992, Noordermeer et al., 1994, Behrens et al., 1998, Jiang and Struhl, 1998). Wnts initiate intracellular accumulation of β-catenin by binding to cell surface receptor complexes consisting of Lrp5/6 and Fzd transmembrane proteins (Bhanot et al., 1996, Yang-Snyder et al., 1996, Wehrli et al., 2000). An unknown kinase(s) then phosphorylates intracellular residues on Lrp5/6 and creates docking sites for Axin (Mao et al., 2001b, Tamai et al., 2004). GSK3β is excluded from this proximal receptor complex by poorly understood mechanisms that may involve the binding of Axin to Lrp5/6 and/or the mobilization of a competitive GSK3 binding protein (GBP) (Li et al., 1999, Farr et al., 2000). β-Catenin molecules that are not phosphorylated by GSK3ß accumulate and enter to the nucleus where they affect gene expression (Behrens et al., 1996). Lef1/Tcf transcription factors are the best-characterized nuclear targets of β-catenin. β-Catenin displaces co-repressors (CoR) from Lef1/Tcf, directly interacts with Lef1/Tcf, and recruits transcriptional co-activators to stimulate expression of many genes, including c-myc and cyclin D1 (He et al., 1998, Shtutman et al., 1999, Billin et al., 2000, Hecht et al., 2000, Sun et al., 2000). Canonical Wnt signaling also represses gene expression by less understood mechanisms that may depend on the availability of other proteins (Cadigan et al., 1998, Baker et al., 1999, Prieve and Waterman, 1999, Jamora et al., 2003, Kahler and Westendorf, 2003). An updated list of genes affected by Wnt signaling can be found on the Wnt Gene Homepage (http://www.stanford.edu/~rnusse/wntwindow.html).

Several extracellular and intracellular proteins negatively regulate canonical Wnt signaling. Dickkopfs (Dkks) and secreted frizzled related proteins (Sfrps) are two families of extracellular factors that antagonize Wnt activities. Dkks limit the availability of Lrp5/6 receptors to Wnts by sequestering Lrp5/6 into complexes with Kremens (Krm) and possibly promoting their internalization to lysosomes (Fig. 1). In contrast, Sfrps bind directly to Wnts and prevent their association with Lrp and Fzd receptors. Inside the cell, APC, Axin, and GSK3ß block β-catenin accumulation in the cytoplasm and natural dominant negative Lef1/Tcf proteins inhibit β-catenin-dependent transcription in the nucleus (Hovanes et al., 2001). The functional importance of these negative regulators in cell growth is best illustrated by their misregulation or inactivation in tumors (Suzuki et al., 2002, Giles et al., 2003).

The realization that the same canonical Wnt signaling pathway that has been extensively studied in developmental and cancer models also participates in postnatal bone formation is one of the most exciting discoveries of the new millennium. Since 2001 when distinct LRP5 mutations were found in humans with low or high bone mass (Gong et al., 2001, Boyden et al., 2002, Little et al., 2002), many components of the canonical Wnt pathway have been studied in models of bone development in vitro and in vivo. In this review, we summarize the available genetic, molecular, and biochemical experiments characterizing the Wnt signaling pathway components in osteoblasts. We will also draw upon data from other models to predict additional mechanisms that may be active in osseous cells.

Section snippets

Wnts

Wnts are 39–46-kDa cysteine-rich, secreted glycoproteins that have been identified in organisms ranging from hydra to humans. Wnt family members are defined by sequence homology to Drosophila wingless (wg) and the murine int-1 proto-oncogene. Human and mouse genomes encode 19 WNT and 18 Wnt genes, respectively (Miller, 2002). Wnts are divided into functional classes based on their ability to induce a secondary body axis in Xenopus embryos and to activate certain signaling cascades. Members of

Wnt receptors and antagonists

Seminal discoveries uncovering the importance of Wnt signaling pathways in osteoblasts identified activating and inactivating mutations in a Wnt receptor, LRP5, as the cause of high bone mass or an osteoporosis syndrome in humans, respectively (Gong et al., 2001, Boyden et al., 2002, Little et al., 2002). These important findings were confirmed in mouse models (Gong et al., 2001, Babij et al., 2003) and pinpoint LRP5 and its ligands as potential therapeutic targets for bone density disorders.

Intracellular components of the canonical Wnt signaling pathway

Stabilization of β-catenin is regarded as the defining event of canonical Wnt signaling. Through a series of incompletely understood events, Wnts prevent GSK from phosphorylating β-catenin and targeting it to proteosomes for degradation. Because of the large number of Wnts and their limited availability as purified, recombinant proteins, investigators often choose to mimic canonical Wnt signaling by increasing intracellular β-catenin levels. This is typically accomplished by either introducing

Transcription factors: Lef1, Tcf1, Tcf3 and Tcf4

The Lef1/Tcf transcription factors are the nuclear effectors of the canonical Wnt signaling pathway and represent the final targets of cellular control over Wnt-dependent growth and survival. Human and mouse Lef1/Tcf family members are encoded by four genes: Tcf1 (Tcf7), Lef1 (Tcf1a), Tcf3 (Tcf7L1) and Tcf4 (Tcf7L2). Note that there is no Tcf2, Lef1 is the second family member. Although Lef1/Tcf transcription factors are largely functionally redundant in vitro, they have distinct roles in

Summary and future directions

The canonical Wnt signaling pathway regulates osteoblast proliferation, survival, and functional lifespan to promote postnatal bone accrual. Downregulation of this pathway by secreted factors promotes osteoblast apoptosis but also appears to be a natural event that occurs during the terminal differentiation to osteocytes. The extracellular agonists and antagonists of canonical Wnt signaling pathways are molecules from which novel therapeutics can be developed to regulate bone density. To fully

References (219)

  • M. El-Tanani et al.

    Differential modulation of transcriptional activity of estrogen receptors by direct protein–protein interactions with the T cell factor family of transcription factors

    J. Biol. Chem.

    (2001)
  • M. El-Tanani et al.

    Ets gene, PEA3 cooperates with beta-catenin–Lef-1 and c-jun in regulation of osteopontin transcription

    J. Biol. Chem.

    (2004)
  • S. Frame et al.

    A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation

    Mol. Cell

    (2001)
  • R.H. Giles et al.

    Caught up in a Wnt storm: Wnt signaling in cancer

    Biochim. Biophys. Acta

    (2003)
  • T. Golan et al.

    The human frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt/beta-catenin signaling cascade

    J. Biol. Chem.

    (2004)
  • Y. Gong et al.

    LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development

    Cell

    (2001)
  • C.A. Gregory et al.

    The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow

    J. Biol. Chem.

    (2003)
  • M. Hadjiargyrou et al.

    Transcriptional profiling of bone regeneration. Insight into the molecular complexity of wound repair

    J. Biol. Chem.

    (2002)
  • P.J. Hey et al.

    Cloning of a novel member of the low-density lipoprotein receptor family

    Gene

    (1998)
  • B. Hoang et al.

    Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis

    J. Biol. Chem.

    (1996)
  • O. Huber et al.

    Nuclear localization of beta-catenin by interaction with transcription factor LEF-1

    Mech. Dev.

    (1996)
  • J. Huelsken et al.

    Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin

    Cell

    (2001)
  • S.M. Hussein et al.

    Smad4 and beta-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2

    J. Biol. Chem.

    (2003)
  • I.E. James et al.

    FrzB-2: a human secreted frizzled-related protein with a potential role in chondrocyte apoptosis

    Osteoarthr. Cartil.

    (2000)
  • M.L. Johnson et al.

    Linkage of a gene causing high bone mass to human chromosome 11 (11q12–13)

    Am. J. Hum. Genet.

    (1997)
  • R.A. Kahler et al.

    Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2-dependent transcriptional activation of the osteocalcin promoter

    J. Biol. Chem.

    (2003)
  • H. Aberle et al.

    Beta-catenin is a target for the ubiquitin–proteasome pathway

    EMBO J.

    (1997)
  • A.R. Afzal et al.

    Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2

    Nat. Genet.

    (2000)
  • A. Ali et al.

    Glycogen synthase kinase-3: properties, functions, and regulation

    Chem. Rev.

    (2001)
  • T. Alliston et al.

    TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation

    EMBO J.

    (2001)
  • M. Almeida et al.

    Potentiation of wnt signaling by activation of the nongenotropic function of the estrogen receptor in osteoblastic cells. American Society for Bone and Mineral Research

    J. Bone Miner. Res.

    (2003)
  • L. Alonso et al.

    Stem cells in the skin: waste not, Wnt not

    Genes Dev.

    (2003)
  • S. Avissar et al.

    Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex

    Nature

    (1988)
  • P. Babij et al.

    High bone mass in mice expressing a mutant LRP5 gene

    J. Bone Miner. Res.

    (2003)
  • G.H. Baeg et al.

    Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless

    Development

    (2001)
  • A. Bafico et al.

    Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow

    Nat. Cell Biol.

    (2001)
  • J.C. Baker et al.

    Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development

    Genes Dev.

    (1999)
  • C. Banerjee et al.

    An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene

    Proc. Natl. Acad. Sci. U. S. A.

    (1996)
  • J. Behrens et al.

    Functional interaction of beta-catenin with the transcription factor LEF-1

    Nature

    (1996)
  • J. Behrens et al.

    Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta

    Science

    (1998)
  • C.N. Bennett et al.

    Wnt signaling promotes osteogenesis in vivo and in vitro. American Society for Bone and Mineral Research

    J. Bone Miner. Res.

    (2003)
  • T. Berndt et al.

    Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent

    J. Clin. Invest.

    (2003)
  • P. Bhanot et al.

    A new member of the frizzled family from Drosophila functions as a Wingless receptor

    Nature

    (1996)
  • J. Billiard et al.

    Receptor tyrosine kinase orphan receptor 2 (Ror2) modulates Wnt signaling pathways in osteoblastic cells. American Society of Bone and Mineral Research

    J. Bone Miner. Res.

    (2003)
  • A.N. Billin et al.

    Beta-catenin–histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator

    Mol. Cell. Biol.

    (2000)
  • P.V. Bodine et al.

    The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

    Mol. Endocrinol.

    (2004)
  • L.M. Boyden et al.

    High bone density due to a mutation in LDL-receptor-related protein 5

    N. Engl. J. Med.

    (2002)
  • J.M. Bradbury et al.

    Alterations of the growth characteristics of the fibroblast cell line C3H 10T1/2 by members of the Wnt gene family

    Oncogene

    (1994)
  • M. Brannon et al.

    XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development

    Development

    (1999)
  • H. Brantjes et al.

    All Tcf HMG box transcription factors interact with Groucho-related co-repressors

    Nucleic Acids Res.

    (2001)
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