Elsevier

Gene

Volume 492, Issue 1, 15 January 2012, Pages 1-18
Gene

Review
Update on Wnt signaling in bone cell biology and bone disease

https://doi.org/10.1016/j.gene.2011.10.044Get rights and content

Abstract

For more than a decade, Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development, bone mass maintenance, and therapeutic potential for regenerative medicine. It is evident that even subtle alterations in the intensity, amplitude, location, and duration of Wnt signaling pathways affects skeletal development, as well as bone remodeling, regeneration, and repair during a lifespan. Here we review recent advances and discrepancies in how Wnt/Lrp5 signaling regulates osteoblasts and osteocytes, introduce new players in Wnt signaling pathways that have important roles in bone development, discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis, and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists, such as sclerostin, Dkk1 and Sfrp1. Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.

Highlights

► An update to a 2004 review on Wnt signaling in osteoblasts and bone disease published in this journal is provided. ► Recent advances in Wnt signaling pathways are discussed. ► New players in Wnt signaling pathways are introduced. ► Progress made in translating basic studies to clinical therapeutics and diagnostics is summarized.

Introduction

Wnts are a large family of 19 secreted glycoproteins that trigger multiple signaling cascades essential for embryonic development and tissue regeneration. Proteins involved in the amplification and transduction of Wnt signals are often altered in cancer or lineage progenitor cells, leading to abnormal cell cycle control and/or altered cell fate decisions (MacDonald et al., 2009, Polakis, 2000). Mutations in several Wnt pathway components also contribute to human skeletal dysplasias. Most notably, mutations in the Wnt co-receptor LRP5 cause low or high bone mass depending on the nature of the alteration (Boyden et al., 2002, Gong et al., 1996, Little et al., 2002) and inactivation of the secreted Wnt antagonist Sclerostin produces high bone mass, sclerosteosis and van Buchem's disease (Balemans et al., 2001, Brunkow et al., 2001). A loss-of-function mutation in LRP6, another Wnt co-receptor, is linked to an inherited disorder characterized by osteoporosis, coronary artery disease, and metabolic syndrome (Mani et al., 2007). Less well known is that inactivating mutations in WTX, an intracellular regulator of β-catenin stability, cause osteopathia striata with cranial sclerosis (OCTS) (Jenkins et al., 2009) and FZD9, a Wnt co-receptor, is deleted in patients with Williams–Beuren syndrome, which is partially characterized by low bone density (Francke, 1999). During the last several years, polymorphisms in these and many more Wnt pathway components were linked to altered bone mineral density in genome wide association studies (Kiel et al., 2007b, Riancho et al., 2011, Rivadeneira et al., 2009, Sims et al., 2008, Veeman et al., 2003). Thus, it has become clear that even subtle alterations in the intensity, amplitude, and duration of Wnt signaling pathways affects skeletal formation during development, as well as bone remodeling, regeneration, and repair during a lifespan. In this review, we provide an update to a 2004 review on Wnt signaling in osteoblasts and bone disease published in this journal (Westendorf et al., 2004). Emphasis is placed on new data from murine genetic studies assessing the requirement for and roles of Wnt pathway components during skeletal development and disease. These observations are discussed in context with current knowledge of molecular and physiological regulation of bone mass. Progress in translating these discoveries to treatments for altered bone mass conditions is also summarized.

Wnts trigger several signaling cascades. The best known is the Wnt/β-catenin pathway (commonly called the canonical pathway), which features the stabilization and nuclear translocation of β-catenin as easily measurable outcomes. In the absence of Wnts, β-catenin associates with cadherins at the plasma membrane. Any excess β-catenin is quickly sequestered by a protein complex containing Axin1/2, Apc, casein kinase (Ck)1, glycogen synthase kinase (Gsk)3β, and Wtx and degraded by ubiquitin-mediated proteolysis (Fig. 1) (For more details see (Westendorf et al., 2004)). When certain Wnts (e.g., Wnt3a) are present, they crosslink cell surface molecules, Lrp5/6 and a Frizzled (Fzd), which mobilizes Gsk3β and Ck1 to the membrane where they phosphorylate serines on Lrp5/6, promote the formation of a signalosome, and recruit Disheveled (Dvl), Axin1/2, and caveolin (Bilic et al., 2007, MacDonald et al., 2009, Niehrs and Shen, 2010, Zhang et al., 2004). This releases β-catenin from the destruction complex, increases its levels, and allows it to enter the nucleus where it can displace co-repressors from transcription factors (e.g., Lef1, Tcf7) and regulate gene expression. Nuclear localization of β-catenin is often used as a metric of enhanced Wnt signaling. Expression levels of target genes (e.g., Axin2, Lef1) are also commonly measured to study Wnt signaling. Although β-catenin is activated by Wnts, it is important to remember that it is also mobilized by other signals (e.g., Igf and Akt activation) and is not exclusive to the canonical Wnt signaling cascade. This point is especially important in bone, as β-catenin deletion triggers bone loss via different mechanisms than Lrp5 inactivation (subsequent sections).

The Wnt/β-catenin pathway stimulates cell proliferation and survival. Enhanced stimulation of the pathway is a feature of many cancers (Polakis, 2000). Under normal physiological settings, multiple proteins keep this cascade in check. In addition to intracellular inhibitors (Axin2), the canonical pathway is neutralized by extracellular factors (Fig. 1). Secreted frizzled related proteins (Sfrps) and Wnt inhibitory factors (Wifs) directly bind Wnts and prevent their interactions with receptors. Other secreted proteins including Dickkopfs (Dkk), Sclerostin (Scl), and Sostdc1 (Wise) bind to Lrp5/6 receptors, inducing receptor internalization and/or reducing their availability to Wnts. Thirdly, some Wnts (e.g., Wnt5a) trigger alternative signaling pathways by co-opting receptor components and thus competing with Wnts (e.g., Wnt3a) that induce β-catenin stabilization. For example, Wnt5a induces the formation of a complex consisting of Lrp5/6, Ror1/2, and Fzd2 (Sato et al., 2010).

In some contexts, Wnts neither stabilize β-catenin nor interact with Lrp5/6. Rather, through Fzds and Dvl, Wnts can trigger alternative intracellular events (Fig. 1 and reviewed by (Gao and Chen, 2010)). Non-β-catenin cascades include the planar cell polarity (PCP) pathway, trimeric G-protein coupled receptor pathways including calcium ion (Ca2 +) signaling, Rho family GTPase pathways, and the Jnk pathways. Dvl has multiple conserved domains that allow it to interact with many binding partners, which determines which downstream pathways are engaged (Gao and Chen, 2010). Furthermore, membrane-spanning receptors such Ror2 and Ryk can activate Dvl-independent signaling (Angers and Moon, 2009).

The most extensively studied non-β-catenin Wnt signaling pathways is the PCP pathway, which enables cells to orient relative to an axis along the plane of a tissue (Henderson and Chaudhry, 2011). PCP signaling governs cell movement in the embryo via convergent extension (Sokol, 1996) and determines cell fates, enabling the creation of asymmetric and highly aligned structures such as hair follicles as well as orchestrating the polarized beating of motile cilia in numerous tissues (Devenport and Fuchs, 2008, Jones et al., 2008). The establishment of polarity in the plane of the epithelium provides directional information during development. Wnt binding to Fzd leads to Dvl-driven sorting of cellular components to either the proximal or distal regions of the cell and orients it within the tissue (Veeman et al., 2003). Thus far, little is known about PCP activation during bone remodeling.

Dvl activation of the Rho GTPase family member Rac1 leads to Jnk activation and stimulation of the transcription factors c-Jun and ATF2 (Li et al., 2005a, Ohkawara and Niehrs, 2011, Sato et al., 2010). Wnt3a causes chondrocyte de-differentiation by activating c-Jun/AP-1 and suppressing Sox-9 expression, supporting a role for a non-β-catenin/Wnt3a pathway in bone development (Hwang et al., 2005). Wnt binding to Fzd can also promote Dvl interactions with the adaptor protein disheveled-associated activator of morphogenesis (Daam)1, which activates the Rho guanine nuclear exchange factor WGEF (Wu and Herman, 2006). WGEF induces RhoA/ROCK pathway activation, which promotes cytoskeletal reorganization to control cell shape and adhesion (Gao and Chen, 2010). Dvl/Daam1 interactions can also cause cytoskeletal reorganization by influencing Profilin independent of RhoA activation (Gao and Chen, 2010).

Evidence is mounting that Wnt activates trimeric G-protein signaling to control a number of downstream signaling pathways. G proteins are required for Wnt activity, but whether there are direct interactions between Fzd and G proteins remain unresolved (Katanaev et al., 2005, Katanaev and Tomlinson, 2006, Liu et al., 2007a, Liu et al., 2007b, Purvanov et al., 2010). Physical interactions between Fzd and G proteins were observed under physiological conditions (Koval and Katanaev, 2011). Thus, Wnt3a stimulated Gαs and Gαi/o, but not Gαq11 association with Fzd receptors in brain tissue. Wnt/Fzd induced cAMP accumulation and PKA activation though Gαs protein (Witze et al., 2008). In contrast, Gαi/o stimulated phospholipase C, intracellular Ca+ 2 release and direct PKC activation. G protein signaling, specifically, Gαq11 activation, was also required for nuclear localization of β-catenin following Wnt3a treatment (Tu et al., 2007). The βγ subunits of the trimeric G protein complex interact with Dvl in vertebrate cells. Fzd7 and G protein βγ subunits are required for Wnt11 to stimulate axis organization, indicating the βγ subunits as well as the α subunit are involved in non-β-catenin G protein-mediated signaling (Angers et al., 2006, Penzo-Mendez et al., 2003).

Osteoblasts, osteocytes, and osteoclasts directly regulate bone mass. Osteoblasts originate from mesenchymal progenitor cells and are responsible for producing proteins, such as type 1 collagen, that form a mineralizable matrix. Runx2, Sp7 (osterix), Wnts, Lrp5, and β-catenin are among the crucial factors required for their specification from mesenchymal precursors and osteo-chondoprogenitors. Wnts and β-catenin subsequently contribute to proliferation and survival of osteoblasts (Westendorf et al., 2004). β-catenin also regulates the communication or coupling of osteoblasts with osteoclast precursors, which originate from hematopoietic stem cells, by controlling expression of osteoprotegerin (Opg), a competitive inhibitor of Rankl and Rank interaction, to affect bone resorption (Glass et al., 2005). Osteocytes are terminally differentiated osteoblasts embedded within the mineralized matrix that communicate changes in mechanical loading and the extracellular environment to osteoblasts and osteoclasts on the bone surface to stimulate fracture repair and influence bone remodeling (Bonewald, 2011).

Wnts and Wnt pathway components are essential for many stages of osteoblast lineage development and maturation. Knowledge in this area has advanced in the last decade due to the availability and utilization of genetic approaches that test the requirement or role for certain molecules in bone development, biology, and disease. These models include germline knockout (KO), conditional knockout (CKO) or knock-in (CKI), and transgenic (Tg) expression. Table 1 summarizes bone phenotypes that result when Wnt pathway components are genetically altered in osteoblast lineage cells or the germline. Table 2 lists bone phenotypes of mice where β-catenin levels are altered in osteoclast lineage cells and their precursors. The CKO, CKI, and Tg strategies allow for tissue-specific and/or inducible expression. Several promoters drive expression of Cre recombinase (for CKO or CKI strategies) or transgenes in osteoblast and osteoclast lineage cells at different stages of maturation (Fig. 2) (Van Koevering and Williams, 2008). In the following sections, we review studies that utilized these technologies to advance our understanding of Wnt pathways in bone biology and disease.

Section snippets

Wnts

Wnts are secreted, cysteine-rich glycoproteins involved in controlling cell proliferation, cell-fate specification, gene expression, and cell survival. Cells recognize Wnts with 10 Frizzled receptors (Fzd) and Lrp molecules (Lrp5/6 and potentially Lrp4). The large number of ligands and receptors creates great combinatorial diversity and contributes to widely variable cellular responses depending on the molecules present. Wnts were historically classified as either “canonical” or “non-canonical”

LDL receptor-related proteins

Low-density lipoprotein receptor-related proteins (Lrp) are evolutionarily conserved plasma membrane receptors with a variety of functions including lipid metabolism, cargo transport, and cellular signaling. Lrp5/6 are low affinity co-receptors for Wnts and high affinity receptors for soluble Wnt antagonists: Scl, Sost-dc1, and Dkk1. Lrp4 is also emerging as a regulator of bone mass density.

Secreted Wnt antagonists: Dkks, Sfrps, Wif1, Sost, and Sost-dc1

Secreted Wnt antagonists generally utilize two distinct mechanisms to inhibit Wnt signaling. Sfrps, Cerberus and Wif1 bind to Wnts and/or Fzds to directly interfere with association of the ligand with its receptor (Fig. 1). In contrast, Dkk, Sost and Sost-dc1 (Wise) bind to the Lrp5/6 co-receptor and inhibit Wnts from associating with the Fzd/Lrp receptor complex. Existing data suggest that some of these inhibitors are viable targets for new anabolic therapeutics.

Ctnnb1 (β-catenin)

β-catenin is a cytoplasmic and nuclear protein encoded by the Ctnnb1 gene. It is a key link in numerous signaling cascades, including the “canonical Wnt pathway”, is essential for embryonic development, and is hyperactivated by mutations in many cancers. Wnt ligation of Lrp5/6 and Frizzled receptors inactivates the β-catenin destruction complex consisting of Apc, Axin, Ck1, Gsk3, Wtx, and the E2 ubiquitin ligase, βTrCP. As β-catenin accumulates, some is transported to the nucleus where it

Emerging areas for Wnts in bone biology

The remarkable advancements in our understanding of the molecular underpinnings of rare bone diseases and in how Wnts control bone formation and osteoblast proliferation, differentiation, and survival have quickly led to the development of multiple therapies for more common diseases of altered bone mass, such as osteoporosis (Rachner et al., 2011). To fully understand the effects of these drugs, it will be crucial to also study how Wnts and Wnt antagonists affect other cells in the bone marrow

Summary and conclusions

In conclusion, much has been learned about the roles of Wnt pathway components in bone development, remodeling, and repair during the last decade though the use of genetic animal models. These studies were fueled by the desire to understand the molecular underpinnings for rare bone diseases and have quickly led to the development of multiple therapies for common diseases of altered bone mass (e.g., postmenopausal osteoporosis) and for regenerative medicine. Despite these rapid and measurable

Acknowledgments

The authors are supported by several NIH grants (, , , , ).

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