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Targeting the Wnt signaling pathway for the development of novel therapies for osteoporosis Expert Rev. Endocrinol. Metab. 5(5), 711–722 (2010)

Maria P Yavropoulou1 and Socrates E Papapoulos†1 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands † Author for correspondence: Tel.: +31 715 262 490 Fax: +31 715 248 136 [email protected] 1

A number of anti-osteoporotic drugs, predominantly inhibitors of bone resorption, are currently used in the management of patients with osteoporosis to reduce the risk of fractures. While the management of the disease has improved significantly, there are still unmet needs, mainly due to a lack of agents able to replace bone that has already been lost. Human and animal genetics have identified the pivotal role of the Wnt signaling pathway in the regulation of bone formation by the osteoblasts and have made it a very attractive target for the development of novel treatments for osteoporosis. In this article, we review evidence that supports the targeting of components of the Wnt signaling pathway for the design of bone-forming treatments for osteoporosis. KEYWORDS : novel treatments • osteoporosis • sclerostin • Wnt signaling

Osteoporosis is a common chronic disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased bone fragility and susceptibility to fractures [1] . Fractures are frequent, their incidence increases with age and they cause significant morbidity and mortality. Therefore, the aim of treatment of osteoporosis is the prevention of fractures by pharmacological and nonpharmacological interventions. The skeleton is renewed throughout life in an orderly fashion through the action of the osteoclasts that resorb bone and the osteoblasts that lay down new bone matrix that subsequently mineralizes. In osteoporosis, there is an imbalance between the rate of bone formation and bone resorption, in favor of resorption, resulting in bone loss, deterioration of bone architecture and increased bone fragility. Consistent with the pathophysiology of the disease, pharmacological interventions are broadly distinguished into inhibitors of resorption and turnover, and stimulators of bone formation. During the past 15 years, there have been significant developments in the pharmacotherapy of osteoporosis, and effective treatments have become available to physicians. A number of currently available drugs, predominantly inhibitors of bone resorption, reduce the risk of osteoporotic fractures. While the results obtained so far have improved the www.expert-reviews.com

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management of patients with osteoporosis significantly, the risk of fragility fractures, particularly nonvertebral fractures, remains high and there are unmet needs in the treatment of the disease that require a broader range of therapies. Advances in our understanding of the pharmacology of existing classes of compounds, and particularly in our knowledge of the local regulation of bone metabolism, may help to achieve this aim. In general, improvement of the treatment of chronic diseases involves several approaches; these include further development of existing classes of interventions, new regimens of drug administration, combination therapies or the identification of novel therapeutic targets. Such approaches are being explored in osteoporosis as well. Examples of the first two are the bisphosphonates, of which four are available worldwide for the treatment of osteoporosis, and these can be given either orally or intravenously at intervals ranging from 1 day to 1 year. Another example are selective estrogen receptor modulators, of which three are approved for the management of osteoporosis. Combination therapies are currently restricted to different ways of administering parathyroid hormone (PTH) with bisphosphonates. Particularly relevant for new approaches are studies of human and animal genetics, which have led to identification of novel, more specific

© 2010 Expert Reviews Ltd

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signaling pathways in bone cells that provide targets for new therapeutics. Such novel targets in osteoclasts include, among others, receptor activator for nuclear factor kB ligand (RANKL) and cathepsin-K. A fully human monoclonal antibody to RANKL (denosumab) was developed and recently approved for the treatment of osteoporosis [2] , while cathepsin-K inhibitors have been evaluated in Phase II studies and one of them (odanacatib) is currently in Phase III clinical development [3,4] . Inhibitors of bone resorption, although highly efficacious, cannot replace bone already lost, underscoring the need for new therapeutic approaches. The PTH paradigm (PTH 1-34 and PTH 1-84) illustrated the possibility of stimulating bone formation in patients with osteoporosis, and increasing understanding of the molecular pathways regulating bone formation has provided potential targets for the development of new boneforming agents. In particular, the recognition of the central role of the Wnt signaling pathway in the regulation of bone formation has made it a very attractive target for new drug development in osteoporosis. We review here the current evidence supporting components of this signaling pathway as targets for osteoporotic treatments. Wnt signaling pathway

Wnt proteins form a family of 19 highly conserved secreted signaling molecules, rich in cysteine, that play an important role in a variety of cellular processes in the body, particularly in cell growth, differentiation and apoptosis [5] . Although the number of Wnt proteins involved in osteoblastogenesis is not yet known, several have been demonstrated to affect bone remodeling through the canonical pathway. Binding of Wnts to the seven-transmembrane domain-spanning frizzled receptor (FzR) and the low-density lipoprotein receptor-related protein (LRP) 5 and 6 co-receptors [6] leads to dephosphorylation and translocation of the cytoplasmic b-catenin into the nucleus. Ten FzR receptors with differences in ligand specificity and tissue expression have been identified so far [7] . The interaction of b-catenin with T-cell-specific transcription factor (TCF)/ lymphoid-enhancing factor 1 transcription factors displaces transcriptional co-repressors [8] , such as Groucho and histone deacetylases, and recruits transcriptional co-activators, such as the histone acetylase p300/CBP and cAMP response elementbinding protein (CREB) [9] . The formation of the heterodimeric b-catenin–TCF/LEF1 is crucial for the activation of Wnt target genes, such as Runx-2 [10,11] . Wnt signaling in bone metabolism

Fundamental to the discovery of the role of the canonical Wnt signaling pathway in bone formation have been studies of patients with rare skeletal disorders, such as osteoporosis–pseudoglioma syndrome, the high bone mass phenotype, sclerosteosis and van Buchem disease. The osteoporosis–pseudoglioma syndrome is due to loss-of-function mutations of LRP5 and is characterized by low bone mass associated with low bone formation and skeletal fragility manifested in childhood [12] . Conversely, the high bone mass phenotype is due to gain-of-function mutations of LRP5 and, as 712

the name indicates, is associated with increased bone mass due to increased osteoblastic activity without a concomitant increase in bone resorption [13,14] . LRP4, expressed in osteoblasts, has also recently been identified as a potent regulator of Wnt signaling in bone [15–18] . Sclerosteosis and van Buchem disease are two sclerosing bone disorders with closely related phenotypes characterized by very high bone mass due to increased bone formation [19,20] . The bone is of good quality and no fractures have been reported in patients with these diseases. Sclerosteosis is due to loss-of-function mutations in a newly cloned gene called SOST, which is located on chromosome 17q12–21 and encodes the protein sclerostin [19] , a negative regulator of canonical Wnt signaling. No mutations within this gene could be found in patients with van Buchem disease, but, instead, a 52-kb deletion 35 kb downstream of the SOST gene was identified. The deleted region was later found to contain regulatory elements for SOST transcription, explaining its ability to induce a phenotype closely resembling that of patients with sclerosteosis [20] . The exclusive expression of sclerostin in mature osteocytes is probably responsible for the phenotype of patients with sclerosteosis and van Buchem disease, which is limited to bone tissue. Wnts also signal through b-catenin-independent (noncanonical) mechanisms to regulate morphogenesis during vertebrate development [21] . Noncanonical Wnts activate FzRs and G-protein-coupled receptors, but they can also bind to tyrosine kinase-like receptors, such as those related to tyrosine kinases and receptor tyrosine kinase-orphan receptor 2 (ROR2) [22] . Genetic ablation of the noncanonical Wnt7b protein in mice results in deficient bone formation, suggesting that noncanonical Wnt signaling may also play a role in bone remodeling [23] . Additionally, noncanonical Wnt signaling has been implicated in responses to mechanical loading involving stimulation of the expression of osteoprotegerin and fluid flow-induced osteogenesis [24,25] . Finally, the ROR2 receptor, which is mainly activated by Wnt 5a, has been reported to regulate differentiation of mesenchymal cells into adipocytes or osteoblasts, and targeting downstream effectors of activated ROR2 receptors may stimulate bone formation [26,27] . Targeting the canonical Wnt signaling pathway

The human disease paradigms suggest that modulation of components of the Wnt signaling pathway may lead to the desired outcome in patients with osteoporosis, namely, the formation of new good quality bone. Various intracellular and extracellular components of the Wnt canonical pathway can, theoretically, be targets for new pharmaceuticals (FIGURE 1) . However, for clinical use, treatments should not only modify the expression of target molecules, but also need to have bone specificity to avoid potential effects on other organs in the body. Therefore, extracellular secreted molecules expressed predominantly in bone tissue present the most attractive targets of new treatments. These include sclerostin, Dickkopf (Dkk)-1 and sFRP-1, which bind to the membrane receptors and inhibit the intracellular signaling of the Wnt canonical pathway. Expert Rev. Endocrinol. Metab. 5(5), (2010)

Wnt signaling & osteoporosis

Secreted antagonists of the Wnt pathway

sFRP inhibitors

Osteocyte-produced sclerostin decreases bone formation by inhibiting the terminal DKK-1 differentiation of osteoblasts and promotWiF-1 ing their apoptosis [28] . Sclerostin was origisFRP DKK-1 nally thought to be an antagonist of bone inhibitors morphogenetic proteins (BMPs) based on Kremen its amino acid sequence similarity to difSCL SCL ferential screening-selected gene aberrative Wnt inhibitors in neuroblastoma family (DAN) of secreted glycoproteins, which share the capacity to antagonize BMP activity [29] . However, in subsequent experiments it was demonFrizzled strated that sclerostin could not antagonize all BMP responses, and had a mechanism of action distinct from that described for classical BMP antagonists, inhibiting bone formation by blocking Wnt signaling in osteoblasts [30–32] by a not yet fully clarified GBP Dvl mechanism. Sclerostin binds to the first GSK3β YWTD-EGF repeats of LRP5 [33,34], and inhibitors this binding is decreased in the mutated Proteolytic system form of LRP5, which is associated with the GSK3β Ubiquitin–proteasome high bone mass phenotype [35] . However, Axin although sclerostin binds to LRP5/6 and CKI Proteasome antagonizes Wnt signaling, it does not PP2A inhibitors appear to compete with Wnts for binding β-catenin APC to this co-receptor. SOST knock-out mice Degradation were demonstrated to have similar skeletal features to patients with sclerosteosis, with significant increases (>50%) in bone mass Histone at both the trabecular and cortical comdeacetylase partments of the lumbar spine and the inhibitors TCF hip [36] . By contrast, transgenic mice overexpressing human SOST exhibited a low LEF Nucleus bone mass phenotype and decreased bone strength [29] . The findings of these human and animal studies led to the development of an DNA antibody against sclerostin, which has been tested mainly in animal models. Figure 1. Potential targets of drug development in the Wnt signaling pathway. Subcutaneous administration of 25 mg/kg Potential targets for drug development are indicated in the black circles. sclerostin antibody twice weekly for 5 weeks APC: Adenomatosis polyposis coli; CK1: Casein kinase-1; DKK: Dickkopf; Dvl: Dishevelled; FRP: Frizzled-related protein; GBP: GSK-3-binding protein; to aged ovariectomized rats resulted in a GSK3: Glucogen synthase kinase 3; LEF: Lymphoid-enhancing factor 1; LRP: Low-density robust increase in bone mass and improvereceptor-related protein; PP2A: Protein phosphatase 2; SCL: Sclerostin; sFRP: Secreted ment in strength at virtually all skeletal sites frizzled-related protein; TCF: T-cell factor; Wif: Wnt inhibitory factor. [37] . Remarkably, this short-term treatment with sclerostin antibody not only completely reversed estrogen cortical thickness and reduction in cortical porosity [37] . These deficiency-induced bone loss, but it further increased bone mass effects of sclerostin antibodies on the mass and quality of cortiand bone strength to levels greater than those of sham-operated cal bone, if confirmed in human studies, could have a significant control animals. Bone biopsies showed that bone formation mark- clinical impact on the prevention of nonvertebral fractures [38,39] . edly increased in trabecular as well as in periosteal, endocortical Similar results were also reported in cynomologus monkeys after and intracortical surfaces, leading to increases in trabecular and 2 months of treatment with a humanized antisclerostin antibody LRP5/6


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Increased bone mineral density (BMD) was observed at the femoral neck, radius and lumbar spine (FIGURE 2) [40] . Furthermore, administration of sclerostin antibody to this nonhuman primate model almost doubled vertebral load to failure, indicating an increase in bone strength [40] . Recently, treatment with a sclerostin antibody was reported to increase fracture callus density and strength in rodent fracture-healing models and 3–5-year-old male cynomologus monkeys who underwent bilateral, transverse fibular osteotomies [41] . Finally, in a mouse model of chronic colitis, a short period of treatment (19 days) with a sclerostin antibody completely reversed the bone loss and decline of several bone mechanical and microstructural properties typically associated with chronic inflammation [42] . The increased bone formation induced by sclerostin antibodies was not associated with an increase in bone resorption. Instead, a decrease in osteoclast surface was observed [37] , suggesting a functional uncoupling between osteoclasts and osteoblasts, as also shown in studies of SOST knock-out mice [36] . The underlying mechanism of this surprising finding is not yet clear, and longerterm studies are needed to examine whether this uncoupling of bone formation to bone resorption is transient or persistent.

Furthermore, the effect of sclerostin antibodies on bone formation appears not to be affected by cotreatment or pretreatment with alendronate [43,44] , and is reversible after discontinuation of the treatment [37] . In the first human placebo-controlled, dose-escalating study of 72 healthy men and postmenopausal women, it was shown that a single injection of a monoclonal antibody against sclerostin markedly increased bone formation markers and BMD, and was well tolerated [45] . Serum procollagen type 1N propeptide levels reached a peak 14–25 days after the antibody administration and returned progressively to baseline values after approximately 2 months. By contrast, the bone resorption marker, serum carboxyterminal cross-linking telopeptide of bone collagen, decreased to a minimum approximately 14 days after antibody injection, and returned to baseline values after approximately 2 months, reflecting the uncoupling in osteoblast and osteoclast activity that was reported in preclinical studies. Clinical Phase II studies with this antibody are currently underway. Also particularly relevant for further development are studies of heterozygous carriers of sclerosteosis who were demonstrated to have BMD values consistently higher than those of healthy individuals without any of

Sclerosteosis

OB Bone thickness


[40] .

Vehicle Vehicle

LC Normal

OC Sclerostin antibodies Sclerostin antibodies

Sclerostin

OCYT Periosteal surface Endocortical surface Bone remodeling time

Figure 2. Sclerostin inhibition and bone formation. (A) Sclerostin is produced by osteocytes and inhibits osteoblast differentiation. Lack of sclerostin leads to increased bone formation. (B) The skulls of a patient with sclerosteosis and a healthy individual. (C–D) Intravenous administration of Scl-Ab in cynomolgus monkeys increased cortical bone formation at the femur midshaft. Fluorescent microscopy images of the femur midshaft illustrate the sclerostin-neutralizing monoclonal antibody (Scl-AbIV) -mediated increase in tetracycline (orange; days 14 and 24) and calcein labels (green; days 47 and 57) on the endocortical and periosteal surfaces. LC: Lining cells; OB: Osteoblasts; OC: Osteoclasts; OCYT: Osteocytes. Adapted with permission from [121,122] . Figure (B) is courtesy of H Hamersma.

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the bone complications, due to bone overgrowth in the skull and the mandible, of the homozygotes [46] . These data suggest that titration of sclerostin activity in vivo can have a favorable effect on bone mass without the skeletal complications of sclerosteosis. These animal and human studies reveal that the stimulation of bone formation by the inhibition of sclerostin has distinct differences from those following PTH administration – the current standard of bone forming therapy of osteoporosis. PTH increases cancellous and endocortical bone formation but has a limited, if any, effect on periosteal bone formation, and increases cortical porosity [47] . Additionally, most of the bone-forming activity of PTH occurs at sites already undergoing bone remodeling rather than at quiescent surfaces [48,49] . These effects are mirrored in the changes of biochemical markers of bone turnover during treatment with daily injections of PTH, which are characterized by early increases in serum procollagen type 1N propeptide values and followed by increases in serum cross-linking telopeptide of bone collagen values. By contrast, inhibition of sclerostin stimulates bone formation at all bone envelopes and reduces cortical porosity with no effect on osteoclastic activity [37] . Available data in humans also showed consistent increases in markers of bone formation with either decreases or no change in markers of bone resorption, and an impressive early increase in BMD [45] . The results of the studies with sclerostin antibody, if confirmed in human studies over longer periods of time, indicate a possible new treatment paradigm for which the term ‘anabolic treatment of osteoporosis’ should be reserved (FIGURE 2) . Dickkopf-1 (Dkk-1)

The Dkk family of secreted glycoproteins consists of four members (Dkk-1–4) that share two conserved cysteine-rich domains at the N- and C- terminal – Cys 1 and Cys 2, respectively [50] . Of these, Dkk-1 and 2 are the most relevant to bone metabolism, and Dkk-1 is the member best studied so far. Dkk-1 interacts with specific regions in the C-terminal of LRP5/6 co-receptors through the Cys 2 domain [50] . It also binds with high affinity to the single-pass transmembrane receptors Kremen-1 and -2, which potentiate its capacity to inhibit Wnt signaling [51] . The molecular mechanism, by which Dkk-1 antagonizes LRP5/6 action, has not yet been completely clarified, and may either promote internalization and subsequent degradation of the ternary complex Dkk-1/LRP5/6/Kremen1/2 [52,53] or may directly compete with the binding of Wnt proteins to LRP5/6 [52] . Dickkopf-1 acts as an antagonist, while Dkk-2 has been reported to act either as an agonist or antagonist of Wnt signaling, depending on the cell type [53] . In maturing osteoblasts, both Dkk-1 and Dkk-2 are overexpressed, and the inhibition of Wnt signaling is a prerequisite for the formation of mineralized bone matrix [54] . In adult mice, Dkk-1 is strongly expressed only in bone tissue, whereas in embryonic mice, it is also required for the regulation of the development of extraskeletal tissues, such as eye and skin [55] . The role of Dkk-1 in bone formation was further supported by genetic studies in mice lacking a single allele of Dkk-1, while homozygous deletion of Dkk-1 leads to embryonic lethality. The www.expert-reviews.com

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Dkk-1 heterozygous-deficient mice developed a marked increase in trabecular bone volume and bone formation rate [56] . By contrast, transgenic mice overexpressing Dkk-1 had severe osteopenia in both cortical and cancellous bone, owing to decreased osteoblast number and activity [57] . Furthermore, studies with transgenic mice that variably express Dkk-1 have revealed an inverse relationship between Dkk-1 expression and trabecular and cortical bone mass, that even a 25% reduction in Dkk-1 expression could significantly increase trabecular bone volume [58] . These data indicate that titrated inhibition of Dkk-1 expression in the adult tissue can favorably affect bone formation. Treatment of ovariectomized rats with a Dkk-1 antisense oligonucleotide for 28 weeks restored bone loss and biomechanical strength of the femurs by increasing the number of osteoblasts and abrogating the effects of estrogen-loss on osteoclastogenesis [59] . Consistent with these results, neutralizing monoclonal antibodies to Dkk-1 given for 8 weeks to estrogen-deficient osteopenic mice induced significant stimulation of new bone formation at endocortical and trabecular bone envelopes, and partial resolution of osteopenia in femur and lumbar vertebrae [60] . Moreover, treatment with a Dkk-1 antisense oligonucleotide reversed most of the catabolic effects of dexamethasone on osteoblast survival and activity, and decreased adipocyte volume and differentiation in the marrow cavity of steroid-treated rats [61] . The development of Dkk-1 neutralizing monoclonal antibodies is currently under intense investigation, although small molecules blocking Dkk-1 association with LRP5/6 or Kremen 1/2 could also offer an alternative approach to enhancing Wnt signaling by blocking Dkk-1 antagonistic activity [62] . Such interventions may not only be useful in the treatment of osteoporosis, but also in the management of other diseases affecting the skeleton in which Dkk-1 is overexpressed, such as multiple myeloma and rheumatoid arthritis [63–65] . Administration of neutralizing antibodies to Dkk-1 increased bone formation in myelomatous mice models [66,67], while inhibition of Dkk-1 in a mouse model of rheumatoid arthritis reversed the bone-destructive pattern of rheumatoid arthritis to the bone forming pattern of osteoarthritis (e.g., formation of osteophytes) [65] . The expression of Dkk-1 in multiple tissues, at least at early stages of development, may pose limitations in its potential use in osteoporosis. However, the development of molecules with tissue selectivity could represent a promising tool for the improvement of therapeutic approaches for bone loss. Secreted frizzled related protein-1

The family of secreted frizzled-related proteins (sFRPs) consists of eight known members, of which the first five (sFRP-1–5) have been identified in mammals [68] . sFRPs bind to Wnt proteins and prevent them from interacting with the LRP5 and frizzled receptors, inactivating both canonical and noncanonical Wnt signaling pathways. Additionally, they can also bind and form nonfunctional complexes with the FzRs [68] . Several studies haveinvestigated the role of sFRPs, mainly sFRP-1 and sFRP-3, in modulating Wnt signaling in bone. 715


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In in vitro studies, sFRP-1 was found to regulate the survival, differentiation and function of osteoblasts and osteocytes [69] . Furthermore, dexamethasone treatment of mesenchymal cells increased expression of sFRP-1, which in turn led to increased cell apoptosis and reduced osteogenic activities of these cells, through transcriptional regulation [70] . Knocking down sFRP-1 by siRNA alleviated cell apoptosis and restored diminished osteogenic differentiation of the dexamethasone-treated mesenchymal cells, suggesting that regulation of Wnt/sFRP signal transduction could also be used as an alternative strategy for the prevention of glucocorticoid-induced osteoporosis [70] . Deletion of the sFRP-1 gene in mice increased trabecular BMD, volume and mineral apposition rate at multiple skeletal sites, but had no effect on cortical bone or extraskeletal tissues [71] . Additionally, it blunted the ability of PTH to increase trabecular bone volume, suggesting overlapping mechanisms of action [72] . Inactivation of the FRZB gene, which encodes sFRP-3, in mouse models of osteoarthritis, increased cortical bone thickness, density and stiffness and stimulated cortical bone formation following loading [73] . However, these mice also demonstrated increased articular cartilage loss. Current research is focused on the development of synthetic small-sized inhibitors of sFRP-1 to stimulate canonical Wnt signaling and enhance bone formation. Diphenylsulfone sulfonamide [74,75] and imino-oxothiazolidines [76] have been identified as such molecules, and were shown to dose-dependently increase bone formation in ex vivo and in vitro assays. Further chemical optimization can lead to the synthesis of molecules with an improved pharmacological profile [77] . The broad tissue expression and the agonistic/antagonistic action of sFRP-1 on Wnt signaling, depending on the concentration and the cell type, may limit its use as a therapeutic target. Therefore, research focuses on the potential development of specific and orally available inhibitors with improved pharmacokinetic characteristics that can be used not only in the treatment of osteoporosis, but also of other diseases associated with increased sFRP-1 expression [78–80] . Intracellular components of the Wnt canonical pathway

Although they are less attractive targets than sclerostin and Dkk-1, intracellular components of the Wnt signaling pathway are also currently being investigated for potential therapeutic use. Among these, the intracellular enzyme glycogen synthase kinase (Gsk) -3b seems to represent the most amenable target (FIGURE 1) . Glycogen synthase kinase 3b

Glycogen synthase kinase is a multifunctional serine/threonine kinase, which comprises two isoforms, Gsk-3a and Gsk-3b, and is expressed in almost every tissue of eukaryotic organisms [81] . Gsk-3b kinase was first recognized as a downstream target of insulin stimulation [82] , but was later found to regulate a variety of developmental intracellular signaling pathways, such as Wnt [83] . In canonical Wnt signaling, cytosolic Gsk-3b phosphorylates b-catenin, axin and adenomatosis polyposis coli (APC), leading to ubiquitination and final degradation of 716

b-catenin from the ubiquitin proteasome proteolytic system. Novel findings, however, demonstrated that a membrane-bound isoform of Gsk-3b also phosphorylates and activates the LRP6 co-receptor, promoting its association with the scaffolding protein axin and, thus, stimulating Wnt signaling [84] . Gsk-3b has also been suggested to control endochondral bone formation through modulation of FGF18 [85] and to contribute to etoposide-induced apoptosis of a mesenchymal cell line through regulation of Bcl-2 expression [86] . Mice with homozygous deficiency of Gsk-3b (Gsk-3b-/-) do not survive owing to severe liver dysfunction [87] . whereas, heterozygous mice (Gsk-3b+/-) have increased trabecular and cortical bone mass compared with their wild-type (Gsk-3b+/+)littermates, without abnormalities in the growth plate or any other extraskeletal organ [88] . Inhibition of Gsk-3b, with selective or nonselective inhibitors, in primary osteoblastic cultures derived from wild-type (Gsk-3b+/+) mice promoted bone formation to levels similar to those of Gsk-3b+/- mice-derived cell cultures, through enhancement of Runx-2 transcriptional activity [89] . These results suggest that inhibition of Gsk-3b may have a beneficial effect on bone formation. In humans, epidemiological studies of patients with bipolar disorders treated with the nonselective Gsk-3b inhibitor lithium, have provided equivocal results regarding BMD and fracture risk [88,90,91] . There are no studies with selective inhibitors of Gsk-3b. The wide distribution of Gsk-3b and its involvement in multiple signaling pathways make it an unattractive target for the treatment of osteoporosis. Ubiquitin–proteasome proteolytic system

The ubiquitin–proteasome system is the intracellular proteolytic system responsible for the final degradation of phosphorylated cytoplasmic b-catenin in the absence of Wnt proteins. The intracellular degradation of proteins is a multistage process that includes the targeting of proteins with attachment of small polypeptides known as ubiquitins, identification of ubiquitinated products from the proteasome complex and final proteolytic degradation in an ATP-dependent fashion [92] . Inhibitors of the ubiquitin–proteasome complex are currently used in the treatment of cancer, and recent evidence has suggested that these molecules may also exert a positive effect on bone formation [93] . Treatment of multiple myeloma patients with the proteasome inhibitor bortezomib, demonstrated an increase in bone formation that was independent of the action of the drug on the progression of the disease [94–96] . In in vitro studies, proteasome inhibitors exerted a positive effect on bone formation through the regulation of the Runx-2 transcription factor [97,98] and the nuclear factor kB (NF-kB) signaling pathway [99,100] in osteoblastic and osteoclastic cell lines, respectively. In vivo studies in rodents further confirmed a bone anabolic effect of various proteasome inhibitors [101] . The underlying molecular mechanisms seem to include both attenuation of osteoclastogenesis and enhancement of osteoblastogenesis, mostly via Wnt-dependent and independent activation of b-catenin/TCF pathways [102] . Furthermore, animal studies Expert Rev. Endocrinol. Metab. 5(5), (2010)


have demonstrated that bortezomib can increase bone mass not only in myelomatous bone but also in normal bone, and in ovariectomy-induced osteoporotic bone tissue [103,104] .


Histone deacetylases

Post-translational modification of histones in the cell nucleus facilitates chromatin remodeling and regulates gene expression. Recruitment of histone acetylases to gene promoters activates RNA polymerase II, and enhances transcription of the target genes. Their activity is counterbalanced by histone deacetylases, which leads to chromatin condensation and repressed gene expression. Inhibitors of histone deacetylases (HDACis), which arrest growth, and promote differentiation and apoptosis preferentially of malignant cells [105,106] , are currently being investigated as anticancer therapeutic agents in clinical trials. The molecular mechanism underlying the HDACis-induced tumor growth arrest appears to involve, at least partly, the upregulation of Wnt signaling activity through increased levels of transcriptionally active b-catenin [107] . However, other mechanisms independent of the Wnt pathway, such as nonhistone targets [107] , have also been reported to contribute to antitumor effects of HDACis, and thus the potential application of these compounds in the treatment of osteoporosis is highly unlikely.

Review

a secreted extracellular Wnt antagonist, is receiving most of the attention. Produced almost exclusively by osteocytes, sclerostin represents a promising therapeutic target, the inhibition of which may increase bone formation without a simultaneous increase in bone resorption. Patients with sclerosteosis and van Buchem disease lack any extraskeletal complications, and heterozygote carriers with sclerosteosis are otherwise healthy individuals with higher BMD values [39] . The latter finding further supports the notion that inhibition of sclerostin activity can be titrated to increase bone formation. Wnt proteins are ubiquitously expressed in the human body, and any manipulation of Wnt signaling activity could result in adverse events from different tissues. For instance, nearly 80% of sporadic colorectal cancers are reported to carry inactivating mutations of the APC gene, which encodes the scaffolding protein APC of the canonical Wnt pathway [113] . Moreover, overexpression of b-catenin in a mouse model demonstrated the development of skeletal tumors in approximately 80% of the animals studied [114] . Additionally, both canonical and noncanonical Wnt pathways have been implicated in the pathogenesis of cardiovascular diseases such as myocardial infarction and cardiac hypertrophy [115,116] . Therefore, any therapeutic manipulation of Wnt signaling should be closely monitored for potential adverse effects.

Role of serotonin

Not directly related to the topic of this article, but relevant to the function of LRP5, is the recent work of Karsenty et al. showing that, in mice, LRP5 residing in the enterochromaffine cells of the duodenum regulates the synthesis of serotonin outside the brain [108] . Earlier studies have shown that serotonin exerts a negative effect on bone formation after binding to specific receptors in osteoblasts [109,110] , and as pharmacological inhibition of serotonin reuptake decreases bone formation and mineral density in mice, it may also increase the risk of fractures in humans [111] . In mice, absence of gut-produced LRP5 is associated with a decrease in circulating serotonin and increased bone formation [108] . Furthermore, inhibition of serotonin synthesis by an antagonist to Tph1, the initial enzyme in gut-derived serotonin synthesis, stimulated bone formation in cancellous bone to the same degree as PTH in rodents, but had no effect on cortical bone [112] . These data reveal an important role of LRP5 produced outside bone in the regulation of bone formation by a Wnt signaling independent mechanism, and have identified circulating serotonin as a potential target for new bone-forming therapies for osteoporosis. Expert commentary

The identification of the pivotal role of canonical Wnt signaling in bone metabolism and the ongoing intensive research on the mechanisms involved in its regulation have provided new and exciting perspectives for pharmaceutical intervention and drug discovery. Several intracellular and extracellular components of this pathway are currently being investigated as potential targets for the development of new agents aiming to increase bone formation in patients with osteoporosis. Among them, sclerostin, www.expert-reviews.com

Five-year view

It is expected that sclerostin inhibitors will be developed as treatments for patients with osteoporosis in studies with clinically relevant outcomes. A 5-year period is, in our view, essential for establishing the efficacy and tolerability of such inhibitors. Other molecules are also expected to be clinically developed, the most obvious choice currently being inhibitors of Dkk-1. Such clinical studies may not only provide a long-awaited real anabolic therapy of osteoporosis, but may also help in better understanding the local regulation of bone remodeling. The first results of the treatment of animals with a sclerostin inhibitor suggested an uncoupling between bone formation and resorption [39] . The intriguing possibility of de novo bone formation in quiescent bone surfaces in adults, without concomitant stimulation of bone resorption, goes beyond our current understanding of the obligatory coupling of the two processes, and the underlying mechanisms will require intensive investigation. Additionally, it may be that sclerostin interacts with components of the Wnt signaling pathway that participate in osteoclastogenesis. There is already evidence that stimulation of the Wnt pathway upregulates osteoprotegerin production and downregulates that of RANKL [117] . Such evidence, coupled with findings indicating that osteoblast activity may be modulated by factors secreted by the osteoclasts [118], can reshape our knowledge of the local regulation of bone metabolism and lead to the development of tailor-made therapies for individuals with osteoporosis. The regulation of sclerostin production will also be intensively studied due to its restricted expression in osteocytes. For many years, this most abundant cell of bone has been considered as the receiver and translator of mechanical signals [119] . However, 717


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this attractive hypothesis has never been vigorously tested due to the lack of suitable experimental approaches. Sclerostin provides such a tool, and questions regarding the translation of mechanical to chemical signals in bone can now be addressed. There is already evidence that mechanical loading alters sclerostin expression in the osteocytes of rodents [120], but there is no information about a skeletal adaptation to mechanical signals following sclerostin inhibition. Finally, the role of individual components of the Wnt signaling pathway and their complex interactions will be further investigated, and that of the noncanonical pathway on skeletal metabolism will be more systematically addressed. In parallel to these studies, it is apparent that research efforts will also focus on the role of extraskeletal LRP5 and serotonin on bone metabolism. A key issue in the development of treatments for

human diseases will be the safety and tolerability of any new intervention. Therefore, the coming 5 years are expected to be dominated by basic and clinical studies on the regulation of bone formation, predominantly by components of the Wnt signaling pathway, in particular, sclerostin. Financial & competing interests disclosure

Studies of sclerostin in the authors’ laboratory are supported by the European Commission; Grant: EU FP7 (TALOS:Health-F2–2008–201099). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Therapies that can replace lost bone in selected patients with osteoporosis are needed. • The Wnt signaling pathway offers a number of potential targets for the development of bone-forming agents. • Sclerostin, due to its specific localization in osteocytes and the exclusive bone phenotype of patients with sclerostin deficiency, is a very attractive target. • Short-term inhibition of sclerostin in animals increased bone formation dramatically at all bone surfaces, including the periosteum, without a concomitant increase in bone resorption. • Longer-term studies of sclerostin inhibition in humans need to establish whether the nature of this effect is permanent or transient. • Potential extraskeletal effects of inhibitors of components of the Wnt signaling pathway need to be vigorously investigated.

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