J Phys Fitness Sports Med, 3(3): 341-345 (2014) DOI: 10.7600/jpfsm.3.341 JPFSM: Short Review Article Wnt signals and bone metabolism

Kazuhiro Maeda*, Mitsuru Saito and Keishi Marumo

Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan

Received: May 1, 2014 / Accepted: May 30, 2014

Abstract Wingless-related MMTV integration site (Wnt) family members are secreted glycoproteins with a molecular weight of approximately 40kDa, and are categorized as cyto- kines involved in various phases of life phenomena, such as ontogenesis, morphogenesis, and carcinogenesis. The glycoproteins are conserved in various species from C. (Caenorhabditis) elegans to mammals, and 19 types of Wnt homologs have been identified in humans. The is broadly classified as either a canonical or noncanonical pathway. The Wnt canonical signaling pathway stimulates bone formation, and this pathway is inhibited by Sclerostin, which is known to be produced by and to inhibit bone formation. Be- cause mechanical loading is reported to inhibit the production of Sclerostin in osteocytes and to enhance bone formation, osteocytes are recognized as mechanosensors in the hard tissue. Recently, clinical administration of anti-Sclerostin was begun for osteoporosis. Here we outline the roles of Wnt signaling in bone formation and describe the current status of use of anti-Sclerostin antibody. Keywords : Wnt signal, Sclerostin, mechanical stress, , , osteoclast

Introduction Mechanisms of Wnt signaling transduction In the early 1980s, Dr. Nusse et al. of the United States Canonical and non-canonical pathways7). The Wnt sig- reported integration site 1 (int-1) as an oncogene associ- naling pathways can be broadly classified into the canoni- ated with the development of murine breast cancer1). In cal pathway involving β-catenin and the non-canonical the late 1980s, an ortholog of int-1 in D. melanogaster pathway not involving β-catenin. Usually, β-catenin is was identified2), which was identical to the known wing- phosphorylated, rapidly degraded and regulated such that less associated with the formation of the drosophila it does not accumulate in the cytoplasm (Fig. 1A). The wings3). This gene was named Wnt after wingless and canonical pathway inhibits GSK-3β, which induces ac- int-1. Furthermore, at the turn of the 21st century, low cumulation of β-catenin in the cytoplasm. Accumulated density lipoprotein receptor-related 5 (LRP5), a β-catenin translocates into the nucleus and induces the receptor for Wnt, was shown to be associated with the expression of target with T cell factor (TCF)/lym- regulation of bone mass4), and the roles of Wnt signal- phocyte enhancer factor 1 (LEF1) (Fig. 1B). The non-ca- ing in bone metabolism have drawn attention ever since. nonical pathway, a pathway not involving β-catenin, acti- Sclerosteosis is a disease characterized by progressive, vates calmodulin-dependent protein kinase II (CaMK II), systemic overgrowth of the bones. In 2001, the Sclerostin protein kinase C (PKC), c-Jun N-terminal kinase (JNK), (SOST) gene was discovered as the gene responsible for etc. (Fig. 1C). Which of the two pathways, canonical or the causation of sclerosteosis5); a loss-of-function muta- non-canonical, is activated is determined by the combina- tion of Sclerostin was shown to induce sclerosteosis. The tion of ligands, receptors, and coreceptors. Ligands, such protein coded by the SOST gene is Sclerostin. Based on as Wnt3a, bind to Frizzled (Fzd) receptors and LRP5/6 this, it was expected that Sclerostin might have the effect complexes to activate the canonical pathway. On the other of inhibiting the development of sclerosteosis. Although hand, ligands such as Wnt5a and Wnt11 activate the Wnt/ Sclerostin was first considered to be an inhibitor of bone calcium (Ca2+) signaling pathway and Wnt/planar cell morphogenetic protein (BMP) signaling, the molecule is polarity (PCP) signaling pathway without inducing accu- currently considered as a factor that binds to LRP5/6 to mulation of β-catenin in the cytoplasm. inhibit the Wnt canonical pathway6). LRP5/6, receptors for the Wnt canonical pathway8). LRP5 and LRP6 are single transmembrane receptors that were *Correspondence: [email protected] originally identified as candidate disease susceptibility 342 JPFSM: Maeda K, et al.

canonical pathway noncanonical pathway Wnt5a A LRP5/6 B Wnt3a C Frizzled Ror1/2 LRP5/6 Frizzled Frizzled

GSK-3 GSK-3

-catenin RhoA Rac -catenin CaMKII P -catenin -catenin Axin APC accumulation of PKC Ub -catenin JNK Ub Ub P -catenin nucleus -catenin Wnt/Ca2+ Wnt/PCP TCF/LEF pathway pathway

degradationof expression of target genes -catenin

Fig. 1 Wnt signal transduction (modified from Reference 7) A. In cytoplasm, β-catenin is phosphorylated by complexes of GSK-3β, APC and Axin and rapidly degraded by the ubiquitin- proteasome system. B. The canonical pathway is activated by ligands, such as Wnt3a, through their binding to Fzd receptors and LRP5/6 complexes. Inhibition of GSK-3β induces accumulation of β-catenin in cytoplasm. Accumulated β-catenin translocates into nucleus and in- duces the expression of target genes with TCF/LEF1. C. The non-canonical pathway is activated by ligands, such as Wnt5a, through their binding to Fzd receptors or Fzd/receptor tyrosine kinase-like orphan receptor (Ror) 1/2 complexes. This binding activates the pathway without β-catenin. genes for type 1 diabetes mellitus9,10). Extracellularly, the DKK family is ubiquitously expressed in the body the receptors have four β-propeller domains (BP1-BP4), and plays important roles not only in the development of which constitute the tertiary structure of con- the head and limbs, but also in the formation of various sisting of β-sheets (Fig. 2A). The β-propeller domains organs13,14). Meanwhile, Sclerostin binds to the BP1 do- which bind to Wnt ligands are considered to be impor- main of LRP5/6 (Fig. 2A). Sclerostin, which is expressed tant for the signal transduction of the canonical pathway. specifically in osteocytes15), inhibits bone formation by Moreover, inhibitors of the Wnt canonical pathway, such inhibiting signal transduction through the Wnt canonical as the Dickkopf (DKK) family proteins and Sclerostin, pathway. Moreover, a group from Amgen (Applied Mo- also competitively inhibit signal transduction of the Wnt lecular Genetics) reports, based on analyses of Sclerostin ligands by binding to these domains. gene-deficient mice, that phenotypes except that of hard tissues were normal in these mice compared to wild-type DKK family proteins and Sclerostin, inhibitors of the mice16). Wnt canonical pathway. The DKK family proteins bind to the LRP5/6 complex to inhibit signal transduction Bone formation and Wnt signaling through the Wnt canonical pathway. DKK1, especially, is known to bind to the BP1 and BP3 domains of LRP5/6 Osteoporosis-pseudoglioma (OPPG) syndrome is a ge- (Fig. 2A). DKK1 was originally reported as a molecule netic disease characterized by juvenile-onset osteoporosis necessary for the formation of the head of Xenopus11). and blindness. Ever since loss-of-function mutations of The molecule has also been shown to be important for the the LRP5 gene were reported to be the cause of the OPPG development of the head and limbs in mammals12). Thus, syndrome, the role of Wnt signaling in bone formation JPFSM: Wnt signals and bone metabolism 343

BP : -propeller domain A Structure of LRP5 and binding EGF : EGF-like domain LDLR : LDL type A repeat domain domain for Wnt inhibitors TM : transmembrane domain ICD : intracellular domain (DKK1 and sclerostin) LDLR BP1 EGF1 BP2 EGF2 BP3 EGF3 BP4 EGF4 ICD

DKK1 DKK1 TM Axin sclerostin several Wnts several Wnts LRP5 G171V activation of high bone reduction of the affinity canonical mass between the BP1 and DKK1 pathway B Role of LRP5 affecting the bone formation system. duodenal theory (left) and osteoblast-lineage cell theory(right) serotonin

Htr1b LRP5 canonical pathway LRP5 CREB Inhibition of -catenin proliferation osteoblast serotonin osteoblast chromaffin cells bone bone duodenum

Fig. 2 LRP5, the receptor for the Wnt canonical pathway A. Structure of LRP5 and binding domain for Wnt inhibitors (DKK1 and Sclerostin) (modified from Reference 8) There is a four-repeat structure consisting of tyrosine (Try)-tryptophan (Trp)-threonine (Thr)-aspartic acid (Asp) repeat (β-propeller domain) and epidermal growth factor (EGF) domain. Moreover, the receptor has the Axin-binding domain associ- ated with protein interaction within the cell. B. Role of LRP5 affecting the bone formation system. Duodenal theory and osteoblast-lineage cell theory (modified from Refer- ence 17) When serotonin produced by chromaffin cells in the duodenum is transported through the bloodstream and binds to the sero- tonin receptors (Htr1b) expressed on , cyclic adenosine monophosphate response element-binding protein (CREB) is inhibited, resulting in inhibition of osteoblast proliferation. Signaling from LRP5 inhibits serotonin synthesis in chromaffin cells in the duodenum. As a result, osteoblast differentiation is enhanced. Meanwhile, LRP5 is also reported to activate the canonical pathway in osteoblast-lineage cells instead of the intestine to enhance osteoblast differentiation. has drawn close attention4). On the other hand, it has also specific LRP5, rather than intestine-specific LRP5, regu- been reported that families with LRP5 G171V, a gain-of- lates the bone mass in hard tissues. Thus, it is still contro- function mutation of LRP5, develop hyperostosis. When versial in which cells LRP5 regulating bone formation is the 171st glycine of LRP5 is replaced by valine, the expressed (Fig. 2B). Future studies are awaited. conformation of the BP1 domain is changed (Fig. 2A). It has been revealed that this change reduces the affinity Sclerostin and bone metabolism between the BP1 domain and DKK1, which enhances the Wnt canonical pathway, leading to the development of Osteocytes and Sclerostin. As described above, Scleros- hyperostosis. A group led by Dr. Karsenty17) reported that tin produced by osteocytes inhibits the Wnt canonical serotonin produced in the chromaffin cells of the duode- pathway and negatively regulates bone formation. Be- num inhibits osteoblast differentiation, and that serotonin cause mechanical loading is reported to inhibit the pro- synthesis is inhibited downstream of LRP5 in the chro- duction of Sclerostin in osteocytes and to enhance bone maffin cells, which enhances osteoblast differentiation. formation19), Sclerostin is considered to be involved in the On the other hand, a group led by Dr. Warman18) showed pathogenesis of disuse osteopenia. Furthermore, there is that while osteocyte-specific LRP5-deficient mice lose a possibility that Sclerostin exerts an effect on osteocytes bone mass, intestine-specific LRP5-deficient mice do and enhances osteoclast differentiation, and it is suggested not lose bone mass. These results indicate that osteocyte- that Sclerostin may positively regulate bone resorption20). 344 JPFSM: Maeda K, et al.

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