**New Aspect of Bone Morphogenetic Protein Signaling and Its Relationship with Wnt Signaling in Bone**

Nobuhiro Kamiya *Center for Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children, Dallas, Texas,* 

*USA* 

#### **1. Introduction**

282 Molecular Interactions

Okuda, H.; Ogura, K.; Kato, A.; Takubo, H. & Watanabe, T. (1998). A Possible Mechanism of

Rosse, C. & Mejino, JLV. (2003). A Reference Ontology for Biomedical Informatics: the

Sai, K.; Saeki, M.; Saito, Y.; Ozawa, S.; Katori, N.; Jinno, H.; Hasegawa, R.; Kaniwa, N.;

Slatter, JG.; Schaaf, LJ.; Sams, JP.; Feenstra, KL.; Johnson, MG.; Bombardt, PA.; Cathcart, KS;

Timizi,SH.; Aitken,S.; Moreira, DA.; Mungall, C.; Seueda, J.; Shah, NH. & Miranker, DP.

Tsukamoto, Y.; Kato Y; Ura. M.; Horii, I.; Ishitsuka, H.; Kusuhara, H. & Sugiyama, Y. (2001).

Tukey, RH.; Strassburg, CP. & Mackenzie, PI. (2002). Pharmacogenomics of Human UDP-

Vossen, M.; Sevestre, M.; Niederalt. C.; Jang, IJ.; Willmann. S. & Edginton, AN. (2007).

Whitehouse, LW.; Menzies, A.; Dawson, B.; Cyr, TD.; By AW.; Black, DB. & Zamecnik, J.

Willmann, S.; Hohn, K.; Edginton, A.; Sevestre, M.; Solodenko, J.; Weiss, W.; Lippert, J. &

Elucidation, *J Pharm Biomed Anal 1994*, Vol. 2, No.11, pp.1425-1441

*Drug Metabolism and Disposition*, Vol.28, No.4, pp.423-433

Biomedical Semantics 2011, Vol.2 (Suppl 1):S3

5-FU, *Pharm Res*, Vol.18, No.8, pp.1190-1202

Aug. 22nd,Epub ahead of print

Pharmacol, *Ther*, Vol. 75, pp.501-515

pp.478-500

No.3, pp.446-450

*2006*, pp.200-211

4, No. 13

Eighteen Patient Deaths Caused by Interactions of Sorivudine, a New Antiviral Drug, with Oral 5-fluorouracil Prodrugs. *J Pharmacol Exp Ther*, Vol. 287, No.2, pp.791-799 Pirmohamed, M. (2011). Pharmacogenetics: past, present and future, Drug Discovery Today,

Foundational Model of Anatomy, Journal of Biomedical Informatics, Vol.36,

Sawada, J.; Komamura, K., Ueno, K.; Kamakura, S.; Kitakaze, M., Kitamura, Y.; Kamatani, N.; Minami, H; Ohtsu, A; Shirao, K.; Yoshida, T. & Saijo, N. (2004). UGT1A1 Haplotypes Associated with Reduced Glucuronidation and Increased Serum Bilirubin in Irinotecanadministered Japanese Patients with Cancer. Clin

Verburg, MT.; Pearson, LK.; Compton, LD.; Miller, LL.; Baker, DS.; Pesheck, CV.; Raymond, S. & Lord, I. (2000). Pharmacokinetics, Metabolism, and Excretion of Irinotecan (CPT-11) Following I.V. Infusion of [14C]CPT-11 in Cancer Patients,

(2011). Mapping between the OBO and OWL Ontology Languages, Journal of

A Physiologically based Pharmacokinetic Analysis of Capecitabine, a Triple Prodrug of 5-FU, in Humans, the Mechanism for Tumor-selective Accumulation of

Glucuronosyltransferases and Irinotecan Toxicity, *Molecular Pharmacology*, Vol.62,

Dynamically Simulating the Interaction of Midazolam and the CYP3A4 Inhibitor Itraconazole using Individual Coupled Whole-Body Physiologically-based Pharmacokinetic (WBPBPK) Models. *Theoretical Biology and Medical Modelling*, Vol.

(1994). Mouse Hepatic Metabolites of Ketoconazole: Isolation and Structure

Schmitt ,W. (2007). Development of a Physiology-based Whole-Body Population Model for Assessing the Influence of Individual Variability on the Pharmacokinetics of Drugs, *Pharmacokinet Pharmacodyn*, Vol. 34, No.3, pp.401-431 Yoshikawa, S.; Kenji, S. & Konagaya, A. (2004). Drug Interaction Ontology (DIO) for Inferences of Possible Drug-Drug Interactions, *Medinfo*, Vol.11, pp.454-458 Zhang, S.; Bodenreider, O. & Golbreich, C. (2006). Experiences in Reasoning with the

Foundational Model of Anatomy in OWL DL, *Pacific Symposium on Biocomputing* 

Bone morphogenetic proteins (BMPs) were discovered and named in 1965 by Marshall Urist, who initially identified the ability of an unknown factor in bone to induce ectopic bones in muscle 1. In the last 45 years, the osteogenic function of BMPs has been extensively examined, mainly using osteoblasts in culture with exogenous treatments of BMPs 2. Based on their potent osteogenic abilities, clinical trials have been initiated to use BMP2 and BMP7 to improve fracture repair 2. The FDA (Food and Drug Administration) has approved BMP2 and BMP7 for clinical use in long bone open-fractures, non-union fractures and spinal fusion. However, recent clinical/pre-clinical studies have shown a negative impact of BMPs on bone formation under certain physiological conditions 3-7, challenging the current dogma. This book chapter will focus on the recent findings of roles of BMP signaling in bone including its relationship with Wnt signaling through Wnt (Wngless, Int-1) receptor SOST (Sclerostin) and DKK1 (Dickkopf1). This new molecular interaction would explain the negative outcomes of BMP's therapy in orthopaedics.

#### **2. Signaling by BMPs**

Marshall Urist made the key discovery that demineralized bone matrix induced bone formation in 1965 1. It took another 24 years for BMPs to be discovered. The combined works of several researchers led to the isolation of BMPs and later the cloning 8-11. BMPs belong to the transforming growth factor- (TGF-) gene superfamily 12. Like other members of the TGF- family, BMPs signal through transmembrane serine/threonine kinase receptors such as BMP type I and type II receptors. Upon ligand binding, type I and II receptors form hetero-multimers 13, and the type II receptor phosphorylates and activates a highly conserved glycine- and serine-rich domain (TTSGSGSG) called a GS box between the transmembrane and kinase domains in the type I receptor. The activated BMP type I receptors relay the signal to the cytoplasm through the Smad (Sma and Mad related protein) pathway by phosphorylating their immediate downstream targets, receptor-regulated Smads (R-Smads; Smad1, Smad5, and Smad8) proteins, which then interact with co-Smad (Smad4) protein and translocate into the nucleus 14. It is also known that non-Smad pathways through p38 MAPK (mitogen-actiated protein kinase) and TAK1 (Transforming growth factor β–activated kinase 1) are also involved in the BMP signaling 15. There are three type I receptors [BMPRIA (BMP receptor type IA, ALK3), BMPRIB (BMP receptor type IB, ALK6) and ACVRI (Activin receptor type I, ALK2) and three type II receptors [BMPRII (BMP receptor type II), ACVRIIA (Activin receptor type IIA) and ACVRIIB (Activin receptor type IIB)], and approximately 30 ligands are identified 16. Type I receptor ACVRI was originally described as an activin receptor, but it is now believed to be a receptor for BMPs. In osteoblasts, BMP2, BMP4, BMP6 and BMP7 and their receptors BMPRIA and ACVRI are abundantly expressed 17. BMPRIA is a potent receptor of BMP2 and BMP4 18, 19, as is ACVRI for BMP7 20. In addition, BMP antagonists Noggin, Chordin, and Gremlin were identified in osteoblasts 21. These antagonists fine-tune BMP signaling in osteoblasts, as BMPs upregulate expression levels of antagonists while inducing BMP signaling 22 **(Table 1, Figure 1)**.

Fig. 1. Potential molecular interaction of BMP signaling in osteoblasts. BMP2, BMP4, BMP6, and BMP7 are osteoinductive and are expressed by osteoblasts. BMP2 and BMP4 are potent ligands for BMPRIA as are BMP6 and BMP7 for ACVRI. Canonical BMP signaling is through the Smad pathway via Smad1, Smad5, and Smad8 (i.e. Smad1/5/8-Smad4 complex), while non-canonical BMP signaling is through non-Smad pathways including TAK1 and p38 MAPK. Target genes are activated by these two pathways in osteoblasts.

pathways through p38 MAPK (mitogen-actiated protein kinase) and TAK1 (Transforming growth factor β–activated kinase 1) are also involved in the BMP signaling 15. There are three type I receptors [BMPRIA (BMP receptor type IA, ALK3), BMPRIB (BMP receptor type IB, ALK6) and ACVRI (Activin receptor type I, ALK2) and three type II receptors [BMPRII (BMP receptor type II), ACVRIIA (Activin receptor type IIA) and ACVRIIB (Activin receptor type IIB)], and approximately 30 ligands are identified 16. Type I receptor ACVRI was originally described as an activin receptor, but it is now believed to be a receptor for BMPs. In osteoblasts, BMP2, BMP4, BMP6 and BMP7 and their receptors BMPRIA and ACVRI are abundantly expressed 17. BMPRIA is a potent receptor of BMP2 and BMP4 18, 19, as is ACVRI for BMP7 20. In addition, BMP antagonists Noggin, Chordin, and Gremlin were identified in osteoblasts 21. These antagonists fine-tune BMP signaling in osteoblasts, as BMPs upregulate

expression levels of antagonists while inducing BMP signaling 22 **(Table 1, Figure 1)**.

Noggin Chordin Gremlin

Type II BMPRIA Type II ACVRI

Smad1/5/8 Smad1/5/8

Smad4

Smad-pathway

Non-Smad-pathway

TAK1 p38 MAPK P P

BMP6 BMP7

P

Smad complex *nucleus*

Smad6/7

*cytoplasm*

Target gene activation

*extracellular*

BMP2 BMP4

Fig. 1. Potential molecular interaction of BMP signaling in osteoblasts. BMP2, BMP4, BMP6, and BMP7 are osteoinductive and are expressed by osteoblasts. BMP2 and BMP4 are potent ligands for BMPRIA as are BMP6 and BMP7 for ACVRI. Canonical BMP signaling is through the Smad pathway via Smad1, Smad5, and Smad8 (i.e. Smad1/5/8-Smad4 complex), while non-canonical BMP signaling is through non-Smad pathways including TAK1 and p38 MAPK. Target genes are activated by these two pathways in osteoblasts.


Table 1. Osteogenic BMPs and their signaling cascades in osteoblasts

#### **3. Molecular interaction of BMP and Wnt**

In addition to BMP signaling, Wnt signaling has been examined for a decade because of its role in bone formation and bone mass 23-27. The physiological impact of Wnt signaling on bone mass was first reported in 2001, by showing that loss-of-function mutations in the coreceptor LRP5 (Low-density lipoprotein receptor-related protein 5) cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG), a low bone mass phenotype in humans 28. The importance of other Wnt ligands and receptors as bone mass effectors has been documented using genetic approaches for DKK1 29, DKK2 30, sFRPs (secreted frizzled-related proteins) 31, Sost/sclerostin 32, Lrp5 33, 34 and Lrp6, all of which are expressed in osteoblasts. However, changes in BMP signaling in bone had not been reported in Wnt-related mutations in mice.

#### **3.1 In vitro relationship**

*In vitro* experiments using pluripotent mesenchymal cell lines or primary osteoblasts to test the interaction between BMP and Wnt signaling in osteoblasts have yielded both synergistic and antagonistic results: the treatment of C2C12 cells and primary osteoblasts with BMP2 induced Wnt3a expression and stabilized Wnt/β-catenin signaling 35-37. The treatment of C3H10T1/2 cells with Wnt3a induced the BMP4 expression levels 38. These suggest a positive autocrine loop 37, 39. In contrast, inhibition of BMP signaling by treatment of primary osteoblasts with dorsomorphin, an inhibitor of BMP type I receptors, increased canonical Wnt signaling 40. Treatment of C2C12 cells with Wnt3a repressed BMP2-dependent *Id1* (Inhibitor of DNA binding 1) expression 41. Similarly, treatment of cultured skull bone with BMP antagonist Noggin increased canonical Wnt signaling 42. Moreover, one study investigated intracellular cross-talk between BMP and Wnt pathways using uncommitted bone marrow stromal cells and provided a potential mechanism whereby BMP-2 antagonizes Wnt3a-induced proliferation in osteoblast progenitors by promoting an interaction between Smad1 and Dvl-1 [i.e. the human homolog of the Drosophila dishevelled gene (dsh) 1] that restricts Wnt/β-catenin activation43. Another interaction via Pten (phosphatase and tensin homolog)-Akt pathway has been reported in hair follicle stem/progenitor cells 44; however, it is less likely in osteoblasts 45. Taken together, there seems to be both positive and negative feedback loops between the two signaling pathways **(Figure 2)**.

1) Positive loop 2) Negative loop

Fig. 2. A potential relationship between the two major signaling BMP and Wnt in osteoblasts based on in vitro studies. 1) Both signaling pathways function in a positive loop. 2) Both signaling pathways function in a negative loop. It is expected that these two signaling pathways may regulate each other in an age-dependent and context-dependent manner. Further studies are desired to investigate the details of each condition.

#### **3.2 In vivo relationship**

*In vivo*, only a few studies have revealed a link between the two signaling pathways. We recently found that loss-of-function of BMP signaling in osteoblasts via BMPRIA upregulates canonical Wnt signaling during embryonic and postnatal bone development, suggesting a negative regulation of Wnt signaling by BMP 40, 42. In these studies, we found that upregulation of Wnt signaling is at least in part mediated by suppression of Wnt inhibitors Sost/sclerostin and Dkk1, and both Sost/sclerostin and Dkk1 are direct targets of BMP signaling. In addition, *Sost* expression was severely downregulated in *Bmpr1a*-deficient bones as assessed by microarray analysis 42. Interestingly, both Smad-dependent and Smadindependent pathways appear to contribute to the Dkk1 expression, whereas Sost/sclerostin requires only Smad-dependent signaling, suggesting differential regulation of these genes by the BMP signaling via BMPRIA 40. BMP and Wnt signaling regulate the development and remodeling of many tissues and interact synergistically or antagonistically in a context- and age-dependent manner *in vivo* 46, 47. It is possible that in bone, BMP signaling inhibits Wnt signaling by upregulating the Sost/sclerostin expression in osteoblasts **(Figure 3)**.

#### **3.3 SOST/Sclerostin and DKK1**

Both SOST and DKK1 are inhibitors for canonical Wnt signaling and have been highlighted because neutralizing antibodies for SOST (AMG785) and DKK1 (BHQ880) have been developed as bone anabolic agents and these potential drugs are under clinical trial 48. It is known that both Dkk1 and Sost/sclerostin inhibit Wnt/β-catenin signaling by binding to coreceptors. As both Dkk1 and Sost/sclerostin are secreted proteins expressed by osteoblasts, their role in regulating bone mass has been investigated using human and mouse genetic approaches.

BMP signal

Wnt signal

Smad1/5/8

Fig. 2. A potential relationship between the two major signaling BMP and Wnt in osteoblasts based on in vitro studies. 1) Both signaling pathways function in a positive loop. 2) Both signaling pathways function in a negative loop. It is expected that these two signaling pathways may regulate each other in an age-dependent and context-dependent manner.

*In vivo*, only a few studies have revealed a link between the two signaling pathways. We recently found that loss-of-function of BMP signaling in osteoblasts via BMPRIA upregulates canonical Wnt signaling during embryonic and postnatal bone development, suggesting a negative regulation of Wnt signaling by BMP 40, 42. In these studies, we found that upregulation of Wnt signaling is at least in part mediated by suppression of Wnt inhibitors Sost/sclerostin and Dkk1, and both Sost/sclerostin and Dkk1 are direct targets of BMP signaling. In addition, *Sost* expression was severely downregulated in *Bmpr1a*-deficient bones as assessed by microarray analysis 42. Interestingly, both Smad-dependent and Smadindependent pathways appear to contribute to the Dkk1 expression, whereas Sost/sclerostin requires only Smad-dependent signaling, suggesting differential regulation of these genes by the BMP signaling via BMPRIA 40. BMP and Wnt signaling regulate the development and remodeling of many tissues and interact synergistically or antagonistically in a context- and age-dependent manner *in vivo* 46, 47. It is possible that in bone, BMP signaling inhibits Wnt

signaling by upregulating the Sost/sclerostin expression in osteoblasts **(Figure 3)**.

Both SOST and DKK1 are inhibitors for canonical Wnt signaling and have been highlighted because neutralizing antibodies for SOST (AMG785) and DKK1 (BHQ880) have been developed as bone anabolic agents and these potential drugs are under clinical trial 48. It is known that both Dkk1 and Sost/sclerostin inhibit Wnt/β-catenin signaling by binding to coreceptors. As both Dkk1 and Sost/sclerostin are secreted proteins expressed by osteoblasts, their role in regulating bone mass has been investigated using human and mouse genetic

Further studies are desired to investigate the details of each condition.

BMP signal

1) Positive loop 2) Negative loop

Wnt signal

**3.2 In vivo relationship** 

**3.3 SOST/Sclerostin and DKK1** 

approaches.

Fig. 3. Possible regulation between BMP and Wnt in Osteoblasts. A proposed model of the relationship between the BMP signaling via BMPRIA and the canonical Wnt signaling in osteoblasts. Both Dkk1 and sclerostin/Sost are downstream targets of the BMP signaling. The BMP signaling upregulates the Sost expression primarily through the Smad-dependent signaling while it upregulates the Dkk1 expression through both the Smad and non-Smad signaling (p38 MAPK). As Dkk1 and sclerostin/Sost act as Wnt signaling inhibitors, BMP signaling in osteoblasts, in turn, leads to a decrease in osteogenesis and bone mass. Dkk1 and sclerostin/Sost play an important role in regulating bone mass as downstream effectors of BMPRIA signaling in bone taking balances between BMP signaling and Wnt signaling.

#### **3.3.1 SOST/Sclerostin**

Sost/sclerostin was originally reported as a member of the BMP antagonist DAN family (i.e. the Dan gene family of BMP antagonists) 49, 50. Although DAN family members modulate both BMP and Wnt signaling in Xenopus 51-53, recent studies suggest a primary role of Sost/sclerostin in Wnt signaling in mouse and humans: Sost/sclerostin is not a BMP antagonist 54 but rather a Wnt inhibitor 55 that binds the Wnt co-receptor low density lipoprotein receptor-related protein 5 and 6 (LRP5 and LRP6) 32, 56. Conventional knockouts of *Sost* (i.e. *Sost* KO) are viable and exhibit increased bone mass 57. In humans, loss-offunction and hypomorphic mutations in *SOST* cause sclerosteosis 58, 59 and Van Buchem disease 60, 61, respectively, with a high bone mass (HBM) phenotype. These mutants share the HBM phenotype with other gain-of-function of LRP5 mutation, due to the defect in DKK1 mediated regulation of LRP5 in humans 62-64 and overexpression of Lrp5 in mice 65. In contrast, loss-of-function of LRP5 leads to OPPG with low bone mass 28, which is similar to the bone phenotype of mice overexpressing *Sost* 50. In addition, recent genome-wide SNPbased analyses identified a significant association between bone mineral density and the *SOST* gene locus 66-68.

#### **3.3.2 DKK1**

Conventional knockouts of *Dkk1* die *in utero* from defective head induction and limb formation 29. Similar to *Sost* KO mice, mice heterozygous for *Dkk1* (*Dkk1*+/– mice), however, exhibit a high bone mass (HBM) phenotype 69, while overexpression of *Dkk1* in osteoblasts causes osteopenia 70. In addition, increased *DKK1* expression in bone marrow has also been associated with lytic bone lesions in patients with multiple myeloma 71. Collectively, these results support the hypothesis that Dkk1 functions as a potent negative regulator of bone mass.

#### **3.3.3 Sost/DKK1 expression in the** *Bmpr1a* **cKO mice**

Conditional knockouts of *Bmpr1a*, which are deficient in the *Dkk1* and *Sost* expression, show a HBM phenotype 40, 42, 72. In particular, Sost expression levels were the most dramatically reduced in the cKO mice during embryonic stages 42. Furthermore, both Sost and Dkk1 expression levels were increased by the addition of BMP2, a potent ligand for BMPRIA, using primary osteoblasts 40. Similarly, both Sost and Dkk1 expression levels were significantly reduced in the *Acvr1* cKO mice 73. In addition, both Sost and Dkk1 expression levels were increased by the addition of BMP7, a potent ligand for ACVRI, using primary osteoblasts 73. These facts support the new concept of molecular interactions between BMP signaling and Wnt signaling that Dkk1 and Sost/sclerostin act physiologically as inhibitors of canonical Wnt signaling as downstream targets of BMP receptors BMPRIA and ACVRI and that BMP signaling can negatively controls Wnt signaling in osteoblasts **(Figure 3)**.

#### **3.4 Effects of Wnt signaling on osteoclasts**

There is accumulating evidence that Wnt signaling also plays a critical role in osteoclastogenesis regulated by osteoblasts through the RANKL (Receptor activator of nuclear factor kappa-B ligand)-OPG (Osteoprotegerin) pathway. Recently, two *in vivo* studies have suggested that the canonical Wnt signaling is important in the regulation of osteoclastogenesis by osteoblasts. One study provided evidence that the Wnt pathway positively regulates the expression of *Opg* in osteoblasts 74. Overexpression of stabilized catenin in osteoblasts, which results in an increase of canonical Wnt signaling level, decreases osteoclast differentiation leading to increased bone volume in mice 74. Another study showed that an osteoblast-specific deletion of -catenin leads to an impaired maturation and mineralization of bones in mice due to the elevated expression of *Rankl* and diminished *Opg* 75. These facts suggest that the canonical Wnt pathway negatively regulates osteoblasts in their supporting function in osteoclastogenesis, and thus upregulation of Wnt signaling in osteoblasts can suppress osteoclast-mediated bone resorption 75. Taken together, it is possible that the treatment of bones with BMPs can reduce Wnt activity in osteoblasts and in turn enhance osteoclast activity.

#### **4. BMP signaling and mouse genetics**

288 Molecular Interactions

contrast, loss-of-function of LRP5 leads to OPPG with low bone mass 28, which is similar to the bone phenotype of mice overexpressing *Sost* 50. In addition, recent genome-wide SNPbased analyses identified a significant association between bone mineral density and the

Conventional knockouts of *Dkk1* die *in utero* from defective head induction and limb formation 29. Similar to *Sost* KO mice, mice heterozygous for *Dkk1* (*Dkk1*+/– mice), however, exhibit a high bone mass (HBM) phenotype 69, while overexpression of *Dkk1* in osteoblasts causes osteopenia 70. In addition, increased *DKK1* expression in bone marrow has also been associated with lytic bone lesions in patients with multiple myeloma 71. Collectively, these results support the hypothesis that Dkk1 functions as a potent negative regulator of bone

Conditional knockouts of *Bmpr1a*, which are deficient in the *Dkk1* and *Sost* expression, show a HBM phenotype 40, 42, 72. In particular, Sost expression levels were the most dramatically reduced in the cKO mice during embryonic stages 42. Furthermore, both Sost and Dkk1 expression levels were increased by the addition of BMP2, a potent ligand for BMPRIA, using primary osteoblasts 40. Similarly, both Sost and Dkk1 expression levels were significantly reduced in the *Acvr1* cKO mice 73. In addition, both Sost and Dkk1 expression levels were increased by the addition of BMP7, a potent ligand for ACVRI, using primary osteoblasts 73. These facts support the new concept of molecular interactions between BMP signaling and Wnt signaling that Dkk1 and Sost/sclerostin act physiologically as inhibitors of canonical Wnt signaling as downstream targets of BMP receptors BMPRIA and ACVRI and that BMP signaling can negatively controls Wnt

There is accumulating evidence that Wnt signaling also plays a critical role in osteoclastogenesis regulated by osteoblasts through the RANKL (Receptor activator of nuclear factor kappa-B ligand)-OPG (Osteoprotegerin) pathway. Recently, two *in vivo* studies have suggested that the canonical Wnt signaling is important in the regulation of osteoclastogenesis by osteoblasts. One study provided evidence that the Wnt pathway positively regulates the expression of *Opg* in osteoblasts 74. Overexpression of stabilized catenin in osteoblasts, which results in an increase of canonical Wnt signaling level, decreases osteoclast differentiation leading to increased bone volume in mice 74. Another study showed that an osteoblast-specific deletion of -catenin leads to an impaired maturation and mineralization of bones in mice due to the elevated expression of *Rankl* and diminished *Opg* 75. These facts suggest that the canonical Wnt pathway negatively regulates osteoblasts in their supporting function in osteoclastogenesis, and thus upregulation of Wnt signaling in osteoblasts can suppress osteoclast-mediated bone resorption 75. Taken together, it is possible that the treatment of bones with BMPs can reduce Wnt activity in osteoblasts

**3.3.3 Sost/DKK1 expression in the** *Bmpr1a* **cKO mice** 

signaling in osteoblasts **(Figure 3)**.

and in turn enhance osteoclast activity.

**3.4 Effects of Wnt signaling on osteoclasts** 

*SOST* gene locus 66-68.

**3.3.2 DKK1** 

mass.

Along with the huge advancement in technologies involving mouse genetics over the last decade, many of the BMP signaling related genes have been knocked out in mice. BMP2, BMP4, BMP6 and BMP7 and their receptor BMPRIA and ACVRI are abundantly expressed in bone. However, conventional knockout mice for these genes result in an early embryonic lethality and thus, it is not possible to investigate bone development and remodeling using these models 76-82. To avoid the embryonic lethality, a strategy of conditional knockout mice using a Cre-loxP system has been employed. A bone-specific conditional deletion of *Bmpr1a* using an *Og2-Cre* mouse, in which a Cre recombination is restricted in differentiated osteoblasts under the osteocalcin promoter, was first reported in 2004 83. Interestingly, this study demonstrated that the response of osteoblasts to BMP signaling is age-dependent; in the mutant mice, bone volume decreased in young mice but increased in aged mice. In addition, the activity of osteoclasts was reduced in the aged osteoblast-specific *Bmpr1a*deficient mice, which may have lead to the complex skeletal phenotype. These facts suggest that the BMP signaling in differentiated osteoblasts can control the balance between bone formation by osteoblasts and resorption by osteoclasts, thereby affecting the final outcome of the amount of bone mass in an age-dependent manner. The increased bone mass in the *Bmpr1a*-deficient mice appeared to be in opposition to the general concept of BMPs as osteogenic inducers; however, the concept is reasonable if the target cell for BMPs as osteogenic inducers is mesenchymal cells or chondrocytes,. It is expected that BMPs have multifaceted functions *in vivo* because different cell types exhibit differing responses to BMPs. In addition, the opposite outcome in the *Bmpr1a*-deficient mice was discussed from the point of molecular interaction in the sections 3.

#### **4.1 BMP signaling in chondrocytes, mesenchymal cells, and osteoblasts**

During skeletogenesis, bones are formed via two distinct processes: intramembranous and endochondral bone formation 84. Intramembranous bone formation occurs primarily in flat bones (*e.g.*, calvarial bones) where mesenchymal cells differentiate directly into osteoblasts 85. Endochondral bone formation occurs primarily in long bones where condensed mesenchymal cells differentiate into chondrocytes to form cartilage templates, and then chondrocytes are replaced by osteoblasts 86. Recently many studies have been designed to investigate the difference in the molecular mechanism by which BMP signaling regulates these cell types. Several Cre mouse lines have been used to target different cell types including osteoblast, chondrocyte, and mesenchymal cells **(Table 2).** BMP signaling in chondrocytes and mesenchymal cells both positively control bone size and mass while BMP signaling in osteoblasts can reduce them.

#### **4.1.1 Chondrocytes**

There are several lines of evidence that show that BMP signaling in chondrocytes is required for bone size and the amount of bone mass. BMP signaling through BMPRIA is essential for postnatal maintenance of articular cartilage, using a *Gdf5*-Cre mouse line specific for chondrocytes in joints 87. Similarly, the critical role of *Bmpr1a* together with *Bmpr1b* in chondrocytes during endochondral bone formation using a *Col2*-Cre mouse line was reported.88. Moreover, in chondrocytes a simultaneous deficiency in Smad 1 and Smad 5,


Table 2. Bone mass observed in genetically engineered mutant mice of BMP signaling

which are BMPs' downstream target molecules, reduces bone mass 90. In parallel, studies focusing on BMP ligands and their antagonists provide further evidence that BMPs are critical for normal development of cartilage. A transgenic mouse line to overexpress *Bmp4* in mesenchymal cells/chondrocytes using a type XI collagen promoter (Col11a2) was generated, and bone mass was increased in the mutant mice 89. Another transgenic mouse line in which *Noggin* was overexpressed in the same cells (*Col11a2*-*Noggin*) demonstrated a decreased bone mass. As Noggin is an antagonist for BMPs (BMP2, BMP4, BMP5, BMP6, and BMP7) with various degrees of affinity 95, these results suggest that BMP signaling positively controls proliferation and differentiation of chondrocytes.

#### **4.1.2 Mesenchymal cells**

290 Molecular Interactions

*Bmpr1a* and *Bmpr1b* Col2-Cre down E12.5-E16.5 Reduced 88 *Bmp4* overexpression Col11a2 up E18.5 Increased 89 *Noggin* overexpression Col11a2 down E18.5 Reduced 89

Prx1-Cre down E10.5-

*Bmp2* cKO Prx1-Cre down 5M Reduced 92

*BMP signal Stage Bone mass Ref.* 

7W, 9 Reduced 87

newborn Reduced 90

newborn, 3W Reduced 91

5M Increased

5M Increased 73

Reduced Increased

83

40, 42, 72

10M

Col1 up E18.5 Reduced 93

Col1 down E17.5, 3W Increased 93

down E18.5, 3W,

down E18.5, 3W,

*Bmpr1a* cKO Ctsk-Cre down 8W Increased 94

which are BMPs' downstream target molecules, reduces bone mass 90. In parallel, studies focusing on BMP ligands and their antagonists provide further evidence that BMPs are critical for normal development of cartilage. A transgenic mouse line to overexpress *Bmp4* in mesenchymal cells/chondrocytes using a type XI collagen promoter (Col11a2) was generated, and bone mass was increased in the mutant mice 89. Another transgenic mouse line in which *Noggin* was overexpressed in the same cells (*Col11a2*-*Noggin*) demonstrated a decreased bone mass. As Noggin is an antagonist for BMPs (BMP2, BMP4, BMP5, BMP6, and BMP7) with various degrees of affinity 95, these results suggest that BMP signaling

Table 2. Bone mass observed in genetically engineered mutant mice of BMP signaling

*Promoter Cremouse* 

*Bmpr1a* cKO Gdf5-Cre down E12.5-E16.5,

*Smad1* and *Smad5* Col2-Cre down E12.5-

*Bmpr1a* cKO Ogl2-Cre down 3M

3.2 kb Col1- CreER

3.2 kb Col1- CreER

positively controls proliferation and differentiation of chondrocytes.

*Chondrocyte*

Double knockout of

Double knockout of

Double knockout of *BMP2* and *BMP4*

*Bmpr1a* cKO

*Acvr1* cKO

*Bmp4* overexpression 2.3 kb

*Noggin* overexpression 2.3 kb

*Mesenchymal cell*

*Osteoblast*

*Osteoclast*

Similar to chondrocytes, a few studies demonstrated a requirement of BMP signaling in mesenchymal cells for proper bone development and remodeling using a mesenchymal cellspecific Cre mouse line, *Prx1-Cre*, in which Cre is active in mesenchymal cells as early as embryonic day 9.5 96. Using the *Prx1-Cre* mouse, the simultaneously conditional deletions of *Bmp2* and *Bmp4* in mesenchymal cells resulted in an impairment of osteogenesis during late embryogenesis 91, 92. In contrast, the conditional deletion of *Bmp2* in mesenchymal cells does not show overt developmental abnormalities; however, the resulted mice lack an initiation of fracture healing 91, 92. Interestingly, *Bmp7*-deficiency in mesenchymal cells did not affect bone mass probably due to the compensation by Bmp4 97. Taken together, it is possible that the defects in the BMP signaling in chondrocytes largely contribute to the phenotypes described above because chondrocytes are derived from mesenchymal cells and play an important role in the process of fracture repair.

#### **4.1.3 Osteoblasts**

As aforementioned, a differentiated osteoblast-specific deletion of *Bmpr1a* caused an increase in bone mass in aged mice 83. Similar to this finding, an overexpression of a BMP antagonist, Noggin, in osteoblasts increases bone volume with a reduced osteoclast number and osteoclastogenesis both at embryonic day 17.5 (E17.5) and at 3 weeks 93. In parallel, the overexpression of *Bmp4* in osteoblasts reduced bone mass presumably due to the increase in the osteoclast number at E18.5 93. Recently, *Bmpr1a* was conditionally disrupted in immature osteoblasts using a tamoxifen inducible Cre driven by a 3.2-kb alpha1(I) collagen chain gene (Col1a1) promoter. In the mutant mice, bone mass was dramatically increased during the

Fig. 4. Increased bone mass in the osteoblast-specific conditional knockout (cKO) mice for BMP receptors BMPRIA or ACVRI at the adult stage. *Bmpr1a* or *Acvr1* cKO mice were generated by crossing a floxed mouse line for *Bmpr1a*(*Bmpr1a*fx/fx) or *Acvr1*(*Acvr1*fx/fx) with a transgenic mouse line harboring a tamoxifen–inducible Cre driven by a 3.2 kb mouse procollagen 1(I) promoter. The Cre recombination was induced specifically in the osteoblasts by 10 weeks of tamoxifen administration from 10 weeks after birth, and bones were removed at 22 weeks. Radiodensity of rib bones was assessed by X-ray. (A) The radiodensity was dramatically increased in the *Bmpr1a* cKO mice (Cre+, *Bmpr1a*fx/fx) compared with controls (Cre–, *Bmpr1a*fx/fx). (B) The radiodensity was dramatically increased in the *Acvr1* cKO mice (Cre+, *Acvr1*fx/fx) compared with controls (Cre–, *Bmpr1a*fx/fx).

bone remodeling stage at 22 weeks as well as the bone developmental stages at E18.5 and 3 weeks 42, 72 **(Figure 4A).** This result is an interesting contrast to previous work that disruption of *Bmpr1a* in differentiated osteoblasts results in decrease of bone mass in young adult stages (3-4 weeks). The increased bone mass in the *Bmpr1a*-deficient mice resulted from severely suppressed bone resorption due to reduced osteoclastogenesis, despite a simultaneous small reduction in the rate of bone formation 72. Levels of RANKL and OPG are changed in the *Bmpr1a*-deficient osteoblasts and fail to support osteoclastogenesis 42, 72. In addition, the conditional disruption of *Acvr1* in osteoblasts also demonstrated a dramatic increase in bone mass, similar to the bone phenotype of *Bmpr1a*-deficient mice **(Figure 4B)**, although osteoclastic activity is still under investigation 73. These findings suggest that BMP signaling may have dual roles in osteoblasts; to stimulate both bone formation by osteoblasts and bone resorption supporting osteoclastogenesis. Disruption of BMP signaling in immature osteoblasts alters the balance of bone turn over to increase the bone mass, which is opposite to what people have expected for the past 4 decades.

#### **4.1.4 Other cell type**

Angiogenesis is another necessary step in new bone formation in skeletal development as well as in bone remodeling after fracture 98, 99. Both BMP2 and BMP7 are known to induce angiogenesis by associating with other growth factors such as VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), and TGF-1 100. A study using an adenovirus vector in muscle demonstrated that BMP9 induces ectopic bone formation similar to BMP2 101, 102. As BMP9 is abundantly expressed in endothelial cells that are primarily cell types for angiogenesis 103, it is possible that BMP signaling in endothelial cells synergizes anabolic bone formation. The mechanism and origin of precursor cells for ectopic bone formation, which is physiologically observed in the patients with FOP (fibrodysplasia ossificans progressiva), is under investigation 104-106 but could be endothelial cells 107.

#### **4.1.5 Possible interpretation**

Mesenchymal cells, chondrocytes, and endothelial cells respond to BMPs by inducing bone mass and size **(Table 3)**. Recent histological findings suggest that the process of endochondral bone formation, which first forms cartilage template prior to the final bone following vessel formation (i.e. angiogenesis), plays a critical role in the process of ectopic bone formation 108. The origin of precursor cells for the ectopic bone is under investigation 105, 106; however, it is possible that formation of ectopic bones by BMPs 1 is largely due to the stimulation of chondrocytes, mesenchymal cells, and/or endothelial cells in soft tissue, which results in an expansion of ectopic cartilage subsequently replaced by osteoblasts. There is another possibility that the BMP signaling directly affects osteoblasts to form ectopic bone. However, this possibility is less likely based on recent evidence that reduced


Table 3. A variety of cell types in bone that mediate bone mass in response to BMPs

bone remodeling stage at 22 weeks as well as the bone developmental stages at E18.5 and 3 weeks 42, 72 **(Figure 4A).** This result is an interesting contrast to previous work that disruption of *Bmpr1a* in differentiated osteoblasts results in decrease of bone mass in young adult stages (3-4 weeks). The increased bone mass in the *Bmpr1a*-deficient mice resulted from severely suppressed bone resorption due to reduced osteoclastogenesis, despite a simultaneous small reduction in the rate of bone formation 72. Levels of RANKL and OPG are changed in the *Bmpr1a*-deficient osteoblasts and fail to support osteoclastogenesis 42, 72. In addition, the conditional disruption of *Acvr1* in osteoblasts also demonstrated a dramatic increase in bone mass, similar to the bone phenotype of *Bmpr1a*-deficient mice **(Figure 4B)**, although osteoclastic activity is still under investigation 73. These findings suggest that BMP signaling may have dual roles in osteoblasts; to stimulate both bone formation by osteoblasts and bone resorption supporting osteoclastogenesis. Disruption of BMP signaling in immature osteoblasts alters the balance of bone turn over to increase the bone mass,

Angiogenesis is another necessary step in new bone formation in skeletal development as well as in bone remodeling after fracture 98, 99. Both BMP2 and BMP7 are known to induce angiogenesis by associating with other growth factors such as VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), and TGF-1 100. A study using an adenovirus vector in muscle demonstrated that BMP9 induces ectopic bone formation similar to BMP2 101, 102. As BMP9 is abundantly expressed in endothelial cells that are primarily cell types for angiogenesis 103, it is possible that BMP signaling in endothelial cells synergizes anabolic bone formation. The mechanism and origin of precursor cells for ectopic bone formation, which is physiologically observed in the patients with FOP (fibrodysplasia

ossificans progressiva), is under investigation 104-106 but could be endothelial cells 107.

Mesenchymal cells, chondrocytes, and endothelial cells respond to BMPs by inducing bone mass and size **(Table 3)**. Recent histological findings suggest that the process of endochondral bone formation, which first forms cartilage template prior to the final bone following vessel formation (i.e. angiogenesis), plays a critical role in the process of ectopic bone formation 108. The origin of precursor cells for the ectopic bone is under investigation 105, 106; however, it is possible that formation of ectopic bones by BMPs 1 is largely due to the stimulation of chondrocytes, mesenchymal cells, and/or endothelial cells in soft tissue, which results in an expansion of ectopic cartilage subsequently replaced by osteoblasts. There is another possibility that the BMP signaling directly affects osteoblasts to form ectopic bone. However, this possibility is less likely based on recent evidence that reduced

**Cell types that can increase bone mass Cell types that can reduce bone mass**  Mesenchymal cells Osteoclasts Chondrocytes Osteoblasts

Table 3. A variety of cell types in bone that mediate bone mass in response to BMPs

which is opposite to what people have expected for the past 4 decades.

**4.1.4 Other cell type** 

**4.1.5 Possible interpretation** 

Osteoblasts Endothelial cells BMP signaling in osteoblasts results in an increase in bone mass. As current methods of systemic and local treatment affect multiple cell types simultaneously in bone, it is important to evaluate the effects of BMPs on more than just osteoblasts.

#### **4.2 Effect of BMP signaling on osteoclasts**

Bone mass is determined by the balance between bone formation and bone resorption. Osteoclasts are multinuclear cells derived from hematopoietic stem cells to secrete enzymes for bone resorption 109. Recent mouse genetic studies revealed the importance of BMP signaling for osteoclastic activity and bone resorption.

#### **4.2.1 Regulation of osteoclast by osteoblast-dependent BMP signaling**

It is expected that BMPs play roles in osteoclastogenesis and their functions, because receptors for BMPs are expressed in these cells 110. Additionally, osteoblasts also play critical roles in bone resorption by regulating osteoclastogenesis because they produce RANK ligand (RANKL), essential to promote osteoclastogenesis, and its decoy receptor, osteoprotegerin (OPG) 111, 112. A balance between RANKL and OPG is important to determine the degree of osteoclastogenesis, i.e. more RANKL production by osteoblasts leads to more osteoclasts; thus more bone resorption is expected. As RANKL is an osteoblastic product and BMPs induce osteoblast maturation, BMPs indirectly stimulate osteoclastogenesis and thus, osteoclastogenesis is impaired when osteoblastogenesis is blocked with BMP antagonists in culture 113. The physiological effects of BMP signaling in osteoblasts on osteoclastogenesis were determined later using an osteoblast-specific gain-of-function or loss-of-function mouse model. For the cases of the osteoblast-specific deletion of *Bmpr1a* and osteoblast-specific over expression of *Noggin*, osteoclastogenesis is highly compromised leading to an increase of bone mass 83, 93. In contrast, osteoblast-specific overexpression of *Bmp4* increased osteoclastogenesis 93. The regulation of RANKL by BMPs was suggested based on an *in vitro* study 114. This concept was recently proven in mouse studies, as *Bmpr1a*-deficient osteoblasts were not able to support osteoclastogenesis due to an imbalance between RANKL and OPG 42, 72. It is therefore concluded that osteoblasts can respond to BMPs by inducing osteogenic (i.e. bone anabolic) action as well as osteoclastogenic (i.e. bone catabolic) action simultaneously presumably dependening on context and timing **(Table 3).**

#### **4.2.2 Regulation of osteoclast by osteoclast-dependent BMP signaling**

BMP receptors are expressed in osteoclasts 110. When BMP signaling through BMPRIA was deficient in osteoclasts using a Catepsin K promoter (CtsK), bone mass was increased as expected 94(Table 2). Interestingly, both bone formation rate and osteoblast number assessed by bone histomorphometry analysis were increased while osteoclast number was reduced in the mutant mice compared to their controls. It is possible that some coupling factors can control osteoblast function in an osteoclast-dependent manner in the mutant mice (i.e. osteoclast-derived coupling factors). Further studies are needed to determine whether such factors mediate BMPRIA-induced coupling from osteoclasts to osteoblasts.

#### **5. Future direction of BMPs and Wnt**

As is discussed in the former part of this review, it is important to understand that BMPs have variable and context-sensitive effects on diverse cell types in bone including chondrocytes, osteoblasts, and osteoclasts. Studies focusing on BMP receptors in chondrocytes including mesenchymal cells suggest that these cells can respond to BMP signaling by increasing bone mass during the endochondral formation process. As discussed in the latter part, BMP signals can consistently inhibit Wnt signaling and bone mass while exerting concordant effects on *Dkk1* and *Sost*. This revision of traditional understanding of the BMP signaling pathway in clinical therapeutics might suggest that in some circumstances, BMP inhibition would be desirable for promoting bone mass. More importantly, if BMP signaling reduces bone mass by inhibiting Wnt signaling through SOST/DKK1 in osteoblasts, small molecule antagonists for BMPs or BMP receptors can conversely increase bone mass and size. Therefore, development of these molecules would be a next step towards disease conditions in which bone mass is reduced such as osteoporosis and bone fracture. Although antibodies for SOST and DKk1 have been developed in order to increase bone mass, the small molecule antagonists which can be an upstream of SOST and DKK1 would be used as more potent therapeutic agents for osteoporosis. Last, the function of the BMP signaling in osteoclasts remains largely unknown in terms of coupling factors and merits future study, although the BMP signaling regulates osteoblast-dependent osteoclastogenesis via the RANKL-OPG pathway.

#### **6. Conclusion**

Understanding the complex roles of the BMP signaling pathway and its molecular interaction with other signaling pathway (i.e. Wnt) in a variety of cell-types in bone including chondrocytes, osteoblasts and osteoclasts, which contribute to normal physiological conditions (i.e. bone development, homeostasis, and remodeling) will not only help to improve current knowledge of the pathological conditions (i.e. bone fracture, osteoporosis, and other congenital and aging-related bone diseases) but may provide novel therapeutically useful strategies.

#### **7. Acknowledgment**

I would like to thank Drs. Yuji Mishina, Jian Q. Feng, Tatsuya Kobayashi, and Henry M. Kronenberg for the generation of multiple transgenic mouse lines and Harry K. W. Kim for encouragement. This work was supported by the Lilly Fellowship Foundation and TSRH Research Foundation (GL170999, GL171041).

#### **8. References**


chondrocytes, osteoblasts, and osteoclasts. Studies focusing on BMP receptors in chondrocytes including mesenchymal cells suggest that these cells can respond to BMP signaling by increasing bone mass during the endochondral formation process. As discussed in the latter part, BMP signals can consistently inhibit Wnt signaling and bone mass while exerting concordant effects on *Dkk1* and *Sost*. This revision of traditional understanding of the BMP signaling pathway in clinical therapeutics might suggest that in some circumstances, BMP inhibition would be desirable for promoting bone mass. More importantly, if BMP signaling reduces bone mass by inhibiting Wnt signaling through SOST/DKK1 in osteoblasts, small molecule antagonists for BMPs or BMP receptors can conversely increase bone mass and size. Therefore, development of these molecules would be a next step towards disease conditions in which bone mass is reduced such as osteoporosis and bone fracture. Although antibodies for SOST and DKk1 have been developed in order to increase bone mass, the small molecule antagonists which can be an upstream of SOST and DKK1 would be used as more potent therapeutic agents for osteoporosis. Last, the function of the BMP signaling in osteoclasts remains largely unknown in terms of coupling factors and merits future study, although the BMP signaling

regulates osteoblast-dependent osteoclastogenesis via the RANKL-OPG pathway.

Understanding the complex roles of the BMP signaling pathway and its molecular interaction with other signaling pathway (i.e. Wnt) in a variety of cell-types in bone including chondrocytes, osteoblasts and osteoclasts, which contribute to normal physiological conditions (i.e. bone development, homeostasis, and remodeling) will not only help to improve current knowledge of the pathological conditions (i.e. bone fracture, osteoporosis, and other congenital and aging-related bone diseases) but may provide novel

I would like to thank Drs. Yuji Mishina, Jian Q. Feng, Tatsuya Kobayashi, and Henry M. Kronenberg for the generation of multiple transgenic mouse lines and Harry K. W. Kim for encouragement. This work was supported by the Lilly Fellowship Foundation and TSRH

[2] Simpson, A. H.; Mills, L.; Noble, B., The role of growth factors and related agents in accelerating fracture healing. *J Bone Joint Surg Br* 2006, 88, (6), 701-5. [3] Aro, H. T.; Govender, S.; Patel, A. D.; Hernigou, P.; Perera de Gregorio, A.; Popescu, G.

[4] Laursen, M.; Hoy, K.; Hansen, E. S.; Gelineck, J.; Christensen, F. B.; Bunger, C. E.,

I.; Golden, J. D.; Christensen, J.; Valentin, A., Recombinant Human Bone Morphogenetic Protein-2: A Randomized Trial in Open Tibial Fractures Treated

Recombinant bone morphogenetic protein-7 as an intracorporal bone growth stimulator in unstable thoracolumbar burst fractures in humans: preliminary

[1] Urist, M. R., Bone: formation by autoinduction. *Science* 1965, 150, (698), 893-9.

with Reamed Nail Fixation. *J Bone Joint Surg Am* 2011.

results. *Eur Spine J* 1999, 8, (6), 485-90.

**6. Conclusion** 

therapeutically useful strategies.

Research Foundation (GL170999, GL171041).

**7. Acknowledgment** 

**8. References** 


[22] Gazzerro, E.; Gangji, V.; Canalis, E., Bone morphogenetic proteins induce the expression

[23] Baron, R.; Rawadi, G.; Roman-Roman, S., Wnt signaling: a key regulator of bone mass.

[24] Glass, D. A., 2nd; Karsenty, G., Molecular bases of the regulation of bone remodeling by the canonical Wnt signaling pathway. *Curr Top Dev Biol* 2006, 73, 43-84. [25] Harada, S.; Rodan, G. A., Control of osteoblast function and regulation of bone mass.

[26] Hartmann, C., A Wnt canon orchestrating osteoblastogenesis. *Trends Cell Biol* 2006, 16,

[27] Krishnan, V.; Bryant, H. U.; Macdougald, O. A., Regulation of bone mass by Wnt

[28] Gong, Y.; Slee, R. B.; Fukai, N.; Rawadi, G.; Roman-Roman, S.; Reginato, A. M.; Wang,

[29] Mukhopadhyay, M.; Shtrom, S.; Rodriguez-Esteban, C.; Chen, L.; Tsukui, T.; Gomer, L.;

[30] Li, X.; Liu, P.; Liu, W.; Maye, P.; Zhang, J.; Zhang, Y.; Hurley, M.; Guo, C.; Boskey, A.;

[31] Bodine, P. V.; Zhao, W.; Kharode, Y. P.; Bex, F. J.; Lambert, A. J.; Goad, M. B.; Gaur, T.;

[32] Li, X.; Zhang, Y.; Kang, H.; Liu, W.; Liu, P.; Zhang, J.; Harris, S. E.; Wu, D., Sclerostin

[33] Ai, M.; Holmen, S. L.; Van Hul, W.; Williams, B. O.; Warman, M. L., Reduced affinity to

[34] Patel, M. S.; Karsenty, G., Regulation of bone formation and vision by LRP5. *N Engl J* 

and limb morphogenesis in the mouse. *Dev Cell* 2001, 1, (3), 423-34.

Dorward, D. W.; Glinka, A.; Grinberg, A.; Huang, S. P.; Niehrs, C.; Izpisua Belmonte, J. C.; Westphal, H., Dickkopf1 is required for embryonic head induction

Sun, L.; Harris, S. E.; Rowe, D. W.; Ke, H. Z.; Wu, D., Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation. *Nat Genet* 2005, 37, (9),

Stein, G. S.; Lian, J. B.; Komm, B. S., The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice. *Mol* 

binds to LRP5/6 and antagonizes canonical Wnt signaling. *J Biol Chem* 2005, 280,

and inhibition by DKK1 form a common mechanism by which high bone massassociated missense mutations in LRP5 affect canonical Wnt signaling. *Mol Cell Biol* 

H.; Cundy, T.; Glorieux, F. H.; Lev, D.; Zacharin, M.; Oexle, K.; Marcelino, J.; Suwairi, W.; Heeger, S.; Sabatakos, G.; Apte, S.; Adkins, W. N.; Allgrove, J.; Arslan-Kirchner, M.; Batch, J. A.; Beighton, P.; Black, G. C.; Boles, R. G.; Boon, L. M.; Borrone, C.; Brunner, H. G.; Carle, G. F.; Dallapiccola, B.; De Paepe, A.; Floege, B.; Halfhide, M. L.; Hall, B.; Hennekam, R. C.; Hirose, T.; Jans, A.; Juppner, H.; Kim, C. A.; Keppler-Noreuil, K.; Kohlschuetter, A.; LaCombe, D.; Lambert, M.; Lemyre, E.; Letteboer, T.; Peltonen, L.; Ramesar, R. S.; Romanengo, M.; Somer, H.; Steichen-Gersdorf, E.; Steinmann, B.; Sullivan, B.; Superti-Furga, A.; Swoboda, W.; van den Boogaard, M. J.; Van Hul, W.; Vikkula, M.; Votruba, M.; Zabel, B.; Garcia, T.; Baron, R.; Olsen, B. R.; Warman, M. L., LDL receptor-related protein 5 (LRP5) affects bone

102, (12), 2106-14.

(3), 151-8.

945-52.

(20), 19883-7.

2005, 25, (12), 4946-55.

*Med* 2002, 346, (20), 1572-4.

*Endocrinol* 2004, 18, (5), 1222-37.

*Curr Top Dev Biol* 2006, 76, 103-27.

*Nature* 2003, 423, (6937), 349-55.

signaling. *J Clin Invest* 2006, 116, (5), 1202-9.

accrual and eye development. *Cell* 2001, 107, (4), 513-23.

of noggin, which limits their activity in cultured rat osteoblasts. *J Clin Invest* 1998,


[50] Winkler, D. G.; Sutherland, M. K.; Geoghegan, J. C.; Yu, C.; Hayes, T.; Skonier, J. E.;

[51] Piccolo, S.; Agius, E.; Leyns, L.; Bhattacharyya, S.; Grunz, H.; Bouwmeester, T.; De

[52] Bell, E.; Munoz-Sanjuan, I.; Altmann, C. R.; Vonica, A.; Brivanlou, A. H., Cell fate

[53] Itasaki, N.; Jones, C. M.; Mercurio, S.; Rowe, A.; Domingos, P. M.; Smith, J. C.;

[54] van Bezooijen, R. L.; Roelen, B. A.; Visser, A.; van der Wee-Pals, L.; de Wilt, E.;

[55] van Bezooijen, R. L.; Svensson, J. P.; Eefting, D.; Visser, A.; van der Horst, G.; Karperien,

[56] Semenov, M.; Tamai, K.; He, X., SOST is a ligand for LRP5/LRP6 and a Wnt signaling

[57] Li, X.; Ominsky, M. S.; Niu, Q. T.; Sun, N.; Daugherty, B.; D'Agostin, D.; Kurahara, C.;

novel BMP antagonist. *Embo J* 2003, 22, (23), 6267-76.

inhibitor. *Development* 2003, 130, (7), 1381-9.

inhibitor. *J Biol Chem* 2005, 280, (29), 26770-5.

*Development* 2003, 130, (18), 4295-305.

Nodal, BMP and Wnt signals. *Nature* 1999, 397, (6721), 707-10.

classical BMP antagonist. *J Exp Med* 2004, 199, (6), 805-14.

containing protein. *Am J Hum Genet* 2001, 68, (3), 577-89.

*Med Genet* 2002, 110, (2), 144-52.

stimulated bone formation. *J Bone Miner Res* 2007, 22, (1), 19-28.

Shpektor, D.; Jonas, M.; Kovacevich, B. R.; Staehling-Hampton, K.; Appleby, M.; Brunkow, M. E.; Latham, J. A., Osteocyte control of bone formation via sclerostin, a

Robertis, E. M., The head inducer Cerberus is a multifunctional antagonist of

specification and competence by Coco, a maternal BMP, TGFbeta and Wnt

Krumlauf, R., Wise, a context-dependent activator and inhibitor of Wnt signalling.

Karperien, M.; Hamersma, H.; Papapoulos, S. E.; ten Dijke, P.; Lowik, C. W., Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a

M.; Quax, P. H.; Vrieling, H.; Papapoulos, S. E.; ten Dijke, P.; Lowik, C. W., Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-

Gao, Y.; Cao, J.; Gong, J.; Asuncion, F.; Barrero, M.; Warmington, K.; Dwyer, D.; Stolina, M.; Morony, S.; Sarosi, I.; Kostenuik, P. J.; Lacey, D. L.; Simonet, W. S.; Ke, H. Z.; Paszty, C., Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. *J Bone Miner Res* 2008, 23, (6), 860-9. [58] Balemans, W.; Ebeling, M.; Patel, N.; Van Hul, E.; Olson, P.; Dioszegi, M.; Lacza, C.;

Wuyts, W.; Van Den Ende, J.; Willems, P.; Paes-Alves, A. F.; Hill, S.; Bueno, M.; Ramos, F. J.; Tacconi, P.; Dikkers, F. G.; Stratakis, C.; Lindpaintner, K.; Vickery, B.; Foernzler, D.; Van Hul, W., Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). *Hum Mol Genet* 2001, 10, (5), 537-43. [59] Brunkow, M. E.; Gardner, J. C.; Van Ness, J.; Paeper, B. W.; Kovacevich, B. R.; Proll, S.;

Skonier, J. E.; Zhao, L.; Sabo, P. J.; Fu, Y.; Alisch, R. S.; Gillett, L.; Colbert, T.; Tacconi, P.; Galas, D.; Hamersma, H.; Beighton, P.; Mulligan, J., Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-

Dikkers, F. G.; Hildering, P.; Willems, P. J.; Verheij, J. B.; Lindpaintner, K.; Vickery, B.; Foernzler, D.; Van Hul, W., Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease. *J Med Genet* 2002, 39, (2), 91-7. [61] Staehling-Hampton, K.; Proll, S.; Paeper, B. W.; Zhao, L.; Charmley, P.; Brown, A.;

Gardner, J. C.; Galas, D.; Schatzman, R. C.; Beighton, P.; Papapoulos, S.; Hamersma, H.; Brunkow, M. E., A 52-kb deletion in the SOST-MEOX1 intergenic region on 17q12-q21 is associated with van Buchem disease in the Dutch population. *Am J* 

[60] Balemans, W.; Patel, N.; Ebeling, M.; Van Hul, E.; Wuyts, W.; Lacza, C.; Dioszegi, M.;


type IA receptor (BMPRIA) increases bone mass. *J Bone Miner Res* 2008, 23, (12), 2007-17.


[73] Kamiya, N.; Kaartinen, V.; Mishina, Y., Loss-of-function of ACVR1 in osteoblasts

[74] Glass, D. A., 2nd; Bialek, P.; Ahn, J. D.; Starbuck, M.; Patel, M. S.; Clevers, H.; Taketo, M.

[75] Holmen, S. L.; Zylstra, C. R.; Mukherjee, A.; Sigler, R. E.; Faugere, M. C.; Bouxsein, M.

[76] Dudley, A. T.; Lyons, K. M.; Robertson, E. J., A requirement for bone morphogenetic

[77] Gu, Z.; Reynolds, E. M.; Song, J.; Lei, H.; Feijen, A.; Yu, L.; He, W.; MacLaughlin, D. T.;

[78] Luo, G.; Hofmann, C.; Bronckers, A. L.; Sohocki, M.; Bradley, A.; Karsenty, G., BMP-7 is

[79] Mishina, Y.; Suzuki, A.; Ueno, N.; Behringer, R. R., Bmpr encodes a type I bone

[80] Mishina, Y.; Crombie, R.; Bradley, A.; Behringer, R. R., Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis. *Dev Biol* 1999, 213, (2), 314-26. [81] Winnier, G.; Blessing, M.; Labosky, P. A.; Hogan, B. L., Bone morphogenetic protein-4 is

[82] Zhang, H.; Bradley, A., Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. *Development* 1996, 122, (10), 2977-86. [83] Mishina, Y.; Starbuck, M. W.; Gentile, M. A.; Fukuda, T.; Kasparcova, V.; Seedor, J. G.;

[84] Kronenberg, H. M., Developmental regulation of the growth plate. *Nature* 2003, 423,

[85] Nakashima, K.; de Crombrugghe, B., Transcriptional mechanisms in osteoblast differentiation and bone formation. *Trends Genet* 2003, 19, (8), 458-66. [86] Mackie, E. J.; Ahmed, Y. A.; Tatarczuch, L.; Chen, K. S.; Mirams, M., Endochondral

[87] Rountree, R. B.; Schoor, M.; Chen, H.; Marks, M. E.; Harley, V.; Mishina, Y.; Kingsley, D.

function and bone remodeling. *J Biol Chem* 2004, 279, (26), 27560-6.

postnatal bone acquisition. *J Biol Chem* 2005, 280, (22), 21162-8.

2007-17.

751-64.

9, (22), 2795-807.

(17), 2105-16.

(6937), 332-6.

*Development* 1999, 126, (11), 2551-61.

*Biochem Cell Biol* 2008, 40, (1), 46-62.

cartilage. *PLoS Biol* 2004, 2, (11), e355.

patterning. *Genes Dev* 1995, 9, (22), 2808-20.

embryogenesis. *Genes Dev* 1995, 9, (24), 3027-37.

type IA receptor (BMPRIA) increases bone mass. *J Bone Miner Res* 2008, 23, (12),

increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1. *Biochem Biophys Res Commun, 2011, 414, (2), 326-30.*

M.; Long, F.; McMahon, A. P.; Lang, R. A.; Karsenty, G., Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. *Dev Cell* 2005, 8, (5),

L.; Deng, L.; Clemens, T. L.; Williams, B. O., Essential role of beta-catenin in

protein-7 during development of the mammalian kidney and eye. *Genes Dev* 1995,

van den Eijnden-van Raaij, J.; Donahoe, P. K.; Li, E., The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

an inducer of nephrogenesis, and is also required for eye development and skeletal

morphogenetic protein receptor that is essential for gastrulation during mouse

required for mesoderm formation and patterning in the mouse. *Genes Dev* 1995, 9,

Hanks, M. C.; Amling, M.; Pinero, G. J.; Harada, S.; Behringer, R. R., Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast

ossification: how cartilage is converted into bone in the developing skeleton. *Int J* 

M., BMP receptor signaling is required for postnatal maintenance of articular

