Osteoblasts and Wnt Signaling: Meeting Report from the 30Th Annual Meeting of the American Society for Bone and Mineral Research

IBMS BoneKEy. 2008 November;5(11):449-453 http://www.bonekey-ibms.org/cgi/content/full/ibmske;5/11/449 doi: 10.1138/20080353 MEETING REPORTS

Osteoblasts and Wnt Signaling: Meeting Report from the 30th Annual Meeting of the American Society for Bone and Mineral Research

September 12-16, 2008, Montréal, Québec, Canada

Joseph Caverzasio Service of Bone Diseases, Department of Rehabilitation and Geriatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland

Each ASBMR Annual Meeting is rich in new molecules regulate Wnt signaling in information for osteoblast biology. As osteoblasts and bone formation is not well- expected from recent advances in this field, understood and these issues were the focus several laboratories concentrated their of several interesting studies reported at the efforts on the Wnt signaling pathway and meeting. sclerostin, which are the two systems that could provide information for the Whereas genetic evidence has clearly development of new molecules to stimulate established a prominent role for Lrp5 in bone formation. Some of the key new bone development, the role for Lrp6 findings are summarized below. remained unclear. The selective ablation of Lrp6 in osteoblasts is associated with lower The Wnt/LRP5 Signaling Pathway bone mass (1). Ablation of both Lrp5 and Remains a Potential Drug Target for Lrp6 in osteoblasts induces a severe Osteo-Anabolism osteopenia and phenocopied mice lacking β- catenin, suggesting that both Lrp5 and Lrp6 With the identification of Lrp5 mutations in are required to fully activate β-catenin in patients with either osteoporosis or mature osteoblasts. osteosclerosis, the Wnt signaling pathway is considered to be of crucial importance for Two studies investigated the role of Kremen the regulation of osteoblast differentiation (Krm1 and Krm2), transmembrane proteins and function. The Wnt canonical pathway is serving as receptors for molecules of the triggered by the binding of Wnt family Dkk family that antagonize Wnt signaling members to a co-receptor complex that through binding to Lrp5 and Lrp6. One study includes Frizzled (Fzd, a G protein-coupled focused on Krm2 (2). Surprisingly, the receptor (GPCR)-like protein) and Lrp5/6. expression pattern of this molecule was The canonical signal is transmitted by the β- exactly opposite to that of Dkk1. It was catenin pathway and the Tcf/Lef1 mainly observed in non-mineralized bone transcriptional factors. Canonical Wnt and not in mineralized cultures. This signaling is important during embryogenesis observation did not suggest that Krm2 is and osteoblastogenesis and is tightly involved in the action of Dkk1 on regulation regulated by several secreted factors, of Lrp5. Thus, the authors created a Krm2 including members of the Dickkopf (Dkk), overexpression mouse model driven by a Wnt-inhibitor factor (Wif), and secreted Col1a1 promoter and found a striking frizzled related protein (sFRP) families and reduction (2/3) in bone mass due to both a sclerostin (Sost). marked reduction in bone formation and increased bone resorption. Cell analysis The molecular mechanism by which suggested that Krm2 is involved in extracellular factors and intracellular inactivation of Lrp6. Clearly, further studies

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IBMS BoneKEy. 2008 November;5(11):449-453 http://www.bonekey-ibms.org/cgi/content/full/ibmske;5/11/449 doi: 10.1138/20080353 are required to determine the mechanism by intracellular pathways (Wnt/Ca2+ and which Krm2 influences the differentiation of Wnt/Planar Cell Polarity (PCP)), osteoblasts. respectively. Recently, the non-canonical Wnt-4 has been found to be regulated by Another study investigated the physiological parathyroid hormone (PTH) and expressed relevance of Krm1 and Krm2 as Wnt in bone lining cells, osteocytes, and modulators for bone remodeling (3). Double, hypertrophic chondrocytes (5). At this year's but not single mutants showed enhanced meeting, the same group reported that the Wnt signaling and osteoblast differentiation differentiation of primary osteoblastic cells is markers and increased trabecular bone increased by Wnt-4 (6). This effect is volume with small changes in bone associated with minimal activation of the resorption parameters, suggesting a major canonical pathway but a clear activation of effect on bone formation. The triple the Wnt/Ca2+ pathway (a 12x increase in knockouts in which Dkk1 was haplo- CamkII phosphorylation and a 2x increase in insufficient failed to show significant JNK phosphorylation). Using specific changes in BV/TV above and beyond the inhibitors to CamkII and JNKs, the authors Krm1/2 double knockouts, suggesting a found that Wnt-4 stimulates bone marker functional interaction between Kremens and gene expression through non-canonical Dkk1 as negative regulators of Wnt/β- means. Their data support a role for non- catenin signaling and trabecular bone canonical Wnts and imply that the non- formation. canonical pathway is also important for regulation of bone. Because of its prominent role in the regulation of bone mass, the Wnt signaling A new molecule for regulation of β-catenin system is of potential interest for anabolic translocation in the nucleus, FHL2, has been drug development. One regulatory system of presented (7), and the paper has recently interest is the sFRP family that functions as been published online (8). FHL2 is a a Frizzled decoy receptor, antagonizing Wnt member of the LIM-only subclass of the LIM activity and therefore, unlike Dkk1 and Sost, protein superfamily. The authors found that suppressing both canonical and non- FHL2 is a key factor involved in canonical pathway activation. It was dexamethasone-induced osteogenic cell reported that deletion of sFRP4 in growing differentiation in mesenchymal stem cells mice strongly favors trabecular bone that are precursors of osteoblasts. formation but has an opposite effect on Immunocytochemical analysis showed that cortical bones without influencing bone FHL2 overexpression induces β-catenin resorption (4). This interesting observation translocation to the nucleus where the two suggests that the Wnt system may proteins co-localize. This study provides differentially influence trabecular and cortical good evidence that FHL2 is an important bone. factor for the differentiation of mesenchymal osteoprogenitors into osteoblasts via Another player in the Wnt system activation of Wnt/β-catenin signaling. investigated and reported at the meeting was Fzd9. Analysis of the skeletal The Molecular Mechanism by Which phenotype of Fzd9-deficient mice indicated Sclerostin Inhibits Osteoblastic Cell a 40% decrease in trabecular bone volume Differentiation Remains Unclear at the age of 24 and 72 weeks, with no effects at 6 weeks. Thus, Fzd9 is an Bone diseases associated with mutations or interesting target for the treatment of bone deletions in Sost indicate that sclerostin is a diseases in adults. major regulator of bone formation. Sclerostin is a member of the DAN family of Wnts are divided into Wnt-1 and Wnt-5a glycoproteins that shares the capacity to signaling classes, which activate canonical inhibit BMP activity. Recent reports (Wnt/β-catenin) and non-canonical documented that sclerostin antagonizes

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IBMS BoneKEy. 2008 November;5(11):449-453 http://www.bonekey-ibms.org/cgi/content/full/ibmske;5/11/449 doi: 10.1138/20080353 canonical Wnt signaling (9;10) by binding to formation. Mice harboring a tamoxifen- the Wnt coreceptor Lrp5. These studies inducible cre recombinase under the control have been performed in Xenopus embryos of the CMV/βactin promoter to selectively and HEK293 cells. This molecular delete Osx after birth displayed interaction had not yet been documented in disappearance of bone trabeculae, thin and osteogenic cells. At this year's meeting, an porous cortical bones, and a massive antagonistic effect of sclerostin on activation accumulation of mineralized cartilage (15). of Lrp5/6 by Wnts was not confirmed in two Calcein incorporation was almost completely different osteoblastic cell systems having absent in lumbar vertebrae and long bones decreased Wnt3a- and BMP2-induced of postnatal Osx-null mutants, indicating that differentiation responses to sclerostin (11). Osx is required for bone formation The absence of a sclerostin antagonizing postnatally. Inactivation of Osx also affected effect on activation of Lrp5/6 contrasted with the development of osteocytes, suggesting the complete blunting action of Dkk1, a well- that Osx plays a key role in regulating and recognized antagonist of Lrp5 activity. The maintaining osteocyte function. Using the study also found that sclerostin by itself can time-specific and site-specific Cre/loxP stimulate different signaling pathways such system, another study also reported that as PKC, ERK1/2 and Akt. Data from this Osx plays a significant role in regulating study and another report (12) also osteoblast differentiation and bone formation documented that sclerostin inhibits in adult bone (16). osteogenic cell differentiation induced by a selective inhibitor of GSK3β acting To better understand the control of downstream of the Wnt/Lrp5 receptor. Thus, transcriptional activation by Osx, Osx- it remains unclear whether the blunting interacting proteins were identified using effect of sclerostin on BMP- and Wnt- affinity purification techniques. A Jumonji C induced osteoblastic cell differentiation is (JmjC)-domain containing protein, NO66, mediated by a direct interaction with Lrp5/6. that interacts with Osx in osteoblast cells Alternative mechanisms must be has been discovered (17). NO66 is considered. expressed in all developing bones and exhibits a JmjC-dependent histone A previous report indicated that continuous demethylase activity. Interaction between administration of PTH suppresses sclerostin NO66 and Osx inhibits gene activation by expression in osteocytes (13). Most of the in Osx. Knockdown of NO66 in osteoblast cells vivo effects of PTH are mediated by cyclic- triggers accelerated osteoblast AMP-dependent signaling downstream of differentiation and mineralization as well as the PTH/PTHrP GPCR. In a mouse model a marked stimulation in expression of Osx lacking Gsα in osteoblasts, sclerostin was target genes including Col1a1, osteocalcin markedly increased (14), suggesting that the and bone sialoprotein. These data provide cAMP pathway also mediates inhibition of the first evidence that NO66 serves as a PTH-induced sclerostin expression. In these negative regulator of Osx target genes in mice, osteoblastic cell differentiation and osteoblasts by modulating histone maturation were markedly impaired with the methylation states. formation of woven instead of lamellar bone. Dlx5 is a homeodomain-containing Progress in Understanding the transcription factor expressed in developing Regulation of Osterix Expression and skeletal elements. Dlx5-null mice have Function delayed cranial ossification and abnormal osteogenesis. In osteoblasts exposed to The transcription factor Osterix (Osx) is BMP2, it was found that Dlx5 precedes Osx essential for osteoblast differentiation and expression and binds to a BMP2-responsive bone formation, as mice lacking Osx die at homeobox sequence, suggesting that Dlx5 birth with a complete absence of is essential for mediating Osx expression by intramembranous and endochondral bone BMP2 (18). Furthermore, data presented

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IBMS BoneKEy. 2008 November;5(11):449-453 http://www.bonekey-ibms.org/cgi/content/full/ibmske;5/11/449 doi: 10.1138/20080353 suggest that Dlx5 is a novel target of p38 Ann N Y Acad Sci. 2007 Nov;1116:354- MAPK that has been shown to be relevant 9. for the osteogenic effect of BMP2. The phosphorylation of Dlx5 by p38 enhances 6. Bergenstock MK, Tamasi J, Partridge Dlx5 transcriptional activity, increasing Osx NC. WNT-4 acts through the non- expression and suggesting a p38-induced canonical pathways to stimulate Dlx5 phosphorylation for regulation of Osx osteoblast and bone marrow stromal transcriptional activity. stell cell differentiation. J Bone Miner Res. 2008 Sep;23(Suppl 1):S90. Conflict of Interest: None reported. 7. Hamidouche Z, Haÿ E, Vaudin P, Peer Review: This article has been peer-reviewed. Charbord P, Marie PJ, Fromigué O. References FHL2 mediates dexamethasone- induced mesenchymal stem cell 1. Zylstra CR, Wan C, VanKoevering KK, osteogenic differentiation by activating Sanders AK, Lindvall C, Clemens TL, Wnt/beta-catenin signaling and Runx2 Williams BO. Osteoblast-specific expression. J Bone Miner Res. 2008 deletion of Lrp6 reveals distinct roles for Sep;23(Suppl 1):S385. [Abstract] Lrp5 and Lrp6 in bone development. J Bone Miner Res. 2008 Sep;23(Suppl 8. Hamidouche Z, Haÿ E, Vaudin P, 1):S2. [Abstract] Charbord P, Schüle R, Marie PJ, Fromigué O. FHL2 mediates 2. Schulze J, Seitz S, Schneebauer M, dexamethasone-induced mesenchymal Amling M, Schinke T. Transgenic over- cell differentiation into osteoblasts by expression of the Wnt antagonist activating Wnt/beta-catenin signaling- Kremen-2 in osteoblasts leads to severe dependent Runx2 expression. FASEB J. impairment of bone formation and 2008 Nov;22(11):3813-22. increased bone resorption. J Bone Miner Res. 2008 Sep;23(Suppl 1):S2. 9. Li X, Zhang Y, Kang H, Liu W, Liu P, [Abstract] Zhang J, Harris SE, Wu D. Sclerostin binds to LRP5/6 and antagonizes 3. Saito H, Ellwanger K, Clément-Lacroix canonical Wnt signaling. J Biol Chem. P, Hesse E, Maltry N, Niedermeyer J, 2005 May 20;280(20):19883-7. Lee RW, Rawadi G, Westphal H, Niehrs C, Baron R. Deletion of the Dkk1 co- 10. Semënov M, Tamai K, He X. SOST is a receptors Kremen 1 and Kremen 2 in ligand for LRP5/LRP6 and a Wnt mice leads to increased bone formation signaling inhibitor. J Biol Chem. 2005 and bone mass. J Bone Miner Res. Jul 22;280(29):26770-5. 2008 Sep;23(Suppl 1):S2. [Abstract] 11. Caverzasio J. Wnt/LRP5-independent 4. Saito H, Hinkle R, Ebert D, Blanton C, inhibition of osteoblastic cell Jaiswal N, Elenich L, Cody D, Baron R, differentiation by sclerostin. J Bone Sabatakos G. Deletion of the Wnt Miner Res. 2008 Sep;23(Suppl 1):S72. signaling antagonist secreted frizzled [Abstract] related protein 4 (sFRP4) in mice induces opposite bone formation 12. Grabenstaetter T, Sakane Y, Jacobi C, phenotypes in trabecular and cortical Lu C, Bauer A, Fernandez C, Ramage bone. J Bone Miner Res. 2008 P, Hartmann L, Leupin O, Kneissel M, Sep;23(Suppl 1):S3. [Abstract] Halleux C. SOST blocks GSK3-beta inhibitor-induced alkaline phosphatase 5. Bergenstock MK, Partridge NC. activity. J Bone Miner Res. 2008 Parathyroid hormone stimulation of Sep;23(Suppl 1):S251. [Abstract] noncanonical Wnt signaling in bone.

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14. Wu JY, Maes C, Chen M, Weinstein LS, 17. Sinha KM, Yasuda H, Coombes MM, Kronenberg HM. Deletion of the G Dent SR, de Crombrugghe B. protein subunit Gsα in early osteoblasts Regulation of the osteoblast-specific leads to accelerated osteoblast transcription factor Osterix by NO66, a maturation and formation of woven bone Jumonji family histone demethylase. J with abnormal osteocytes, resulting in Bone Miner Res. 2008 Sep;23(Suppl severe osteoporosis. J Bone Miner Res. 1):S87. 2008 Sep;23(Suppl 1):S71. [Abstract] 18. Ortuño MJ, Ulsamer A, Ruiz S, 15. Zhou X, Zhang Z, Feng JQ, Darnay BG, Susperregui AG, Osses N, Rosa JL, Kim J, de Crombrugghe B. Osterix is Ventura F. BMP2 induces Osterix required for skeletal growth and bone expression through up-regulation of homeostasis after birth. J Bone Miner Dlx5 and its phosphorylation by p38 Res. 2008 Sep;23(Suppl 1):S22. MAPK. J Bone Miner Res. 2008 [Abstract] Sep;23(Suppl 1):S88.

16. Baek W, Lee M, Oh J, Kim S, Akiyama H, de Crombrugghe B, Kim J. Osterix, a

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