MEETING REPORTS Osteoblasts and Wnt Signaling

IBMS BoneKEy. 2009 October;6(10):393-397 http://www.bonekey-ibms.org/cgi/content/full/ibmske;6/10/393 doi: 10.1138/20090404

MEETING REPORTS

Osteoblasts and Wnt Signaling: Meeting Report from the 31st Annual Meeting of the American Society for Bone and Mineral Research

September 11-15, 2009 in Denver, Colorado

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

Wnt Signaling, Serotonin and Bone metabolism, a group investigated the phenotype of transgenic mice expressing Just after the 2008 ASBMR Annual Meeting lefΔN from the 2.3-Col1a1 promoter (3). in Montreal, a group led by Gerard Karsenty Lymphoid enhancer factor 1 (Lef1) is a Wnt- reported that Lrp5 controls bone formation responsive transcription factor that by regulating serotonin in the duodenum (1). associates with the nuclear co-regulator β- This information markedly changed the catenin. Lef1 and the N-terminal truncated concept that the low bone mass isoform of Lef1, lefΔN (expressed mainly in osteoporosis pseudoglioma (OPPG) and mature osteoblasts), are capable of binding high bone mass (HBM) phenotypes were to DNA and regulating gene expression. A due to cell autonomous alteration in Wnt significant increase in osteoblast activity and signaling in osteoblasts. Thus, whereas the in trabecular bone density of the proximal role of Wnt signaling in osteoblastogenesis tibia was detected in transgenic mice. There from precursor mesenchymal cells is well- was also a significant increase in mRNA of accepted, the importance of this pathway in OPG and in the OPG/RANKL ratio mRNA. the activation of osteoblasts for controlling However, osteocalcin mRNA in the cortex bone formation remains unclear and needs was decreased 4-fold in lefΔN transgenic reconsideration. mice. From these observations, the study concluded that the constitutive expression of From the information mentioned above, one lefΔN promotes osteoblast activity in group investigated the relationship between trabecular bone and bone development and circulating serotonin and bone mass inhibits end-stage osteoblast maturation in measured by high-resolution pQCT in a cortical bone and normal bone homeostasis. population-based sample of 215 women that These data further support a cell had no medication that could interfere with autonomous effect of the Wnt pathway in serum serotonin levels (2). After correction osteoblasts with different effects on for the effect of serotonin on body mass trabecular and cortical bone, probably index, the serum serotonin level was a through differences in either the cell significant negative predictor of total femoral autonomous effect of Wnt proteins in neck and vertebral BMD, and of trabecular osteoblasts of trabecular and cortical bone thickness of the radius. Thus, these data or in Wnt antagonists present at each bone provide support for a physiological role of site. Data also confirm an important effect of circulating serotonin in regulating bone this pathway in regulating bone resorption density and structure. through the OPG/RANKL system.

Wnt Signaling and Osteoblasts A recent report indicated that osteoblast- targeted disruption of glucocorticoid In order to better understand the cell signaling is associated with a reduction of autonomous role of the Wnt pathway for Wnt7b and Wnt10b mRNA and β-catenin regulation of osteoblasts and bone protein levels in transgenic (Col2.3-

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11βHSD2) versus control osteoblasts (4). osteocytes downstream of Sost plays a This change in Wnt expression and crucial role. A detailed analysis of the loxP/loxP signaling was associated with alteration in Ctnnb1 ; Dmp1-Cre phenotype is osteoblastogenesis, suggesting that Wnt needed to better understand the type of signaling by osteoblasts is essential for interaction between Sost and β-catenin- mesenchymal cell differentiation towards the dependent Wnt signaling in osteocytes for osteoblastic lineage. To define the individual controlling bone metabolism. roles of Wnt7b and Wnt10b in the control of osteoblast differentiation, the same group Previous findings suggested that Sost may knocked down Wnt7b or Wnt10b in MC3T3- interact with Lrp5 in osteoblasts to control E1 cells and investigated the phenotype of bone formation. As mentioned above, this these cells (5). Knockdown of Wnt7b concept has recently been challenged by resulted in complete failure of mineralized work suggesting that Lrp5 controls bone formation whereas alteration of cell formation by inhibiting serotonin synthesis in differentiation was less severe when Wnt10b the duodenum (1). To further investigate this was knocked down. Further investigation relationship, Sost; Lrp5 double knockout indicated that Wnt7b could compensate for mice were generated (8). As expected, Sost- the absence of Wnt10b since its expression deficient mice displayed high bone mass was increased several-fold with time and Lrp5-deficient mice displayed low bone incubation. Associated with blunted mass phenotypes. Compared with wild type mineralization in the absence of Wnt7b, controls, Sost deficiency rescued the Lrp5 osteocalcin expression was markedly mutant osteopenic phenotype. However, the reduced, suggesting an alteration in net bone gain in Lrp5 mutants due to Sost osteoblast differentiation. From these deficiency was much smaller compared to observations, the study concluded that mice possessing a normal Lrp5 gene. This Wnt7b is probably an important Wnt protein lower bone gain was also observed in mice for increasing the differentiation of lacking just one Lrp5 wild-type allele. The osteoblastic cells. This conclusion is also authors concluded from their study that Sost supported by a recent study showing that targets Lrp5-dependent and -independent Wnt7b, expressed by osteogenic cells in pathways to control bone formation in vivo. vivo, induces osteoblast differentiation in Further studies will be required to assess vitro (6). the cellular and molecular mechanisms by which Sost interacts with either Lrp5 or the Interaction Between the Wnt Signaling serotonin pathways to control osteoblastic Pathway and Sclerostin cell activity and bone formation.

To investigate whether the Wnt pathway is Recent genetic studies in humans and mice an important target of Sost for controlling have clearly shown that sclerostin is a bone formation, a group studied whether potent secreted inhibitor of bone formation. Sost deletion could rescue the osteoporotic In vitro studies suggested that sclerostin phenotype in mice lacking β-catenin may inhibit osteoblastic activity by binding to (Ctnnb1) in osteocytes (Ctnnb1loxP/loxP; the Wnt co-receptor low density lipoprotein- Dmp1-Cre mice) (7). Osteocyte-specific loss related (LRP) 5/6. To assess whether of Ctnnb1 resulted in osteoporosis, while sclerostin binds to these molecules at the Sost deficiency induced a high bone mass cellular level, a series of tandem affinity phenotype. The increased bone mass in purification screens using HEK293 and response to Sost deficiency was markedly UMR106 cells were performed (9). Results decreased in mice lacking β-catenin. The identified LRP4 and glypicans as novel reason for this blunting effect remains sclerostin binding partners. Interaction with unclear. This blunting effect was also LRP6 and, with lower confidence, LRP5, observed with heterozygous Ctnnb1 deletion was confirmed. Sclerostin-mediated from osteocytes, which by itself did not inhibition of Wnt-induced signaling was cause a low bone mass phenotype. From increased in the presence of LRP4 in these observations, this study concluded HEK293 and UMR106 cells and diminished that β-catenin-dependent Wnt signaling in in HEK293 cells with knockdown of LRP4.

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DKK1 had no effect in these experiments, LRP5 in PTH-induced changes in bone suggesting that LRP5 was not involved. formation with mechanical loading. Whereas Finally, silencing of LRP4 by lentiviral- administration of PTH nearly doubled the mediated shRNA increased osteoblastic cell bone formation response to loading in WT differentiation in MC3T3-E1 cells and mice (+48% to +90%), it was reduced in completely blocked sclerostin-inhibitory HBM mice (from +26% to 12%). The authors action on in vitro bone mineralization. From concluded that there is probably an overlap these observations, the authors concluded in signaling between the HBM LRP5 that the interaction between sclerostin and receptor and PTH that acts to suppress the LRP4 is required for the inhibitory effect of formation response when both are sclerostin on bone formation, and that LRP4 stimulated simultaneously. is a potent target for controlling bone formation. The Non-Canonical Wnt Signaling Pathway and Cytokine Interaction with The Wnt Signaling Pathway, Mechanical Wnt Signaling in Bone Pathophysiology Loading and Bone Formation ROR2 is a recently described alternative Mechanical loading is an important stimulus receptor for Wnt proteins that has been for bone formation but the molecular events shown to influence bone development and involved in mechanical signal transduction metabolism via non-canonical Wnt signaling are not well understood. The role of LRP5 in with a knockout phenotype resembling that mechanical loading-related stimulation of of Wnt5a (13). It was reported that ROR2 is cortical and cancellous bone in mice was one of the genes highly expressed in investigated (10) using either transgenic osteosarcoma (14). Knockdown of ROR2 in LRP5G171V, Lrp5(-/-) or wild type mice. the osteosarcoma cell lines U2OS and Mechanical stimulation was performed on Saos-2 caused suppression of cell the right tibia whereas decreased loading proliferation and invasive ability. Increased was obtained with ablation of the right sciatic expression of ROR2 in osteosarcoma was nerve. Essentially, the authors found that associated with overexpression of WNT5B. two weeks of loading engendered an The authors concluded that ROR2 and osteogenic response in all groups with a WNT5B are promising therapeutic targets greater response in cortical and cancellous for the treatment of osteosarcoma. bone in LRP5G171V mice. From these observations, they concluded that the Proinflammatory cytokines such as tumor absence of the LRP5 gene has no effect on necrosis factor (TNF) are known to reduce bones' osteogenic response, an observation bone mass in periarticular bone by that does not confirm data from a previous increasing bone resorption. Whether TNF report indicating that the Wnt coreceptor also influences bone formation in vivo is not LRP5 is essential for skeletal well understood and was investigated at the mechanotransduction (11). The presence of meeting (15). For this analysis, the authors the HBM G171V mutation, however, is used transgenic mice expressing human associated with a higher osteogenic TNF and investigated changes in bone mass response to loading and some protection compared with WT mice at different time from bone loss due to disuse. intervals. They observed that BMD was similar until 4 weeks, after which an Another group also investigated the effect of accelerated accrual of bone mass due to mechanical loading on bone formation in increased osteoblast number in WT mice wild type (WT) and LRP5G171V (HBM) mice was blunted in TgTNF mice with lower in similar experimental conditions (12). BV/TV, TbN, TbTh and Tb connectivity and Surprisingly, the authors found a smaller no difference in osteoclast number and CTx. stimulation of bone formation in loaded bone Associated with this in vivo effect of TNF on from HBM (+26%) compared with WT bone development, mesenchymal stem cells (+48%) mice, a result that is not consistent (MSCs) from 12-week-old TgTNF mice with data presented in the above paper. cultured in differentiating medium had a These authors also investigated the role of reduced expression of Osx, RUNX2,

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AlkPhos, BSP, osteocalcin and delayed architecture, probably by interfering with the mineralization. TgTNF cells also showed action of Wnt protein(s) in bone cells. reduced Wnts 10b, 5a, and 5b, β-catenin mRNA and DKK1, suggesting alteration in Conflict of Interest: None reported.

Wnt signaling. To determine if TNF Peer Review: This article has been peer-reviewed. suppressed Wnt signaling in vivo, TgTNF mice were crossed with a β-catenin- References activated transgene driving expression of nuclear β-galactosidase reporter (BAT-gal) 1. Yadav VK, Ryu JH, Suda N, Tanaka KF, transgenic mice expressing the lacZ Gingrich JA, Schütz G, Glorieux FH, transgene under the control of β-catenin/T Chiang CY, Zajac JD, Insogna KL, cell factor responsive elements. LacZ Mann JJ, Hen R, Ducy P, Karsenty G. activity was assessed at 4 weeks when Lrp5 controls bone formation by osteoblast differentiation was expected to inhibiting serotonin synthesis in the accelerate. TgTNF/BAT-Gal mice showed a duodenum. Cell. 2008 Nov 90% reduction in Wnt signaling compared 28;135(5):825-37. with WT/BAT-Gal mice as measured in trabecular bone sections. From these 2. Moedder U, Achenbach S, Amin S, observations, the authors concluded that Riggs B, Melton L, Khosla S. Serum proinflammatory cytokines such as TNF can serotonin levels are related to bone blunt osteoblast proliferation and accelerate density and structural parameters in accrual of bone in young mice by blocking women. J Bone Miner Res. Wnt signaling. 2009;24(Suppl 1). [Abstract]

Wnt Inhibitors and the Regulation of 3. Secreto F, Hoeppner L, Whitney T, Bone Mass Razidlo D, Stensgard B, Li X, Evans G, Hefferan T, Yaszemski M, Westendorf J. As part of Lexicon's Genome5000 program Lef1∆N dependent increases in bone to characterize mouse knockout phenotypes mass result from enhanced osteoblast of pharmaceutically relevant genes, activity. J Bone Miner Res. secreted frizzled related protein-4 (sFRP4) 2009;24(Suppl 1). [Abstract] KO mice were generated (16). sFRPs are known to antagonize Wnt signaling either by 4. Zhou H, Mak W, Zheng Y, Dunstan CR, functioning as decoy receptors for Wnts or Seibel MJ. Osteoblasts directly control by forming non-functional complexes with lineage commitment of mesenchymal frizzled (17). A previous study reported that progenitor cells through Wnt signaling. J osteoblast-targeted overexpression of Biol Chem. 2008 Jan 25;283(4):1936- sFRP4 in mice resulted in low trabecular 45. bone mass compared to wild type littermates (18). In the current study's sFRP4 KO 5. Shao X, Fong-Yee C, Dunstan C, Jia W, analysis, the authors found an increase in Chen D, Seibel M, Zhou H. Differential trabecular bone mass, which is concordant regulation of osteoblast differentiation by with the previous observation (18). In Wnt7b and Wnt10b. J Bone Miner Res. addition to the increased trabecular bone, 2009;24(Suppl 1). [Abstract] ablation of sFRP4 was associated with an increased cortical bone diameter but a 6. Tu X, Joeng KS, Nakayama KI, reduced cortical bone thickness. Associated Nakayama K, Rajagopal J, Carroll TJ, with these changes in bone mass and McMahon AP, Long F. Noncanonical structure, bone breaking strength (maximal Wnt signaling through G protein-linked load) was elevated by 42% in vertebral PKCdelta activation promotes bone bodies but reduced 23% in the femoral formation. Dev Cell. 2007 shaft. These data, together with previous Jan;12(1):113-27. observations, clearly indicate that sFRP4 influences both cortical and trabecular bone 7. Kramer I, Merdes M, Jeker H, Keller H, Kneissel M. Sost deficiency dependent

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bone gain is blunted in osteocyte expression. J Bone Miner Res. 2009; specific beta-catenin mutant mice. J 24(Suppl 1). [Abstract] Bone Miner Res. 2009;24(Suppl 1). [Abstract] 13. Caverzasio J. Non-canonical Wnt signaling: what is its role in bone? IBMS 8. Kramer I, Merdes M, Keller H, Kneissel BoneKEy. 2009 Mar;6(3):107-15. M. Sost exerts its action via Lrp5 dependent and independent pathways 14. Morioka K, Matsuda K, Kawano H, to control bone formation in vivo. J Bone Nakamura K, Kawaguchi H, Nakamura Miner Res. 2009;24(Suppl 1). [Abstract] Y. Identification of receptor tyrosine kinase-like orphan receptor 2 (ROR2)/ 9. Oliver L, Halleux C, Morvan F, Hu S, Lu WNT5B signaling as a therapeutic target C, Bauer A, Kneissel M. LRP4 is a novel against osteosarcoma. J Bone Miner osteoblast and osteocyte expressed Res. 2009;24(Suppl 1). [Abstract] specific facilitator of SOST-mediated inhibition of in vitro bone formation. J 15. Gilbert L, Lu X, Chen H, Weitzmann M, Bone Miner Res. 2009;24(Suppl 1). Nanes M. Systemic bone loss in TNF [Abstract] expressing mice is due to suppression of Wnt signaling and osteoblast 10. Saxon L, Jackson B, Lanyon L, Price J. differentiation independent of osteoclast Absence of LRP5 in mice has no effect activity. J Bone Miner Res. on bone's adaptive response to loading 2009;24(Suppl 1). [Abstract] or disuse but the high bone mass G171V LRP5 gain of function mutation 16. Brommage R, Liu J, Lee E. Elevated is associated with an increase in trabecular bone mass with reduced loading's osteogenic effect and cortical bone thickness in sFRP4 protection from disuse. J Bone Miner knockout mice. J Bone Miner Res. Res. 2009;24(Suppl 1). [Abstract] 2009;24(Suppl 1). [Abstract]

11. Sawakami K, Robling AG, Ai M, Pitner 17. Kawano Y, Kypta R. Secreted ND, Liu D, Warden SJ, Li J, Maye P, antagonists of the Wnt signalling Rowe DW, Duncan RL, Warman ML, pathway. J Cell Sci. 2003 Jul 1;116(Pt Turner CH. The Wnt co-receptor LRP5 13):2627-34. is essential for skeletal mechanotransduction but not for the 18. Nakanishi R, Akiyama H, Kimura H, anabolic bone response to parathyroid Otsuki B, Shimizu M, Tsuboyama T, hormone treatment. J Biol Chem. 2006 Nakamura T. Osteoblast-targeted Aug 18;281(33):23698-711. expression of Sfrp4 in mice results in low bone mass. J Bone Miner Res. 2008 12. Hackfort B, Alvarez G, Akhter M, Cullen Feb;23(2):271-7. D. Differential effects of parathyroid hormone (PTH) and tibial compression on bone formation with variation in Lrp5

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