Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading. A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading. Over the past few years the Wnt/β-catenin-signaling pathway has been shown to be an important component of bone mass accrual, regulation, and maintenance. Key to this understanding were the identification of inactivating mutations in LRP5 resulting in an osteoporosis pseudoglioma syndrome (1Gong Y. Slee R.B. Fukai N. Rawadi G. Roman-Roman S. Reginato A.M. Wang 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. Cell. 2001; 107: 513-523Abstract Full Text Full Text PDF PubMed Scopus (1853) Google Scholar) and gain of function mutations in the LRP5 gene that give rise to a high bone mass phenotype in humans (2Boyden L.M. Mao J. Belsky J. Mitzner L. Farhi A. Mitnick M.A. Wu D. Insogna K. Lifton R.P. N. Engl. J. Med. 2002; 346: 1513-1521Crossref PubMed Scopus (1333) Google Scholar, 3Little R.D. Carulli J.P. Del Mastro R.G. Dupuis J. Osborne M. Folz C. Manning S.P. Swain P.M. Zhao S.C. Eustace B. Lappe M.M. Spitzer L. Zweier S. Braunschweiger K. Benchekroun Y. Hu X. Adair R. Chee L. FitzGerald M.G. Tulig C. Caruso A. Tzellas N. Bawa A. Franklin B. McGuire S. Nogues X. Gong G. Allen K.M. Anisowicz A. Morales A.J. Lomedico P.T. Recker S.M. Van Eerdewegh P. Recker R.R. Johnson M.L. Am. J. Hum. Genet. 2002; 70: 11-19Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 4Van Wesenbeeck L. Cleiren E. Gram J. Beals R.K. Benichou O. Scopelliti D. Key L. Renton T. Bartels C. Gong Y. Warman M.L. De Vernejoul M.C. Bollerslev J. Van Hul W. Am. J. Hum. Genet. 2003; 72: 763-771Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Both of these conditions can be mimicked in mice (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar, 6Kato M. Patel M.S. Levasseur R. Lobov I. Chang B.H. Glass Jr., D.A. Hartmann C. Li L. Hwang T.H. Brayton C.F. Lang R.A. Karsenty G. Chan L. J. Cell Biol. 2002; 157: 303-314Crossref PubMed Scopus (941) Google Scholar). LRP5 and LRP6 have been shown to be co-receptors (7Wehrli M. Dougan S.T. Caldwell K. O'Keefe L. Schwartz S. Vaizel-Ohayon D. Schejter E. Tomlinson A. DiNardo S. Nature. 2000; 407: 527-530Crossref PubMed Scopus (723) Google Scholar, 8Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 9Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1088) Google Scholar, 10Pinson K.I. Brennan J. Monkley S. Avery B.J. Skarnes W.C. Nature. 2000; 407: 535-538Crossref PubMed Scopus (890) Google Scholar) with the frizzled family of receptors and are involved in signaling through the canonical Wnt/β-catenin pathway (11Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (883) Google Scholar, 12Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1733) Google Scholar). Upon binding of extracellular Wnt to LRP5/6 and frizzled coreceptor complex, dishevelled (Dsh) is activated, causing axin recruitment to the membrane (where it interacts with LRP5/6) and inactivation of glycogen synthase kinase-3β (GSK-3β) 7The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; HBM, high bone mass; PG, prostaglandin; MAR, mineral apposition rate; SFRP1, secreted frizzled-related protein 1; WISP2, Wnt1-inducible signaling pathway protein 2; eNOS, endothelial nitric-oxide synthase; Cxn43, connexin 43; FzD2, frizzled 2; CCND1, cyclin D1. 7The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; HBM, high bone mass; PG, prostaglandin; MAR, mineral apposition rate; SFRP1, secreted frizzled-related protein 1; WISP2, Wnt1-inducible signaling pathway protein 2; eNOS, endothelial nitric-oxide synthase; Cxn43, connexin 43; FzD2, frizzled 2; CCND1, cyclin D1. that results in stabilization and accumulation of β-catenin in the cytoplasm (13Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (489) Google Scholar). Stabilized β-catenin translocates to the nucleus and together with T-cell factor/lymphoid enhancer binding factor transcription factors activates transcription (12Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1733) Google Scholar, 13Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (489) Google Scholar, 14Willert J. Epping M. Pollack J.R. Brown P.O. Nusse R. BMC Dev. Biol. 2002; 2: 8Crossref PubMed Scopus (318) Google Scholar). Regulation of GSK-3β is a key step in the pathway, and inhibitors of this enzyme can lead to β-catenin stabilization and initiation of target gene expression independent of Wnt binding. Signaling through this pathway is also regulated at the extracellular level by a number of modulator proteins (15Mao B. Niehrs C. Gene (Amst.). 2003; 302: 179-183Crossref PubMed Scopus (264) Google Scholar, 16Mao B. Wu W. Davidson G. Marhold J. Li M. Mechler B.M. Delius H. Hoppe D. Stannek P. Walter C. Glinka A. Niehrs C. Nature. 2002; 417: 664-667Crossref PubMed Scopus (859) Google Scholar, 17Jones S.E. Jomary C. BioEssays. 2002; 24: 811-820Crossref PubMed Scopus (349) Google Scholar, 18Mao B. Wu W. Li Y. Hoppe D. Stannek P. Glinka A. Niehrs C. Nature. 2001; 411: 321-325Crossref PubMed Scopus (894) Google Scholar, 19Bafico A. Liu G. Yaniv A. Gazit A. Aaronson S.A. Nat. Cell Biol. 2001; 3: 683-686Crossref PubMed Scopus (662) Google Scholar, 20Semenov M.V. Tamai K. Brott B.K. Kuhl M. Sokol S. He X. Curr. Biol. 2001; 11: 951-961Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 21Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Development. 2003; 130: 4295-4305Crossref PubMed Scopus (271) Google Scholar, 22Li X. Zhang Y. Kang H. Liu W. Liu P. Zhang J. Harris S.E. Wu D. J. Biol. Chem. 2005; 280: 19883-19887Abstract Full Text Full Text PDF PubMed Scopus (1042) Google Scholar). We have found that the increased peak bone mass that results in mice overexpressing the high bone mass (HBM) mutated gene product (LRP5 G171V) is due primarily to osteoblast-induced positive bone balance (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar). The bone mineral density in these animals plateaus and is maintained through 1.5 years of age without apparent abnormalities in bone shape or size. Furthermore, the LRP5 G171V mutation in mice results in a skeleton that has enhanced structural strength (femur, femoral neck, tibiae, and vertebral body), material properties (vertebral body), and bone mass/ash weight (ulnae) than their non-transgenic littermates (23Akhter M.P. Wells D.J. Short S.J. Cullen D.M. Johnson M.L. Haynatzki G.R. Babij P. Allen K.M. Yaworsky P.J. Bex F. Recker R.R. Bone (NY). 2004; 35: 162-169Crossref PubMed Scopus (123) Google Scholar). The denser and stronger bones in LRP5 G171V transgenic mice have been suggested to be due to greater sensitivity of bone to normal mechanical stimuli resulting in an altered response to weight-related forces (23Akhter M.P. Wells D.J. Short S.J. Cullen D.M. Johnson M.L. Haynatzki G.R. Babij P. Allen K.M. Yaworsky P.J. Bex F. Recker R.R. Bone (NY). 2004; 35: 162-169Crossref PubMed Scopus (123) Google Scholar). This pattern is quite similar to that seen clinically in humans where affected members have increased bone mineral density and bones that are of normal shape and size. In the LRP5 G171V HBM kindred the most significant changes in bone density are associated with the load bearing bones (24Johnson M.L. J. Musculoskelet. Neuronal Interact. 2004; 4: 135-138PubMed Google Scholar, 25Johnson M.L. Picconi J.L. Recker R.R. Endocrinologist. 2002; 12: 445-453Crossref Scopus (28) Google Scholar). Furthermore, we have shown that HBM transgenic mice (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar) have increased sensitivity to mechanical load (25Johnson M.L. Picconi J.L. Recker R.R. Endocrinologist. 2002; 12: 445-453Crossref Scopus (28) Google Scholar, 26Cullen D.M. Akhter M.P. Mace D. Johnson M.L. Babij P. Recker R.R. J. Bone Miner. Res. 2002; 17: 332Google Scholar), whereas Sawakami et al. (27Sawakami K. Robling A.G. Ai M. Pitner N.D. Liu D. Warden S.J. Li J. Maye P. Rowe D.W. Duncan R.L. Warman M.L. Turner C.H. J. Biol. Chem. 2006; 281: 23698-23711Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) have shown in vivo that bone formation response as a result of loading of LRP5-/- mice was nearly abolished compared with wild type mice. In addition, Hens et al. (28Hens J.R. Wilson K.M. Dann P. Chen X. Horowitz M.C. Wysolmerski J.J. J. Bone Miner. Res. 2005; 20: 1103-1113Crossref PubMed Scopus (165) Google Scholar) have shown the activation of the Wnt/β-catenin-signaling pathway in osteoblasts from TOPGAL mice containing a lacZ gene driven by a T-cell factor promoter. Finally, Clement-Lacroix et al. (29Clement-Lacroix P. Ai M. Morvan F. Roman-Roman S. Vayssiere B. Belleville C. Estrera K. Warman M.L. Baron R. Rawadi G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17406-17411Crossref PubMed Scopus (399) Google Scholar) have shown that the GSK-3β inhibitor LiCl increases bone formation in LRP5-/- mice. Collectively, these data strongly suggest that LRP5 and the Wnt/β-catenin-signaling pathway play a critical role in the bone response to mechanical loading. Although the molecular mechanisms by which mechanical loading affects bone mineral density have not been fully elucidated, various in vitro and in vivo models of mechanical loading have attributed it to increased cell proliferation, activation of cell signaling, and transcriptional activation of a number of genes. Rapid signaling responses have also been reported including changes in intracellular Ca+2, release of prostaglandins (PGE2 and PGI2), nitric oxide release, and increases in cAMP levels. (30Pavalko F.M. Gerard R.L. Ponik S.M. Gallagher P.J. Jin Y. Norvell S.M. J. Cell Physiol. 2003; 194: 194-205Crossref PubMed Scopus (81) Google Scholar, 31Hughes-Fulford M. Sci. STKE 2004. 2004; : RE12Google Scholar, 32Danciu T.E. Adam R.M. Naruse K. Freeman M.R. Hauschka P.V. FEBS Lett. 2003; 536: 193-197Crossref PubMed Scopus (144) Google Scholar, 33Nomura S. Takano-Yamamoto T. Matrix Biol. 2000; 19: 91-96Crossref PubMed Scopus (220) Google Scholar, 34Pommerenke H. Schmidt C. Durr F. Nebe B. Luthen F. Muller P. Rychly J. J. Bone Miner. Res. 2002; 17: 603-611Crossref PubMed Scopus (79) Google Scholar, 35Ziros P.G. Gil A.P. Georgakopoulos T. Habeos I. Kletsas D. Basdra E.K. Papavassiliou A.G. J. Biol. Chem. 2002; 277: 23934-23941Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The studies described in this report demonstrate that a number of target genes of the Wnt/β-catenin-signaling pathway are up-regulated in response to mechanical loading and that the LRP5 G171V mutation results in exaggerated increases in expression of several of these genes. We further show that activation of the pathway by treatment with a GSK-3β inhibitor results in an anabolic bone formation response and that use of this inhibitor in combination with mechanical loading produces a synergistic effect on the expression of Wnt/β-catenin pathway target genes. These results strongly implicate the Wnt/β-catenin-signaling pathway as being a critical component of the bone response to mechanical loading. Reagents—Calcein, hematoxylin, and eosin were obtained from Sigma-Aldrich. Fetal bovine serum (FBS), heat-inactivated FBS, α-minimum essential medium, Dulbecco's modified Eagle's medium, penicillin, streptomycin, l-glutamine, glutamax, and Geneticin were purchased from Invitrogen. Bovine serum albumin was purchased from Serologicals Proteins Inc. (Kankakee, IL). In Vivo Loading of Tibiae in LRP5 G171V Transgenic and Non-transgenic Littermates—All animal protocols were conducted with approval of the Wyeth and Creighton University Institutional Animal Care and Use Committees. The heterozygous LRP5 G171V transgenic mice have been described and show a statistically significant increase in bone density (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar). Non-transgenic littermates were used as controls. There were a total of 15 animals/sex/genotype in each group. At 17 weeks of age all animals were anesthetized to permit proper leg positioning before loading. Using a 4-point bending device (23Akhter M.P. Wells D.J. Short S.J. Cullen D.M. Johnson M.L. Haynatzki G.R. Babij P. Allen K.M. Yaworsky P.J. Bex F. Recker R.R. Bone (NY). 2004; 35: 162-169Crossref PubMed Scopus (123) Google Scholar), the mechanical loading regimen (∼2500 microstrain) 8Microstrain is defined as a unit of strain that is the percentage of change in length or relative deformation (10,000 microstrain = 0.01 strain = 1% deformation). delivered to the right tibiae (the left tibiae served as the non-loaded controls) was 6N for females and 7N for males (36 cycles, 2 Hz), which ensured that all mice experienced similar levels of maximal compressive and tensile strains during bending loads. RNA from the right tibiae was collected at 4 or 24 h after application of load. Tibiae from 5 mice were pooled to compose a single group. Three replicate groups for each treatment/genotype were analyzed. Cell Culture—MC3T3-E1 osteoblastic cells, used in the in vitro mechanical loading experiments, were cultured in αMEM supplemented with 10% heat inactivated fetal bovine serum, 1% glutamax, and 1% penicillin/streptomycin. Wnt3A-conditioned media was obtained from an overexpressing Wnt3A stable murine L-cell line (ATCC, Manassas, VA) that was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% l-glutamine, and 0.4 mg/ml Geneticin. To obtain Wnt3A-conditioned media, cells were seeded into 100-mm dishes and cultured for 4 days in growth medium without Geneticin, the medium was removed and sterile-filtered, and fresh medium was added to the plates and cultured for an additional 3 days. The medium was then removed and sterile-filtered and combined with the initial batch of cultured media. Control-conditioned medium was obtained in a similar fashion using the parental L-M(TK-) cell line (ATCC, Manassas, VA). The Wnt3A-conditioned media activated canonical Wnt signaling in MC3T3-E1 cells as determined using a T-cell factor-luciferase reporter transiently transfected into these cells (10 μl of conditioned media showed a 10-fold induction of reporter activity compared with control media-treated and untreated cells (data not shown). RNA Isolation—The mouse tibiae were dissected free of soft tissue, and the proximal and distal metaphysis were removed leaving the diaphysis. The tibiae were then cut transversely with bone cutters to expose the trabecular bone and the bone marrow. The trabecular bone and marrow cavities were flushed with ice-cold sterile phosphate-buffered saline to remove the marrow, and the clean bone was placed in liquid nitrogen. A Bessman tissue pulverizer (Fisher) rinsed in 100% ethanol and precooled in liquid nitrogen was used to reduce the tibiae to a powder. Total RNA (2 μg/tibia) was isolated from non-loaded and loaded bones using the ToTALLY RNA kit (Ambion, Austin TX) as per the manufacturer's instructions. Ten μg of total RNA was treated with 4 units of DNase I (Ambion) to remove any genomic DNA contamination. To isolate RNA from MC3T3-E1 cells the cultures were washed twice with 2 ml each of phosphate-buffered saline, and then the RNA was isolated using the QIAshredder and the RNeasy kit (Qiagen, Valencia, CA) as described by the manufacturer. The RNA was treated with 27 units of DNase I (Qiagen) on the RNA isolation column provided in the kit as described by the manufacturer. Quantitative Real-time RT-PCR was RNA was to at for 2 h were on an DNA using of The conditions for were 2 at at then each of 15 at and at on plates with of the and for each The expression for each mouse gene was to murine A of used in this can be found in and in a and and GSK-3β inhibitor M.P. D.A. P.S. L. D. Brown M.J. D. Smith J.C. Chem. Biol. 2000; 7: Full Text Full Text PDF PubMed Scopus Google Scholar) at or was for the days the right of the in mice. each treatment that animals was on and on for mineral apposition the were and the bones were in ethanol for 24 The of the was whereas the was used for The were with and eosin for of To mineral apposition of single and bone were and the was used to were on at were using the analysis of were in for h, in and β-catenin was using a mouse was using the was using the kit in of the mouse bone after in of on the in Vivo to wild type mice were with or twice for days. There were 15 animals per group. The right tibiae were loaded at 6N for at 2 The left tibiae served as controls. The animals were at 4 h and the tibia was isolated and in liquid nitrogen. Tibiae were pooled from each to per group. The RNA was from tibiae and were by on from the tibiae on and genes. In cells were at cells per in a type I and then cultured for days or before loading the cells were washed twice with 2 ml of α-minimum essential medium before 2 ml of fresh media containing bovine serum glutamax, and penicillin/streptomycin. before mechanical the medium was and ml of α-minimum essential serum albumin with or without Wnt3A-conditioned media or media was added to each The cells were subjected to mechanical to 3400 microstrain (2 for 5 h using a strain unit RNA was or 24 h from both the as as the controls. data are as the data non-loaded loaded was To strain and GSK-3β inhibitor or of analysis of was A was then each of the GSK-3β inhibitor or Wnt3A with strain the strain of in Vivo of has been reported that the LRP5 G171V transgenic mice have increased bone formation compared with non-transgenic mice (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar), and mechanical loading increases bone the that this mutation to altered of the Wnt/β-catenin pathway, we the that of the pathway have exaggerated changes in expression in response to loading compared with normal mice. We a preliminary analysis of gene expression from explant cultures from LRP5 G171V non-transgenic mice and several genes including Wnt10B, secreted frizzled-related protein cyclin and Wnt1-inducible signaling pathway protein 2 associated with the mutation (data not shown). We the expression of these Wnt/β-catenin target genes were regulated by mechanical loading. analysis of tibia from LRP5 G171V mice after in vivo mechanical loading by bending resulted in increases in the expression of genes including synthase synthase and endothelial nitric-oxide synthase S.C. S. G. J. PubMed Scopus Google Scholar, Cullen D.M. J.A. J. Bone Miner. Res. 12: PubMed Scopus Google Scholar) The expression of these genes was increased 4 h in wild type and LRP5 G171V transgenic mice however, the response was greater in the This response was 24 h (data not shown). In non-transgenic transcription of SFRP1, CCND1, connexin 43 and genes was increased at both 4 and 24 h after in vivo mechanical loading A and RNA levels of frizzled 2 was increased at 4 h to line by 24 of LRP5 was not affected by load in this whereas LRP6 expression was increased in LRP5 G171V animals at 4 h and In LRP5 G171V transgenic a significant and increase in the transcription of the Wnt/β-catenin target genes was including which was not regulated by mechanical loading in wild type mice A and the pattern demonstrated in the non-transgenic there was an increased expression of at both and application of mechanical load increased expression of and the response was greater in the LRP5 G171V