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Essential Role of β-Catenin in Postnatal Bone Acquisition*

Open AccessPublished:March 31, 2005DOI:https://doi.org/10.1074/jbc.M501900200
      Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the β-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of β-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of β-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the β-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-κB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/β-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.
      The Wnt signaling pathway is implicated in the regulation of bone mineral density (
      • Gong 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.
      ,
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ,
      • Kato M.
      • Patel M.S.
      • Levasseur R.
      • Lobov I.
      • Chang B.H.
      • Glass II, D.A.
      • Hartmann C.
      • Li L.
      • Hwang T.H.
      • Brayton C.F.
      • Lang R.A.
      • Karsenty G.
      • Chan L.
      ,
      • Little 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.
      ,
      • Babij 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.
      ). Wnt ligands bind and activate a specific cellular receptor complex composed of a member of the frizzled family of seven transmembrane-spanning proteins and either LRP5 or LRP6 (
      • He X.
      • Semenov M.
      • Tamai K.
      • Zeng X.
      ). LRP5 and LRP6 are members of a distinct subfamily of the low density lipoprotein receptor proteins (
      • He X.
      • Semenov M.
      • Tamai K.
      • Zeng X.
      ). Loss of LRP5 causes osteoporosis pseudoglioma syndrome in humans, which is characterized by low bone density at an early age (
      • De Paepe A.
      • Leroy J.G.
      • Nuytinck L.
      • Meire F.
      • Capoen J.
      ,
      • Gong Y.
      • Vikkula M.
      • Boon L.
      • Liu J.
      • Beighton P.
      • Ramesar R.
      • Peltonen L.
      • Somer H.
      • Hirose T.
      • Dallapiccola B.
      • De Paepe A.
      • Swoboda W.
      • Zabel B.
      • Superti-Furga A.
      • Steinmann B.
      • Brunner H.G.
      • Jans A.
      • Boles R.G.
      • Adkins W.
      • van den Boogaard M.J.
      • Olsen B.R.
      • Warman M.L.
      ), whereas point mutations in LRP5 are associated with extremely high bone density in an autosomal dominant inheritance pattern in humans and mice (
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ,
      • Little 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.
      ,
      • Babij 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.
      ,
      • Van 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.
      ).
      Binding of cognate receptors by Wnt ligands can initiate several downstream signaling cascades (
      • He X.
      • Semenov M.
      • Tamai K.
      • Zeng X.
      ).
      R. Nusse, personal communication.
      1R. Nusse, personal communication.
      Activation of the “canonical” pathway involves the stabilization of cytoplasmic levels of β-catenin. The main function of the adenomatous polyposis coli (APC)
      The abbreviations used are: APC, adenomatous polyposis coli; OPG, osteoprotegerin; μCT, microcomputed tomography; RANKL, receptor activated by nuclear factor-κB ligand; RT, reverse transcription; OC, osteocalcin.
      2The abbreviations used are: APC, adenomatous polyposis coli; OPG, osteoprotegerin; μCT, microcomputed tomography; RANKL, receptor activated by nuclear factor-κB ligand; RT, reverse transcription; OC, osteocalcin.
      protein appears to be the normal degradation of β-catenin. In the absence of APC, β-catenin levels are elevated, which ultimately contributes to increased proliferation (
      • Polakis P.
      ). This pathway requires either LRP5 or LRP6 for activity (
      • He X.
      • Semenov M.
      • Tamai K.
      • Zeng X.
      ), but LRP5 and LRP6 may have other cellular functions that are not directly related to regulation of Wnt signaling. For example, mice lacking LRP5 display defects in cholesterol and glucose metabolism (
      • Fujino T.
      • Asaba H.
      • Kang M.J.
      • Ikeda Y.
      • Sone H.
      • Takada S.
      • Kim D.H.
      • Ioka R.X.
      • Ono M.
      • Tomoyori H.
      • Okubo M.
      • Murase T.
      • Kamataki A.
      • Yamamoto J.
      • Magoori K.
      • Takahashi S.
      • Miyamoto Y.
      • Oishi H.
      • Nose M.
      • Okazaki M.
      • Usui S.
      • Imaizumi K.
      • Yanagisawa M.
      • Sakai J.
      • Yamamoto T.T.
      ). It remains unclear whether the bone defects seen in humans and mice lacking the Lrp5 gene are due solely to defects in Wnt signaling or if other downstream signaling cascades are involved.
      In this work, we have shown that osteoblast-specific deletion of the β-catenin gene leads to early onset, severe osteoporosis and is associated with defective osteoblast differentiation in vitro. In contrast, loss of Apc leads to early onset, severe osteopetrosis leading to lethality early in life. Interestingly, we show that these osteoblast-specific deletions are associated with alterations in the regulation of osteoprotegerin (OPG) and receptor activated by nuclear factor-κ B ligand (RANKL) and with altered osteoclastogenesis in vivo. Thus, this work provides the first genetic evidence that dysregulation of β-catenin in osteoblasts leads to defects in bone development and supplies the first link between Wnt/β-catenin signaling in osteoblasts and functional alterations of the osteoclast regulated by the OPG/RANKL signaling axis.

      EXPERIMENTAL PROCEDURES

      Mouse Crosses—OC-cre mice (
      • Zhang M.
      • Xuan S.
      • Bouxsein M.L.
      • von Stechow D.
      • Akeno N.
      • Faugere M.C.
      • Malluche H.
      • Zhao G.
      • Rosen C.J.
      • Efstratiadis A.
      • Clemens T.L.
      ) were mated with homozygous conditional mutants carrying modified Apc (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ) or β-catenin (
      • Brault V.
      • Moore R.
      • Kutsch S.
      • Ishibashi M.
      • Rowitch D.H.
      • McMahon A.P.
      • Sommer L.
      • Boussadia O.
      • Kemler R.
      ) alleles to generate OC-cre/Apcflox/+ and OC-cre/β-cateninflox/+ progeny, which were used in subsequent matings. All experiments performed were in compliance with the guiding principles of the “Care and Use of Animals” available at www.nap.edu/books/0309053773/html and approved prior to use by the Van Andel Research Institute Institutional Animal Care and Use Committee.
      Genotype Analysis—DNA was prepared from tail biopsies using an AutoGenprep 960 automated DNA isolation system. PCR-based strategies were then used to genotype these mice (details available upon request).
      Demineralized Bone Histology—Tissue samples were fixed in formalin overnight, decalcified in Immunocal decalcifying agent (Decal, Baltimore, MD) overnight, and then dehydrated through a graded alcohol series in a Ventana Renaissance processor (Ventana Medical Systems, Tucson, AZ). Tissues were paraffin-embedded, and 5-μm sections were adhered to glass slides. Slides were de-paraffinized and stained with hematoxylon and eosin or left unstained for immunohistochemistry.
      Mineralized Bone Histology—Femurs were fixed in ethanol at room temperature, dehydrated, and embedded in methylmethacrylate. 3-μm sections were cut with a Microm microtome and stained with modified Mason-Goldner trichrome stain. The number of osteoblasts and osteoclasts per bone perimeter were measured at standardized sites under the growth plate using a semiautomatic method (Osteoplan II; Kontron, Munich, Germany) at a magnification of ×200. These parameters comply with guidelines of the nomenclature committee of the American Society of Bone and Mineral Research (
      • Parfitt A.M.
      • Drezner M.K.
      • Glorieux F.H.
      • Kanis J.A.
      • Malluche H.
      • Meunier P.J.
      • Ott S.M.
      • Recker R.R.
      ).
      Osteoblast Isolation and Culture—Osteoblasts were isolated from calvaria of newborn mice by serial digestion in α-minimal essential medium (Mediatech, Herndon, VA) containing 10% bovine serum albumin, 25 mm HEPES, pH 7.4, 0.2 mg/ml collagenase type I (Worthington, Lakewood, NJ), 0.7 mg/ml collagenase type 2 (Worthington), and 1 mm CaCl2. Calvaria were digested for 15 min at 37 °C with constant agitation. The digestion solution was collected, washed with fresh medium, and digested an additional five times. Digestions 3–6 (containing the osteoblasts) were centrifuged, washed with α-minimal essential medium containing 10% fetal bovine serum, 1% pen/strep, and plated overnight at 37 °C. The next day, the cells were trypsinized and 1.1 × 105 cells were plated on 6-cm dishes. The medium was supplemented with 5 mm β-glycerophosphate (Sigma) and 100 μg/ml ascorbic acid (Sigma) (mineralization medium), which was replaced every other day.
      Immunohistochemistry—β-Catenin was detected in cells using a mouse monoclonal antibody (BD Transduction Laboratories) at a 1:100 dilution. The signal was detected with the Vectastain ABC kit (Vector Labs, Burlingame, CA) and visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Vector Labs). Slides were counterstained with hematoxylin.
      Von Kossa Staining—Cultures were maintained in differentiation medium for the days indicated and fixed in 10% neutral buffered formalin. The cells were washed with water, dehydrated, and allowed to air dry. Silver nitrate (2%) was added to the cells for 20 min. The cells were washed with water and then incubated with 5% sodium carbonate for 3 min.
      Microcomputed Tomography (μCT)—High resolution images of the femur were acquired using a desktop microtomographic imaging system (MicroCT40; Scanco Medical AG, Basserdorf, Switzerland). The femur was scanned at 45 keV with an isotropic voxel size of 6 μm, and the resulting two-dimensional cross-sectional images are shown in gray scale. Scanning was started in the mid-epiphysis and extended proximally for ∼3.6 mm (600 CT slices/specimen).
      OPG Serum Enzyme-linked Immunosorbent Assay—Serum was collected from animals, and serum OPG levels were quantitated using the mouse OPG/TNFSRSF11B immunoassay according to the manufacturer's specifications (R&D Systems, Minneapolis, MN).
      Semiquantitative RT-PCR—mRNA was extracted from cells using TRIzol (Invitrogen) extraction protocol. Briefly, the cells were homogenized in TRIzol and mRNA was extracted using chloroform. The RNA was precipitated by isopropanol, and 5 μg of mRNA was used to synthesize first strand cDNA using an Invitrogen superscript cDNA synthesis kit. RNA controls and reverse transcription controls were used for all reactions. First strand cDNA was amplified using the sequence-specific primer sets listed below at the indicated annealing temperatures (30 cycles), and the products were resolved on 1–2% agarose gels. 1-kb or 100-bp ladders (Invitrogen) were used as markers. Primers used were: OPG, (5′-GTGAAGCAGGAGTGCAAC-3′ and 5′-GCAAACTGTGTTTCGCTC-3′ at 54 °C annealing temperature); RANKL, (5′-TGTACTTTCGAGCGCAGATG-3′ and 5′-ACATCCAACCATGAGCCTTC-3′ at 59 °C); osteocalcin, (5′-CAAGTCCCACACAGCAGCTT-3′ and 5′-AAAGCCGAGCTGCCAGAGTT-3′ at 58 °C); Runx-2, (5′-CCAAATTTGCCTAACAGAATG-3′ and 5′-GAGGCTGTGGTTTCAAAGCA-3′ at 56 °C); and β-actin (5′-CTGAACCCTAAGGCCAACCGTG-3′ and 5′-GGCATACAGGGACAGCACAGCC-3′ at 56 °C).

      RESULTS

      Mice with Osteoblast-specific Deletions of β-Catenin or Apc Die by 4 Weeks of Age—Because global inactivation of either the β-catenin gene (
      • Haegel H.
      • Larue L.
      • Ohsugi M.
      • Fedorov L.
      • Herrenknecht K.
      • Kemler R.
      ,
      • Huelsken J.
      • Vogel R.
      • Brinkmann V.
      • Erdmann B.
      • Birchmeier C.
      • Birchmeier W.
      ) or Apc (
      • Fodde R.
      • Edelmann W.
      • Yang K.
      • van Leeuwen C.
      • Carlson C.
      • Renault B.
      • Breukel C.
      • Alt E.
      • Lipkin M.
      • Khan P.M.
      • Kucherlapati R.
      ,
      • Moser A.R.
      • Shoemaker A.R.
      • Connelly C.S.
      • Clipson L.
      • Gould K.A.
      • Luongo C.
      • Dove W.F.
      • Siggers P.H.
      • Gardner R.L.
      ) results in early embryonic death, we crossed mice containing conditionally inactivatable alleles of β-catenin (β-catenin-flox) (
      • Brault V.
      • Moore R.
      • Kutsch S.
      • Ishibashi M.
      • Rowitch D.H.
      • McMahon A.P.
      • Sommer L.
      • Boussadia O.
      • Kemler R.
      ) or Apc (Apc-flox) (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ) to mice expressing cre under the control of the osteocalcin (OC) promoter (
      • Zhang M.
      • Xuan S.
      • Bouxsein M.L.
      • von Stechow D.
      • Akeno N.
      • Faugere M.C.
      • Malluche H.
      • Zhao G.
      • Rosen C.J.
      • Efstratiadis A.
      • Clemens T.L.
      ) to disrupt these genes in osteoblasts. Previous analysis of the OC-cre strain via crossing it to strains carrying cre reporter transgenes indicated that expression of cre recombinase was specific to cells of the osteoblast lineage with no detectable expression or function in other cell lineages (
      • Zhang M.
      • Xuan S.
      • Bouxsein M.L.
      • von Stechow D.
      • Akeno N.
      • Faugere M.C.
      • Malluche H.
      • Zhao G.
      • Rosen C.J.
      • Efstratiadis A.
      • Clemens T.L.
      ). As expected, deletion of the β-catenin gene led to loss of β-catenin protein (Fig. 1, a and b). Consistent with its role in mediating β-catenin degradation (
      • Polakis P.
      ), deletion of Apc was associated with significantly elevated levels of β-catenin as assessed by immunohistochemistry (Fig. 1, b and c). By 1 week of age mice homozygous for either mutation could be identified by their reduced size (Fig. 1, d–g). OC-cre;β-catenin-flox/flox (Δβ-catenin, or Δβ-cat) mice died within 5 weeks (Fig. 1h). Growth retardation was even more severe in the OC-cre;Apc-flox/flox (ΔAPC) mice (Fig. 1g), and these mice generally succumbed within 2 weeks (Fig. 1i). At weaning, only 75% of the expected number of Δβ-catenin mice and only 10% of the expected number of ΔAPC mice were identified (Fig. 1, h and i). No defects in the incisors were observed either grossly or histologically, and the teeth erupted normally in both mutants (data not shown). The cause of the postnatal lethality in these mutant mice is currently unclear but is certainly not because of aberrant expression of the osteocalcin promoter in extraosseous tissues (
      • Zhang M.
      • Xuan S.
      • Bouxsein M.L.
      • von Stechow D.
      • Akeno N.
      • Faugere M.C.
      • Malluche H.
      • Zhao G.
      • Rosen C.J.
      • Efstratiadis A.
      • Clemens T.L.
      ). Importantly, mice carrying osteoblast-specific deletions of both the Apc and β-catenin genes (ΔAPC/Δβ-catenin) display growth and survival characteristics similar to those lacking only the β-catenin gene (data not shown), suggesting that the severe phenotype induced by loss of Apc is due to dysregulation of β-catenin signaling.
      Figure thumbnail gr1
      Fig. 1Comparison of mutant and control mice. a–c, immunohistochemical detection of β-catenin protein expression in cultured osteoblasts. a, Δβ-catenin osteoblasts, 40×; b, cre-/flox/flox osteoblasts; 40×; c, ΔAPC osteoblasts, 40×. d, appearance of Δβ-catenin mice at postnatal day 10; e, ΔAPC mice at postnatal day 11. Growth curve of Δβ-catenin (f) and ΔAPC (g) mice compared with control littermates. Survival curve for Δβ-catenin (h) and ΔAPC (i) mice.
      Δβ-Catenin and ΔAPC Mice Have Dramatic Defects in Bone Development—Analysis of femurs from Δβ-catenin mice by μCT revealed striking reductions in both the trabecular and cortical bone compartments (Fig. 2, a and b). In contrast, analysis of ΔAPC mice revealed a significant accumulation of bone matrix in the femur, to the point where the marrow space was almost completely filled (Fig. 2, d and e). Bone in the metaphyseal region was poorly mineralized, whereas osteoid in the diaphysis was more completely mineralized and entirely filled the marrow cavity. μCT images of femurs from ΔAPC/Δβ-catenin mice were similar to those seen in Δβ-catenin mice, again suggesting that loss of APC induced phenotypes in a β-catenin-dependent manner (Fig. 2c).
      Figure thumbnail gr2
      Fig. 2μCT of femurs from Δβ-catenin, ΔAPC, and control mice. a, 31-day-old wild-type mouse; b, 31-day-old Δβ-catenin mouse; c, 31-day old ΔAPC/Δβ-catenin mouse; d, 12-day-old wild-type mouse; e, 12-day-old ΔAPC littermate. All panels show images ranging from the midshaft (left side) to the epiphysis (right side).
      Examination of undecalcified sections from tibia of the Δβ-catenin mice disclosed a dramatic reduction in mineralized cortical and trabecular bone (Fig. 3, a–d), consistent with changes observed by μCT. By contrast, the ΔAPC mice had dramatically increased bone deposition associated with disturbances in bone architecture and composition (Fig. 3, e–h). For example, the growth plate of ΔAPC mice lacked a secondary ossification center and was misshapen (Fig. 3g), possibly because of the rapid rate of bone formation and the lack of osteoclasts (see below) that would normally function to shape the ends of the bone. Further analysis of decalcified bone sections from 4-week-old Δβ-catenin and 2-week-old ΔAPC mice showed marked abnormalities in all bone examined, including vertebrae, long bones, and calvaria (Fig. 4).
      Figure thumbnail gr3
      Fig. 3Mineralized histology of bones from Δβ-catenin and ΔAPC mice. a and b, 30-day-old wild-type mouse; c and d, 30-day-old Δβ-catenin littermate. d, inset, a small portion of bone lined by a group of osteoclasts (*). e and f, 12-day-old wild-type mouse; g and h, 12-day-old ΔAPC littermate. Note the dramatic increase in mineralized bone (blue staining).
      Figure thumbnail gr4
      Fig. 4Histological analysis of decalcified bone from Δβ-catenin and ΔAPC mice. a, d, and g, femur; b, e, and h, vertebrae; c, f, and i, skull from 32-day-old Δβ-catenin (a–c) and ΔAPC (g–i) mice as compared with age-matched (d–f) control littermates. The skull sections were taken from equivalent positions immediately dorsal to the hippocampus. Calibration marks are indicated.
      Effects of Dysregulation of β-Catenin on Osteoblast Differentiation in Vitro—To examine the cellular mechanisms responsible for these disturbances in the mutant mice, we studied the effect of conditional deletion of the β-catenin and Apc genes in calvarial osteoblasts in vitro. Cells derived from mice carrying the floxed alleles were infected with adenovirus expressing the cre recombinase (Cre+) or a control adenovirus directing the expression of green fluorescent protein (Cre–) and then differentiated in the presence of β-glycerol phosphate and ascorbate (mineralizing medium). No obvious qualitative differences in proliferation rates or osteoblast density were observed in osteoblasts mutant for either gene (data not shown). However, consistent with the osteopenic phenotype of the Δβ-catenin mice, osteoblasts deficient in β-catenin showed delayed and diminished expression of osteocalcin as well as a marked reduction in calcified nodule formation (von Kossa staining). Interestingly, Runx-2 expression levels were similar in control and β-catenin osteoblasts (Fig. 5a), suggesting that early events in the osteoblast differentiation program (
      • Karsenty G.
      ) do not depend on signaling through β-catenin. In contrast, ΔAPC osteoblasts appeared to mature and mineralize normally in vitro, although osteocalcin expression levels increased somewhat prematurely as compared with controls (Fig. 5b). It appears, therefore, that excess β-catenin signaling does not severely impact the ability of osteoblasts to differentiate, at least in vitro. Thus, the more dramatic effects of disruption of the β-catenin gene observed in vivo may be due to non-cell autonomous functions of APC in osteoblasts.
      Figure thumbnail gr5
      Fig. 5In vitro differentiation of β-catenin or Apc flox/flox osteoblasts infected with either adenovirus-green fluorescent protein (Cre–) or adenovirus-Cre (Cre+). a, β-catenin-deficient (Cre+) and control (Cre–) osteoblasts were stained for mineralization by Von Kossa staining after 15 days in mineralization medium. mRNA was collected after 5, 10, and 15 days (d) in culture. RT-PCR for osteocalcin (OCN) and Runx2 are shown. RT-PCR for β-actin is included as a control. b, assays identical to those in panel a were performed on APC-deficient (Cre+) and control osteoblasts (Cre–).
      Dysregulation of β-Catenin in Osteoblasts Is Associated with Abnormal Osteoclastogenesis—Initial histological analysis (Fig. 3) suggested a disturbance in osteoclastogenesis in both mutants. Indeed, quantitation of osteoclasts in representative long bone sections showed that osteoclast numbers were dramatically increased in the Δβ-catenin mutants but entirely absent in the ΔAPC mice (Fig. 6a). In addition, osteoblasts were dramatically decreased in Δβ-catenin mutants at 4 weeks of age and were absent in ΔAPC mice at 2 weeks of age (data not shown). However, the dramatic deposition of osteoid material in ΔAPC mice suggests they were present at an earlier age. These findings suggested that dysregulated β-catenin signaling in osteoblasts not only causes cell-autonomous osteoblast defects but can also impact bone resorption by altering the numbers of osteoclasts. To explore this possibility further, we measured the expression of RANKL, the major osteoclast differentiation factor, and the osteoclast inhibitory factor, OPG (
      • Hofbauer L.C.
      • Schoppet M.
      ). In Δβ-catenin osteoblasts, OPG mRNA expression was decreased compared with controls after 5 and 10 days of differentiation in mineralization medium, whereas RANKL expression was increased (Fig. 6b). The reverse pattern was observed in the ΔAPC osteoblasts (Fig. 6c). In addition, levels of serum OPG in the ΔAPC mice were 3-fold higher than controls (Fig. 6d). Serum OPG levels were similar to controls in both Δβ-catenin and ΔAPC/Δβ-catenin mice (data not shown). Taken together, these observations suggest that alterations in β-catenin signaling in osteoblasts brought about by each mutation lead to marked disturbances in osteoclast differentiation.
      Figure thumbnail gr6
      Fig. 6Alterations in β-catenin signaling in osteoblasts affect osteoclastogenesis. a, histomorphometry was performed on a representative OC-cre;β-catenin-flox/flox mouse (Δβ-catenin) and a 31-day-old littermate (control) and on an OC-cre;Apc-flox/floxAPC) 12-day-old mouse and a littermate control. The numbers of osteoclasts observed per 100 mm peripheral bone surface are shown. b and c, calvarial osteoblasts derived from either β-catenin-flox/flox (b) or Apc-flox/flox (c) newborn mice were infected with either adenovirus-green fluorescent protein (Cre–) or adenovirus-Cre (Cre+). Semiquantitative RT-PCR was performed to evaluate expression of osteoprotegerin (OPG) and RANKL. RT-PCR for β-actin was performed as a control (shown in ). c, assays identical to those in panel b were performed on Apc-deficient (Cre+) and control osteoblasts (Cre–). d, the levels of OPG in the serum of 2–3-week-old ΔAPC mice (n = 3) are shown relative to control littermates (n = 3). OPG levels in ΔAPC mice are significantly increased (p <0.01).

      DISCUSSION

      In this study we used conditional mutagenesis to disrupt selectively the genes encoding β-catenin and Apc in mouse osteoblasts. In both models, osteoblast-specific disruption of the canonical Wnt signaling pathway led to postnatal death. In the case of the ΔAPC mice, we speculate that deficiencies in hematopoiesis may be responsible. As shown, ΔAPC mice develop bone in which the vast majority of the marrow component is absent. In other situations where osteopetrosis is observed in humans and mice, it is often accompanied with hepatosplenomegaly associated with extramedullary hematopoiesis. Interestingly, despite severe osteopetrosis, we have not observed any evidence of extramedullary hematopoiesis in these mice. Importantly, mice carrying osteoblast-specific deletions of both the Apc and the β-catenin genes are phenotypically similar to those lacking only the β-catenin gene (Fig. 2), suggesting that the majority of the phenotype induced by loss of APC is because of dysregulated β-catenin signaling. In the case of Δβ-catenin mice, we occasionally observe animals with paralysis, consistent with osteoporotic-related fractures, accounting for some portion of the lethality observed. However, we find that many of these animals die very suddenly between 26 and 30 days of age (Fig. 1) without prior evidence of paralysis. One explanation is that catastrophic fractures may occur leading to full paralysis and sudden death. Alternatively, defects in hematopoietic cell regulation or other systemic defects may play a role.
      The skeletal phenotypes observed in these mice allow two important conclusions regarding the role of Wnt signaling in skeletal development. First, Wnt signaling controls postnatal bone acquisition by determining β-catenin levels in osteoblasts, and the previously identified alterations in bone mass in humans carrying mutations in LRP5 (
      • Gong 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.
      ,
      • Boyden L.M.
      • Mao J.
      • Belsky J.
      • Mitzner L.
      • Farhi A.
      • Mitnick M.A.
      • Wu D.
      • Insogna K.
      • Lifton R.P.
      ,
      • Little 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.
      ,
      • Van 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.
      ) result from aberrant β-catenin signaling. However, it is important to note that the bone phenotypes resulting from inactivation of osteoblast β-catenin signaling, although consistent with the alterations seen in patients carrying mutations in LRP5, were more severe. One explanation for this difference is the potential role of LRP6 in β-catenin regulation in osteoblasts. We speculate that the continued presence of LRP6 in the context of LRP5 deficiency allows residual Wnt signaling through β-catenin. In agreement with this idea, we (
      • Holmen S.L.
      • Giambernardi T.A.
      • Zylstra C.R.
      • Buckner-Berghuis B.D.
      • Resau J.H.
      • Hess J.F.
      • Glatt V.
      • Bouxsein M.L.
      • Ai M.
      • Warman W.W.
      • Williams B.O.
      ) and others (
      • Kelly O.G.
      • Pinson K.I.
      • Skarnes W.C.
      ) have shown that mice carrying mutations in both Lrp5 and Lrp6 show synergistic defects in bone development. In an alternative model, functions of β-catenin independent of Wnt signaling contribute to the observed phenotype. For example, β-catenin also plays a key role in mediating cell-cell adhesion through its interaction with E-cadherin (
      • Nelson W.J.
      • Nusse R.
      ). E-cadherin is a single-pass transmembrane protein that forms homotypic dimers with E-cadherin proteins expressed on adjacent cells (
      • Nelson W.J.
      • Nusse R.
      ). The intracellular portion of E-cadherin binds directly to either β-catenin or the related plakoglobin protein, which then associates with α-catenin. The association of α-catenin with the actin cytoskeleton completes the coupling of cell-cell adhesion to the cytoskeleton. Although some studies have suggested that plakoglobin and β-catenin are interchangeable in mediating these cell adhesion complexes (
      • Wheelock M.J.
      • Johnson K.R.
      ), it is possible that loss of β-catenin could also affect cell-cell adhesion in osteoblasts. However, the fact that ΔAPC mice exhibit the converse phenotype suggests that the functions of β-catenin in canonical Wnt signaling underlie the observed phenotypes. Also, our observations that mice carrying osteoblast-specific mutations in both Apc and β-catenin genes have phenotypes similar to those seen in mice mutant for only the β-catenin gene suggests that the osteopetrosis seen in ΔAPC animals is due to dysregulation of β-catenin signaling. A more definitive picture of the role of cadherin-mediated adhesion in osteoblast differentiation may be provided by creating mice carrying osteoblast-specific knockouts of the α-catenin gene. This will disrupt cadherin-based adhesion in osteoblasts without directly affecting the canonical Wnt signaling pathway.
      The second conclusion from our studies is that β-catenin signaling in the mature osteoblast controls postnatal bone acquisition through both cell-autonomous and non-autonomous mechanisms. Thus, β-catenin signaling may not be required for the initial commitment of cells to the osteoblast lineage, based on Runx2 expression (
      • Kato M.
      • Patel M.S.
      • Levasseur R.
      • Lobov I.
      • Chang B.H.
      • Glass II, D.A.
      • Hartmann C.
      • Li L.
      • Hwang T.H.
      • Brayton C.F.
      • Lang R.A.
      • Karsenty G.
      • Chan L.
      ,
      • Karsenty G.
      ), but appears to be essential for the performance of the more mature osteoblast (e.g. osteocalcin expression and mineralization) (
      • Aronow M.A.
      • Gerstenfeld L.C.
      • Owen T.A.
      • Tassinari M.S.
      • Stein G.S.
      • Lian J.B.
      ,
      • Owen T.A.
      • Aronow M.
      • Shalhoub V.
      • Barone L.M.
      • Wilming L.
      • Tassinari M.S.
      • Kennedy M.B.
      • Pockwinse S.
      • Lian J.B.
      • Stein G.S.
      ). However, the marked alterations in osteoclast numbers and reciprocal patterns of expression of OPG and RANKL in the context of dysregulated β-catenin signaling suggest that non-cell autonomous effects on osteoclast progenitors also impact overall bone acquisition. Further support for this latter effect has come from a recent report indicating that the OPG gene may be a direct transcriptional target for complexes containing the β-catenin protein (
      • Jackson A.
      • Vayssiere B.
      • Garcia T.
      • Newell W.
      • Baron R.
      • Roman-Roman S.
      • Rawadi G.
      ).
      These combined effects of Wnt signaling through the canonical pathway in mature osteoblasts are therefore critical in the control of normal bone acquisition, and this pathway represents a plausible pharmaceutical target for treating osteoporosis and other bone disorders.

      Acknowledgments

      We thank Tetsuo Noda for the Apc-flox embrymonic stem cells and Rolf Kemler for making β-catenin-flox mice available through the Jackson Laboratories. We thank Pam Swiatek and the Van Andel Research Institute (VARI) Mouse Germ Line Modification Core for generating Apc-flox mice from these ES cells. We also thank Kyle Furge for advice on statistics, David Nadziejka for critically reading the manuscript, Phil Sohn for help formatting figures, Jim Resau and the VARI Laboratory of Analytical, Cellular, and Molecular Microscopy for assistance, and Bryn Eagleson, Jason Martin, Sylvia Marinelli, and the rest of the VARI vivarium staff for expert animal husbandry. We acknowledge the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for supporting these cores (Michigan Animals Models Consortium Grant 085P1000815).

      References

        • Gong 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: 513-523
        • Boyden 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: 1513-1521
        • Kato M.
        • Patel M.S.
        • Levasseur R.
        • Lobov I.
        • Chang B.H.
        • Glass II, D.A.
        • Hartmann C.
        • Li L.
        • Hwang T.H.
        • Brayton C.F.
        • Lang R.A.
        • Karsenty G.
        • Chan L.
        J. Cell Biol. 2002; 157: 303-314
        • Little 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: 11-19
        • Babij 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: 960-974
        • He X.
        • Semenov M.
        • Tamai K.
        • Zeng X.
        Development. 2004; 131: 1663-1677
        • De Paepe A.
        • Leroy J.G.
        • Nuytinck L.
        • Meire F.
        • Capoen J.
        Am. J. Med. Genet. 1993; 45: 30-37
        • Gong Y.
        • Vikkula M.
        • Boon L.
        • Liu J.
        • Beighton P.
        • Ramesar R.
        • Peltonen L.
        • Somer H.
        • Hirose T.
        • Dallapiccola B.
        • De Paepe A.
        • Swoboda W.
        • Zabel B.
        • Superti-Furga A.
        • Steinmann B.
        • Brunner H.G.
        • Jans A.
        • Boles R.G.
        • Adkins W.
        • van den Boogaard M.J.
        • Olsen B.R.
        • Warman M.L.
        Am. J. Hum. Genet. 1996; 59: 146-151
        • Van 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: 763-771
        • Jackson A.
        • Vayssiere B.
        • Garcia T.
        • Newell W.
        • Baron R.
        • Roman-Roman S.
        • Rawadi G.
        Bone. 2005; 36: 585-598
        • Polakis P.
        Curr. Opin. Genet. Dev. 1999; 9: 15-21
        • Fujino T.
        • Asaba H.
        • Kang M.J.
        • Ikeda Y.
        • Sone H.
        • Takada S.
        • Kim D.H.
        • Ioka R.X.
        • Ono M.
        • Tomoyori H.
        • Okubo M.
        • Murase T.
        • Kamataki A.
        • Yamamoto J.
        • Magoori K.
        • Takahashi S.
        • Miyamoto Y.
        • Oishi H.
        • Nose M.
        • Okazaki M.
        • Usui S.
        • Imaizumi K.
        • Yanagisawa M.
        • Sakai J.
        • Yamamoto T.T.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 229-234
        • Parfitt A.M.
        • Drezner M.K.
        • Glorieux F.H.
        • Kanis J.A.
        • Malluche H.
        • Meunier P.J.
        • Ott S.M.
        • Recker R.R.
        J. Bone Miner. Res. 1987; 2: 595-610
        • Haegel H.
        • Larue L.
        • Ohsugi M.
        • Fedorov L.
        • Herrenknecht K.
        • Kemler R.
        Development. 1995; 121: 3529-3537
        • Huelsken J.
        • Vogel R.
        • Brinkmann V.
        • Erdmann B.
        • Birchmeier C.
        • Birchmeier W.
        J. Cell Biol. 2000; 148: 567-578
        • Fodde R.
        • Edelmann W.
        • Yang K.
        • van Leeuwen C.
        • Carlson C.
        • Renault B.
        • Breukel C.
        • Alt E.
        • Lipkin M.
        • Khan P.M.
        • Kucherlapati R.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8969-8973
        • Moser A.R.
        • Shoemaker A.R.
        • Connelly C.S.
        • Clipson L.
        • Gould K.A.
        • Luongo C.
        • Dove W.F.
        • Siggers P.H.
        • Gardner R.L.
        Dev. Dyn. 1995; 203: 422-433
        • Brault V.
        • Moore R.
        • Kutsch S.
        • Ishibashi M.
        • Rowitch D.H.
        • McMahon A.P.
        • Sommer L.
        • Boussadia O.
        • Kemler R.
        Development. 2001; 128: 1253-1264
        • Shibata H.
        • Toyama K.
        • Shioya H.
        • Ito M.
        • Hirota M.
        • Hasegawa S.
        • Matsumoto H.
        • Takano H.
        • Akiyama T.
        • Toyoshima K.
        • Kanamaru R.
        • Kanegae Y.
        • Saito I.
        • Nakamura Y.
        • Shiba K.
        • Noda T.
        Science. 1997; 278: 120-123
        • Zhang M.
        • Xuan S.
        • Bouxsein M.L.
        • von Stechow D.
        • Akeno N.
        • Faugere M.C.
        • Malluche H.
        • Zhao G.
        • Rosen C.J.
        • Efstratiadis A.
        • Clemens T.L.
        J. Biol. Chem. 2002; 277: 44005-44012
        • Karsenty G.
        Endocrinology. 2001; 142: 2731-2733
        • Hofbauer L.C.
        • Schoppet M.
        J. Am. Med. Assoc. 2004; 292: 490-495
        • Holmen S.L.
        • Giambernardi T.A.
        • Zylstra C.R.
        • Buckner-Berghuis B.D.
        • Resau J.H.
        • Hess J.F.
        • Glatt V.
        • Bouxsein M.L.
        • Ai M.
        • Warman W.W.
        • Williams B.O.
        J. Bone Min. Res. 2004; 19: 2033-2040
        • Kelly O.G.
        • Pinson K.I.
        • Skarnes W.C.
        Development. 2004; 131: 2803-2815
        • Nelson W.J.
        • Nusse R.
        Science. 2004; 303: 1483-1487
        • Wheelock M.J.
        • Johnson K.R.
        Curr. Opin. Cell Biol. 2003; 15: 509-514
        • Aronow M.A.
        • Gerstenfeld L.C.
        • Owen T.A.
        • Tassinari M.S.
        • Stein G.S.
        • Lian J.B.
        J. Cell. Physiol. 1990; 143: 213-221
        • Owen T.A.
        • Aronow M.
        • Shalhoub V.
        • Barone L.M.
        • Wilming L.
        • Tassinari M.S.
        • Kennedy M.B.
        • Pockwinse S.
        • Lian J.B.
        • Stein G.S.
        J. Cell. Physiol. 1990; 143: 420-430