Volume 8, Issue 5, Pages 751-764 (May 2005) Canonical Wnt Signaling in Differentiated Osteoblasts Controls Osteoclast Differentiation  Donald A. Glass, Peter Bialek, Jong Deok Ahn, Michael Starbuck, Millan S. Patel, Hans Clevers, Mark M. Taketo, Fanxin Long, Andrew P. McMahon, Richard A. Lang, Gerard Karsenty  Developmental Cell  Volume 8, Issue 5, Pages 751-764 (May 2005) DOI: 10.1016/j.devcel.2005.02.017 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 TCF Transcription Factors Are Expressed in Osteoblasts (A) Left: Schematic representation of the TOPGAL transgene. Three canonical LEF/TCF binding sites and a minimal c-fos promoter upstream of the LacZ gene. Middle: β-galactosidase staining of an E18.5 TOPGAL embryo showed blue staining in limbs and ribs (10×). Right: Histological analysis of forelimb of E18.5 TOPGAL embryo, counterstained with eosin; osteoblasts stained blue (arrows) (400×). (B) In situ hybridization analysis for Runx2 (a, f, k, p, u, z), Lef1 (b, g, l, q, v, aa), Tcf1 (c, h, m, r, w, bb), Tcf3 (d, i, n, s, x, cc), and Tcf4 (e, j, o, t, y, dd) expression at E14.5 (a–e, p–t), E16.5 (f–j, u–y), and E18.5 (k–o, z–dd) on wt mandibles (a–o) and forelimbs (p–dd). Tcf1 is coexpressed with Runx2 in the mandible and forelimbs at all developmental time points analyzed, while Tcf4 expression is observed starting at E16.5. Lef1 expression is detected in forelimb chondrocytes at E14.5. Tcf3 expression was not detected at any time point analyzed (50×). (C) Real-time PCR analysis for Tcf1, Tcf4, and Lef1 of wt osteoblast RNA, normalized to 18S. Only Tcf1 and Tcf4 were detected in osteoblasts. (D) Western blot analysis for TCF-1, TCF-4, LEF-1, and Runx2 on nuclear extracts from primary osteoblasts and control tissues (intestine for TCF-4; thymus for all others). TCF-1 and TCF-4 are detected in osteoblasts. (E) Left: Schematic representation of activating (top) and repressive (bottom) isoforms of Tcf1. The location of the β-catenin-interacting domain (BID) and real-time PCR primers for detection of Tcf1 isoforms (Tcf1ACTIV and Tcf1ALL, respectively) are indicated. Right: Real-time PCR analysis for Tcf1ACTIV and Tcf1ALL expression in osteoblasts and thymus, normalized to 18S. Activating isoforms of Tcf1 are more abundant in osteoblasts than in thymus. Error bars represent standard error. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Activation of β-Catenin in Osteoblasts Leads to an Osteoclast Defect (A) PCR analysis of genomic DNA from calvaria (c), skin (s), and thymus (t) of wt and βcat(ex3)ob mice. A lower migrating band is observed when exon 3 is absent from βcat(ex3)ob calvaria (arrow). (B) Immunohistochemical analysis of newborn wt (top) and βcat(ex3)ob (bottom) calvaria using an anti-β-catenin antibody. Increased levels of β-catenin are seen in cytoplasms and nuclei of βcat(ex3)ob osteoblasts (arrows). (C) Survival curve of βcat(ex3)ob mice. βcat(ex3)ob mice survived only when fed a liquid diet. (D) Left: Lower incisors are absent in a 3-week-old βcat(ex3)ob mouse. Right: X-ray analysis of 3-week-old wt (top) and βcat(ex3)ob (bottom) skulls shows that lower incisors formed (arrow) but failed to erupt through the mandible of the βcat(ex3)ob mouse (20×). (E) Histomorphometric analysis of 3-week-old wt and βcat(ex3)ob vertebrae shows increased bone volume (BV/TV) in βcat(ex3)ob mice (25×). (F) Alcian blue staining in 3-week-old wt and βcat(ex3)ob tibia shows cartilaginous remnants in βcat(ex3)ob mice (50×). (G) TRAP staining in 3-week-old wt and βcat(ex3)ob vertebrae. βcat(ex3)ob osteoclasts are smaller and fewer in number (400×). (H) Urinary deoxypyridinoline crosslinks (Dpd) elimination is decreased in 3-week-old βcat(ex3)ob mice compared to wt. (I) Number of osteoblasts in 3-week-old wt and βcat(ex3)ob vertebrae is similar. (J) Left: Rib osteomata (arrows) are present in 3-week-old βcat(ex3)ob mice (20×). Right: Histological analysis of wt (top) and βcat(ex3)ob (bottom) ribs. Note the densely packed irregular bone trabeculae lined by osteoblasts in the βcat(ex3)ob mouse (200×). (K and L) Calvarial RNA microarray results identify many genes dysregulated greater than 2-fold in βcat(ex3)ob mice and in Lrp5−/− mice compared to wt. Error bars represent standard error. Asterisk indicates statistical significance. p < 0.05. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Osteoprotegerin Expression Is Increased in βcat(ex3)ob Osteoblasts (A) In situ hybridization for Opg (a–f) at E14.5 (a, b), E16.5 (c, d), and E18.5 (e, f) on wt (a, c, e) and βcat(ex3)ob (b, d, f) forelimbs. Opg expression is increased in βcat(ex3)ob forelimbs (50×). (B) Real-time PCR analysis for Opg (black bars) and Rankl (white bars) of wt and βcat(ex3)ob osteoblasts, normalized to 18S. Opg expression is increased in βcat(ex3)ob osteoblasts. (C) Northern blot analysis of wt and βcat(ex3)ob long bone and osteoblast RNA. Blots were sequentially probed for Opg, Rankl, and Gapdh as a control. Note the increased Opg expression in βcat(ex3)ob cells. (D) Northern blot analysis of wt, βcat(ex3)ob, and Lrp5−/− long bone RNA for Opg; Gapdh serves as a control. Opg expression is increased in βcat(ex3)ob but unchanged in Lrp5−/− bones. (E) ELISA analysis for OPG in serum from wt and βcat(ex3)ob mice. Note the increased levels of OPG in βcat(ex3)ob mice. (F) In situ hybridization for Runx2 (a–f), α1(I) Collagen (g–l), and α2(I) Collagen (m–r) at E14.5 (a, b, g, h, m, n), E16.5 (c, d, i, j, o, p), and E18.5 (e, f, k, l, q, r) on wt (a, c, e, g, i, k, m, o, q) and βcat(ex3)ob (b, d, f, h, j, l, n, p, r) forelimbs. α1(I) Collagen and α2(I) Collagen expression is increased in βcat(ex3)ob forelimbs (50×). (G) Northern blot analysis of wt and βcat(ex3)ob long bone RNA. Blots were sequentially probed for Runx2, Osterix, ATF4, Fra1, and ΔFosB. No detectable change in expression levels of these genes is observed. (H) Real-time PCR analysis for α1(I) Collagen of wt and βcat(ex3)ob osteoblasts, normalized to 18S. α1(I) Collagen expression is increased in βcat(ex3)ob osteoblasts. (I) Northern blot analysis for α1(I) Collagen of wt and βcat(ex3)ob long bone RNA; Gapdh serves as a control. α1(I) Collagen expression is increased in βcat(ex3)ob bones. Error bars represent standard error. Asterisk indicates statistical significance. p < 0.05. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Low Bone Mass, Increased Osteoclast Activity, and Decreased Opg Expression in Mice Lacking β-Catenin in Osteoblasts (A) PCR analysis of genomic DNA from wt, βcatob+/−, and Δβcatob calvaria. A lower migrating band is observed when exons 2–6 are removed from βcatob+/− and Δβcatob calvaria (arrow). (B) Immunohistochemical analysis of newborn wt (left) and Δβcatob (right) calvaria using an anti-β-catenin antibody. Decreased levels of β-catenin are seen in cytoplasms and nuclei of Δβcatob osteoblasts. (C) Histomorphometric analysis of 1-month-old wt and Δβcatob vertebrae shows decreased bone volume (BV/TV) in Δβcatob mice (25×). (D) TRAP staining of 1-month-old wt and Δβcatob vertebrae. Δβcatob osteoclasts are larger and greater in number (400×). (E) Dpd elimination is increased in Δβcatob mice. (F) Number of osteoblasts in 1-month-old wt and Δβcatob vertebrae is similar. (G) Calcein double labeling of 1-month-old wt and Δβcatob vertebrae. Bone formation rate (BFR) is similar in both mice (400×). (H) Northern blot analysis of wt and Δβcatob long bone and osteoblast RNA. Blots were sequentially probed for Opg, Rankl, and Gapdh as a control. Opg expression is decreased in Δβcatob cells. (I) Real-time PCR analysis for Opg (black bars) and Rankl (white bars) in wt and Δβcatob osteoblasts, normalized to 18S. Opg expression in decreased in Δβcatob osteoblasts. (J) In situ hybridization for Runx2 (a, b), Osterix (c, d), α1(I) Collagen (e, f), α2(I) Collagen (g, h), and Osteocalcin (i, j) at E16.5 on forelimbs of wt (a, c, e, g, i) and Δβcatob (b, d, f, h, j) embryos. (K) Real-time PCR analysis for Osterix (black bars) and Osteocalcin (white bars) in wt and Δβcatob osteoblasts, normalized to 18S. Error bars represent standard error. Asterisk indicates statistical significance. p < 0.05. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The Bone Resorption Defect in β-Catenin Mutant Mice Is Osteoblast Autonomous (A) TRAP (+) cells were counted following coculture of wt or Δβcatob bone marrow monocytes (BMMs) with either wt or Δβcatob osteoblasts. Δβcatob osteoblasts increased osteoclast differentiation regardless of the BMM genotype. (B) TRAP (+) cell number following coculture of wt or βcat(ex3)ob BMMs with either wt or βcat(ex3)ob osteoblasts. βcat(ex3)ob osteoblasts decreased osteoclast differentiation regardless of the BMM genotype. (C) TRAP (+) cell number following coculture of wt BMMs with either wt or βcat(ex3)ob osteoblasts, with or without an anti-OPG antibody. βcat(ex3)ob osteoblasts decreased osteoclast differentiation of wt BMMs while the anti-OPG antibody prevented this effect. (D) BrdU incorporation of newborn wt, Δβcatob, and βcat(ex3)ob calvarial sections. The percentage of proliferating cells was comparable between wt, Δβcatob, and βcat(ex3)ob mice. (E) TUNEL analysis of newborn wt, Δβcatob, and βcat(ex3)ob calvarial sections. The percentage of apoptotic cells was comparable between wt, Δβcatob, and βcat(ex3)ob mice. Error bars represent standard error. Asterisk indicates statistical significance. p < 0.05. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Regulation of Osteoclast Differentiation by β-Catenin Requires TCF Proteins (A) Histomorphometric analysis of 1-month-old wt and Tcf1−/− vertebrae shows decreased bone volume (BV/TV) in Tcf1−/− mice (25×). (B) TRAP staining of 1-month-old wt and Tcf1−/− vertebrae. Osteoclast number is increased in Tcf1−/− mice (400×). (C) Dpd elimination is increased in Tcf1−/− mice. (D) Number of osteoblasts in 1-month-old wt and Tcf1−/− vertebrae is similar. (E) Calcein double labeling of 1-month-old wt and Tcf1−/− vertebrae. Bone formation rate (BFR) is similar in both mice (400×). (F) Northern blot analysis of wt and Tcf1−/− osteoblast RNA. Blots were sequentially probed for Opg, Rankl, and Gapdh. Note the decreased Opg expression in Tcf1−/− cells. (G) Histomorphometric analysis of 1-month-old wt, βcatob+/−, Tcf1+/−, and βcatob+/−;Tcf1+/− vertebrae shows decreased bone volume (BV/TV) only in βcatob+/−;Tcf1+/− mice (25×). (H) TRAP staining of 1-month-old wt and βcatob+/−;Tcf1+/− vertebrae. βcatob+/−;Tcf1+/− osteoclasts are larger and greater in number (400×). (I) Number of osteoblasts in 1-month-old wt and βcatob+/−;Tcf1+/− vertebrae is similar. (J) Calcein double labeling of 1-month-old wt and βcatob+/−;Tcf1+/− vertebrae. Bone formation rate (BFR) is similar in both mice (400×). (K) Real-time PCR analysis for Opg in wt and βcatob+/−;Tcf1+/− bones, normalized to 18S. Note the decreased Opg expression is βcatob+/−;Tcf1+/− bones. Error bars represent standard error. Asterisk indicates statistical significance. p < 0.05. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Osteoprotegerin Expression Is Regulated by β-Catenin and TCF Proteins (A) Schematic representation of the proximal 3.6 kb of the Osteoprotegerin promoter with the three putative canonical TCF binding sites indicated by black boxes. (B) Electromobility shift assay (EMSA). Osteoblast nuclear extracts (NE) were incubated with labeled Opg site 3 (lane 1), site 2 (lane 2), or site 1 oligonucleotides (lane 3). A protein-DNA complex formed only upon incubation of NE with site 1. (C) EMSA. Osteoblast NE were incubated with antibodies against TCF-1 (lanes 2 and 5), TCF-4 (lanes 3 and 5), LEF-1 (lane 7), Runx2 (lane 8), or with preimmune serum (lanes 1, 4, and 6) prior to incubation with labeled Opg site 1 oligonucleotides. Only antibodies against TCF-1 and TCF-4 decreased binding to the Opg site 1 oligonucleotides. (D) Chromatin immunoprecipitation for Runx2, TCF-1, TCF-4, and β-catenin from primary osteoblast cultures. Antibodies against the TCF proteins and β-catenin can immunoprecipitate site 1 of the Opg promoter, but not the Osteocalcin promoter (OSE) or the Opg coding sequence (CDS). (E) ROS17/2.8 cells were transiently transfected with wt 3.6 kb Opg promoter-luc (pOpg 3.6-luc) or Opg promoter-luc reporter constructs with either site 1, site 3, or sites 2 and 3 mutated (pOpgm1-luc, pOpgm3-luc, or pOpgm2+3-luc, respectively). Only the mutation of site 1 decreased Opg promoter activity. (F) COS7 cells were transiently transfected with either pOpg 3.6-luc, pOpgm1-luc, pOpgm3-luc, or pOpgm2+3-luc reporter constructs with or without HA-Lef1 and/or β-cateninactiv. LEF-1 increased transactivational activity of pOpg 3.6-luc, pOpgm3-luc, and pOpgm2+3-luc, which was further increased with β-catenin. In contrast, LEF-1 failed to activate pOpgm1-luc. Error bars represent standard error. Developmental Cell 2005 8, 751-764DOI: (10.1016/j.devcel.2005.02.017) Copyright © 2005 Elsevier Inc. Terms and Conditions