1. Volume 11, Issue 2, Pages 161-171 (February 2010)
Suppression of Wnt Signaling by Dkk1 Attenuates PTH-Mediated Stromal Cell Response and New Bone Formation 
Jun Guo, Minlin Liu, Dehong Yang, Mary L. Bouxsein, Hiroaki Saito, R.J. Sells Galvin, Stuart A. Kuhstoss, Clare C. Thomas, Ernestina Schipani, Roland Baron, F. Richard Bringhurst, Henry M. Kronenberg 
Cell Metabolism 
Volume 11, Issue 2, Pages (February 2010)
DOI: /j.cmet
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
PTH Suppresses Dkk1 mRNA Expression, and Overexpression of Dkk1 in Osteoblasts Attenuates Wnt Signaling in Bone
(A) The cultured rudiments were treated with vehicle or human PTH(1–34) (PTH, 100 nM) for 6 hr and then processed for in situ hybridization with 35S-labeled Dkk1 antisense riboprobe. White spots (arrows) seen with dark field microscopy show intensity of Dkk1 mRNAs in osteoblastic cells.
(B) E16.5 tibiae were treated with vehicle (blue bars) or PTH (pink bars) for indicated hours, and then total RNA was isolated for qRT-PCR analysis with the primers specific for Dkk1. Data are expressed as percent vehicle-treated control; error bars represent standard error (∗p < 0.05 versus vehicle).
(C) Targeting construct for generation of transgenic mouse lines by pronuclear injection.
(D) In situ hybridization analysis of E18.5 and 6-week tibiae dissected from WT or Dkk1 transgenic mice (Tg) with S35-labeled riboprobes specific for Dkk1 transgene mRNA (Tg-specific) or for both endogenous and transgenic Dkk1 mRNA (Dkk1 mRNA).
(E) High-magnification bright field of Dkk1 in situ hybridization in tibial primary spongiosa from 6-week-old WT and Tg mice. Black spots indicate Dkk1 mRNAs in osteoblastic cells (arrows).
(F) qRT-PCR analysis of mRNA expression in 12-week-old humeri with primers that detect both endogenous and transgenic Dkk1 mRNA.
(G) qRT-PCR analysis of mRNA expression in 6-week-old tibiae of TOPGAL mice with (Tg) or without (WT) transgenic Dkk1 with primers for Axin2 mRNA (filled bar) and TOPGAL mRNA (open bar). Data are expressed as percent mRNA level of WT, and error bars represent standard error (n = 6, ∗p < 0.05 versus WT) (see also Figures S1 and S2).
Cell Metabolism  , DOI: ( /j.cmet )
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3. Figure 2 Low Bone Mass in Dkk1 Mice
(A) von Kossa stain of plastic sections from 12-week-old proximal tibiae.
(B) μCT analysis of 12-week-old distal femurs and vertebrae.
(C) Histomorphometric analysis of 12-week-old tibiae. Abbreviations: BFR, bone formation rate; BV/TV, bone volume/total volume; Conn.density, connectivity density; MAR, mineral apposition rate; N.Ob/BS, osteoblast number per bone surface; N.Oc/BS, osteoclast number per bone surface; Ob.S/BS, osteoblast surface per bone surface; Oc.S/BS, osteoclast surface per bone surface; SMI, structure model index; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness. Results are given as mean ± SD. Statistical analysis was performed by Student's t test. For all panels, ∗p < 0.05 and ∗∗p < 0.01 versus WT (n = 6–8) (see also Figure S3).
Cell Metabolism  , DOI: ( /j.cmet )
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4. Figure 3
Attenuated Stromal Cell Response and New Bone Formation in Dkk1 Mice with Secondary Hyperparathyroidism
(A) Both von Kossa and Masson-Goldner trichrome stain were performed on plastic sections of proximal tibiae of 6-week-old mice fed a low-calcium diet or control diet for 3 weeks. von Kossa stain reveals mineral in bone, and Masson-Goldner trichrome stain demonstrates extensive peritrabecular fibroblast-like cell accumulation (arrowheads) and substantial woven bone-like newly formed bone (with high density of osteocytes and less mineral as stained in red, arrows), particularly in the metaphyseal trabecular regions as indicated in a lower-magnification view, in WT but not in Tg mice fed the low-calcium diet.
(B) The bar graph shows measurements of percent of the bone volume formed by the “fibrotic” cells of the peritrabecular stromal cell response. Data are represented as percent fibroblastic volume (Fb.V)/bone volume (BV), and error bars represent standard error, n = 6; ∗p < 0.05 versus control diet (see also Figures S4 and S5).
Cell Metabolism  , DOI: ( /j.cmet )
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5. Figure 4
Strikingly Curtailed Bone Formation and Increased Bone Resorption in Dkk1 Mice after Continuous PTH Infusion
(A and B) (A) Masson-Goldner trichrome stain on plastic sections and (B) TRAP activity stain on paraffin sections from proximal tibiae of 14-week-old WT and Tg mice receiving continuous infusion of human PTH(1–34) (100 μg/kg/day) (PTH) or of vehicle for 2 weeks. PTH infusion increased metaphyseal trabecular bone formation and stromal cell response (arrows in [A] in WT but not in Tg mice).
(C) Serum PINP and CTX measured in 14-week-old WT and Tg mice receiving continuous PTH infusion for 2 weeks. Error bars represent standard error, n = 6; ∗p < 0.05 versus day 0; and b, p < 0.05 versus WT.
Cell Metabolism  , DOI: ( /j.cmet )
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6. Figure 5
Extensive Peritrabecular Stromal Cell Responses Caused by Constitutively Active PTH Receptor Were Strikingly Curtailed in Dkk1 Mice, and PTH Activates Wnt Signaling Even in the Presence of Dkk1
(A) Masson-Goldner trichrome stain of plastic sections from 8-week proximal tibiae revealed extensive peritrabecular fibroblast-like cell accumulation (arrowheads) in Jansen transgenic mice that express a constitutively active PTH receptor, but not in mice expressing both transgenes at the same time. The bar graph shows fibroblastic measurements in the metaphyseal trabecular regions as an indication of peritrabecular stromal cell response. Data are represented as percent Fb.V/BV, and error bars represent standard error, n = 6; ∗p < 0.05 versus Jansen alone.
(B) PTH activates Wnt signaling in vivo even in Dkk1 mice. Total RNA was isolated for analysis of TOPGAL and Axin2 mRNAs from tibiae of 6-week-old TOPGAL mice with (Tg) or without (WT) transgenic Dkk1 receiving subcutaneous injection of human PTH(1–34) at a dose of 2 mg/kg 24 hr before sacrifice. Data are represented as percent WT vehicle injection. Error bars represent standard error, n = 6; ∗p < 0.05 versus vehicle injection.
Cell Metabolism  , DOI: ( /j.cmet )
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7. Figure 6
Effect of PTH and Wnt3a on Wnt Target Genes and β-Catenin in Primary Osteoblasts
(A and B) qRT-PCR analysis of Wnt target genes (Axin2, Tcf1, and TOPGAL) in primary osteoblasts derived from 6-week-old WT and Dkk1 transgenic (Tg) mice that were crossed with TOPGAL mice. Primary osteoblasts were cultured in 6-well plates with αMEM medium supplemented with 10% FBS. At 70% confluence, primary cells were refed with fresh medium and 48 hr later were treated for 6 hr with vehicle (Veh), hPTH (PTH, 100 nM), Wnt3a (Wnt, 10 ng/ml), Dkk1 (500 ng/ml), and PTH plus Wnt3a or plus Dkk1 (A), or with Dkk1 antibody (Ab, 25 μg/ml), PTH plus Dkk1 antibody (PTH+Ab), Wnt3a plus Dkk1 antibody (Wnt+Ab), or PTH plus Wnt3a and Dkk1 antibody (PTH+Wnt+Ab) (B), as indicated. a, p < 0.05 versus vehicle; b, p < 0.05 versus Wnt3a or versus Wnt3a plus Dkk1 antibody; c, p < 0.05 versus PTH. Same experiment was repeated three times with similar results.
(C) Western blot analysis of nuclear β-catenin. Primary osteoblasts from WT tibiae were treated with hPTH (100 nM) and/or Wnt3a (10 ng/ml), Dkk1 (500 ng/ml) for 16 hr, and nuclear extracts were analyzed by western blotting with anti-β-catenin antibody and densitometry with AlphaImager Relative levels (bar graph) have been obtained from three independent experiments. ∗p < 0.05 versus vehicle; ˆp < 0.05 versus Wnt3a alone (see also Figure S6).
Error bars represent standard error.
Cell Metabolism  , DOI: ( /j.cmet )
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8. Figure 7 Effect of PTH on β-Catenin, Lef1, and Dkk1 in MC3T3E1 Cells
(A and B) PTH had no significant effect on nuclear β-catenin translocation. Nuclear β-catenin localization was evaluated in MC3T3E1 cells stimulated with vehicle, PTH (100 nM) Wnt3a (25 ng/ml), or PTH plus Wnt3a for 24 hr, as indicated. β-catenin was detected by immunofluorescence confocal microscopy with anti-β-catenin antibody (A). MC3T3E1 cells were treated with PTH, Fsk, and/or Wnt3a at the indicated concentrations for 24 hr, and nuclear extracts were analyzed by western blotting with anti-β-catenin antibody and densitometry with AlphaImager Relative levels have been obtained from three independent experiments (B).
(C) Immunoprecipitation and western blot analysis of PKA-mediated phosphorylation of β-catenin. Total cell lysates from MC3T3E1 cells treated with PTH (100 nM) or Fsk (10 μM) for 20 min after serum-free culture for 24 hr were immunoprecipitated with anti-β-catenin antibody, and the immune complexes were analyzed by western blotting with antibodies against phosporylated PKA substrate (PKAS) or β-catenin.
(D) Western blot analysis of Lef1 expression. MC3T3E1 cells were treated with PTH (100 nM), Fsk (10 μM), Wnt3a (30 ng/ml), or Fsk plus Wnt3a (F+W) for 24 hr, and total cell lysates were analyzed by western blotting with anti-Lef1 antibody.
(E) qRT-PCR analysis of Dkk1 and Lef1 expression. MC3T3E1 cells were treated with PTH (100 nM) for the indicated time periods. Error bars represent standard error, and similar results have been obtained from three independent experiments. ∗p < 0.01 versus basal (0 hr) (see also Figure S7).
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2010 Elsevier Inc. Terms and Conditions