Research ArticleBone

Wnt1 is an Lrp5-independent bone-anabolic Wnt ligand

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Science Translational Medicine  07 Nov 2018:
Vol. 10, Issue 466, eaau7137
DOI: 10.1126/scitranslmed.aau7137
  • Fig. 1 Characteristics of patients with heterozygous WNT1 mutations.

    (A) Number of patient demographics: sex, age, genotype, and fracture history [Vert. Fx (n), number of vertebral fractures; Per. Fx (n), peripheral fractures] of all patients. (B) Radiographies showing typical fractures of the spine (left), distal femur (middle), and proximal femur (right) in patients (numbers 1, 3, 5). Arrows point to the fracture lines. (C) DXA measurement (Z scores) at the lumbar spine and (D) at the hip. (E) HR-pQCT at the distal tibia. (F) Table of mean values of bone parameters and percent change compared to controls (% ref) at the distal tibia (in bold are the most affected parameters). Tb.N, trabecular number; Tb.Th, trabecular thickness; Ct.Th, cortical thickness. (G and H) Age-related changes of bone volume per tissue volume (BV/TV) and cortical thickness (Ct.Th) at the distal tibia. (I) Trichrome-Goldner staining of an iliac crest biopsy from patient 3. Scale bars, 1 mm (left) and 50 μm (right). (J) Bone histomorphometry of the biopsy. Tb.Sp, trabecular separation; O.Th, osteoid thickness; OS/BS, osteoid surface per bone surface; OV/BV, osteoid volume per bone volume; Ob.S/BS, osteoblast surface per bone surface; Oc.S/BS, osteoclast surface per bone surface. (K) Serum osteocalcin and (L) BAP measured in all patients at initial presentation compared to the reference range (gray boxes). (M) DPD per creatinine measured in the urine. (N to P) Evolution of bone parameters in patient 1 over 6-year treatment with denosumab and teriparatide, (N) osteocalcin, (O) BAP, and (P) DPD and (Q) timeline (years) showing the clinical history. Gray boxes indicate the reference range for each parameter.

  • Fig. 2 Wnt1 inactivation in osteoblasts phenocopies WNT1 mutations in humans.

    (A) Strategy used to generate Wnt1 conditional deletion. The localization of the primers used for genotyping is indicated by the blue arrows for the floxed and by the orange arrows for the recombined alleles. (B) Genotyping of various tissues isolated from mice homozygote for the Wnt1 floxed allele intercrossed or not with the Runx2-Cre deleter. Genotyping of bone marrow–derived osteoblasts is shown. (C) X-ray of 24-week-old Wnt1fl/fl and Runx2-Cre;Wnt1fl/fl male littermates; the arrows indicate the presence of fractures. (D) μCT scan of the indicated bones and von Kossa staining of the tibia of Runx2-Cre;Wnt1fl/fl; the arrows indicate the presence of fractures. (E) Frequency distribution of the fractures in the various bones of 24-week-old Runx2-Cre;Wnt1fl/fl mice. n = 4 mice carrying a total of 21 detectable fractures (Fx). (F) Longitudinal section of μCT-scanned femora of Wnt1fl/fl and Runx2-Cre;Wnt1fl/fl. (G) Quantification of the trabecular BV/TV and (H) of the cortical thickness. (I) Force necessary to fracture femurs as determined by three-point bending test. n = 4 (Wnt1fl/fl) and n = 3 (Runx2-Cre;Wnt1fl/fl) for (G) to (I). Data are the means ± SEM. **P < 0.01; *P < 0.05 (unpaired t test).

  • Fig. 3 The inducible Tet-off Wnt1 transgene is functional.

    (A) Schematization of the Tet-off system used to generate the transgenic mice. The tTA driver is controlled by the Col1a1 promoter gene to regulate the expression of Wnt1 transgene in the osteoblastogenic lineage. Wnt1 expression is silenced in the presence of DOX (+Dox) and induced when removing it (−Dox) from the food. (B to D) Quantitative real-time fluorescence polymerase chain reaction (QPCR) analysis of Wnt1 expression (B) in long bones (LB) and calvariae (Calv) of 6-week-old male control or Wnt1 transgenic (Wnt1Tg) mice 2 days after removing the DOX (−) compared to littermates maintained with DOX (+) (n ≥ 3) and in various organs of 6-week-old control (C) or Wnt1Tg (D) males after removing DOX for 1 week [note that the y-axis scales differ between (C) and (D); n ≥ 2]. WAT, white adipose tissue; BAT, brown adipose tissue; bm, bone marrow; Calv, calvariae. (E) Enzyme-linked immunosorbent assay (ELISA) quantification of circulating Wnt1 in control and Wnt1Tg males 3 weeks (3) and 9 weeks (9) after DOX removal, starting from the age of 3 weeks (w). n = 3. (F) Low (top) and high (bottom) magnification of Wnt1 immunostaining in tibiae of 6-week-old control and Wnt1Tg mice 1 week after DOX removal; white arrowheads indicate the presence of osteoblasts lining trabecular bone (b). (G) X-ray of 12-week-old control and Wnt1Tg male mice (9 weeks after DOX removal). Data are the means ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05 [unpaired t test (E) or one-way analysis of variance (ANOVA) (B)].

  • Fig. 4 High bone mass is induced when switching on Wnt1 in growing mice.

    (A) von Kossa staining of vertebrae and tibiae of 6-week-old control and Wnt1Tg male mice after removing DOX for 3 weeks. (B to D) Histomorphometric analysis of the trabecular bone parameters of 6-week-old mice after removing DOX for 3 weeks (−) compared to mice kept under DOX (+). The same analysis was performed in 12-week-old mice after inducing the transgene for 9 weeks. n ≥ 3. (B) Bone volume per tissue volume (BV/TV). (C) Trabecular thickness (Tb.Th). (D) Trabecular numbers (Tb.N). (E) Representative μCT of longitudinal (top panels) and of transversal sections at midshaft (bottom panels) scans of femora of 12-week-old control and Wnt1 transgenic mice 9 weeks after removing DOX. (F) Quantification of cortical thickness in 6-week-old mice 3 weeks after removing the DOX or in 12-week-old mice 9 weeks after induction. n ≥ 4. Data are the means ± SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 [one-way ANOVA (B to D) or unpaired t test (F)].

  • Fig. 5 Wnt1 is a bone-anabolic Wnt ligand.

    (A) Representative toluidine blue staining of vertebral sections of 12-week-old male mice after removing DOX for 9 weeks. (B to E) Histomorphometric analysis of the cellular components of the bones after removing DOX for 9 weeks (−) compared to mice kept under DOX (+). (B) Osteoclast numbers per bone perimeter (Oc.N/B.Pm). (C) Osteoclast surface area per bone surface (Oc.S/BS). (D) Osteoblast numbers per bone perimeter (Ob.N/B.Pm). (E) Osteoblast surface area per bone surface (Ob.S/BS). (F) Representative low-magnification and (G) high-magnification calcein double labeling of sections of the vertebrae of 12-week-old control and Wnt1Tg, 9 weeks after removing the DOX. (H to J) Dynamic histomorphometry analysis of (H) mineralizing surface per bone surface, (I) MAR, and (J) BFR in the 12-week-old mice, 9 weeks after inducing the transgene. n ≥ 3. Data are the means ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05 (one-way ANOVA).

  • Fig. 6 Wnt1 induces bone formation in aging mice.

    (A) von Kossa staining of sections of vertebra of 1-year-old mice maintained for 9 weeks with DOX-free food. (B) Histomorphometric analysis of the bone parameters (BV/TV, Tb.Th, and Tb.N). (C) Histomorphometric quantification of osteoblast surface area and number and (D) of osteoclast surface area and number. (E) Quantification of the BFR in the spine of the mice. BS, bone surface area. (F) μCT imaging of the femora, longitudinal section, and detail of the trabecular bone. (G) μCT quantification of the endochondral BV/TV, the trabecular thickness, and the trabecular number. (H) μCT imaging of transversal section at the midshaft of the femur. (I) Quantification of the cortical thickness in (H). (J) μCT imaging of the calvaria. (K) Quantification of the calvarial thickness (Calv.) and porosity (Calv. porosity). n ≥ 3 (B to E), n ≥ 8 (G and I), and n ≥ 7 (K). Data are the means ± SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01 (unpaired t test).

  • Fig. 7 The bone-anabolic effect of Wnt1 is rapid.

    (A) von Kossa staining of sections of vertebrae (top) and tibiae (bottom) of 6-week-old Wnt1 transgenic male mice maintained for 1 week with DOX-free food. (B) Histomorphometric analysis of the bone parameters (BV/TV, Tb.Th, and Tb.N). n ≥ 6. (C) ELISA quantification of circulating Wnt1. n = 3. (D) Quantification of osteoclast number and osteoclast surface area and (E) of osteoblast number and osteoblast surface area. n ≥ 5. (F) QPCR analysis of the transgene expression in the calvaria of the Wnt1Tg mice 2 and 7 days after removing DOX; data are normalized to the control mice (red line). (G) QPCR analysis of the expression of markers of bone formation and (H) of potential Wnt/β-catenin target genes in the calvaria of the Wnt1Tg mice 2 and 7 days after removing DOX, normalized to control mice (red line). n ≥ 6 (F to H). Data are the means ± SEM. ****P < 0.0001; *P < 0.05 [unpaired t test (B to F) or one-sample t test (hypothetical value, 1; *P < 0.05; G and H)].

  • Fig. 8 The bone-anabolic effect of Wnt1 is independent of Lrp5.

    (A) von Kossa staining of sections of vertebrae and tibiae of 6-week-old male mice maintained for 1 week with DOX-free food. (B) Histomorphometric analysis of the bone parameters (BV/TV, Tb.Th, and Tb.N) in the vertebrae and (C) in the tibiae. (D) Quantification of osteoclast number and osteoclast surface area and (E) of osteoblast number and osteoblast surface area in the vertebrae. (F) Comparison of the BV/TV between Wnt1Tg and Wnt1Tg;Lrp5−/− mice 1 and 3 weeks after removing the DOX. (G) Comparison of the increased cortical thickness 3 weeks after removing the DOX. (H) Comparison of the BFR 3 weeks after removing the DOX. The values in (F), (G) and (H) are reported as fold changes of their respective controls (red lines, nontransgenic or Lrp5−/− mice). n = 5 (B, D, and E), n ≥ 3 (C, F, and H), n ≥ 4 (G). Data are the means ± SEM. ****P < 0.0001; **P < 0.01; *P < 0.05 (unpaired t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/466/eaau7137/DC1

    Materials and Methods

    Fig. S1. Age-related decreased bone mass in the radius of patients with WNT1 mutation.

    Fig. S2. Inactivation of Wnt1 in osteoclasts does not affect bone remodeling.

    Fig. S3. Histology of the fractures in Runx2-cre;Wnt1fl/fl mice.

    Fig. S4. Wnt1 is a general bone-anabolic molecule.

    Fig. S5. Wnt1 induces bone formation in adult mice.

    Fig. S6. Wnt1 induction protects aging female from bone loss.

    Fig. S7. Wnt1 expression induces a rapidly increased bone mass in adult mice.

    Fig. S8. Wnt1 is not directly stimulating osteoblast differentiation.

    Fig. S9. Wnt1 is not up-regulated during osteoblast differentiation.

    Table S1. Individual subject-level data (Excel file).

    References (5058)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Age-related decreased bone mass in the radius of patients with WNT1 mutation.
    • Fig. S2. Inactivation of Wnt1 in osteoclasts does not affect bone remodeling.
    • Fig. S3. Histology of the fractures in Runx2-cre;Wnt1fl/fl mice.
    • Fig. S4. Wnt1 is a general bone-anabolic molecule.
    • Fig. S5. Wnt1 induces bone formation in adult mice.
    • Fig. S6. Wnt1 induction protects aging female from bone loss.
    • Fig. S7. Wnt1 expression induces a rapidly increased bone mass in adult mice.
    • Fig. S8. Wnt1 is not directly stimulating osteoblast differentiation.
    • Fig. S9. Wnt1 is not up-regulated during osteoblast differentiation.
    • References (5058)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Individual subject-level data (Excel file).

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