Lrp5 functions in bone to regulate bone mass

Journal name:
Nature Medicine
Volume:
17,
Pages:
684–691
Year published:
DOI:
doi:10.1038/nm.2388
Received
Accepted
Published online

Abstract

The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.

At a glance

Figures

  1. Generation and characterization of HBM-causing Lrp5 knock-in mice.
    Figure 1: Generation and characterization of HBM-causing Lrp5 knock-in mice.

    (a) Schematic depicting the targeting and genotyping strategies for Lrp5 HBM alleles. The 5′ targeting arm contains a neomycin-resistance cassette (NeoR) flanked by loxP sites (arrowheads). The 3′ targeting arm begins in intron 2 and extends into intron 4, and is followed by a thymidine kinase (TK) cassette. Site-directed mutagenesis altered specific amino acid residues encoded by Lrp5 exon 3 (asterisks). The relative locations and orientation of primers used for PCR genotyping and their expected amplimer sizes are noted. (b) Autoradiographs of a northern blot containing whole-bone total RNA from mice with different Lrp5 genotypes initially hybridized with a radioactive Lrp5 cDNA probe (top) and subsequently with a glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA probe which serves as a loading control (bottom). (c) Photograph of an agarose gel containing PCR amplimers derived from genomic DNA of mice with different Lrp5 genotypes. (d) Graphs depicting the areal bone mineral density (aBMD) measured by dual-energy X-ray absorptiometry (DEXA) in mice with different Lrp5 genotypes; mice followed until the age of 16.5 weeks (top and middle). Graphs depicting the percentage of trabecular bone volume in the total volume (BV/TV) of the distal femora and fifth lumbar vertebrae of 16.5-week-old male and female mice with different Lrp5 genotypes (bottom). (e) Representative μCT scan images obtained from 16.5-week-old mice with different Lrp5 genotypes. Scale bars, 1 mm. (f) Graphs depicting biomechanical properties of whole femora in a three-point bending assay from 16.5-week-old mice with different Lrp5 genotypes. (g) Representative images of new bone formation assessed by double calcein labeling of mice with different Lrp5 genotypes. Bone formation rates/bone surface area (BFR/BS); mineral apposition rates (MAR); mineralizing surface/bone surface (MS/BS). The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus WT mice; #P < 0.05 versus heterozygous Lrp5 HBM mice.

  2. Effect of activating Lrp5 NeoR-containing HBM alleles.
    Figure 2: Effect of activating Lrp5 NeoR-containing HBM alleles.

    (a) Graphs depicting femoral and vertebral trabecular BV/TV in WT mice and in mice with Lrp5 NeoR-containing HBM alleles. (b,c) Graphs depicting femoral trabecular BV/TV (b) and vertebral trabecular BV/TV (c) in mice with (shaded bars) and without (unshaded bars) inherited NeoR-containing Lrp5 HBM alleles (+/AN or +/GN, and +/A or +/G, respectively), and with and without Vil1-Cre (+V and –V, respectively) or Dmp1-Cre (+D and –D, respectively) transgenes. (d) Photographs of agarose gels containing PCR amplimers derived from mouse genomic DNA extracted from either duodenum or femur cortex of mice with different Lrp5 and Cre-transgene genotypes. PCR amplimers correspond to the sizes depicted in Figure 1a. Top, amplimers from the Dmp1-Cre cross. Bottom, amplimers from the Vil1-Cre cross. WT allele (arrowheads), AN or GN allele (double arrowheads) A or G allele (arrows). (e) Graphs depicting fluorochrome-derived bone formation parameters in the distal femur from 9-week-old female mice that were administered double calcein labeling. Group notations (x axis) follow those described for panel b. (f) Graphs depicting the proportion of distal femur trabecular bone surface covered by osteoclasts (Oc.S; left) and osteoblasts (Ob.S; right). (g) Graphs depicting femoral and vertebral trabecular BV/TV in 12-week-old mice with (shaded bars) and without (unshaded bars) inherited NeoR-containing Lrp5 HBM alleles (+/AN and +/A), respectively, and with and without the Prrx1-Cre transgene (+P and −P, respectively). MS/BS, MAR, BFR/BS are as defined in Figure 1. The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus NeoR-containing littermates that did not inherit a Cre transgene.

  3. Generation and characterization of mice with a conditional knockout allele of Lrp5.
    Figure 3: Generation and characterization of mice with a conditional knockout allele of Lrp5.

    (a) Schematic depicting the creation of the Lrp5 floxed allele. loxP sites (arrowheads), the neomycin-resistance cassette (NeoR), flippase (Flp) recognition target (FRT) sites (diamonds) and the diphtheria toxin (DT) cassette are shown. The relative locations and orientation of the three primers (arrows) used for PCR genotyping and their expected amplimer sizes are noted. (b) Photograph of agarose gel depicting PCR amplimers for WT (+), floxed (f) and knockout (−) Lrp5 alleles from genomic DNA of mice with different Lrp5 genotypes. (c) Photographs of agarose gels containing PCR amplimers derived from mouse genomic DNA extracted from either duodenum or femur cortex of floxed Lrp5 mice, with or without the Dmp1-Cre transgene (top) and with or without the Vil1-Cre transgene (bottom). (d) Graphs depicting whole-body aBMD (left), femoral trabecular BV/TV (middle) and vertebral trabecular BV/TV (right) in 16-week-old mice homozygous for WT or floxed Lrp5 alleles, with or without the Dmp1-Cre transgene (+D and –D, respectively). (e) Graphs depicting whole-body aBMD and tibial trabecular BV/TV in 3-month-old and 12-month-old mice heterozygous or homozygous for floxed Lrp5 alleles with or without the Vil1-Cre transgene (+V and –V, respectively). The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus floxed Lrp5 littermates that did not inherit the Cre transgene.

  4. Effect of Lrp5 genotype on 5HT concentration and on Tph1 expression.
    Figure 4: Effect of Lrp5 genotype on 5HT concentration and on Tph1 expression.

    (a) Graphs depicting whole-blood 5HT measured by HPLC in 6-month-old Lrp5 WT and knockout mice that had been backcrossed to C57BL6/J mice. Shown are 5HT measurements in Lrp5 WT and HBM-causing knock-in (G/G) mice on a mixed 129Sv/C57BL/6J background (far left); in 3-month-old Lrp5 WT, knockout and HBM knock-in (+/A) mice on a mixed 129Sv/C57BL/6J genetic background (middle left); and in 3-month-old male (middle right) and 13-month-old female (far right) WT and knockout littermates on a mixed 129SvEvBrd/ C57BL/6J-Tyrc-Brd background. (b) Graphs depicting the quantity of 5HT extracted from several regions of the intestine, beginning in the duodenum and proceeding through the jejunum, ileum and proximal colon in 3-month-old male (left) and in 13-month-old female (right) Lrp5 WT and knockout littermates on a mixed 129SvEvBrd/ C57BL/6J-Tyrc-Brd background. (c) Scattergram depicting vertebral trabecular BV/TV and whole blood 5HT measurements in individual Lrp5 WT (open symbols) and knockout (filled symbols) littermates. Correlations between BV/TV and whole blood serotonin were r2 = 0.13 (P = 0.16) for male mice, and r2 = 0.02 (P = 0.53) for female mice. (d) Graphs depicting normalized Tph1 transcript levels in duodenum RNA extracts from Lrp5 WT, knockout, and HBM-causing knock-in (+/A) mice on a mixed 129Sv/C57BL/6J genetic background (left) and duodenum and colon RNA extracts from Lrp5 WT and knockout mice on a 129SvEvBrd/ C57BL/6J-Tyrc-Brd background (right) with Gapdh serving as the internal control. The mean Tph1 expression level for Lrp5 WT duodenum is set as 100%. The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus WT mice.

  5. Bone mass in WT and Tph1-/- mice.
    Figure 5: Bone mass in WT and Tph1−/− mice.

    (a) Graphs depicting femoral trabecular BV/TV in WT and Tph1−/− mice on either FVB/N or C57BL/6 backgrounds (left) or on a mixed 129SvEvBrd/C57BL/6J-Tyrc-Brd background (right). (b) Graphs depicting the vertebral trabecular BV/TV of the fifth lumbar vertebrae in the same mice described in panel a. (c) Graphs depicting lumbar spine aBMD, as measured by DEXA, in the same mice described in panel a. The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus WT mice using an unpaired t test; none of these differences remain significant after correcting for multiple testing.

  6. Bone mass after pharmacologic inhibition of Tph1 activity.
    Figure 6: Bone mass after pharmacologic inhibition of Tph1 activity.

    (a) Graph depicting dose-dependent changes in 5HT content, compared to vehicle-treated controls, in 9-week-old WT female C57BL/6 mice after receiving daily doses of LP-923941 for 7 days. A daily dose of 250 mg kg−1 lowered 5HT content in whole blood and in intestine, but not in brain. (b) Graph depicting changes in the intestinal 5HT content in sham-operated (SHM) and ovariectomized (OVX) mice that received vehicle or LP-923941 (250 mg per kg per day) for 6 weeks. Ovariectomy alone reduced 5HT content in the duodenum and colon by ~12% (P < 0.05) compared with SHM mice. Treatment with LP-923941 significantly reduced serotonin content equally in all regions of the intestine in SHM and in OVX mice. (c) Effect of treatment with LP-923941 (250 mg per kg per day) or teriparatide (80 μg per kg per day), which is the 1–34 residue amino-terminal fragment of human parathyroid hormone (PTH), on serum P1NP levels (left), vertebral trabecular BV/TV (middle left), midshaft femur cortical thickness (Ct.th; middle right) and midshaft femoral volumetric BMD (right) in SHM and in OVX mice. (d) Effect of treating SHM and OVX rats with LP-923941 (50 or 250 mg per kg per day) or teriparatide (80 μg per kg per day) for 6 weeks on jejunal 5HT content (left), femoral trabecular BV/TV (middle) and vertebral trabecular BV/TV (right). The numbers of mice studied are indicated. Error bars show means ± s.d. *P < 0.05 versus vehicle-treated mice.

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Author information

Affiliations

  1. Orthopedic Research Laboratories, Department of Orthopedic Surgery, Children's Hospital, Boston, Massachusetts, USA.

    • Yajun Cui,
    • Christina M Jacobsen &
    • Matthew L Warman
  2. Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

    • Yajun Cui &
    • Ronald A Conlon
  3. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA.

    • Paul J Niziolek
  4. Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Paul J Niziolek &
    • Alexander G Robling
  5. Department of Biomedical Engineering, Indiana University School of Medicine, Indianapolis, Indiana, USA.

    • Paul J Niziolek &
    • Alexander G Robling
  6. F.M. Kirby Neurobiology Center, Children's Hospital, Boston, Massachusetts, USA.

    • Bryan T MacDonald &
    • Xi He
  7. Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA.

    • Bryan T MacDonald &
    • Xi He
  8. Center for Skeletal Disease Research, Van Andel Research Institute, Grand Rapids, Michigan, USA.

    • Cassandra R Zylstra,
    • Daniel R Robinson,
    • Zhendong Zhong &
    • Bart O Williams
  9. Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.

    • Natalia Alenina,
    • Susann Matthes &
    • Michael Bader
  10. Lexicon Pharmaceuticals, The Woodlands, Texas, USA.

    • Robert Brommage,
    • Qingyun Liu,
    • Faika Mseeh,
    • David R Powell,
    • Qi M Yang &
    • Brian Zambrowicz
  11. Merck Sharp & Dohme Research Laboratories, Oss, The Netherlands.

    • Han Gerrits &
    • Jan A Gossen
  12. Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts, USA.

    • Matthew L Warman
  13. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Matthew L Warman

Contributions

Y.C. created and did studies on the mice with the Lrp5 HBM alleles and measured serum serotonin levels by competitive ELISA. P.J.N. did radiographic imaging and biomechanical testing on the mice with HBM-associated alleles. B.T.M. contributed to the serotonin and Tph1 qRT-PCR measurements in HBM-causing and Lrp5 knockout mice. C.R.Z. did multiple studies using the conditional Lrp5 knockout mice. N.A. studied the Tph1−/− mice, and with S.M. measured whole blood serotonin levels from HBM-causing and Lrp5-knockout mice by HPLC. D.R.R. generated the conditional Lrp5 knockout strain and Z.Z. participated in conditional inactivation of this allele using different Cre transgenes. C.M.J. carried out the Prrx1-Cre experiments. R.B., F.M. and Q.M.Y. organized studies on Lrp5- and Tph -knockout mice, and also organized the mouse pharmacology experiment. H.G. and J.A.G. organized the rat pharmacology experiment. R.A.C., X.H., M.B., D.R.P., Q.L., B.Z., B.O.W., A.G.R. and M.L.W. designed experiments and provided reagents and financial support. M.L.W. prepared the first draft of the manuscript. All co-authors contributed detailed methods and results, and revised and approved the manuscript.

Competing financial interests

Employees of Lexicon Pharmaceuticals (R.B., Q.L., F.M., D.R.P., Q.M.Y. and B.Z.) and Merck Sharp & Dohme Research Laboratories (H.G. and J.A.G.) have received compensation in the form of salary and stock options.

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