Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells.
Genetic mutations in the LRP5 gene affect Wnt signaling and lead to changes in bone mass in humans. Our in vivo and in vitro results show that activated mutation T253I of LRP5 enhances osteogenesis and inhibits adipogenesis. Inactivating mutation T244M of LRP5 exerts opposite effects.


BACKGROUND
Mutations in the Wnt co-receptor, LRP5, leading to decreased or increased canonical Wnt signaling, result in osteoporosis or a high bone mass (HBM) phenotype, respectively. However, the mechanisms whereby mutated LRP5 causes changes in bone mass are not known.


METHODS
We studied bone marrow composition in iliac crest bone biopsies from patients with the HBM phenotype and controls. We also used retrovirus-mediated gene transduction to establish three different human mesenchymal stem cell (hMSC) strains stably expressing wildtype LRP5 (hMSC-LRP5(WT)), LRP5(T244) (hMSC-LRP5(T244), inactivation mutation leading to osteoporosis), or LRP5(T253) (hMSC-LRP5(T253), activation mutation leading to high bone mass). We characterized Wnt signaling activation using a dual luciferase assay, cell proliferation, lineage biomarkers using real-time PCR, and in vivo bone formation.


RESULTS
In bone biopsies, we found increased trabecular bone volume and decreased bone marrow fat volume in patients with the HBM phenotype (n = 9) compared with controls (n = 5). The hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) transduced high level of Wnt signaling. Wnt3a inhibited cell proliferation in hMSC-LRP5(WT) and hMSC-LRP5(T253), and this effect was associated with downregulation of DKK1. Both hMSC-LRP5(WT) and hMSC-LRP5(T253) showed enhanced osteoblast differentiation and inhibited adipogenesis in vitro, and the opposite effect was observed in hMSC-LRP5(T244). Similarly, hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) formed ectopic mineralized bone when implanted subcutaneously with hydroxyapatite/tricalcium phosphate in SCID/NOD mice.


CONCLUSIONS
LRP5 mutations and the level of Wnt signaling determine differentiation fate of hMSCs into osteoblasts or adipocytes. Activation of Wnt signaling can thus provide a novel approach to increase bone mass by preventing the age-related reciprocal decrease in osteogenesis and increase in adipogenesis.
Laboratory for Molecular Endocrinology (KMEB), Department of Endocrinology and Metabolism, University Hospital of Odense, Odense C, Denmark.
Weimin
W
Qiu
Tom E
TE
Andersen
Jens
J
Bollerslev
Susanne
S
Mandrup
Basem M
BM
Abdallah
Moustapha
M
Kassem
D050156:Adipogenesis / Q000235:genetics*; D000328:Adult; D000818:Animals; D015519:Bone Density / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D026502:LDL-Receptor Related Proteins / Q000235:genetics*; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D009154:Mutation; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000235:genetics*; D010641:Phenotype; D014161:Transduction, Genetic; D051153:Wnt Proteins / Q000378:metabolism; D060508:Wnt3 Protein; D060509:Wnt3A Protein
C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins; C555482:LRP5 protein, human; D060492:Low Density Lipoprotein Receptor-Related Protein-5; C555483:Lrp5 protein, mouse; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse