Patients With High Bone Mass Phenotype Exhibit Enhanced Osteoblast Differentiation and Inhibition of Adipogenesis of Human Mesenchymal Stem Cells

Abstract

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. Introduction: 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. Materials and 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-LRP5WT), LRP5T244 (hMSC-LRP5T244, inactivation mutation leading to osteoporosis), or LRP5T253 (hMSC-LRP5T253, 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-LRP5WT and hMSC-LRP5T253 but not hMSC-LRP5T244 transduced high level of Wnt signaling. Wnt3a inhibited cell proliferation in hMSC-LRP5WT and hMSC-LRP5T253, and this effect was associated with downregulation of DKK1. Both hMSC-LRP5WT and hMSC-LRP5T253 showed enhanced osteoblast differentiation and inhibited adipogenesis in vitro, and the opposite effect was observed in hMSC-LRP5T244. Similarly, hMSC-LRP5WT and hMSC-LRP5T253 but not hMSC-LRP5T244 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.