Hydrogen Peroxide Suppresses Lgr4 Expression in Osteoblasts

Objectives: A rare nonsense mutation of LGR4 (Leucine-rich repeat-containing G-protein coupled receptor 4) was found to be strongly associated with low BMD (bone mineral density) and osteoporotic fracture. Since oxidative stress is involved in systemic and alveolar bone loss, we aimed to evaluate the possible role of Lgr4 in the oxidative stress-induced dysfunction of osteoblasts.
Methods: To induce oxidative stress, osteoblastic MC3T3-E1 cells were cultured in the presence or absence hydrogen peroxide (H₂O₂) at various concentration. Cell Viability were investigated by MTT assay and Hoechst 33342 nuclear staining. The expression of Lgr4 and osteogenic-related gene were evaluated by quantitative RT-PCR. Statistical significance was determined by Students’s t-test (p<0.05).
Results: We found that high concentration of H₂O₂ (1mM) significantly suppressed Lgr4 mRNA expression within 12 hours. This was associated with the impaired function of osteoblasts including the reduction of the expression of Runx2, Osx, Col1a1, Alp, Bsp and Opg, and the reduction of cell viability in MC3T3-E1 cells. As functional aspects, BMP2 significantly enhanced Lgr4 expression but H₂O₂ dramatically suppressed Lgr4 expression even in the presence of BMP2. While TGF-β1 suppressed Lgr4 expression, TGF-β1 treatment in the presence of H₂O₂ did not further suppress the Lgr4 expression. Moreover, the suppression of Osx expression upon H₂O₂ treatment was further suppressed under Lgr4 knockdown. This result implies that suppression of Osx by H₂O₂ may depends on the Lgr4 expression.
Conclusions: We demonstrated that H₂O₂ suppresses Lgr4 expression in osteoblasts. Since it is possible that Lgr4 suppression by oxidative stress may impair osteoblast function, the findings might offer a new tarts in therapeutic approaches for bone disorders.