Effect of Hyperglycemia on the Expression of Sclerostin in Osteoblastic Cells

Abstract

Sclerostin is an inhibitor of Wnt/β-catenin signaling and exerts negative effects on bone formation. Hyperglycemia is a representative symptom of diabetes mellitus. Recent studies demonstrated that osteoporotic fracture risk and serum sclerostin level is increased in diabetic patients. However, the molecular mechanism how sclerostin expression is enhanced in diabetic patients remains elusive. Therefore, in this study, the effect of hyperglycemia on the expression of sclerostin in the osteoblast lineage cells was examined. C2C12 cells were cultured in osteogenic medium (DMEM supplemented with 50 ng/ml BMP2 and 5% FBS). Osteocytic MLO-Y4 cells were cultured in αMEM supplemented with 5% calf serum and 5% FBS. To examine the effect of hyperglycemia, glucose concentration in culture medium was adjusted to 40 or 100 mM (vs 5 mM in control medium). Expression of sclerostin was examined by quantitative RT-PCR, Western blot and Sost promoter reporter assays. Top-Flash reporter was used to examine the transcriptional activity of β-catenin/Tcf/Lef complex. Reactive oxygen species (ROS) production was examined by using DCF-DA reagent. Tumor necrosis factor α (TNFα) expression was examined by ELISA and quantitative RT-PCR. TNFα knockdown was induced by transient transfection of cells with TNFα siRNAs. Effect of ROS on sclerostin expression was examined by treating cells with 1 μM H2O2 or 20 mM N-acetylcysteine. High glucose concentration in culture medium increased the mRNA and protein levels of sclerostin and luciferase activity of Sost promoter reporter in both C2C12 and MLO-Y4 cells. High glucose suppressed Wnt3a-induced Top-Flash reporter activity and expression levels of osteoblast marker genes in C2C12 cells. High glucose increased ROS production and TNFα expression levels. Treatment of cells with H2O2 also enhanced expression levels of TNFα and sclerostin. In addition, N-acetylcysteine treatment or knockdown of TNFα attenuated high glucose-induced sclerostin expression. These results suggest that hyperglycemia increase sclerostin expression via increasing the production of ROS and TNFα.