TSAb inhibits osteogenic differentiation of MC3T3-E1 cells and exacerbates bone loss in Graves' disease mice

Graves' disease (GD) is an important risk factor for secondary osteoporosis (OP). Thyrotropin-receptor stimulating antibody (TSAb) is a pathogenic antibody detected in patients with GD. However, few studies have examined the effects of TSAb on bone. Consequently, this study aimed to explore the effect of TSAb on osteoblast differentiation and its possible mechanisms. MC3T3-E1 cells were treated with different concentrations of TSAb. The relative survival rate of cells was assessed using the cell counting kit-8 (CCK-8) assay. Osteoblast differentiation markers were determined using western blotting and immunofluorescence assays. To further evaluate the roles of TSAb in osteogenesis in vivo, a GD-induced OP mouse model was generated by Ad-TSHR289 immunization followed by intragastric administration of methimazole (MMI). Femurs were collected for micro-CT scanning and histomorphometry analysis. The viability of MC3T3-E1 cells did not significantly change with increasing TSAb concentrations. The protein levels of osteoblast differentiation markers (OCN, Col1a1, Runx2, and OPN) in MC3T3-E1 cells treated with 1 and 10 ng/mL TSAb were significantly reduced. Furthermore, TSAb significantly promoted the AKT/mTOR pathway. Moreover, inhibition of this signaling pathway attenuated the phosphorylation of AKT and mTOR enhanced by TSAb and reversed osteoblast differentiation. GD mice treated with MMI exhibited reduced bone mass and degraded bone formation. TSAb exacerbates bone loss in GD mice. These findings demonstrated that TSAb inhibits osteoblast differentiation by activating the AKT/mTOR pathway. This study revealed a novel function of TSAb in regulating osteoblast activity.