The dysregulation of ROS production and osteoclastogenesis is involved in the progress of osteoporosis. To identify novel and effective targets to treat this disease, it is important to explore the underlying mechanisms. In our study, we firstly tested the effect of the Nrf2 activator RTA-408, a novel synthetic triterpenoid under clinical investigation for many diseases, on osteoclastogenesis. We found that it could inhibit osteoclast differentiation and bone resorption in a time- and dose-dependent manner. Further, RTA-408 enhanced the expression and activity of Nrf2 and significantly suppressed RANKL-induced reactive oxygen species (ROS) production. Nrf2 regulates the STING expression and STING induces the production of IFN-β. Here, we found that RTA-408 could suppress STING expression, but that it does not affect Ifnb1 expression. RANKL-induced degradation of IκBα and the nuclear translocation of P65 was suppressed by RTA-408. Although this compound was not found to influence STING–IFN-β signaling, it suppressed the RANKL-induced K63-ubiquitination of STING via inhibiting the interaction between STING and the E3 ubiquitin ligase TRAF6. Further, adenovirus-mediated STING overexpression rescued the suppressive effect of RTA-408 on NF-κB signaling and osteoclastogenesis. In vivo experiments showed that this compound could effectively attenuate ovariectomy (OVX)-induced bone loss in C57BL/6 mice by inhibiting osteoclastogenesis. Collectively, we show that RTA-408 inhibits NF-κB signaling by suppressing the recruitment of TRAF6 to STING, in addition to attenuating osteoclastogenesis and OVX-induced bone loss in vivo, suggesting that it could be a promising candidate for treating osteoporosis in the future.

Osteoporosis develops because of impaired bone formation and/or excessive bone resorption. Although the pharmacological treatment of osteoporosis has been extensively developed, alternative treatments are still needed. Here, we showed that oridonin (ORI), a diterpenoid isolated from Rabdosia rubescens, can suppress osteoclastogenesis and enhance osteogenesis. ORI inhibited the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and bone resorption through the inhibition of p65 nuclear translocation. ORI-induced inhibition of this translocation led to an increase in osteoblast differentiation and mineralization through the promotion of Smad1/Smad5 phosphorylation. Further analyses demonstrated that the inhibition of p65 nuclear translocation is due to the suppression of IκBα phosphorylation and the induced proteasomal degradation of interferon-related development regulator 1 (Ifrd1), a transcriptional corepressor that is involved in the suppression of NF-κB nuclear translocation. Moreover, mice treated with ORI at catabolic and anabolic windows showed a considerable attenuation of ovariectomy (OVX)-induced osteoporosis. Taken together, our findings reveal that ORI protects against OVX-induced bone loss via inhibiting osteoclastic bone resorption but enhancing osteoblastic bone formation through abolishing both Ifrd1-mediating and IκBα-mediated p65 nuclear translocation. These results show the potential of ORI for treatment of osteoporosis and highlight Ifrd1 as a another novel promising target for anti-osteoporotic drugs. © 2017 American Society for Bone and Mineral Research.

High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-β-mediated osteoblastic differentiation. Our results indicated that TGF-β activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-β, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers.Western blotting analysis indicated that TGF-β induced protein expression of E3 ubiquitinprotein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132.Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-β-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-β inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair. K E Y W O R D Salkaline phosphatase, bone morphogenic protein, extracellular signal-regulated kinase, osteogenic differentiation, RUNX2