SOG inhibited RANKL-induced osteoclast formation
To evaluate the effect of SOG on RANKL-mediated differentiation, cells were cultured with treatment of M-CSF, RANKL, and indicated concentrations of SOG. Firstly, the potential cytotoxicity of SOG was evaluated. MTT assay showed that up to 200 μM of concentration, SOG did not exhibit obvious cytotoxic effects compared to cells without SOG treatment (Figure 1b). Further, the effects of SOG on osteoclast formation were determined. As shown in figure 1c, the number and size of osteoclasts were significantly inhibited by SOG treatment in a concentration-dependent manner.
Osteoclast differentiation goes through several stages including early, middle and late stage. To determine at which stage SOG could affect osteoclast formation, 200 μM SOG was added to BMMs at different stage of osteoclastogenesis (Figure 1e) and treatment lasted for 24 h. As figure 1d shown, SOG treatment alleviated the number and size of osteoclast at all stages of cell differentiation. However, SOG stronger inhibited osteoclast formation at middle and late stage (day 3 and day 4) compared to early stage (day1 and day2) (Figure 1d). Taking together, these results suggested that SOG suppressed RANKL-induced osteoclastogenesis, especially at middle-late stage of cell differentiation.
SOG impairs osteoclastic resorption and F-actin ring formation.
Next, the effects of SOG treatment on osteoclast-mediated bone resorption and F-actin ring formation of osteoclasts were examined. It is acknowledged that the formation of well-defined F-actin ring was indispensable for osteoclastic function4. Figure 2a showed that the number and size of F-actin rings were effectively diminished with the increase of the concentration of SOG. Treatment with SOG also dose-dependently decrease the formation of bone resorption pits, in terms of pits area (Figure 2b). SOG treatment almost abolished resorption pits formation at the concentration of 200 μM. To investigate at which stage SOG exerted its inhibitory effects on bone resorption function of osteoclasts, 200 μM SOG was added to BMMs at day 1, 2, 3 and 4 during osteoclastogenesis for 24 h. Consistent with results shown at figure 1d, SOG exhibited stronger inhibition on bone resorption pits formation at middle-late stage (day 3 and day 4 after RANKL treatment) compared to that at early stage (Figure 2c). Collectively, these results indicated that SOG remarkably suppressed bone resorption function of osteoclasts, especially at middle-late stage of cell differentiation.
SOG repressed the induction of osteoclast specific genes
RANKL stimulation up-regulates osteoclast-related genes, which regulats the differentiation, maturation, and function of osteoclasts. NFATc1, a master transcription factors for osteoclastogenesis, regulats the expression of osteoclast specific genes17. To investigate the impacts of SOG on osteoclast-related genes, we used RT-PCR to examine the mRNA expression of these genes, including NFATc1, c-Fos, CTSK, TRAP, DC-STAMP. As figure 3a shown, mRNA expression of these genes was obviously enhanced by RANKL stimulation. Meanwhile, SOG treatment strongly suppressed mRNA level of these genes (Figure 3a and 3b). The expression of key transcription factors c-Fos and NFATc1 was significantly suppressed at both mRNA and protein levels with SOG treatment (Figure 3c).
In addition, to investigate the impacts of SOG on osteoclast-related genes at different stage of cell differentiation, BMMs were treated with SOG on day 1, 2, 3, 4 of osteoclastogenesis. We found that mRNA levels of target genes (TRAP, CTSK, DC-STAMP) were decreased with SOG at all stages of osteoclastogenesis (Figure 3d). However, mRNA levels of CTSK, TRAP and DC-STAMP were more remarkably suppressed at middle-late stage (Figure 3d). Meanwhile, the protein and mRNA levels of c-Fos and NFATc1 were suppressed by SOG at all stages of cell differentiation, but SOG existed stronger inhibition on protein and mRNA levels of c-Fos and NFATc1 at middle-late stage (day 3 and day 4 after RANKL treatment)(Figure 3e and 3f). These results suggested that SOG inhibited osteoclastogenesis by suppressing expression of c-Fos/NFATc1-mediated osteoclast specific genes.
The effect of SOG on NF-κB, MAPK and AKT-GSK3β pathways at initial stage of osteoclastogenesis
It has been reported that NF-κB, MAPK and AKT-GSK3β pathways play a vital role in early induction of NFATc118,19. To further explore the molecular mechanism of SOG modulating NFATc1 signaling, the effects of SOG on RANKL-initiated transient activation of NF-κB, MAPK and AKT as well inactivation of GSK3β were evaluated by western blot assay. As figure 4a shown, RANKL-stimulation induced transient phosphorylation of NF-κB and MAPKs including p38, ERK1/2 and JNK, which was not altered with SOG treatment. As well, RANKL-induced transient activation AKT and subsequent GSK3β inactivation also were not affected with SOG treatment (Figure 4b). These results suggested that SOG treatment might not influence the initial induction of NFATc1 via modulation of NF-κB, MAPK and AKT-GSK3β signaling.
The effects of SOG on the RANKL-induced calcineurin /NFATc1 and AKT-GSK3β/NFATc1 signaling pathway during osteoclastogenesis
Activation of NFATc1 was regulated by calcineurin via dephosphorylation of NFATc1 at middle stage of osteoclastogenesis20. To investigate whether SOG could affect RANKL-induced middle-late signaling pathways, RANKL-stimulated BMMs were treated with SOG for 24 h at different periods of osteoclastogenesis, respectively. We found that the expression of calcineurin catalytic subunit PP2B-Aα was not obvious changed with treatment of SOG (Figure 5a, 5b).
GSK3β was negative regulator of NFATc1 through phosphorylation of serine residues of NFATc121. AKT mediates inactivation of GSK3β via phosphorylating serine 9 residue of GSK3β， which was required for osteoclastogenesis. As figure 5a shown, the relative levels of phosphorylation of AKT and GSK3β were alleviated with SOG treatment during middle-late stages of osteoclast differentiation. These results suggested that SOG might suppress NFATc1 activation via inhibition of AKT-mediated GSK3β inactivation.
Further, SB415286, a selective GSK3β inhibitor, was used to confirm whether SOG could suppress RANKL-induced osteoclast differentiation through inhibition of AKT-mediated GSK3β inactivation. As shown in figure 5b, addition of SB415286 partly weakened the inhibitory effect of SOG on osteoclasts formation at middle-late stage of cell differentiation.
Taken together, these data suggested that SOG repressed RANKL-induced osteoclast differentiation partly by inhibition of AKT-mediated GSK3β inactivation at middle-late stage of osteoclastogenesis.
The effects of Knockdown of 5-LO on RANKL-induced osteoclastogenesis
Previous study showed that SOG inhibited 5-lipoxygenase (5-LO) catalytic activity with an IC50 value of 7.45 μM in vitro16. In order to explore the potential role of 5-LO in suppression of SOG on osteoclast differentiation, the expression of 5-LO in BMMs was knock-down by siRNA. As figure 6a shown, 5-LO silencing by siRNA obviously inhibited osteoclast formation compared to siRNA control. In addition, RANKL-induced NFATc1 expression level were significantly attenuated in 5-LO siRNA transfected BMMs compared to siRNA control (Figure 6b). However, knockdown of 5-LO did not change phosphorylation levels of AKT and GSK3β (Figure 6b). Taken together, these data indicated that 5-LO knockdown remarkably suppressed RANKL-induced osteoclastogenesis, but not through the AKT/GSK3β signaling pathway. SOG might also inhibit osteoclastogenesis through inhibition of 5-LO catalytic activity.
SOG treatment improved LPS-induced bone loss
Given that the inhibitory effects of SOG on osteoclastogenesis in vitro, we further evaluated the potential therapeutic effects of SOG on LPS-induced bone loss in mice model. 2D and 3D reconstruction images of Micro-CT revealed that LPS group developed a significant osteoporosis in femur compared with sham group (Figure 7a). And, ZOL group, as positive group, markedly reduced LPS-induced bone loss. Similarly, SOG treatment also significantly improved bone destruction in LPS-treated mice. Moreover, Alone SOG group had no obvious change in bone mass compared to sham group. Quantitative analysis of bone microparameters showed that SOG group increased the values of BMD, BV/TV and Tb.N and decreased in Tb.Sp value compared with LPS-treated group (Figure 7b). H&E staining further confirmed that SOG treatment significantly reduced LPS-induced bone destruction in mice model (Figure 7c). In addition, bone histomorphometric analyses with TRAP staining revealed that SOG group decreased the number of TRAP-positive osteoclasts compared with LPS group (Figure 7c). These observations indicated that SOG attenuated LPS-induced bone loss in mice.