S6K/FLNC/ITGβ3 signaling pathway regulates osteoclastogenesis and the inhibition of osteoclastogenesis by columbianadin

Background: Over-activation of osteoclastogenesis is a significant factor contributing to bone loss, leading to increased resorption of bone. Columbianadin (CBN), a compound derived from Angelicae Pubescentis Radix, has traditionally been used in Chinese medicine to treat bone-related disorders. However, the specific effects of CBN on bone loss are still poorly understood.

Study design: This study aims to identify a novel target for inhibiting osteoclast differentiation and to elucidate the effects and underlying mechanisms of CBN on osteoclastogenesis.

Methods: We employed a transcriptomics approach to identify genes that undergo significant changes during osteoclast differentiation. These findings were validated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (WB) analysis. Subsequently, we utilized adenoviral transfection to investigate the effects of target genes on osteoclast differentiation. Additionally, we employed proteomics to elucidate the signaling pathways that regulate osteoclast differentiation. We examined the effect of CBN on S6K/FLNC/ITGβ3 signaling pathway and osteoclast differentiation.

Results: Our results revealed a dramatic increase in filamin C (FLNC) levels during osteoclast differentiation. Inhibition of FLNC expression significantly suppressed markers of osteoclast differentiation such as tartrate-resistant acid phosphatase (TRAP), nuclear factor-activated T cell 1 (NFATc1), and c-Fos, as well as inhibited the activity of bone resorption. We further conducted a proteomic analysis and found S6 K protein might be involved in this process. Then we utilized an S6K-specific inhibitor (PF-4,708,671) and demonstrated that inhibiting the S6 K protein reduced FLNC expression and the interaction between FLNC and integrin β3 (ITGβ3) in osteoclasts. Finally, we found that CBN inhibited osteoclast differentiation and bone loss in ovariectomized mice by targeting the S6K/FLNC/ITGβ3 signaling pathway.

Conclusion: FLNC was identified as a critical protein in osteoclastogenesis. The S6K/FLNC/ITGβ3 signaling pathway played a significant role in osteoclast differentiation and bone loss. Furthermore, CBN exhibited anti-osteoporotic effects by inhibiting the S6K/FLNC/ITGβ3 signaling pathway.