N6-Methyladenosine Modification of Rab27A Promotes Osteoporosis by Influencing Osteoclast Differentiation in the Ovariectomized Mouse Model

The development of novel therapies for osteoporosis has attracted increasing attention. Rab27A is upregulated during osteoclast differentiation, and N6-methyladenosine (m6A) modification has emerged as an important epigenetic regulator in orthopedic diseases. Bioinformatic analyses predicted that Rab27A contains multiple m6A modification sites and interacts with several m6A-associated proteins. Knockdown experiments were performed to evaluate the role of Rab27A in osteoclast differentiation and osteoporosis using an ovariectomized (OVX) mouse model. Rab27A depletion attenuated osteoporosis and increased the bone formation percentage from 14.73% to 28.13% (P < 0.05) in OVX mice. Western blotting and quantitative PCR demonstrated that Rab27A knockdown reduced the protein and mRNA expression of the osteoclast markers TRAP, NFATc1, and c-FOS, thereby suppressing osteoclast differentiation of bone marrow-derived macrophages (BMMs) induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). TRAP staining further showed that Rab27A depletion significantly reduced TRAP activity from 24.1% to 12.9% (P < 0.01) following M-CSF and RANKL induction. RNA immunoprecipitation assays confirmed interactions between Rab27A and the m6A reader proteins YTHDC1 and IGF2BP2. Nuclear and cytoplasmic fractionation demonstrated that YTHDC1 facilitates the cytoplasmic export of m6A-modified Rab27A. In addition, q-PCR and western blot analyses showed that knockdown of either YTHDC1 or IGF2BP2 reduced Rab27A expression and decreased the expression of TRAP, NFATc1, and c-FOS, thereby suppressing osteoclast differentiation induced by M-CSF and RANKL. Collectively, these findings indicate that YTHDC1-mediated cytoplasmic export and IGF2BP2-mediated stabilization of m6A-modified Rab27A promote osteoclast differentiation and contribute to bone loss in the OVX mouse model.