Delivery of miR-29a/29c-3p by serum exosomes promotes osteogenesis through TET3-dependent Sox9 demethylation and PI3K/Akt activation

Osteoporosis (OP) is characterized by impaired bone formation, largely attributed to dysfunctional osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Circulating factors, particularly exosomes acting as natural nanocarriers, play crucial roles in regulating BMSCs function within the bone microenvironment. However, the specific mechanisms by which serum exosomes contribute to osteogenic impairment in OP remain elusive. Serum exosomes were isolated from ovariectomized (OVX) rats and characterized. Their impact on BMSCs osteogenesis was evaluated. Global miRNA sequencing identified dysregulated miRNAs in OVX-derived exosomes. The roles of miR-29a-3p and miR-29c-3p were investigated using gain- and loss-of-function approaches in vitro and in vivo. Bioinformatic analysis and experimental validation identified Ten-Eleven Translocation 3 (TET3) as a direct target. TET3 deficiency was modeled in OVX mice. Transcriptomic analysis, bisulfite sequencing PCR, and chromatin immunoprecipitation sequencing were employed to delineate the mechanism of action of TET3. Exosomes derived from OVX rat serum significantly inhibited osteogenic differentiation of BMSCs. MiRNA sequencing revealed a pronounced downregulation of miR-29a-3p and miR-29c-3p within these exosomes. Functionally, overexpression of miR-29a/29c-3p rescued bone formation defects both in vitro and in vivo, while their inhibition suppressed osteogenesis. Mechanistically, TET3, a key DNA demethylase, was confirmed as a direct target of miR-29a/29c-3p. Crucially, TET3 deficiency in OVX mice stimulated BMSCs osteogenesis and bone remodeling. Further mechanistic dissection demonstrated that TET3 represses osteogenesis by directly increasing DNA methylation at the Sox9 promoter, thereby suppressing Sox9 expression, and concurrently inhibiting the PI3K/AKT signaling pathway. Our study defines a novel exosome-mediated pathway in OP: Deficiency of serum exosome-delivered miR-29a/29c-3p elevates TET3, which epigenetically represses Sox9 via promoter hypermethylation and inhibits PI3K/AKT signaling. This exosome/miR-29/TET3/Sox9 axis unveils promising therapeutic targets for OP intervention, particularly leveraging exosome-based modulation or epigenetic editing.