The FOXO3/ZC3H13/SLC3A2 cascade modulates the osteogenic differentiation and ferroptosis of BMSCs

Impaired osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the pathogenesis of osteoporosis. ZC3H13, a crucial m6A writer, promotes BMSC osteogenic differentiation by inhibiting ferroptosis. However, its molecular underpinnings remain largely unexplored. Expression analysis was performed by quantitative PCR and immunoblotting. Cell proliferation was assessed by EdU assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) expression/activity assays and Alizarin Red staining. Ferroptosis alteration was evaluated by detecting ROS, Fe²⁺, and MDA levels. RNA immunoprecipitation (RIP), methylated RIP (MeRIP), luciferase, and mRNA stability assays were used to validate the regulation of ZC3H13 in SLC3A2. The relationship between FOXO3 and ZC3H13 was confirmed by chromatin immunoprecipitation (ChIP) and luciferase experiments. ZC3H13 was downregulated in the bone marrow of osteoporosis patients. ZC3H13 deficiency inhibited BMSC proliferation and osteogenic differentiation while enhancing ferroptosis. Conversely, ZC3H13 overexpression promoted proliferation, differentiation and suppressed ferroptosis. Mechanistically, ZC3H13 mediated the m6A modification and stability of SLC3A2 in an IGF2BP2-dependent manner. ZC3H13 silencing suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through SLC3A2 reduction. Furthermore, FOXO3 acted as a transcriptional activator of ZC3H13. Depletion of FOXO3 suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through downregulation of ZC3H13. Additionally, FOXO3 regulated SLC3A2 expression via ZC3H13. This study delineates a novel FOXO3/ZC3H13/SLC3A2 regulatory cascade that safeguards BMSCs from ferroptosis and is compromised in osteoporosis, providing a new conceptual framework for therapeutic development.