METTL5 Regulates Osteogenesis Through Antioxidant Defense

Objectives: METTL5, a methyltransferase responsible for m⁶A modification of 18S ribosomal RNA, has been implicated in genetic disorders characterized by skeletal and cognitive abnormalities. However, its role in bone development remains unclear. This study aims to investigate the effects of METTL5 deficiency on osteogenesis, which is essential for advancing understanding of related diseases.
Methods: Apart from the Mettl5^-/- mice, Prx1Cre;Mettl5^fl/fl and LysmCre;Mettl5^fl/fl mice were generated for conditional knockout in limb mesenchyme and bone marrow, respectively. Skeletal phenotypes were assessed by micro-CT and Von Kossa staining. Osteoblast and osteoclast activity were evaluated using TRAP, ALP, and immunofluorescence staining. Bone marrow mesenchymal stem cells (BMSCs) were isolated for osteogenic differentiation assays, western blotting, and qRT-PCR. Ribosome profiling and RNA sequencing were conducted to identify translational and transcriptional changes. NAC, a widely applied antioxidant, was administrated to rescue the skeletal and cognitive phenotypes.
Results: Mettl5^-/- mice and Prx1Cre;Mettl5^fl/fl mice exhibited reduced bone mass and decreased osteoblast activity, while osteoclast activity remained unaffected. Conversely, LysmCre;Mettl5^fl/fl mice showed no significant differences in bone parameters or osteoclast activity compared to controls. As METTL5 regulates translation, ribosome profiling identified OSER1 as a key translationally downregulated target in Mettl5^-/- BMSCs, and its overexpression partially rescued osteogenic defects. RNA sequencing revealed transcriptional downregulation of antioxidant-related genes, including Sod1, Cat, and Gpx4, and compromised resistance to oxidative stress in Mettl5^-/- cells. Supplementation with NAC enhanced osteogenic differentiation, and improved bone mass in Mettl5^-/- mice. Furthermore, NAC improved cognitive function in Mettl5^-/- mice, as evidenced by water maze and restored neuronal activation in the hippocampus.
Conclusions: METTL5 deficiency impairs osteogenic differentiation by disrupting antioxidant regulation through the downregulation of OSER1. Antioxidant supplementation with NAC partially rescues osteogenic and cognitive deficits, highlighting its potential therapeutic strategy for the skeletal and neurological defects associated with METTL5 mutations.