METTL3 regulated by histone lactylation promotes ossification of the ligamentum flavum by enhancing the m6A methylation of BMP2.

Abstract

Ossification of the ligamentum flavum (OLF) is characterized by ligamentum flavum thickening and subsequent thoracic canal stenosis. Emerging evidence has demonstrated the involvement of N6-methyladenosine (m6A) methylation in OLF pathogenesis. This study investigates the regulatory role of METTL3-mediated m6A methylation of BMP2 in OLF progression. Clinical ligamentum flavum tissues were analyzed for m6A levels using dot blot analysis. Osteogenic differentiation was assessed through quantitative real-time PCR (qPCR), alkaline phosphatase staining, alizarin red S staining, and western blot analysis. Mechanistic insights were obtained through methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), and luciferase reporter assays. The regulatory role of histone lactylation on METTL3 expression was examined using LDHA knockdown, sodium lactate (Nala) treatment, and 2-deoxy-D-glucose (2-DG) administration in OLF cells. Our findings revealed significant upregulation of METTL3 expression and m6A levels in OLF patients. METTL3 was shown to enhance osteogenic differentiation and m6A methylation of BMP2, which was specifically recognized by IGF2BP1. Furthermore, increased histone lactylation was observed in OLF patients, with enrichment in the METTL3 promoter region facilitating its transcriptional activation. LDHA knockdown-mediated inhibition of endogenous lactylation suppressed osteogenic differentiation, a phenotype that was rescued by METTL3 overexpression. In conclusion, this study elucidates that histone lactylation-mediated upregulation of METTL3 promotes OLF progression through IGF2BP1-dependent m6A methylation of BMP2, providing novel insights into potential therapeutic strategies for OLF management.