SOX4 facilitates osteogenic differentiation through suppressing NLRP3 transcription-mediated pyroptosis.

SOX4 participates in cell differentiation, but its regulatory function and underlying mechanism in the osteogenic differentiation of MC3T3-E1 cells remain elusive. Immunofluorescence staining was used to detect SOX4 subcellular localization and co-localization with NLRP3. Alizarin Red staining was applied to evaluate osteogenic differentiation. RT-PCR and Western blot assays quantified mRNA and protein levels of SOX4, osteogenic markers (OPN, OCN, RUNX2), inflammatory cytokines, and pyroptosis-related proteins (NLRP3, caspase-1, GSDMD). JASPAR predicted SOX4 binding sites in the NLRP3 promoter and ChIP-qPCR was used to validate it. Dual-luciferase reporter assay was applied to assesse transcriptional regulation. Rescue experiments were performed via co-transfection of SOX4 and NLRP3 overexpression vectors. SOX4 was upregulated during MC3T3-E1 osteogenic differentiation. SOX4 overexpression reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), increased IL-10, and inhibited pyroptosis. SOX4 directly bound to the NLRP3 promoter to suppress its transcription. NLRP3 overexpression reversed SOX4-induced enhancement of osteogenic differentiation and marker expression. SOX4 promotes MC3T3-E1 osteogenic differentiation by directly inhibiting NLRP3 transcription, thereby regulating inflammation and pyroptosis. The online version contains supplementary material available at 10.1007/s10616-026-01040-5.