TRIM25-Mediated Ubiquitination and Degradation of SOX8 Promotes Ligament Fibroblast Osteogenic Differentiation and Regulates OPLL Progression by Inhibiting OSR2 Transcription.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a pathological condition characterized by ectopic ossification of spinal ligaments, primarily driven by abnormal osteogenic differentiation of ligament fibroblasts with stem cell-like properties. The SOX transcription factor family is crucial in regulating cell stemness and differentiation. Among them, SOX8 is known to influence osteoblast differentiation, but its role in OPLL remains unclear. METHODS: SOX8 expression was analyzed in non-OPLL and OPLL ligament tissues and cells. Its role in osteogenic differentiation was assessed using ALP/Alizarin Red staining, qPCR, Western blotting, and subcutaneous ectopic ossification models in nude mice. Mass spectrometry and co-immunoprecipitation identified SOX8-interacting E3 ubiquitin ligases, with ubiquitination assays assessing their effects on SOX8 stability. RNA-seq, GTRD analysis, and dual-luciferase reporter assays revealed SOX8 target genes. Functional recovery experiments were conducted to explore the role of these interactions in the osteogenic differentiation of ligament fibroblasts. RESULTS: SOX8 expression was downregulated in OPLL ligament tissues and cells. Functional analyses showed that SOX8 inhibits osteogenic differentiation of ligament fibroblasts both in vitro and in vivo. Mechanistically, TRIM25, an E3 ubiquitin ligase, was found to interact with SOX8, promoting its ubiquitination and degradation. Rescue experiments showed that SOX8 knockdown or overexpression reversed the osteogenic effects of TRIM25 knockdown or overexpression in ligament fibroblasts. Additionally, OSR2 was identified as a transcriptional target of SOX8, with SOX8 promoting OSR2 transcription. OSR2 knockdown negated the inhibitory effects of SOX8 overexpression on osteogenic differentiation. CONCLUSIONS: SOX8 serves as a critical negative regulator of osteogenic differentiation in ligament fibroblasts. TRIM25 promotes ectopic ossification in OPLL by enhancing SOX8 ubiquitination and degradation, while SOX8 inhibits osteogenic differentiation through transcriptional activation of OSR2. These findings highlight the TRIM25/SOX8/OSR2 axis as a key regulator in OPLL ectopic ossification, suggesting it to be a potential target for non-surgical treatment.