SATB2 promotes osteogenic differentiation of OPLL-derived posterior longitudinal ligament cells via Wnt/β-catenin signaling in association with RUNX2.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a prevalent pathological condition characterized by ectopic ossification of spinal ligaments, leading to myelopathy through spinal cord compression. SATB2 is a multifunctional regulator of osteogenic differentiation, and prior studies in other osteogenic systems have suggested links between SATB2, RUNX2, and Wnt/β-catenin signaling [1, 2]; however, whether these regulatory relationships operate in OPLL-derived posterior longitudinal ligament cells (OPLL cells) remains incompletely understood. In this study, we investigated the role of SATB2 in osteogenic differentiation by examining the contribution of RUNX2 and associated changes in Wnt/β-catenin signaling. Our data support that, in OPLL cells, SATB2 overexpression is associated with Wnt/β-catenin-related signaling changes, increased RUNX2 expression, and enhanced osteogenic differentiation of OPLL cells.

METHODS: OPLL cells were transfected with a SATB2-overexpressing plasmid and subjected to osteogenic induction. ALP staining and Alizarin Red S staining were used to assess osteogenic differentiation. RT-qPCR, Western blotting and immunofluorescence were performed to evaluate osteogenic markers and Wnt/β-catenin-related factors. TOPFlash reporter assays were used to assess β-catenin/TCF transcriptional activity. XAV939 was used to inhibit Wnt/β-catenin signaling, and RUNX2 was silenced using siRNA. For the dose-comparison experiment, OPLL cells were treated with XAV939 at final concentrations of 5 or 10 µM. The 10 µM concentration was used for the Western blot experiment and subsequent pathway-inhibition experiments. Co-immunoprecipitation was performed to examine the association between SATB2 and RUNX2 proteins.

RESULTS: SATB2 overexpression was associated with increased total β-catenin expression, reduced phosphorylated β-catenin and DKK1 expression, and increased TOPFlash reporter activity. In contrast to the expected response of a canonical β-catenin/TCF target gene, AXIN2 expression was unexpectedly reduced after SATB2 overexpression. Thus, while the β-catenin and TOPFlash findings were consistent with enhanced β-catenin/TCF transcriptional activity, the AXIN2 response was discordant with the classical feedback pattern of canonical Wnt signaling. RUNX2 knockdown attenuated the SATB2-associated activation of Wnt/β-catenin signaling and osteogenic differentiation. In XAV939-treated cells, SATB2 overexpression partially attenuated the inhibitory effect of Wnt pathway blockade on osteogenic marker expression. Co-immunoprecipitation showed that SATB2 and RUNX2 were present in the same protein complex in OPLL cells. We isolated cells from the posterior longitudinal ligament at sites close to the ossification border and the center of the non‑ossified region in OPLL patients, and induced osteogenic differentiation. No statistically significant differences were detected in the assessed osteogenic readouts between these cell populations (Supplementary Figure S2).

CONCLUSION: Our findings support that, in OPLL cells, SATB2 is associated with Wnt/β-catenin-related signaling changes, increased RUNX2 expression, and enhanced osteogenic differentiation. These data extend prior observations from other osteogenic models to an OPLL-relevant cellular context and suggest that SATB2-associated osteogenic signaling may contribute to OPLL pathogenesis. Further studies are required to define the precise mechanism linking SATB2 to DKK1/Wnt regulation and to determine whether these effects are specific to the OPLL disease context.