KDM4D-RPS5 Complex Promoted Osteo/Dentinogenic Differentiation of DPSCs via CNR1-Maintained Mitochondrial Functional Homeostasis.

Despite significant advances in dental pulp stem cell (DPSC)-based regeneration of the pulp-dentine complex, regulating the directed differentiation of these cells remains a key challenge. The present study investigated the role and underlying mechanism of the KDM4D-RPS5 complex in modulating the odontogenic differentiation of DPSCs, with the goal of providing insights to inform strategies for tooth tissue regeneration and repair. To assess the osteo/dentinogenic differentiation capacity of DPSCs, multiple techniques were employed, including alkaline phosphatase (ALP) activity assays, Alizarin Red S staining, quantitative calcium analysis, and detection of osteo/dentinogenic marker expression. Gene expression levels were quantified using quantitative real-time polymerase chain reaction and Western blot. Chromatin immunoprecipitation and co-immunoprecipitation (Co-IP) assays were performed to investigate the underlying molecular mechanisms. Mitochondrial morphology in DPSCs was observed via transmission electron microscopy (TEM), while the oxygen consumption rate was measured using a Seahorse XF Analyser, and mitochondrial membrane potential was assessed with a JC-10 assay. Finally, the in vivo efficacy of odontogenic differentiation was validated through a subcutaneous transplantation assay in nude mice. We first demonstrated that KDM4D significantly promoted the osteo/dentinogenic differentiation of DPSCs. Furthermore, KDM4D bound directly to RPS5 via a specific structural domain to form a functional complex; disruption of this binding site abolished its capacity to drive differentiation. Mechanistically, ChIP assays revealed that the KDM4D-RPS5 complex epigenetically activated the downstream gene CNR1 by demethylating H3K9me2 at its promoter, thereby facilitating DPSC differentiation. Additionally, mitochondrial functional analysis showed that overexpression of KDM4D, RPS5, or CNR1 enhanced mitochondrial membrane potential and augmented energy metabolism, further supporting the differentiation process. KDM4D bound to RPS5 to form a protein complex, which regulated the demethylation of CNR1 H3K9me2 and further influenced the osteo/dentinogenic differentiation of DPSCs by promoting mitochondrial energy metabolism. These findings identify the KDM4D-RPS5-CNR1 axis as a promising therapeutic target for enhancing DPSC-based dental tissue regeneration.