Objective: Skeletal mandibular protrusion, is prevalent in the Japanese population. Despite therapeutic interventions during the developmental stages, some individuals experience recurrence from mandibular overgrowth. Therefore, it is imperative to identify the factors contributing to mandibular overgrowth. Recent studies have identified nonsynonymous mutations in BEST3 in Japanese patients with mandibular prognathism. However, the specific role of BEST3 in bone metabolism remains unclear. This study aimed to investigate the effect of BEST3 on bone differentiation. Mouse mesenchymal-derived ST2 cells were transfected with BEST3, and the expression of genes associated with mineralization, osteoblast differentiation, and osteoclast differentiation was analyzed. Methods: Mouse ST2 cells were maintained in RPMI1640 medium. Osteoblastic differentiation was induced using α-MEM supplemented with ascorbic acid and β-glycerophosphate. Mineralization was assessed by Alizarin Red S staining. A BEST3 expression vector (pBEST3) was constructed using cDNA derived from HEK293 cells and transfected with Xfect for transient overexpression. Gene expression was evaluated using RT-qPCR of total RNA, and RANKL expression was quantified by ELISA. Results: The overexpression of BEST3 did not influence osteoblast differentiation or mineralization. Furthermore, the expression of bone formation-related genes, including Bmp2, Bmp4, Alp, Col1a1, and Bglap, remained largely unaffected despite the overexpression of BEST3. In contrast, there was a significant upregulation in the expression of osteoclast differentiation factors, notably RANKL (Tnfsf11) and M-CSF (Csf1). Overexpression of BEST3 was correlated with increased RANKL protein levels. Conclusion: Overexpression of ectopic BEST3 in osteoblast-like ST2 cells promotes RANKL gene and protein expression.
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