Odaph Promotes Osteoblast Proliferation and Differentiation by Targeting the AMPK/mTOR Signaling Axis to Activate Autophagy

Background: Odontogenesis-associated phosphoprotein (Odaph) is essential for tooth development. However, its role in osteoblast function and bone remodeling remains unclear. Recent studies suggest that Odaph may influence bone integrity, particularly in the maxillofacial region, thereby implicating it in craniofacial skeletal disorders. The study is designed to clarify the regulatory roles of Odaph in the proliferation, differentiation, and Autophagy of osteoblasts, with particular emphasis on its participation in the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway.

Methods: The MC3T3-E1 osteoblast cell line was employed as an in vitro model, and the effects of Odaph overexpression on cell proliferation, differentiation, and migration were assessed via qPCR, Western blotting, CCK-8 assay, EdU staining, Alkaline Phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. RNA Sequencing (RNA-seq) was carried out to screen for differentially expressed genes, and subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG)/GO enrichment analyses were conducted to verify the participation of the AMPK/mTOR signaling pathway. Autophagy was assessed via Western blotting, fluorescence double staining, transmission electron microscopy, and Autophagy tandem lentiviral detection. For exploring the function of Autophagy in osteogenic differentiation, the Autophagy inhibitor 3-MA was used to treat the cells. Furthermore, a mouse model was utilized to confirm the impacts of Odaph overexpression on osteogenesis and Autophagy in vivo.

Results: Overexpression of Odaph markedly enhanced the proliferation, migration, and osteogenic differentiation of MC3T3-E1 cells, which was supported by the increased expression of osteogenic markers runt-related transcription factor 2 (RUNX2), Collagen I (COL1), and ALP. RNA-seq analysis demonstrated that genes regulated by Odaph were notably enriched in the AMPK/mTOR signaling pathway. Further validation demonstrated that Odaph increased AMPK phosphorylation while suppressing mTOR activity. Odaph overexpression also enhanced the expression of autophagy-related proteins LC3B-II and Beclin1 while reducing p62 levels, whereas 3-MA treatment markedly attenuated these pro-osteogenic effects. Consistently, animal experiments confirmed that Odaph overexpression enhanced osteogenesis in vivo, accompanied by increased AMPK activation and Autophagy induction.

Conclusions: Odaph enhances osteoblast function through Autophagy induction mediated by the AMPK/mTOR axis. These results reveal a new regulatory mechanism in bone biology and indicate that Odaph could serve as a potential therapeutic target for maxillofacial bone conditions, including jaw osteopenia and periodontal bone loss.

MTOR signaling pathway; Odontogenesis-associated phosphoprotein; autophagy; osteoblasts; osteogenesis.