The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i^−/−) arrested tooth root formation, indicated by truncated Hertwig’s epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i^−/− mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i^−/− mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-β (TGF-β) signaling in Atp6i^−/− odontoblasts. Through treating pulp precursor cells with Atp6i^−/− or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i^−/− OC conditioned medium. Importantly, ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i^−/− mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

Runt-related transcription factor 1 (Runx1) plays a key role in cartilage formation, but its function in articular cartilage formation is unclear. We generated non-inducible and inducible Runx1-deficient mice (Runx1^f/fCol2α1-Cre and Runx1^f/fCol2α1-CreER mice) and found that chondrocyte-specific Runx1-deficient mice developed a spontaneous osteoarthritis (OA)-like phenotype and showed exacerbated articular cartilage destruction under OA, characterized by articular cartilage degradation and cartilage ossification, with decreased Col2α1 expression and increased Mmp13 and Adamts5 expression. RNA-sequencing analysis of hip articular cartilage from the Runx1^f/fCol2α1-Cre mice compared to that from wild-type mice and subsequent validation analyses demonstrated that Runx1 is a central regulator in multiple signaling pathways, converging signals of the Hippo/Yap, TGFβ/Smad, and Wnt/β-catenin pathways into a complex network to regulate the expression of downstream genes, thereby controlling a series of osteoarthritic pathological processes. RNA-sequencing analysis of mutant knee joints showed that Runx1’s role in signaling pathways in articular cartilage is different from that in whole knee joints, indicating that Runx1 regulation is tissue-specific. Histopathologic analysis confirmed that Runx1 deficiency decreased the levels of YAP and p-Smad2/3 and increased the levels of active β-catenin. Overexpression of Runx1 dramatically increased YAP expression in chondrocytes. Adeno-associated virus-mediated Runx1 overexpression in the knee joints of osteoarthritic mice showed the protective effect of Runx1 on articular cartilage damaged in OA. Our results notably showed that Runx1 is a central regulator of articular cartilage homeostasis by orchestrating the YAP, TGFβ, and Wnt signaling pathways in the formation of articular cartilage and OA, and targeting Runx1 and its downstream genes may facilitate the design of novel therapeutic approaches for OA.