State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, China.
Department of Cariology and Endodontics, School of Stomatology, Wuhan University, Wuhan, China.
J Cell Biochem. 2024 Jul;125(7):e30577. doi: 10.1002/jcb.30577. Epub 2024 May 9.
Odontoblast differentiation is a key process in dentin formation. Mouse dental papilla cells (mDPCs) are pivotal in dentinogenesis through their differentiation into odontoblasts. Odontoblast differentiation is intricately controlled by transcription factors (TFs) in a spatiotemporal manner. Previous research explored the role of RUNX2 and KLF4 in odontoblast lineage commitment, respectively. Building on bioinformatics analysis of our previous ATAC-seq profiling, we hypothesized that KLF4 potentially collaborates with RUNX2 to exert its biological role. To investigate the synergistic effect of multiple TFs in odontoblastic differentiation, we first examined the spatiotemporal expression patterns of RUNX2 and KLF4 in dental papilla at the bell stage using immunostaining techniques. Notably, RUNX2 and KLF4 demonstrated colocalization in preodontoblast. Further, immunoprecipitation and proximity ligation assays verified the interaction between RUNX2 and KLF4 in vitro. Specifically, the C-terminus of RUNX2 was identified as the interacting domain with KLF4. Functional implications of this interaction were investigated using small hairpin RNA-mediated knockdown of Runx2, Klf4, or both. Western blot analysis revealed a marked decrease in DSPP expression, an odontoblast differentiation marker, particularly in the double knockdown condition. Additionally, alizarin red S staining indicated significantly reduced mineralized nodule formation in this group. Collectively, our findings highlight the synergistic interaction between RUNX2 and KLF4 in promoting odontoblast differentiation from mDPCs. This study contributes to a more comprehensive understanding of the regulatory network of TFs governing odontoblast differentiation.
成牙本质细胞分化是牙本质形成的关键过程。小鼠牙乳头细胞（mDPCs）通过分化为成牙本质细胞在牙本质发生中起着关键作用。成牙本质细胞分化受转录因子（TFs）的时空调控。先前的研究分别探讨了 RUNX2 和 KLF4 在成牙本质细胞谱系决定中的作用。基于我们之前 ATAC-seq 分析的生物信息学分析，我们假设 KLF4 可能与 RUNX2 合作发挥其生物学作用。为了研究多个 TF 在成牙本质细胞分化中的协同作用，我们首先使用免疫染色技术检查了钟状期牙乳头中 RUNX2 和 KLF4 的时空表达模式。值得注意的是，RUNX2 和 KLF4 在前期成牙本质细胞中表现出共定位。此外，免疫沉淀和邻近连接测定在体外验证了 RUNX2 和 KLF4 之间的相互作用。具体而言，RUNX2 的 C 端被确定为与 KLF4 相互作用的结构域。使用小发夹 RNA 介导的 Runx2、Klf4 或两者的敲低研究了这种相互作用的功能意义。Western blot 分析显示，DSPP 表达，一种成牙本质细胞分化标志物，特别是在双敲低条件下，明显下降。此外，茜素红 S 染色表明该组矿化结节形成明显减少。总之，我们的研究结果强调了 RUNX2 和 KLF4 之间在促进 mDPCs 成牙本质细胞分化中的协同相互作用。这项研究有助于更全面地了解调节成牙本质细胞分化的 TF 调控网络。
