Brodie Tooth Development Genetics and Regenerative Medicine Research Laboratory, Department of Oral Biology, University of Illinois at Chicago, Chicago, IL, 60612, USA.
Sci Rep. 2021 Nov 11;11(1):22076. doi: 10.1038/s41598-021-01359-3.
Dentin phosphophoryn synthesized and processed predominantly by the odontoblasts, functions as both structural and signaling protein. Mechanistic studies revealed that DPP stimulation of DPSCs positively impacted the differentiation of DPSCs into functional odontoblasts. Results show that NF-κB signaling and transcriptional activation of genes involved in odontoblast differentiation were influenced by DPP signaling. Specifically, RelA/p65 subunit of NF-κB was identified as being responsible for the initiation of the differentiation cascade. Confocal imaging demonstrated the nuclear translocation of p65 with DPP stimulation. Moreover, direct binding of nuclear NF-κB p65 subunit to the promoter elements of Runx2, Osx, OCN, MMP1, MMP3, BMP4 and PTX3 were identified by ChIP analysis. Pharmacological inhibition of the NF-κB pathway using TPCA-1, a selective inhibitor of IKK-2 and JSH-23, an inhibitor that prevents nuclear translocation and DNA binding of p65 showed impairment in the differentiation process. Functional studies using Alizarin-Red staining showed robust mineral deposits with DPP stimulation and sparse deposition with defective odontoblast differentiation in the presence of inhibitors. In vivo expression of NF-κB targets such as OSX, OCN, PTX3 and p65 in odontoblasts and dental pulp cells from DSPP null mouse was lower when compared with the wild-type. Overall, the results suggest an important role for DPP-mediated NF-κB activation in the transcriptional regulation of early odontogenic markers that promote differentiation of DPSCs.
牙本质磷蛋白主要由成牙本质细胞合成和加工，具有结构和信号蛋白双重功能。机制研究表明，DPP 刺激 DPSCs 可正向影响 DPSCs 向功能性成牙本质细胞的分化。结果表明，DPP 信号影响 NF-κB 信号和牙本质分化相关基因的转录激活。具体来说，NF-κB 的 RelA/p65 亚基被鉴定为起始分化级联反应的关键。共聚焦成像显示 DPP 刺激时 p65 发生核转位。此外，通过 ChIP 分析鉴定到 NF-κB p65 亚基与 Runx2、Osx、OCN、MMP1、MMP3、BMP4 和 PTX3 启动子元件的直接结合。使用 TPCA-1（一种 IKK-2 的选择性抑制剂）和 JSH-23（一种阻止 p65 核转位和 DNA 结合的抑制剂）抑制 NF-κB 通路的药理抑制作用显示，在分化过程中存在缺陷。用 Alizarin-Red 染色进行的功能研究表明，DPP 刺激下有大量矿化沉积物，而在抑制剂存在下成牙本质细胞分化缺陷时矿化沉积物稀疏。与野生型相比，DSPP 缺失小鼠的成牙本质细胞和牙髓细胞中 NF-κB 靶基因 OSX、OCN、PTX3 和 p65 的表达水平较低。总之，这些结果表明 DPP 介导的 NF-κB 激活在早期牙源性标志物的转录调控中发挥重要作用，从而促进 DPSCs 的分化。
