Department of Orthodontics, School of Stomatology, China Medical University, Shenyang, China.
Department of Tissue Engineering, School of Fundamental Sciences, China Medical University, Shenyang, China.
J Periodontal Res. 2020 Jan;55(1):68-76. doi: 10.1111/jre.12689. Epub 2019 Aug 26.
Our study aimed to elucidate the regulatory molecules related to the osteogenic differentiation of periodontal ligament cells (PDLCs).
Periodontal ligament cells are a favorable source for cell-based therapy in periodontal bone engineering and regeneration due to their potential multilineage differentiation ability. However, the molecular mechanism and signaling pathways related to the osteogenic differentiation of PDLCs are still unclear.
Osteoblast-specific protein expression levels were examined by ELISA in osteogenic-induced PDLCs (induced-PDLC group). A microarray assay and a bioinformatics analysis were carried out to reveal significantly expressed genes and the related pathways in induced-PDLCs, and these findings were then confirmed by qRT-PCR and a luciferase reporter assay. Finally, overexpressing and silencing gene systems were established to identify the specific transcriptional relationship and function of the target genes on the osteogenic differentiation of PDLCs.
Osteogenically differentiated PDLCs with high levels of osteoblast-specific proteins were established. The upstream stimulatory factor 2 (USF2) and activating transcription factor 4 (ATF4) mRNA levels were upregulated the most through the MAPK signaling pathway in the induced-PDLC group. USF2 could bind to the transcriptional initiation region of ATF4 and regulate its transcriptional activities. Additionally, the overexpression of USF2 promoted osteoblast-specific gene expression and the Alizarin red staining of PDLCs, while simultaneously overexpressing USF2 and silencing ATF4 reversed the favorable osteogenic effect of the induced-PDLCs by reducing osteoblast-specific gene expression and the Alizarin red staining level.
Our study demonstrated that USF2 could enhance the osteogenic differentiation of PDLCs by regulating ATF4 transcriptional activities, which provides a new strategy to utilize USF2 and ATF4 as potential target molecules for periodontal bone regeneration.
本研究旨在阐明与牙周膜细胞（PDLCs）成骨分化相关的调节分子。
由于牙周膜细胞具有多向分化潜能，因此是牙周骨工程和再生细胞治疗的理想来源。然而，PDLCs 成骨分化相关的分子机制和信号通路仍不清楚。
通过 ELISA 检测成骨诱导 PDLCs（诱导-PDLC 组）中成骨特异性蛋白的表达水平。进行微阵列分析和生物信息学分析，以揭示诱导-PDLCs 中显著表达的基因及其相关通路，并通过 qRT-PCR 和荧光素酶报告基因检测进一步验证。最后，建立过表达和沉默基因系统，以鉴定目标基因在 PDLCs 成骨分化中的特定转录关系和功能。
成功建立了具有高骨特异性蛋白水平的成骨分化 PDLCs。MAPK 信号通路中，上游刺激因子 2（USF2）和激活转录因子 4（ATF4）mRNA 水平上调最明显。USF2 可与 ATF4 的转录起始区结合，并调节其转录活性。此外，过表达 USF2 可促进成骨特异性基因表达和 PDLCs 的茜素红染色，同时过表达 USF2 和沉默 ATF4 可通过降低成骨特异性基因表达和茜素红染色水平，逆转诱导-PDLCs 的有利成骨作用。
本研究表明，USF2 可通过调节 ATF4 转录活性增强 PDLCs 的成骨分化，为利用 USF2 和 ATF4 作为牙周骨再生的潜在靶分子提供了新策略。
