USF2 enhances the osteogenic differentiation of PDLCs by promoting ATF4 transcriptional activities
USF2 enhances the osteogenic differentiation of PDLCs by promoting ATF4 transcriptional activities
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USF2通过促进ATF4转录活性增强PDLCs的成骨分化
DOI：
10.1111/jre.12689
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发表时间：
2019-08-26
影响因子：
3.5
通讯作者：
Liu, Yi
中科院分区：
文献类型：
--
作者：
Liu, Fan;Wang, Xiaohong;Liu, Yi
目的研究牙周膜细胞（PDLCs）成骨分化的调控分子。背景牙周膜细胞具有多向分化潜能，是牙周骨工程和再生中细胞治疗的理想来源。然而，PDLCs向成骨细胞分化的分子机制和信号通路尚不清楚。方法采用ELISA法检测成骨诱导PDLC（诱导PDLC组）成骨细胞特异性蛋白表达水平。通过基因芯片分析和生物信息学分析，揭示诱导后PDLCs中显著表达的基因和相关通路，并通过qRT-PCR和荧光素酶报告基因分析证实这些发现。最后，建立过表达和沉默基因系统，以确定目的基因在PDLCs成骨分化中的特异性转录关系和功能。结果获得了成骨分化的PDLCs，其成骨细胞特异性蛋白表达水平较高。上游刺激因子2（USF 2）和转录激活因子4（ATF 4）的mRNA水平上调最多的诱导PDLC组通过MAPK信号通路。USF 2可与ATF 4的转录起始区结合，调控其转录活性。此外，USF 2的过表达促进成骨细胞特异性基因表达和PDLCs的茜素红染色，而同时过表达USF 2和沉默ATF 4通过降低成骨细胞特异性基因表达和茜素红染色水平逆转了诱导PDLCs的有利成骨作用。结论USF 2可通过调控ATF 4的转录活性促进PDLCs向成骨细胞分化，为USF 2和ATF 4作为牙周骨再生的靶分子提供了新的思路。
Objective Our study aimed to elucidate the regulatory molecules related to the osteogenic differentiation of periodontal ligament cells (PDLCs). Background 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. Methods 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. Results 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. Conclusions 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.