USF2 enhances the osteogenic differentiation of PDLCs by promoting ATF4 transcriptional activities.

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.

Conclusion: 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.