EZH1-DNMT1 axis inhibits the expression of TFPI2 to promote osteogenic differentiation of periosteum-derived stem cells and accelerate fracture repair

Background: The periosteum lies in a dynamic environment with a niche of periosteum-derived stem cells (PDSCs) for their reparative needs. Here, we report that epigenetic repression of tissue factor pathway inhibitor 2 (TFPI2) mediates the osteogenic potential of PDSCs and the ensuing fracture repair.

Methods: Significantly overexpressed TFPI2 after fracture was screened using the GSE152677 dataset, and the expression of TFPI2 in bone tissues of post-fracture mice was verified by RT-qPCR and immunohistochemistry. Loss- and gain-of-function assays were conducted using adenoviruses. Primary mouse PDSCs were extracted, and their osteogenic potential was assessed using ALP staining, alizarin red staining, and western blot analysis. The epigenetic modifiers of TFPI2 were verified using ChIP-qPCR, Co-IP, and qMSP.

Results: TFPI2 expression was elevated after fracture, whereas enhancer of zeste homolog 1 (EZH1) expression was significantly downregulated. Inhibition of TFPI2 expression promoted fracture repair in mice, which was correlated with enhanced osteogenic differentiation of PDSCs. EZH1 repressed TFPI2 expression by modifying trimethylation of histone H3 at lysine 27 (H3K27me3). EZH1 promoted TFPI2 promoter DNA methylation by recruiting DNA-methyltransferase 1 (DNMT1), leading to transcriptional repression of TFPI2. Overexpression of DNMT1 and EZH1 significantly promoted recovery in fractured mice, which was reversed by inhibition of TFPI2.

Conclusions: These results suggest that artificial overexpression EZH1 mediates TFPI2 inhibition by recruiting DNMT1, promoting osteogenic differentiation of PDSCs to accelerate fracture repair.

DNMT1; EZH1; Fracture repair; Periosteum-derived stem cells; TFPI2.