Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway.

Jun Ying,Cuicui Wang,Peijian Tong,Regis O’Keefe,Jianjun Guan,Yousef Abu-Amer,Taotao Xu,Jie Shen,Hongting Jin

doi:10.1172/jci.insight.131816

Abstract

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair.

Highlights

Bone fractures commonly result in prolonged disability and increased socioeconomic burden [1]
We show that ablation of DNA methyltransferase 3b (Dnmt3b) is associated with reduced chondrogenic and osteogenic differentiation in mesenchymal progenitor cells (MPCs), leading to decreased cartilaginous and bony callus formation and impaired fracture repair in mice that is due in part to the upregulation of Rbpjκ expression
We previously identified Dnmt3b as the most regulated epigenetic factor responsive to fracture repair process

Summary

Introduction

Bone fractures commonly result in prolonged disability and increased socioeconomic burden [1]. Bone fracture injuries disrupt marrow sinusoidal architecture and surrounding soft tissues, and blood clots form in the fracture area that initiate an early local inflammatory process [5]. During this early hematoma period, endogenous mesenchymal progenitor cells (MPCs), recruited from multiple sources, undergo precisely regulated multidirectional differentiation to generate the cell lineages involved in bone regeneration and vascularization [6,7,8]. MPC-derived chondrocytes and osteoblasts are essential cell populations necessary for cartilaginous and bony callus formation Differentiation of these cells along their lineages results in fracture union and return of the mechanical integrity of the skeleton [9, 10]. Extensive studies define the cellular and molecular contributions to fracture repair, the effect of epigenetic regulations in fracture repair, MPC differentiation, is still largely unknown

Methods

Results

Conclusion