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Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway
Jun Ying, … , Regis O’Keefe, Jie Shen
Jun Ying, … , Regis O’Keefe, Jie Shen
Published February 13, 2020
Citation Information: JCI Insight. 2020;5(3):e131816. https://doi.org/10.1172/jci.insight.131816.
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Research Article Bone biology

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway

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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.

Authors

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

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Figure 6

Notch inhibition via dual antiplatelet therapy restored fracture repair in Dnmt3bprx1 mice.

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Notch inhibition via dual antiplatelet therapy restored fracture repair ...
(A) Notch pathway inhibition efficacy was determined by real-time qPCR. The mRNA expression of Dnmt3b, HeyL, and Hey1 was measured in fracture calluses of dual antiplatelet therapy–treated (DAPT-treated) and DMSO-treated control mice and Dnmt3bPrx1 mice 3 days after fracture (dpf). (B) Alcian blue/Hematoxylin/Orange G (ABH/OG) staining of fracture callus sections of DAPT- and DMSO-treated control and Dnmt3bPrx1 mice at the indicated times (n = 5). Scale bar: 500 μm. (C) Histomorphometry quantification of the cartilage and bone areas was performed on ABH/OG-stained fracture callus sections (n = 5). (D) Microcomputed tomography assessment of mineralized bone in fracture calluses of DAPT- and DMSO-treated control mice and Dnmt3bPrx1 mice at the indicated times (n = 5). (E) Quantification of bony callus volumes 10 and 14 dpf (n = 5). Data are presented as mean ± SD. *P < 0.05 by 2-way ANOVA test.

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