DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation
Ding Xiao, … , Yousef Abu-Amer, Jie Shen
Ding Xiao, … , Yousef Abu-Amer, Jie Shen
Published December 5, 2023
Citation Information: J Clin Invest. 2024;134(3):e168558. https://doi.org/10.1172/JCI168558.
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Research Article Bone biology

DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation

Abstract

Challenging skeletal repairs are frequently seen in patients experiencing systemic inflammation. To tackle the complexity and heterogeneity of the skeletal repair process, we performed single-cell RNA sequencing and revealed that progenitor cells were one of the major lineages responsive to elevated inflammation and this response adversely affected progenitor differentiation by upregulation of Rbpjk in fracture nonunion. We then validated the interplay between inflammation (via constitutive activation of Ikk2, Ikk2ca) and Rbpjk specifically in progenitors by using genetic animal models. Focusing on epigenetic regulation, we identified Rbpjk as a direct target of Dnmt3b. Mechanistically, inflammation decreased Dnmt3b expression in progenitor cells, consequently leading to Rbpjk upregulation via hypomethylation within its promoter region. We also showed that Dnmt3b loss-of-function mice phenotypically recapitulated the fracture repair defects observed in Ikk2ca-transgenic mice, whereas Dnmt3b-transgenic mice alleviated fracture repair defects induced by Ikk2ca. Moreover, Rbpjk ablation restored fracture repair in both Ikk2ca mice and Dnmt3b loss-of-function mice. Altogether, this work elucidates a common mechanism involving a NF-κB/Dnmt3b/Rbpjk axis within the context of inflamed bone regeneration. Building on this mechanistic insight, we applied local treatment with epigenetically modified progenitor cells in a previously established mouse model of inflammation-mediated fracture nonunion and showed a functional restoration of bone regeneration under inflammatory conditions through an increase in progenitor differentiation potential.

Authors

Ding Xiao, Liang Fang, Zhongting Liu, Yonghua He, Jun Ying, Haocheng Qin, Aiwu Lu, Meng Shi, Tiandao Li, Bo Zhang, Jianjun Guan, Cuicui Wang, Yousef Abu-Amer, Jie Shen

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Figure 7

Rbpjk inhibition by epigenetic modification using CRISPR/dCas9/Dnmt3a editing system rescues fracture nonunion in RA mice.

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Rbpjk inhibition by epigenetic modification using CRISPR/dCas9/Dnmt3a ed...
(A) PCL scaffold with dCas9-Dnmt3a–engineered C3H10T1/2 cells (blue arrow) was applied to the fractured bone in RA mice. (B) ABH/OG staining of callus sections from 14-dpf wild-type RA fractures grafted with PCL scaffolds that were fabricated with dCas9-Dnmt3a-scramble (control) or dCas9-Dnmt3a-Rbpjk1-9 C3H10T1/2 cells (Rbpjk epigenetically modified) (n = 5). Scale bars: 100 mm. (C) Histomorphometric quantifications of bone area based on ABH/OG staining (n = 5). (D) Micro-CT analyses of newly formed bony callus from 14-dpf wild-type RA fractures grafted with PCL scaffolds that were fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells (n = 5). Scale bars: 0.5 mm. (E) Bony callus volume and BV/TV measures of 14-dpf wild-type RA fractures grafted with PCL scaffolds fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells (n = 5). (F) Biomechanical torsion testing of 28-dpf wild-type RA fractures grafted with PCL scaffolds fabricated with control or Rbpjk epigenetically modified C3H10T1/2 cells. Maximum torque and displacement at maximum torque were recorded during testing (n = 8). Data presented as mean ± SD. *P < 0.05 by 2-tailed Student’s t test.