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 1

scRNA-seq analysis of fracture callus reveals distinct differentiation trajectories of progenitors in control and RA mice.

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scRNA-seq analysis of fracture callus reveals distinct differentiation t...
(A and B) Twenty cell clusters from control and RA fracture callus at 4 and 7 dpf (n = 3). A tSNE projection of 25,467 single-cell transcriptomes, annotated post hoc and colored by clustering (A), or by key cell type–specific markers (B). (C) Cluster signature genes. Expression of top differentially expressed genes (rows) scaled across the cells (columns) in each cluster (color bar on top corresponds to color scheme in A), grouped by lineage and conditions (group identity indicated at the bottom). Key genes of some clusters are highlighted on the right. (D) Number of cells in each cluster. Color scheme as in A. (E) Differentiation trajectory of mesenchymal lineage cells constructed by Monocle and colored by pseudotime order (left) and Seurat clusters (right). Cell fractions of each cluster of different conditions (control, unshaded; RA, shaded) shown by pie charts. (F) Pair-wise comparison of cellular compositions of mesenchymal subset by conditions (Control_4d vs. Control_7d; RA_4d vs. RA_7d; Control_4d vs. RA_4d; Control_7d vs. RA_7d), visualized by alluvial plots. (G) The enriched KEGG pathway analysis of genes unregulated and downregulated in RA fracture callus compared with those in controls.