NDR2 is critical for osteoclastogenesis by regulating ULK1-mediated mitophagy
Xiangxi Kong, Zhi Shan, Yihao Zhao, Siyue Tao, Jingyun Chen, Zhongyin Ji, Jiayan Jin, Junhui Liu, Wenlong Lin, Xiao-jian Wang, Jian Wang, Fengdong Zhao, Bao Huang, Jian Chen
Xiangxi Kong, Zhi Shan, Yihao Zhao, Siyue Tao, Jingyun Chen, Zhongyin Ji, Jiayan Jin, Junhui Liu, Wenlong Lin, Xiao-jian Wang, Jian Wang, Fengdong Zhao, Bao Huang, Jian Chen
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Research Article Development Metabolism

NDR2 is critical for osteoclastogenesis by regulating ULK1-mediated mitophagy

Abstract

Bone homeostasis primarily stems from the balance between osteoblasts and osteoclasts, wherein an augmented number or heightened activity of osteoclasts is a prevalent etiological factor in the development of bone loss. Nuclear Dbf2-related kinase (NDR2), also known as STK38L, is a member of the Hippo family with serine/threonine kinase activity. We unveiled an upregulation of NDR2 expression during osteoclast differentiation. Manipulation of NDR2 levels through knockdown or overexpression facilitated or hindered osteoclast differentiation, respectively, indicating a negative feedback role for NDR2 in the osteoclastogenesis. Myeloid NDR2-dificient mice (Lysm+NDR2fl/fl) showed lower bone mass and further exacerbated ovariectomy-induced or aging-related bone loss. Mechanically, NDR2 enhanced autophagy and mitophagy through mediating ULK1 instability. In addition, ULK1 inhibitor (ULK1-IN2) ameliorated NDR2 conditional KO–induced bone loss. Finally, we clarified a significant inverse association between NDR2 expression and the occurrence of osteoporosis in patients. The NDR2/ULK1/mitophagy axis is a potential innovative therapeutic target for the prevention and management of bone loss.

Authors

Xiangxi Kong, Zhi Shan, Yihao Zhao, Siyue Tao, Jingyun Chen, Zhongyin Ji, Jiayan Jin, Junhui Liu, Wenlong Lin, Xiao-jian Wang, Jian Wang, Fengdong Zhao, Bao Huang, Jian Chen

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Figure 5

NDR2 KO enhanced ULK1 stabilization.

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NDR2 KO enhanced ULK1 stabilization. (A and C) Cellular immunofluorescen...
(A and C) Cellular immunofluorescence staining and statistical analysis of ULK1. RANKL (25 ng/mL, 5 days), ULK1 (green), F-actin (red), and DAPI (blue). (B and D) Fluorescence staining and statistical analysis of tissue sections for ULK1. ULK1 (green) and DAPI (blue). (E) The HeLa cell lines were subjected to overexpression of ULK1-HA plasmid and subsequent transfection with NDR2-Flag plasmid in a concentration gradient, followed by Western blot. (F) The HeLa cell lines were transfected with ULK1-HA and NDR2-Flag plasmids, followed by the detection of the interaction between ULK1 and NDR2 proteins through Co-IP. (G) The Western blot of Lysm–NDR2fl/fl and Lysm+NDR2fl/fl BMMs were analyzed. (H) The HeLa cell lines were transfected with ULK1-HA and NDR2-Flag plasmids, followed by the detection of the ubiquitination of ULK1 through Co-IP. (I and K) Immunofluorescence staining and statistical analysis (n = 6) of LC3 was performed on cellular samples, followed by. RANKL (25 ng/mL, 2 days), LC3 (green), and DAPI (blue). (J and L) Fluorescence staining and statistical analysis were performed on tissue sections to assess LC3 expression (n = 3). ULK1 (green) and DAPI (blue). Statistical analyses were determined by 2-way ANOVA (C, D, K, and L). ***P < 0.001 and ****P < 0.0001. Data were presented as mean ± SD.