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 4

NDR2 regulated osteoclastogenesis via ULK1.

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NDR2 regulated osteoclastogenesis via ULK1. (A) Transmission electron mi...
(A) Transmission electron microscope (TEM) image of osteoclastic progenitor cells. The image below is magnified. The yellow arrowheads mark the autophagosome. RANKL (25 ng/mL) was administered to stimulate the cells for 48 hours. (B) Detection of autophagy-associated marker proteins in Lysm–NDR2fl/fl and Lysm+NDR2fl/fl mouse-derived bone marrow macrophages (BMMs) at different time points induced by osteoclasts. (C) ULK1 siRNA was transfected into BMMs with Lysm+NDR2fl/fl genotype, followed by stimulation with RANKL after 12 hours. Protein extraction was performed 48 hours later for Western blot. (D) TRAP staining images of BMMs derived from Lysm–NDR2fl/fl and Lysm+NDR2fl/fl after stimulation of RANKL. Enlarge image below. (E) The differential expression of osteoclast-related genes was assessed by qPCR following ULK1 siRNA treatment (n = 6). (F and G) The levels of osteoclast-related proteins and autophagy were altered following the addition of ULK1 inhibitors, namely ULK1-IN2 (200 nmol) and SBI6965 (20 nmol). (H and I) TRAP staining images with or without ULK1 inhibitors. (J and K) Osteoclast-related gene changes were statistically analyzed with or without ULK1 inhibitors. (L) The osteogenic differentiation of the MC3T3-E1 cell line was induced for 21 days, followed by alizarin red staining and induced for 14 days with alkaline phosphatase staining. Statistical analyses were determined by 1-way ANOVA (J and K), and 2-way ANOVA (E). **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were presented as mean ± SD.