Research ArticleDevelopmentMetabolism Open Access | 10.1172/jci.insight.180409

Xiangxi Kong,1,2 Zhi Shan,1,2 Yihao Zhao,1,2 Siyue Tao,1,2 Jingyun Chen,3 Zhongyin Ji,1,2 Jiayan Jin,1,2 Junhui Liu,1,2 Wenlong Lin,4 Xiao-jian Wang,4 Jian Wang,5 Fengdong Zhao,1,2,5 Bao Huang,1,2 and Jian Chen1,2,5

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

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1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

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1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Zhao, Y. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Tao, S. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Chen, J. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Ji, Z. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Jin, J. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Liu, J. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Lin, W. in: JCI | PubMed | Google Scholar |

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Wang, X. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Wang, J. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Zhao, F. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Huang, B. in: JCI | PubMed | Google Scholar

1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

2Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, China.

3Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

4Institute of Immunology and Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.

5Department of Wound Healing, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.

Address correspondence to: Jian Chen, Bao Huang, or Fengdong Zhao, No. 3, Qingchun Road East, Hangzhou, 310016, China. Phone: 0571.86006667; E-mail: chenjian-bio@zju.edu.cn (Jian Chen); huangbao@zju.edu.cn (BH); zhaofengdong@zju.edu.cn (FZ).

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Find articles by Chen, J. in: JCI | PubMed | Google Scholar

Authorship note: XK, ZS, and YZ contributed equally to this work and should be regarded as the co–first authors.

Published November 19, 2024 - More info

Published in Volume 10, Issue 1 on January 9, 2025
JCI Insight. 2025;10(1):e180409. https://doi.org/10.1172/jci.insight.180409.
© 2024 Kong et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published November 19, 2024 - Version history
Received: February 16, 2024; Accepted: November 13, 2024 View PDF 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.

Introduction

The maintenance of bone mass and physiological function relies on a dynamic equilibrium between osteoclas-mediated (OC-mediated) bone resorption and osteoblast-mediated (OB-mediated) bone formation (1). OCs originate from hematopoietic stem cells within the bone marrow mononuclear cells (BMMs), with their formation and function being regulated by macrophage–colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) (2). Notably, excessive activation of OCs plays a crucial role in the pathogenesis of diseases such as osteoporosis, rheumatoid arthritis–induced bone destruction, and cancer-related bone metastasis (3). Therefore, it is imperative to investigate the regulatory mechanisms underlying OC formation and to identify novel targets for preventing and treating disorders related to bone metabolism.

NDR2 (nuclear Dbf2-related kinase) serine/threonine kinase, also referred to as STK38L, has been identified as a novel member of the Hippo family (4). In addition to a central kinase catalytic domain, NDR2 also features a conserved N-terminal regulatory (NTR) domain and a hydrophobic motif at the C-terminus (5). NDR2 has been recognized as protein kinase that involves in a variety of biological processes, encompassing morphological alterations, centrosome duplication, cell cycle regulation, apoptosis induction, embryonic development, neurodevelopmental processes, and cancer biology (6–13). While several components of the Hippo signaling pathway, such as YAP/TAZ/TEAD complex, RASSF2, MST2, and Ajuba, are known to play crucial roles in OC differentiation (14), the specific involvement of NDR1/2 in this process remains elusive.

Autophagy is a self-clearing process and the degradation of the packaged contents within these autophagosomes facilitates recycling and renewal of cellular components necessary for metabolic updates and turnover of certain organelles (15, 16). OCs secrete β3 integrin and generate actin rings that attach to the bone surface, resulting in the formation of ruffled borders within resorption lacunae. These cells also release autophagy-associated proteins such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), which play crucial roles in bone resorption (17). Key autophagy-related proteins including ATG5, ATG7, ATG4B, and LC3 are essential for the formation of osteoclastic ruffled borders (18, 19). Manipulating autophagic activity effectively modulates the extent of osteoclastic differentiation. TET2 facilitates OC differentiation by downregulating BCL2 expression and positively modulating BECN1-dependent autophagy (20).

The ULK1 complex, consisting of ULK1, ATG13, ATG101, and FIP200 and considered the initiator of autophagy, is activated by upstream kinases during energy or nutrient deprivation to regulate downstream substrates such as ATG9, P62, and PI3KC3-C1 complexes (21). However, there is limited research on the regulation of ULK1 in OC differentiation. Multiple posttranslational modifications, including phosphorylation, acetylation, and ubiquitination, have been demonstrated to modulate ULK1 activity, with particular emphasis on its phosphorylation (22). ULK1 can be phosphorylated by mTORC1, AMPK, and p38 MAPK-related kinases (23, 24). Our previous studies have demonstrated that NDR2 can also induce ubiquitination and degradation of retinoic acid inducible gene I (RIG-I), thereby exerting antiviral immunity (25). Recently, NDR2 was found to phosphorylate ULK1 at Ser495, which resulted in proteasomal degradation of ULK1 and inhibition of autophagy (26). The potential role of the kinase NDR2 in modulating autophagy level and affecting OC differentiation and function warrants further investigation.

We have discovered that the degradation of ULK1 is mediated by NDR2-induced phosphorylation, resulting in alterations in the expression of downstream autophagy-related proteins. This subsequently restricts the levels of autophagy and mitophagy within OCs, consequently affecting OC differentiation and bone resorption. Our findings hold significant implications for further investigating the role of kinase phosphorylation and ULK1-mediated autophagy in osteoporosis-related diseases, while also offering targets and a theoretical foundation for preventing and treating bone metabolic disorders.

Results

NDR2 inhibited osteoclastogenesis and had little effect on osteogenesis. To elucidate the role of NDR2 in osteoclastogenesis, we initially assessed the expression level of NDR2 during OC differentiation. Both protein and gene expression levels of NDR2 were found to be upregulated in response to OC differentiation (Figure 1, A and B, and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.180409DS1) in a concentration-dependent manner (Figure 1, C and D, and Supplemental Figure 1B). Cellular immunofluorescence staining revealed an increase in fluorescence intensity of NDR2 following RANKL stimulation (Figure 1, E and F), suggesting that elevated levels of NDR2 may play a crucial role in osteoclastogenesis. In order to further ascertain whether NDR2 acts as a driving force or inhibitory factor for OC differentiation, BMMs were treated with NDR2 siRNA (Supplemental Figure 1, C and D). The group treated with NDR2 siRNA exhibited a higher number and larger area occupied by OCs compared with the control group (Figure 1, G and H). Considering the superior knockdown efficiency achieved by using NDR2 siRNA-1, subsequent experiments were conducted employing this siRNA. Consistent with TRAP staining results, Western blot demonstrated significant upregulation of OC-related markers (Nfatc1, c-Fos) upon knockdown of NDR2 (Supplemental Figure 1E). Furthermore, BMMs transfected with lentivirus overexpressing NDR2 displayed notable capacity for inhibiting osteoclastogenesis (Figure 1, I and J). These findings indicate that, while there is an initial increase in expression levels of NDR2 during OC differentiation, subsequent negative feedback mechanisms inhibit further progression toward mature osteoclastic phenotypes.

Figure 1

NDR2 inhibited osteoclastogenesis and had little effect on osteogenesis. (A) Proteins were extracted from BMMs at different time points of osteoclast induction, and Western blot was performed. The concentrations of M-CSF and RANKL used were 50 ng/mL and 25 ng/mL, respectively. (B) The mRNA levels of the NDR2 were assessed at various time points during osteoclast differentiation using qPCR (n = 6). (C) NDR2 protein expression was examined in BMMs treated with different concentrations of RANKL for 48 hours. (D) qPCR analysis was performed to assess the mRNA levels of the NDR2 after 48 hours of treatment with varying concentrations of RANKL (n = 6). (E) Representative images of NDR2 fluorescence staining were obtained, with NDR2 shown in green, F-actin in red, and DAPI in blue. (F) Statistical analysis was conducted to evaluate the intensity of NDR2 fluorescence (n = 3). (G) BMMs were transfected with NDR2 siRNA for 12 hours, followed by induction treatment with RANKL (25 ng/mL) for 5 days and subsequent TRAP staining. (H) Statistical analysis was performed on the area of TRAP+ osteoclasts (n = 3). (I) BMMs were subjected to TRAP staining 5 days after RANKL stimulation. (J) Western blotting was conducted on MC3T3-E1 cells that were subjected to a 7-day period of osteogenic induction in order to evaluate the protein levels associated with bone formation. (K) Alizarin red staining was used to visualize mineralized nodules after 21 days of osteogenic induction, while alkaline phosphatase staining was utilized to detect early-stage osteoblastic differentiation after 14 days. Statistical analyses were determined by 2-tailed Student’s t test (F) or 1-way ANOVA (B, D, and H). ***P < 0.001, ****P < 0.0001. Data were presented as mean ± SD.

To eliminate the influence of NDR2 on OBs, we employed the MC3T3-E1 cell line for inducing osteogenic differentiation. Knockdown of NDR2 exhibited a marginal effect on osteoblastogenesis-related genes (Runx2, Alpl, Bglap, Spp1) and proteins (RUNX2) (Supplemental Figure 1, F and G), while alkaline phosphatase (ALP) and alizarin red S (ARS) staining revealed no substantial differences between the experimental groups (Figure 1K). In conclusion, NDR2 exerts negative feedback inhibition on osteoclastogenesis without affecting OBs.

Lysm+NDR2fl/fl mice exhibited decreased bone mass and enhanced osteoclastogenesis. The Lysm+NDR2fl/fl mice were generated by crossing NDR2fl/fl mice with Lysm-cre mice. The μ-CT results reveal a decrease in bone mass in Lysm+NDR2fl/fl mice compared with NDR2fl/fl counterparts. Specifically, there was a reduction observed in the bone volume/tissue volume (BV/TV) and trabecular number (Tb.N), accompanied by an increase in trabecular separation (Tb.Sp) (Figure 2, A and B). Additionally, conditional KO of NDR2 resulted in a significant reduction in cortical bone thickness (Ct.Th) (Figure 2, C and D). Similar observations of spontaneous bone loss were observed in both trabecular and cortical bone for female mice, which mirrored the findings seen in male mice (Supplemental Figure 2, A–D). TRAP staining revealed an increased presence of OCs in distal femoral sections of Lysm+NDR2fl/fl mice, and quantitative histomorphological analysis demonstrated upregulation of both the ratios of OC area/bone tissue area (Oc.S/BS) and OC number/bone tissue area (Oc.N/BS) (Figure 2, E–G).

Figure 2

Lysm+NDR2fl/fl mice exhibited decreased bone mass and enhanced osteoclastogenesis. (A) Femurs from 2-month-old Lysm–NDR2fl/fl and Lysm+NDR2fl/fl littermates were imaged using μCT techniques. (B) Parameters related to trabecular bone in the proximal femur (n = 6) were analyzed, including bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). (C) Representative 2D images of cortical bone were captured. (D) Statistical analysis was performed on cortical bone thickness (Ct.Th). (E) H&E staining and TRAP staining were conducted on sections of the femur. (F and G) Histomorphological analysis was carried out on TRAP staining, specifically osteoclast number per unit of bone surface area (N.Oc/BS) and osteoclast surface area per unit of bone surface area (Oc.S/BS; n = 6 per group). (H) Bone marrow–derived macrophages (BMMs) extracted from Lysm–NDR2fl/fl and Lysm+NDR2fl/fl littermates were induced for osteoclast differentiation at different concentrations of RANKL. After 5 days, TRAP staining was performed. (I) Western blotting was used to detect differences in expression levels of osteoclast-related proteins between the 2 groups after RANKL stimulation. (J) The activity of osteoclasts was assessed using hydroxyapatite-coated plates. (K) Statistical analysis was conducted to assess the extent of bone erosion area (n = 3). Statistical analyses were determined by 2-tailed Student’s t test (B, D, F, G, and K). **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were presented as mean ± SD.

To further elucidate whether the reduced bone mass observed in Lysm+NDR2fl/fl mice resulted from enhanced osteoclastogenesis, bone marrow–derived macrophages (BMMs) derived from NDR2fl/fl and Lysm+NDR2fl/fl mice were isolated for subsequent in vitro investigations. Under different RANKL concentrations, OCs derived from NDR2-KO mice appeared earlier and fused more, especially at lower concentrations (Figure 2H). The expression levels of OC differentiation–related markers (Nfatc1, Acp5, and c-Fos) were significantly higher in comparison with those observed in NDR2fl/fl-derived BMMs (Figure 2I). The demineralization assay indicated enhanced OC activity and a larger area of bone plate erosion following NDR2 knockdown (Figure 2, J and K). Consistent with these findings, there was also a propensity toward proosteoclast activity at the genetic level, exhibiting significant genetic differences even without RANKL induction (Supplemental Figure 2E). Therefore, it can be inferred that NDR2 knockdown accelerates bone loss by promoting osteoclastogenesis and enhancing OC activity.

Lysm+NDR2fl/fl mice aggravated OVX-induced and aging-related bone loss. Osteoporosis is a prevalent complication in postmenopausal women, with estrogen withdrawal being the primary factor contributing to OC hyperactivity (27). To investigate the potential involvement of NDR2 in estrogen deficiency–induced osteoporosis, ovariectomy was performed on 12-week-old female Lysm+NDR2fl/fl mice. Surprisingly, μCT 2D and 3D images reveal a significant reduction in bone trabeculae after OVX in the Lysm+NDR2fl/fl group compared with the NDR2fl/fl group, as evidenced by a decrease in BV/TV (%) and Tb.Th (mm–1) and an increase in Tb.Sp (mm) (Figure 3, A and B). The histomorphological staining results further confirm a significant increase in the number of OCs in vivo following NDR2 KO (Figure 3, C–E), indicating that NDR2 exacerbates osteoclastogenesis and promotes bone loss after OVX modeling. Osteocalcin (OCN), a noncollagenous protein expressed and secreted by OBs, serves as an indicator of bone formation in vivo. OCN fluorescence staining revealed no significant difference in OB numbers between groups, indicating that there was no effect on bone formation (Supplemental Figure 3, A and B).

Figure 3

Lysm+NDR2fl/fl mice aggravated OVX-induced and aging-related bone loss. (A) OVX was performed on 2-month-old Lysm–NDR2fl/fl and Lysm+NDR2fl/fl mice, and the mice were sacrificed after 8 weeks for femur sample collection. μCT was utilized to illustrate the alterations in femoral trabeculae. (B) Analysis of parameters associated with trabecular bone in the proximal femur was conducted (n = 6). (C) H&E staining and TRAP staining were performed on sections of the femur. (D and E) Histomorphological analysis of TRAP staining was carried out (n = 6). (F) Representative μCT images of 18-month-old Lysm–NDR2fl/fl and Lysm+NDR2fl/fl mouse femurs. (G) Analysis of parameters related to trabecular bone in the proximal femur (n = 6). (H) H&E staining and TRAP staining were conducted on sections of the femur. (I) Histomorphological analysis of TRAP staining (n = 6). Statistical analyses were determined by 2-tailed Student’s t test (G and I), and 2-way ANOVA (B, D, and E). ns indicated no statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were presented as mean ± SD.

Aging is also a significant contributing factor to the development of osteoporosis (28, 29); however, its underlying mechanism remains intricate and not yet fully elucidated. In the 18-month Lysm+NDR2fl/fl group compared with NDR2fl/fl group, there was observed a decrease in bone mass (Figure 3, F and G, and Supplemental Figure 3, E and F), accompanied by an increased number of OCs (Figure 3, H and I). These findings suggest that NDR2 KO further exacerbates age-related bone loss. Furthermore, we discovered a significant downregulation of NDR2 expression levels in both the OVX model and aging groups (Supplemental Figure 3, C, D, G, and H), indicating that the diminished inhibitory effect of NDR2 on osteoclastogenesis may serve as an important factor in the pathogenesis of osteoporosis.

NDR2 regulated osteoclastogenesis via ULK1. To further elucidate the underlying mechanisms of NDR2 in regulating OC differentiation, we initially investigated the classical pathway of osteoclastogenesis. Following RANKL induction, there was a marginal alteration in MAPK/NF-κB levels upon NDR2 knockdown, with even slight downregulation observed in Erk and p38 phosphorylation levels. These findings suggest that the augmented osteoclastogenesis resulting from NDR2 knockdown is not mediated through these canonical pathways (Supplemental Figure 4A).

Autophagy plays a pivotal role in osteoclastogenesis; the inhibition of autophagy was found to impede OC differentiation, consistent with previous research (Supplemental Figure 4, B and C). We investigated whether NDR2 knockdown could modulate autophagy in OCs, and we subsequently assessed the level of autophagy in proosteoclast cells. The Lysm+NDR2fl/fl group exhibited an increased number of autophagosomes in proosteoclast cells (Figure 4A), indicating a significant upregulation of autophagy in this group. Furthermore, the upregulation of LC3, an essential autophagy marker, following NDR2 KO suggests the induction of active autophagy (Figure 4B). ULK1 (UNC-51–like kinase 1), a core component of the ULK1 complex, plays a crucial role in initiating autophagy (30). After NDR2 knockdown, the expression levels of ULK1 were elevated compared with those in the NDR2fl/fl group, whereas they were downregulated upon NDR2 overexpression (Supplemental Figure 4E). Therefore, we postulated that NDR2 modulates autophagy by influencing ULK1, thereby exerting regulatory control over osteoclastogenesis. The alterations observed in other autophagy markers may be attributed to variations in ULK1 levels.

Figure 4

NDR2 regulated osteoclastogenesis via ULK1. (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.

ULK1 siRNA was employed to further investigate whether NDR2 knockdown promotes osteoclastogenesis through ULK1. Knockdown of ULK1 resulted in reduced levels of the autophagy marker LC3B and inhibition of OC-related markers (Figure 4C). TRAP staining demonstrated that the OC-promoting effect of NDR2 knockdown could be rescued by ULK1 siRNA (Figure 4D). Consistent with the protein results, downregulation of OC-related gene expression was observed upon treatment with ULK1 siRNA (Figure 4E and Supplemental Figure 4D). An ULK1-specific inhibitor (ULK1-IN2) was also utilized to confirm that NDR2 knockdown–induced overactivity in osteoclastogenesis is mediated by ULK1, which significantly decreased autophagy levels and inhibited OC differentiation (Figure 4, F, H, and J). Similarly, SBI6965, another selective inhibitor for ULK1, exhibited a similar inhibitory effect on autophagy and OC differentiation (Figure 4, G, I, and K). Based on these findings, we concluded that upregulation of ULK1 induced by NDR2 knockdown enhanced osteoclastogenesis via promotion of autophagy. Simultaneously, ALP and ARS staining were performed to validate the effect of these 2 inhibitors on osteoblastogenesis. The results demonstrate that SBI6965 exhibited an inhibitory effect on osteoclastogenesis at the concentration that effectively suppressed OB differentiation, whereas ULK1-IN2 did not exhibit such an effect (Figure 4L).

NDR2 KO enhanced ULK1 stabilization and promoted mitophagy. To explore how NDR2 regulates ULK1 to affect osteoclastogenesis, we initially conducted fluorescence staining for ULK1 and observed an enhanced fluorescence intensity following NDR2 knockdown (Figure 5, A and C). During OC differentiation, autophagy levels progressively increased, leading to the degradation of ULK1 along with autophagosomes in lysosomes. Consequently, the level of ULK1 decreased in the late stage compared with the early stage. Subsequently, tissue section staining confirmed higher expression of ULK1 in the Lysm+NDR2fl/fl group compared with the NDR2fl/fl group, with further enhancement of ULK1 fluorescence intensity after OVX and aging modeling (Figure 5, B and D, and Supplemental Figure 5, A and B). Additionally, gradient overexpression of NDR2 in HeLa cells resulted in reduced levels of ULK1 protein (Figure 5E), and coimmunoprecipitation (Co-IP) analysis validated the interaction between NDR2 and ULK1 proteins (Figure 5F).

Figure 5

NDR2 KO enhanced ULK1 stabilization. (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 Wes