Keywords
Osteoporosis; Osteoclast differentiation; Cathepsin K; NVP-BHG712

Introduction

The dynamic balance between bone formation and resorption is necessary to maintain bone homeostasis(Chen, Wang, et al., 2018); Matsuoka, Park, Ito, Ikeda, and Takeshita (2014); (Zaidi, 2007). Due to internal and external factors, the disturbance of this dynamic balance leads to an increase in bone resorption activity and the occurrence of compensatory bone diseases, such as postmenopausal osteoporosis (PMPO) and rheumatoid arthritis (RA)(Eastell et al., 2016). Therefore, inhibition of osteoclast resorption activity may be an effective method for treating pathological bone loss in these diseases(Chen, Zhi, Cao, et al., 2017; Chen, Zhi, Pan, et al., 2017; Chen, Zhi, et al., 2018; Zhi et al., 2018). Osteoclasts are multinucleated giant cells that differentiate from mononuclear macrophages; osteoclasts are located on the surface of bone tissue and perform bone resorption functions by synthesizing and secreting a variety of organic acids and proteases to dissolve bone salts and degrade organic matter(Boissy, Saltel, Bouniol, Jurdic, & Machuca-Gayet, 2002). In the process of osteoclast differentiation, they are stimulated by two essential cytokines, namely, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL)(Fumoto, Takeshita, Ito, & Ikeda, 2014; Park, Lee, & Lee, 2017). The binding of the cytokine M-CSF to the receptor c-Fms maintains the survival and proliferation as well as the differentiation of BMMs into osteoclast precursor cells. The cytokine RANKL binds to the receptor RANK to promote the differentiation of osteoclast precursor cells into mature osteoclasts(Fumoto et al., 2014), and this interaction induces osteoclast differentiation and the expression of function-related genes, including CTSK, MMP9 and CTR.
CTSK is an important osteoclast enzyme that degrades type I collagen, and CTSK inhibitors have been used in the treatment of osteoporosis for many years(Ho et al., 2009; Hou et al., 1999; Reiser, Adair, & Reinheckel, 2010; Schilling et al., 2007). Odanacatib, which is a drug that inhibits CTSK function in osteoclasts, has been abandoned by the Desert East Company because it increases the occurrence of cardiovascular events(McClung et al., 2019; Mullard, 2016; Saag et al., 2021). A large amount of evidence has shown that CTSK inhibitors can effectively inhibit bone resorption by osteoclasts(Lotinun et al., 2013; Mukherjee & Chattopadhyay, 2016). Therefore, the identification of potential CTSK inhibitors will facilitate the development of treatments for bone metabolic diseases.
NVP-BHG712, which is as a specific EphB4 inhibitor, has attracted much attention because of its effects in inhibiting tumor metastasis(Becerikli et al., 2015; Li et al., 2021; Troster et al., 2018). Angiogenesis plays a key role in bone formation and osteoporosis(Filipowska, Tomaszewski, Niedźwiedzki, Walocha, & Niedźwiedzki, 2017; Kusumbe, Ramasamy, & Adams, 2014; Ramasamy, Kusumbe, Wang, & Adams, 2014). A recent study reported that NVP-BHG712 inhibited angiogenesis(You et al., 2017). However, the role of NVP-BHG712 in osteoclast formation remains unclear. In our study, the results suggested that NVP-BHG712 may act as a potent inhibitor of osteoclast formation to improve bone loss in ovariectomized mice.

Methods

Molecular docking . To screen CTSK-targeting compounds, we downloaded crystal conformations from the Protein Data Bank database and screened CTSK-targeting compounds from more than 9,000 Selleckchem compounds. The binding pocket of CTSK (Fig. 1A) to which the compounds bind was identified according to the resolution, and the conformation of CTSK while bound to the known ligands 5TDI, 4DMY, 3KWZ, and 4X6H was selected according to the binding strength. According to the physical characteristics of the compounds, the compound NVP-BHG712, which binds to the CTSK molecule, was selected.
Animals . All animals care, ethical principles and experimental procedures were approved by the Cheeloo College of Medicine of Shandong University (Shandong, China). Animal studies are reported in compliance with the ARRIVE guidelines and with the recommendations made by theBritish Journal of Pharmacology . C57BL/6JN mice (6-8 weeks of age) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). To evaluate the effect of NVP-BHG712 on bone mass in OVX females, OVX females underwent ovariectomy at 8 weeks of age. The mice were randomly divided into 6 groups (5 mice in each group): the Sham group, OVX group, and 5 mg kg-1, 10 mg kg-1, 20 mg kg-1, and 40 mg kg-1 NVP-BHG712 groups. In the Sham group, the ovaries of the mice were only exposed to the surrounding adipose tissue, while in the OVX group and different NVP-BHG712 groups, both ovaries were removed from the mice. After one week of postoperative recovery, the Sham and OVX groups were treated with 0.5% CMC-Na, and the NVP-BHG712 group was treated with NVP-BHG712 dissolved in 0.5% CMC-Na. After 6 weeks, bilateral femurs and blood were collected from the mice for analyses.
Cell culture . The RAW264.7 cell line was purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China, RRID: CVCL_0493) and authenticated by STR profiling. The cells were cultured in DMEM medium (Gibco, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin‒streptomycin (Solarbio, Beijing, China) at 37°C in a humidified atmosphere with 5% CO2.
BMMs were obtained as described previously(Gooi, Chia, Vrahnas, & Sims, 2019; Maridas, Rendina-Ruedy, Le, & Rosen, 2018; Stern et al., 2012) and were cultured in α-MEM (Gibco, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin‒streptomycin (Solarbio, Beijing, China) at 37°C in a humidified atmosphere with 5% CO2.
CCK-8 . According to the reagent standard, BMMs were incubated in 96 well plates (1x104 cells per well) for 24 hours. BMMs were cultured in induction medium containing M-CSF (30 ng/ml) (R&D systems, Mn, USA) and RANKL (50 ng/ml) (R&D systems). Then, different concentrations of NVP-BHG712 (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 μM) were added and incubated for 72 hours. Then, CCK-8 solution (Beyotime Biotechnology, Shanghai, China) was added to each well and incubated for another 2 h before the absorbance was measured.
TRAP staining . TRAP staining for the detection of mature osteoclasts was performed using a tartrate resistant acid phosphatase kit (Servicebio) according to the manufacturer’s instructions. The number of TRAP+ cells per mm2 was counted.
F-actin ring staining . BMMs were fixed in 4% paraformaldehyde and stained with F-actin (Beyotime Biotechnology). DAPI was used to stain the nuclei. Images were acquired using light and fluorescence microscopes.
Bone resorption . BMMs were seeded on bone plates (Corning, NYC, USA) and treated with M-CSF, RANKL and NVP-BHG712. The cells were removed from the bone plates on day 7. Images were acquired using light and fluorescence microscopes.
Western blotting . BMMs were harvested and subjected to Western blotting assays, which were performed as described previously(Hu et al., 2019; Sun et al., 2019), and the following antibodies were used for immunoblotting: anti-CTSK (Abcam, ab19027, RRID: AB_2261274), anti-MMP9 (Abcam, ab76003, RRID: AB_1310463), anti-CTR (Abcam, ab11042, RRID: AB_297696), anti-β-actin (Proteintech, 66009-1-Ig, RRID: AB_2687938), anti-rabbit IgG (Cell Signaling Technology (CST), #7074, RRID: AB_2099233), and anti-mouse IgG (CST, #7076, RRID:AB_330924).
RNA isolation and qPCR . BMMs were harvested, and total RNA was isolated using a RNeasy kit (Spark Jade). An aliquot of 1 μg of total RNA was subjected to reverse transcription with a SPARK script II reverse transcription PCR kit (Spark Jade) according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green Master Mix (Roche). mRNA expression levels were calculated by the method using GAPDH as an internal control and further normalized to the mean expression level of the control group. The primers were as follows: mouse-MMP9: forward, 5’-CGTCGTGATCCCCACTTACT-3’, and reverse, 5’-AACACACAGGGTTTGCCTTC-3’; mouse-CTR: forward, 5’-CCTGCAGA TGCCCAGTGAAG-3’, and reverse, 5’-TGTGTAGTAGCCCTGCTCCC-3’; mouse-IP3R1: forward, 5’-AACTGTGGGACCTTCACCAG-3’, and reverse, 5’-AACTCTCGCCAGTTTCTGG-3’; mouse-IP3R2: forward, 5’-GTTACAGGATGTCGTGGCCT-3’, and reverse, 5’-ATTCGCCGTAATGTGCTACC-3’; mouse-IP3R3: forward, 5’-CAATGAGCACCACGAGAAGA-3’, and reverse, 5’-AACTTGACAGGGGTCACCAG-3’; mouse-NFATc1: forward, 5’-GGGTCAGTGTGACCGAAGAT-3’, and reverse, 5’-GGAAGTCAGAAGTGGGTGGA-3’; mouse-OC-STAMP: forward, 5’-ATGAGGACCATCAGGGCAGCCACG-3’, and reverse, 5’-GGAGAAGCTGGGTCAGTAGTTCGT-3’; mouse-DC-STAMP: forward, 5’-AAAACCCTTGGGCTGTTCTT-3’, and reverse, 5’-GGCTGCTTTGATCGTTTCTC-3’; mouse-Atp6v1c1: forward, 5’-CACGTTGGCTGTCTTGGTTG-3’, and reverse, 5’-CTTCGCGTTAGCAAACTTCC-3’; mouse-αv-integrin: forward, 5’-ACAAGCTCACTCCCATCACC-3’, and reverse, 5’-ATATGAGCCTGCCGACTGAC-3’; mouse-GAPDH: forward, 5’-TCAAGAAGGTGGTGAAGCAG-3’, and reverse, 5’-AGTGGGAGTTGCTGTTGAAGT-3’.
ELISA and microenzyme labeling method . Mouse serum was collected, and the tartrate-resistant acid phosphatase 5b (TRACP-5b) level was measured with a TRACP-5b ELISA Kit (Elabscience, Wuhan, China). Alkaline phosphatase (ALP) activity was measured with an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Microcomputed tomography . Bone density and bone volume of the right femur were measured using a Micro-CT SkyScan 1176 (Bruker, Germany). Briefly, samples were fixed in 4% paraformaldehyde and incubated in 70% ethanol. Scanning was performed using a voxel size of 9 mm, X-ray tube potential of 58 kV and X-ray intensity of 431 μA. For specimen scanning, the volumes of interest were evaluated using CT Analyzer software. Representative 3D images created using NRecon software.
H&E staining . Tissues were collected, cleaned to remove excess tissue, fixed in 4% paraformaldehyde and demineralized in 10% EDTA for 10–14 days before being embedded in paraffin wax. Sections were cut to 5 μm thicknesses using a Leica RM2165 and subsequently stained with H&E or TRAP.
Statistical Design . SPSS version 26.0 (IL, USA) and GraphPad Prism 9 (CA, USA) were used for statistical analysis. Two-tailed unpaired Student’s t tests were used for comparisons of two groups, and one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used for comparisons of more than two groups. P values less than 0.05 were considered statistically significant.

Results