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