Small molecule inhibitor RepSox prevented ovariectomy-induced osteoporosis by suppressing osteoclast differentiation and bone resorption

2.1 Media and reagents

RepSox, Triton X-100, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), and a tartrate-resistant acid phosphatase (TRAP) staining kit were purchased from Sigma-Aldrich (St. Louis, MO). Alpha modification of Eagle s medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin solution were purchased from Gibco-BRL (Gaithersburg, MD). A cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Tokyo, Japan). Recombinant mouse macrophage colony-stimulating factor (M-CSF) protein and receptor activator of nuclear factor κB ligand (RANKL) were obtained from R&D Systems (Minneapolis, MN), reconstituted in sterile phosphate buffer saline containing 0.1% bovine serum albumin (BSA), and stored at ‒20℃. The specific primary antibodies and secondary antibodies used in the experiments were obtained from Abcam (Cambridge, UK).

2.2 Cell culture

Bone marrow macrophages (BMMs) were isolated from the whole bone marrow of six-week-old female C57BL/6 mice as described previously (Li et al., 2015). In brief, cells were separated from femoral and tibial bone marrow and cultured in a T75 flask with α-MEM containing 10% FBS, 1% penicillin/streptomycin, and 30 ng/ml M-CSF. MC3T3-E1 cells were incubated in α-MEM with 10% FBS in a humidified atmosphere of 5% CO₂ at 37℃. The medium was changed every 2 days.

2.3 Clinical bone tissue specimen assay

Ethical approval for this investigation was obtained from the Research Ethics Committee of the Fifth Affiliated Hospital of Wenzhou Medical University, and an informed consent was obtained from all patients in the study. The bone tissue specimens were isolated from patients with osteoarthritis and OP during an operation, as described previously (Jiang, Zhang, Jiang, Chen, & Dai, 2008). The patients’ clinical information is listed in Supporting Information Table S1. Part of the bone tissue was ground into powder, and total RNA was extracted using a Qiagen RNeasy Mini kit (Qiagen, Valencia, CA) and then subjected to complementary DNA (cDNA) synthesis. Real-time polymerase chain reaction (PCR) was performed using a SYBR Premix Ex Tag kit (TaKaRa Biotechnology, Dalian, China) and an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA).

2.4 Cell viability assay

The cytotoxic effect of RepSox on the BMMs or MC3T3-E1 cells was assessed using CCK-8 assays, according to the manufacturer's protocol. The BMMs were seeded in 96-well plates at a density of 8 × 10³ cells/well in triplicate and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF; after 24 hr, the cells were treated with different concentrations of RepSox for another 48 or 96 hr. MC3T3-E1 cells were cultured in 96-well plates in complete α-MEM at a density of 2 × 10³ cells/well with increasing concentrations of RepSox for 48 or 96 hr. Ten microliters of CCK-8 buffer was added to each well, the cells were incubated at 37℃ for 2 hr, and the absorbance was measured at 450 nm on an ELX800 microplate reader (Bio-Tek, Vermont). Cell viability was calculated relative to that of the control cells from the optical density (OD) using the following formula: cell viability = (experimental group OD − zeroing OD)/(control group OD − zeroing OD).

2.5 In vitro osteoclastogenesis assay

The BMMs were plated into a 96-well plate at a density of 8 × 10³ cells/well (in triplicate) in complete α-MEM and incubated for 24 hr. Next, 30 ng/ml M-CSF, 100 ng/ml RANKL, and different concentrations of RepSox (0, 125, 250 or 500 nM) were added to each well. The cell culture medium was replaced every 2 days until mature osteoclasts had formed. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and stained for TRAP using the staining kit. TRAP-positive cells with more than three nuclei were regarded as osteoclasts. The number and the area of osteoclasts (percentage of the well) per well were measured using ImageJ software.

2.6 In vitro bone absorption assay

The BMMs were seeded at a density of 2.4 × 10⁴ cells/cm² onto bovine bone slices (in triplicate) and incubated with 30 ng/ml M-CSF and 100 ng/ml RANKL until mature osteoclasts had formed, followed by treatment with 0, 125, 250, or 500 nM RepSox for 48 hr. Adherent cells were completely removed from the slices. Resorption pits were imaged using a scanning electron microscope (FEI Quanta 250), and the bone resorption area was quantified using ImageJ software.

2.7 RNA extraction and quantitative PCR assay

Quantitative PCR was used to calculate the relative specific gene expression during osteoclast and osteoblast formation. Briefly, the BMMs were seeded in 12-well plates at a density of 10⁵ cells/well and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF for 24 hr. After that, the BMMs were treated with 100 ng/ml RANKL and 500 nM RepSox for 0, 1, 3, or 5 days or with different concentrations of RepSox (0, 250 or 500 nM) for 5 days. MC3T3-E1 cells were seeded in 12-well plates and cultured in complete α-MEM. When the cells were confluent, they were treated with osteogenic medium (α-MEM supplemented with 10% FBS, 50 μg/ml ascorbic acid, 10 mM glycerophosphate, 100 nM dexamethasone and 1% penicillin/streptomycin) and different concentrations of RepSox for 7, 14, or 21 days. Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen) and followed by cDNA synthesis using reverse transcriptase (TaKaRa Biotechnology). Real-time PCR was performed using a SYBR Premix Ex Tag kit (TaKaRa, Biotechnology) and an ABI 7500 Sequencing Detection System (Applied Biosystems) according to the manufacturers’ protocols. The cycling conditions used were 40 cycles of denaturation at 95℃ for 15 s and amplification at 60℃ for 1 min. GAPDH was used as an internal control gene, and all reactions were run in triplicate. The mouse primer sequences are listed in Table 1, and the human primer sequences are listed in Table 2.

2.8 Western blotting

The BMMs were seeded in 6-well plates and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF. After the cells reached 90% confluence, they were harvested by pretreatment with or without 500 nM RepSox for 1 hr and then stimulated with 100 ng/ml RANKL for 0, 10 or 30 min. Total protein was extracted from cultured cells by using radio immunoprecipitation assay (RIPA) lysis buffer. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Thirty micrograms of each protein lysate was resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Next, the membranes were blocked with 5% nonfat dried skimmed milk powder dissolved in TBST (TBS with 1% Tween-20) for 2 hr and then washed three times with TBST. The membranes were incubated with primary antibodies at 4℃ overnight and secondary antibodies for 1 hr at room temperature. Antibody reactivity was detected by exposure in an Odyssey image scanner (Li-COR. Inc.). Quantitative analysis of the band intensity was done using ImageJ software.

2.9 Luciferase reporter gene assay

The BMMs stably expressed the AP-1 luciferase reporter gene, as described previously (Weber & Markillie, 2003). Briefly, the BMMs were seeded into 12-well plates and cultured with medium for 24 hr. Then, cells were pretreated with or without different concentrations of RepSox for 1 hr followed by stimulation with 100 ng/mL RANKL for 24 hr. Luciferase activity was measured using the Promega Luciferase Assay System (Promega, Madison, WI) and normalized to that of the control group.

2.10 Osteoclastogenesis rescue assay

For the rescue assay, the BMMs were seeded into a 96-well plate at a density of 8 × 10³ cells/well and cultured with complete α-MEM medium containing 30 ng/ml M-CSF. After 24 hr, 100 ng/ml RANKL and 250 nM RepSox with or without various concentrations of anisomycin (a specific JNK activator) were added to each well. After 5–7 days, mature osteoclasts had formed. The cells were fixed and stained with a TRAP staining kit, and the number of TRAP-positive cells were counted.

2.11 Immunofluorescence microscopy analysis

The BMMs were seeded in 6-well plates containing coverslips and cultured for 24 hr. Then, the cells were harvested by pretreatment with 500 nM RepSox for 1 hr followed by stimulation with 100 ng/ml RANKL for 0, 10 or 30 min. After the treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with 5% BSA in PBS for 30 min at room temperature. After blocking, the cells were incubated with a rabbit monoclonal antibody to p-Smad3 (1:200) overnight at 4℃. After further washing three times with PBS, the cells were incubated with Alexa Fluor 488-goat anti-rabbit IgG (1:200) for 1 hr. The nuclei were stained with DAPI. All images were captured on a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).

2.12 OVX-induced OP model

All experiments and animal care were performed in accordance with the guidelines and procedures authorized by the Animal Experimental Ethical Committee of Wenzhou Medical University. An OVX-induced OP model was used to evaluate the effect of RepSox on OP in vivo, as described previously (Guo et al., 2017). Briefly, 32 healthy 8-week-old female C57BL/6 mice were randomly divided into four groups: sham, vehicle, low, and high. The mice in the vehicle, low, and high groups underwent an OVX, and the sham group mice received only an abdominal incision. After 1 week, the mice in the low and high groups were injected intraperitoneally with RepSox at a dose of 2 and 5 mg/kg, respectively, twice per week for 4 weeks. The mice in the sham and vehicle groups received only an injection of the same volume of PBS. The mice in each group were weighed before injection. At the end of the experiment, the mice were killed and both the femurs and lumbar vertebrae were excised and fixed in 4% paraformaldehyde for histological and microcomputed tomography (micro-CT) analysis. Biochemical analysis of serum was used in our study.

2.13 Micro-CT scanning

The fixed femurs and lumbar vertebrae were analyzed using a high-resolution micro-CT (Skyscan 1072) instrument according to the manufacturer's protocols. The trabecular bone volume per total volume (BV/TV), mean trabecular thickness (Tb.Th), mean trabecular number (Tb.N), and mean trabecular separation (Tb.Sp) were measured, as reported previously (Ouyang et al., 2014).

2.14 Histological and histomorphometric analysis

The fixed femurs were decalcified in 10% EDTA for 3 weeks and then embedded in paraffin. Histological sections were prepared for TRAP and hematoxylin and eosin (H&E) staining. The samples were observed and photographed under a high-quality microscope. The mean number of TRAP-positive multinucleated osteoclasts normalized with bone area (OcN/BA) and the ratio of osteoclast area to bone area (OcA/BA) were calculated in each sample.

2.15 Analysis of serum markers

Procollagen type 1 N-terminal peptide (P1NP), a serum marker of bone formation, and the bone resorption marker C-terminal collagen cross-links (CTX-1) were measured in the serum of mice using an ELISA kit (Westang Biological Technology Co., Ltd., Shanghai, China) according to the protocols.

2.16 Alkaline phosphatase and Alizarin Red staining

MC3T3-E1 cells were seeded in 12-well plates and cultured in complete α-MEM. When the cells were confluent, the culture medium was replaced by osteogenic medium with different concentrations of RepSox, as described previously (Kim, Song, & Hwang, 2014). The medium was changed every 2 days. Seven and 21 days later, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, and alkaline phosphatase (ALP) or Alizarin Red staining was performed using a staining kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturers’ instructions.

2.17 Scratch-wound healing assay

MC3T3-E1 cells were seeded in 6-well plates and cultured to confluence, scraped in a straight line with a p200 pipet tip, and treated with RepSox at concentrations of 0, 250, 500, and 1,000 nM for 12 and 24 hr. The wound area was observed under an optical microscope.

2.18 Statistical analysis

The data are presented as the mean ± standard deviation (SD), and each experiment was carried out at least three times. The results were analyzed using SPSS 21.0 software (IBM Corp.). Analysis of variance with Dunnett’s post hoc test were used for comparisons between the groups. The results with p < 0.05 indicated statistical significance among the groups.

3.1 Expression of TGFβRI in bone samples and bone cells

The expression of TGFβRI in bone specimens was initially evaluated. Compared with the osteoarthritis samples, the OP bone samples expressed a higher level of TGFβRI messenger RNA (mRNA; Figure 1a). In addition, the osteoclast- and osteoblast-specific genes were also increased (Figure 1b,c). Next, we determined the expression of TGFβRI in osteoclast and osteoblast cells. During osteoclast differentiation, the expression of TGFβRI and osteoclastogenesis marker genes increased at 1, 3, and 5 days compared with that at day 0 (Figure 1d). In the process of osteoblast differentiation, TGFβRI was constantly expressed during osteogenesis, and the expression of osteoblastogensis marker genes was increased at 7, 14, and 21 days compared to at 0 days (Figure 1e). These data show that TGFβRI was expressed both in osteoblasts and osteoclasts, and TGFβRI expression was enhanced in OP and during osteoclast differentiation.

3.2 RepSox inhibits RANKL-induced osteoclast differentiation

The chemical structure of RepSox is shown in Figure 2a. To investigate the effect of RepSox on osteoclast formation, we treated primary BMMs with M-CSF and RANKL in the absence or presence of various concentrations of RepSox. As shown in Figure 2b, BMMs differentiated into mature, TRAP-positive, multinucleated osteoclasts in the control group. In contrast, the formation of TRAP-positive osteoclasts was significantly inhibited by the presence of RepSox at various concentrations (Figure 2c). The number of osteoclasts (TRAP-positive cells with more than three nuclei) was 223.60 ± 12.77 per well in the control group, 157.40 ± 9.32 per well after treatment with 125 nM RepSox, 84.40 ± 8.67 per well after treatment with 250 nM RepSox and 21.55 ± 3.16 per well after treatment with 500 nM RepSox (p < 0.01). To exclude the possibility that the inhibitory effect of RepSox on osteoclast formation was due to a cytotoxic effect, a cell viability assay was performed. We demonstrated that cell viability was not affected by RepSox at concentrations lower than 500 nM, which significantly inhibited osteoclastogenesis (Figure 2d). The calculated IC₅₀ for RepSox in BMMs was 183.2 μM at 48 hr and 175.15 μM at 96 hr. Together, these results demonstrate that RepSox suppressed osteoclast differentiation in a dose-dependent manner without toxicity to BMMs.

3.3 RepSox inhibits osteoclastic bone resorption activity in vitro

Since RepSox suppresses osteoclast differentiation efficiently, we determined whether RepSox also inhibits osteoclastic bone resorption. Consequently, mature osteoclasts were used to explore the effect of RepSox on bone resorption. Mature osteoclasts treated with different concentrations of RepSox for 48 hr did not affect the viability of cells (Figure 3b). As shown in Figure 3a,c, large bone resorption pits were observed in the control group. However, after treatment with different concentrations RepSox, the resorption area was decreased to 50% in the 125 nM group, to 20% in the 250 nM group, and very few resorption pits were observed in the 500 nM group (p < 0.01). These data indicated a potential inhibitory effect of RepSox on osteoclastic bone resorption.

3.4 RepSox suppresses RANKL-induced osteoclast-specific gene expression

Because RepSox inhibited osteoclast formation and bone resorption, we further explored the underlying mechanisms. Therefore, the expression of osteoclastic-specific genes was examined. In response to RANKL stimulation, TGFβRI and osteoclast-related genes, including NFATc-1, c-Fos, Cathepsin K, TRAP, DC-STAMP, ATP6V0D2 and ATP6V0A3, were upregulated in the control group. In contrast, a general suppressive effect on gene expression was observed in the RepSox-treated groups. RepSox suppressed the osteoclastic marker gene expression in both dose-dependent (Figure 4a) and time-dependent (Figure 4b) manners. Together, these data further demonstrate that RepSox suppresses osteoclast formation in vitro.

3.5 RepSox does not affect osteoblast differentiation, migration or osteoblastic-specific gene expression in vitro

Although RepSox impaired osteoclast formation, it is important to consider if RepSox has any effect on osteoblasts. Therefore, MC3T3-E1 cells were used for osteoblastic differentiation and mineralization research. Cell viability was significantly different in the 96-hr treatment group (p < 0.05), but there was no significant difference in the 48-hr treatment group (Figure 5a). ALP staining clearly showed that there was not only no significant difference but also a slight promotion between the control and RepSox-treated groups (Figure 5b). In addition, Alizarin Red staining also demonstrated that the total mineralized area was similar between the control group and the RepSox-treated groups (Figure 5c). In addition, RepSox did not affect osteoblasts migration (Figure 5d). We further explored the expression of osteoblastic-specific genes, including ALP, BMP-2, Runx2, OCN, Col-1a1, ANGPT1 and Osx. The osteoblast marker mRNA expression was not affected by RepSox at concentrations of 62.5, 125, 250, 500 and 1,000 nM at 7 days (Figure 5e). These results indicated that RepSox does not affect osteoblast differentiation or mineralization in vitro.

3.6 RepSox suppresses the Smad3 and JNK/AP-1 signaling pathways during osteoclastogenesis

To further explore the potential mechanisms on how RepSox affects osteoclast formation, critical signaling pathways involved in osteoclast differentiation were investigated. Mitogen-activated protein kinase (MAPK) signaling pathways, including the extracellular signal-regulated kinase (ERK), JNK and p38 pathways, are some of the key pathways during osteoclast formation; thus, we investigated MAPK activation. In our study, we initially observed that phosphorylation of Smad3 was increased after stimulation with RANKL for 10 min, but RepSox reduced this Smad3 phosphorylation. In addition, the phosphorylation of ERK, JNK, and p38 was increased after stimulation with RANKL for 10 min, and RepSox reduced the phosphorylation of JNK but not ERK or p38 activation (Figure 6a). Quantitative analysis confirmed an inhibitory effect of RepSox on Smad3 and JNK phosphorylation (Figure 6b). Then, the levels of c-Fos, a protein downstream of JNK, were also decreased by RepSox treatment (Figure 6c,d). Next, by using AP-1 luciferase reporter gene assays, the transcriptional activity of AP-1 was obviously increased when treated with RANKL. However, RepSox suppressed AP-1 luciferase activity in a dose-dependent manner (Figure 6e). Furthermore, a rescue assay showed that osteoclast formation was inhibited in the presence of only RepSox; however, after anisomycin was added to the cells, impaired osteoclastogenesis was rescued, and mature osteoclasts were observed (Figure 6f,g). These results suggest that RepSox inhibits c-Jun N-terminal kinase/activator protein-1 (JNK/AP-1) during osteoclast differentiation.

Subsequently, we analyzed the level of Smad3 protein using immunofluorescence staining. The intensity of Smad3 in BMMs was increased when treated with 100 ng/ml RANKL for 10 min, but treatment with RepSox clearly decreased RANKL-induced Smad3 expression (Figure 6h), which was consistent with the western blotting results. To further confirm that RepSox suppressed the osteoclast formation by affecting Smad3 activation, we further used the specific inhibitor of Smad3 (SIS3) to validate this. As shown in Figure 6i, osteoclast formation was significantly restrained when the cells were treated with SIS3. Statistical analysis showed that when the BMMs were treated with SIS3 at 1, 2, and 3 μM concentrations, the number of osteoclasts was significantly lower than that of the control group (Figure 6j). These data suggest that RepSox may affect osteoclast formation by suppression of the Smad3 and JNK/AP-1 signaling pathways.

3.7 RepSox prevents OVX-induced bone loss in vivo

Given that RepSox suppression affected osteoclast formation and function but not osteoblast differentiation in vitro, we hypothesized that RepSox could prevent osteoclastic osteolysis in vivo and may become a new therapeutic drug for osteolytic diseases. The use of RepSox, at least as a TGFβRI inhibitor, for other diseases, such as prostate cancer, may have a protective effect on bone mass (Wan et al., 2012). Thus, the effect of RepSox on OVX-induced OP was measured, and the histomorphology of all mice was analyzed. Initially, three-dimensional reconstructed images of femurs, lumbar vertebrae, and cancellous bones were analyzed (Figure 7a). After OVX, excessive femur bone loss was observed in the vehicle group by micro-CT, and the BV/TV (1.82 ± 0.43%) was significantly reduced compared with the sham group (4.36 ± 0.34%) (p < 0.01; Figure 7c), which demonstrated that our OVX mouse model was successful. When treated with RepSox, further bone loss was prevented. In the low-dose group, in which RepSox was injected at 2 mg/kg twice a week for 4 weeks, the BV/TV was 2.41 ± 0.40%, approximately 20% higher than that in the vehicle group. A more significant difference was observed in the high-dose group, in which RepSox was injected at 5 mg/kg twice a week for 4 weeks, the BV/TV was increased approximately 70% compared with the vehicle group. In addition, other indices such as Tb.N was increased, and Tb.Sp was reduced. A similar effect can also be observed in the lumbar tissues, as shown in Figure 7b. In addition, RepSox did not affect the weight or internal organs of the mice in any group (Supporting Information Figure S1 and S2). These data together indicate a protective effect of RepSox against bone loss.

To further confirm the effect of RepSox on preventing bone loss, histological analysis was performed. As shown in Figure 7d, in contrast to the massive amount of TRAP-positive multiple osteoclasts lined along the eroded bone surface in the vehicle group, the number of osteoclasts was reduced in the RepSox treatment groups. The OcN/BA was 45.53 ± 3.14% in the vehicle group and 20.21 ± 2.83% in the high-dose group; meanwhile, the OcA/BA was 0.12 ± 0.03% in the vehicle group and 0.06 ± 0.02% in the high-dose group (Figure 7e), which is also consistent with the results of the inhibitory effect of RepSox on osteoclastic bone resorption in vitro. Serum CTX-1, a marker of bone resorption, was lower in RepSox treated mice, whereas there was no difference in P1NP, a marker of bone formation (Figure 7f). In summary, these data demonstrated that RepSox inhibited osteoclast formation, function, and osteoclastic osteolysis both in vitro and in vivo.