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Vx-11e protects against titanium-particle-induced osteolysis and osteoclastogenesis by supressing ERK activity
Biochemical and Biophysical Research Communications 514 (2019) 1244e1250
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Vx-11e protects against titanium-particle-induced osteolysis and osteoclastogenesis by supressing ERK activity Chen Li a, 1, Zuoxing Wu a, 1, Guixin Yuan d, Zhanfei Fang d, Xixi Lin a, Ruoyu Pu f, Yanbin Kang b, Li Li e, Siyuan Shao a, Jiaxin Ding a, Jinmin Zhao b, Qian Liu b, **, An Qin a, c, * a
Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Nanning, Guangxi, 530021, China Research Centre for Regenerative Medicine, Department of Trauma Orthopedic and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Guangxi, 530021, China c Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China d Department of Orthopedics, The Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China e Pharmaceutic College, Guangxi Medical University, Nanning, Guangxi, 530021, China f Department of Obstetrics and Gynaecology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2019 Accepted 6 May 2019 Available online 17 May 2019
Wear particle-induced osteolysis around the prosthesis is the most common long-term complication after total joint replacement surgery which often leads to aseptic loosening of the prosthesis. Osteoclasts play key roles in the osteolytic process. Currently there is a lack of clinically effective measures to prevent or treat peri-prosthetic osteolysis and thus identification of new agents that can inhibit the enhanced osteoclastic bone resorption is warranted. Through this study, we discovered that the specific and potent ERK1/2 inhibitor, Vx-11e, can protect against calvarial osteolysis caused by titanium (Ti) particles in vivo. Low doses of Vx-11e mildly reduced osteoclast resorption whilst no calvarial osteolysis was observed with high dose Vx-11e treatment. Histological examination showed fewer osteoclasts and reduced bone erosion in the Vx-11e treated groups. In vitro cellular analyses showed that Vx-11e inhibited osteoclast formation from BMM precursors in response to RANKL, as well as bone resorption by mature osteoclasts. Mechanistically, Vx-11e impaired RANKL-induced ERK1/2 signaling by inhibiting its kinase activity thereby blocking the phosphorylation of downstream substrates. Moreover, Vx-11e significantly reduced the expression of RANKL-mediated genes such as ACP5/TRAcP, CTR, MMP-9, CTSK. Collectively, our data provides evidence for the potential therapeutic use of Vx-11e for the treatment of osteolysis diseases caused by extremely actived osteoclastogenesis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Osteolytic Vx-11e Osteoclast RANKL
1. Introduction Total joint replacement is widely used in arthritis, arthrosis, femoral head necrosis and other diseases [1]. In the United States, the number of arthroplasty cases reached 70 million and showed an increasing trend [2]. With the number of patients receiving this
* Corresponding author. Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China. ** Corresponding author. Research Centre for Regenerative Medicine, Guangxi Medical University, Guangxi, China. E-mail addresses: [email protected] (Q. Liu), [email protected] (A. Qin). 1 Chen Li and Zuoxing Wu contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.05.054 0006-291X/© 2019 Elsevier Inc. All rights reserved.
treatment constantly increasing, so too has the number of patients with failed arthroplasty requiring revision surgery. Although there are substantial advances in biomaterials and prosthesis design, loosening of the prosthesis caused by osteolysis remain the most difficult problem to overcome [3]. Wear particles such as titanium particles generated from prosthesis is considered one of the main causes of TJA (total joint arthroplasty) failure [4]. The pathophysiology of wear particle-induced osteolysis is complex but can be generalized as the local induction of inflammation near and around the prosthesis leading to release of proinflammatory signals that result in the recruitment of immune cells, lymphocytes and osteoclast precursors such as monocytes and macrophages [5]. Subsequently, various pro-inflammatory
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cytokines are released by these cells into the tissues surrounding the loose prosthesis [5,6]. These factors such as TNF-a, IL-6 and IL17 stimulate osteoblasts to release receptor activator of nuclear factor-kB ligand (RANKL) which then promotes the osteoclastogenesis of BMMs and bone resorption leading to peri-prosthetic bone loss [7,8]. Osteoclasts are therefore key players in mediating the osteolytic bone destruction induced by wear particles, and are prime targets for pharmacological intervention. Stimulation with RANKL, monocytic precursors of the monocyte/macrophage lineage fuse into osteoclasts [9]. Binding of RANKL to its cognate receptor RANK on the membranes of monocytic precursor cells induces the rapid activation of a myriad of signaling pathways, of which the NF-kB and MAPK pathways are the most important [10]. Together, these pathways induce the expression and activation of NFATc1, the master transcriptional factor necessary for osteoclastogenesis [11]. Thus, the targeted suppression of NF-kB or MAPK signaling pathways might alleviate enhanced osteoclast bone resorption providing a new strategy for the prevention of osteoclast-mediated bone diseases. Vx-11e is a potent, selective, and orally bioavailable ERK1/2 inhibitor and ERK pathway plays an important role in osteoclast differentiation, thus we hypothesize that Vx-11e could effectively inhibit osteoclastogenesis. According to our results, we found that Vx-11e has a significant effect on inhibiting osteoclastogenesis in vitro. Futhermore, we explored that Vx-11e can protect mice against titanium particle-induced calvarial osteolysis. To sum up, we suggest that Vx-11e has the potential to be exploited as an effective drug in the treatment of osteoclast-mediated osteolysis diseases. 2. Materials and methods 2.1. Media and reagents ERK inhibitor, Vx-11e, was obtained from Selleckchem (Houston, Texas, USA) and dissolved in DMSO. a-MEM and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA). TRAcP Staining Kit and Cell Counting Kit-8 (CCK8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant mouse MCSF and RANKL were obtained from R&D Systems (Minneapolis, MN, USA). Specific antibodies against phosphorylated and total forms of ERK1/2, RSK (p90) p38 and JNK were obtained from Cell Signaling Technology (Danvers, MA, USA), IkBa, and b-actin were obtained from Abcam (China).
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2.3. mCT, histology and histomorphometric analysis Prior to histological assessment, 3D images of whole calvaria from each group were acquired using a high resolution mCT scanner (SkyScan 1076) and associated analysis software (SkyScan, Aartselaar, Belgium). All calvaria were scanned with the following parameters: pixel size, 14.4 mm; X-ray voltage, 50 kV; electric current, 800 mA. A square region of interest (ROI) around the midline suture was chosen for qualitative and quantitative analysis after reconstruction. The percentage of bone volume/tissue volume (BV/TV, %), number of porosities, and percentage of total porosity of each sample were measured in the ROI as previously reported [13]. After micro-CT scanning, the PFA-fixed calvarias were decalcified and embedded in paraffin for H&E and TRAcP staining. The histological sections were examined under a microscope at 50 , 100 , and 200 magnifications. The number of TRAcP-positive osteoclasts was quantified using ImageJ software (NIH, USA). 2.4. Cell culture, osteoclast differentration, and bone resorption in vitro BMMs (bone marrow macrophage) were isolated from femurs and tibias of 4-week-old C57BL/6 J mice by marrow flushing as previously described [14]. The extracted cells were cultured in complete a-MEM (a-MEM with 10% FBS and 100U/mL penicillin/ streptomycin) containing 30 ng/mL M-CSF. For osteoclast differentiation, M-CSF depend BMMs were seeded in 96-well plates at a density of 6 103 cells/well. After sticked, cells were treated without or with serial dilutions of Vx-11e (150 nMe37.5 nM) in the stimulation of RANKL (50 ng/mL) until multinucleated ‘pancake’ shaped osteoclasts were formed in RANKL-treated only control groups. Cells were stained with TRAcP Staining Kit after fixed with 4% paraformaldehyde for 10 min. The number of TRAcP-positive osteoclasts with 3 or more nuclei was quantified by ImageJ software. For bone resorption assay, small pre-osteoclasts cultured with RANKL for at least 3e4 days were dissociated from culture plates and re-seeded onto hydroxyapatite-coated 96-well plates (Corning Inc, NY, USA). Cell was allowed to adhere to the wells for a few hours to overnight after which they were treated with 37.5 or 75 nM Vx-11e for 2 days. Adherent cells were removed using 5% sodium hypochlorite solution and the plates were rinsed with water and air-dried. The results of resorption were observed under microscope and the pit area normalized to total well area was quantified by ImageJ software.
2.2. Murine model of Ti particle-induced calvarial osteolysis 2.5. Cell viability assay To evaluate the potential therapeutic efficacy of Vx-11e in vivo, we generated a mouse calvarial osteolysis model. The experimental protocol was approved by the Animal Ethics Committee of Guangxi Medical University. Thirty two healthy male C57BL/6 J mice were randomly divided into four groups (n ¼ 8 per group): Shamoperated (with PBS injection), Vehicle (Ti particles with PBS injection), low-dose Vx-11e (Ti particles treated with 2 mg/kg Vx-11e), and high-dose Vx-11e (Ti particles treated with 5 mg/kg Vx-11e). Titanium particles were soaked in 75% alcohol for two days and then dried at 37 C [12]. To induce calvarial osteolysis 30 mg of sterilized Ti particles were uniformly coated under the periosteal membrane around the suture of the calvaria. To minimize infections, mice were intraperitoneally injected with 1:100 dilution of penicillin/streptomycin in PBS (100 mL final volume). Two days after Ti particles implantation, PBS (Vehicle group) or Vx-11e (2 mg/kg and 5 mg/kg groups) were injected under the scalp every two days for 2 weeks. After fourteen days, mice were euthanized, and the calvarias were harvested for micro-CT (mCT) and histological analysis.
CCK-8 Cell Proliferation/Cytotoxicity kit was assessed to explore the effect of Vx-11e to the BMMs activity. MeCSFedependent BMMs seeded in 96-well plates at a density of 6 103 cells/well in complete a-MEM were treated with serial dilutions of Vx-11e (from 600 nM to 18.6 nM) for 48 h s, and then 10 mL of CCK-8 reagent was added to each well and incubated at 37 C for 2 h s, the absorbance was measured at 490 nm on an ELX800 Absorbance Microplate Reader (BioTek; Winooski, VT, USA). 2.6. Podosomal actin belt immunofluorescence BMM-derived osteoclasts cultured as described above were fixed with 4% paraformaldehyde and then permeabilized with 0.1% (v/v) Triton X-100 for 5 min s. After extensive washed with PBS, cells were incubated with Alexa-Fluor 647 phalloidin (Invitrogen; San Diego, CA, USA) diluted in 0.2% (w/v) BSA-PBS for 1 h at room temperature. Cells were counterstained with DAPI to visualize cell
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nuclei. Fluorescence images were acquired using the LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany) and processed using the associated Zeiss ZEN software. The number of podosomal actin belts was quantified using ImageJ software. 2.7. RNA extraction and quantitative PCR assay Total RNA were extracted from BMM-derived osteoclasts cultured with RANKL in the absence or presence of serial dilutions of Vx-11e (from 150 nM to 37.5 nM) for 5 days using TRIzol reagent. Complementary DNAs (cDNAs) were synthesized using M-MLV reverse transcriptase with 1 mg extracted RNA template and oligo-dT primers (Promega, USA). The resultant cDNA was then used as template in the SYBR Premix Ex Tag Kit (TaKaRa, Shiga Prefecture, Japan) master mix according to manufacturer's instuctions and qPCR reactions performed on the ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA, USA) using the following cycling conditions: initial denaturation at 93 C for 2 min s; followed by 40 repeated cycles of 93 C for 60 s s, 55 C for 60 s s, and 72 C for 60 s s; and a final extension at 72 C for 7 min s. The following primer sets based on mouse sequences were used: matrix metalloproteinase-9 (MMP-9)Forward:50 -CGTGTCTGGAGATTCGACTTGA-3'; Reverse:50 -TTGGAAACTCACACGCCAGA-30 ), 0 ACP5/TRAcP(Forward:5 -TGTGGCCATCTTTATGCT-3'; Reverse:50 GTCATTTCTTTGGGGCTT-30 ), cathepsinK(CTSK)(Forward:forward:50 GGGAGAAAAACCTGAAGC-3'; reverse: 50 -ATTCTGGGGACTCAGAGC30 ), and calcitonin receptor (CTR)(Forward:50 -TGCAGACAACTCTTGGTTGG; Reverse:50 -TCGGTTTCTTCTCCTCTGGA).
differentiation and therefore inhibition of ERK activity will negatively impact osteoclastogenesis. Thus, we first examined the in vitro effect of the ERK inhibitor, Vx-11e, on RANKL-induced osteoclast differentiation from BMM precursors. To this end, stimulated with RANKL, BMMs were treated with or without indicated concentrations of Vx-11e for osteoclastogenesis, after which cells were fixed and TRAcP stained. As shown in graphical Highlight, BMMs stimulated with RANKL only differentiated efficiently into abundant typical ‘pancake’-shaped multinucleated osteoclasts which are highly TRAcP positive. In contrast, BMMs treated with Vx-11e showed a dose-dependent decrease in the formation of large TRAcP-positive osteoclasts (graphical Highlight). To ensure that the anti-osteoclastogenic effect of Vx-11e was not the result of cell cytotoxicity, we evaluated the viability of the BMM precursor cells in the presence of various concentrations of Vx-11e using the CCK-8 cell proliferation/cytotoxicity assay. As demonstrated in the graphical Highlight, no cytotoxic effects were observed on BMM precursor cells 48 h s after treatment with Vx-11e, even at concentrations of up to 600 nM. Thus it can be concluded that the inhibitory effect of Vx-11e on osteoclast formation at concentrations of up to 150 nM is not a result of cellular cytotoxicity. 3.2. Vx-11e impaired BMM precursor cell fusion and osteoclast bone resorption in vitro
To examine early RANKL-induced signaling pathways, total cellular proteins (TCPs) were extracted from BMMs pre-treated with Vx-11e for 4 h s followed by RANKL stimulation for0, 5, 10, 20, 30, or 60 min. Cells were lysed in a NP-40 lysis buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 1 mM EDTA; and 10% glycerol) supplemented with protease and phosphatase inhibitor cocktail. Protein concentrations were assessed using the BCA protein assay reagent (Beyotime, China) in accordance with manufacturer's protocol, and then 25 mg of TCPs were resolved on 8% SDS-PAGE gels and electrotransferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA) overnight at 4 C. Membranes were blocked with 5% (w/v) BSA in TBST (0.1% Tween 20 in Tris-buffered saline) for 1hr and then incubated with primary antibodies (diluted 1:1000 in 1% (w/v) BSATBST) overnight at 4 C. Membranes were then washed and subsequently incubated with the appropriate secondary fluorescence antibodies conjugated to IRDye 800CW (diluted 1:5000 in 1% (w/v) BSA-TBST) (LI-COR Biociences; Lincoln, NE, USA) for 2 h s. Antibody immunoreactivity was detected by exposing the membrane blots in an Odyssey Infrared Imaging System (LI-COR).
As noted, Vx-11e inhibited the formation of typical large multinucleated osteoclasts suggesting that the precursor cell fusion process is impaired. To further show an impairment of precursor cell fusion, we examined the actin cytoskeleton and nuclei number in BMM-derived osteoclasts treated without or with Vx-11e. Osteoclasts cultured on plastic plates have a belt-like structure of podosomes called the polysomal actin belt at the periphery and circumscribe the cells. Osteoclasts treated with Vx-11e also displayed the polysomal actin belt but were much smaller and less nucleated compared to RANKL-only controls, further suggesting that the fusogenic process is impaired (Fig. 1A). The actin cytoskeleton is also crucial for the bone resorptive function of the osteoclasts. When polarized towards bone resorption, the polysomal actin belt condenses into a tight F-actin ring surrounding the sealing zone from which resorption will take place. Thus based on the previous observations, it was inferred that the bone resorptive activity of mature osteoclasts would also be affected by Vx-11e. As expected, mature osteoclasts exposed to Vx11e exhibited significantly reduced bone resorptive ability (Fig. 1B). When compared with control untreated osteoclasts which almost completely absorbed the total area of the HA-coated well, osteoclasts treated with 75 or 150 nM absorbed only ~60% and 20% of the total well area, respectively (Fig. 1C). These findings demonstrated that Vx-11e impaired osteoclast bone resorption and F-actin ring formation in vitro.
2.9. Statistical analysis
3.3. Vx-11e reduced the expression level of RANKL-mediated genes
All data presented are representative of at least three independent experiments, and are expressed as mean ± standard deviation. The data were analyzed by SPSS 16.0, and Student's t-test was used for comparison between groups with p < 0.05, **p < 0.01 and ***p < 0.001 considered statistically significant.
Osteoclast differentiation and function are related to the expression level of RANKL-mediated genes. Real-time PCR was conducted on RNA extracted from osteoclasts treated with different doses of Vx-11e. As shown in Fig. 2, the expression of MMP-9, ACP5/ TRAcP, CTSK, and CTR was significantly reduced at 150 nM. The expression of MMP-9 and CTSK was dose-dependently reduced whereas TRAcP was only significantly reduced at 150 nM (Fig. 2AeD). The impaired expression of these genes in the presence of Vx-11e coincided with the decreased osteoclast formation and bone resorption observed earlier. Thus our real-time PCR results confirmed the inhibitory effect of Vx-11e on osteoclast differentiation and bone resorption in vitro.
2.8. Western blotting analysis
3. Results 3.1. Vx-11e attenuated RANKL-induced osteoclastogenesis in vitro ERK is one of the key early signaling cascades activated following RANKL stimulation and is required for efficient osteoclast
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Fig. 1. Vx-11e impaired precursor cell fusion and mature osteoclast bone resorption in vitro. (A) Effects of Vx-11e on BMM precursor fusion. MeCSFedependent BMMs stimulated with RANKL in the absence or presence of indicated concentrations of Vx-11e were fixed and immuno-stained for actin (Alexa-Fluor 647 phalloidin) and nuclei (DAPI). (B) Vx-11e inhibits mature osteoclast bone resorption. Equal numbers of BMM-derived osteoclasts stimulated with 50 ng/mL RANKL for at least 3e4 days were seeded onto hydroxyapatite-coated wells and then treated with indicated concentrations of Vx-11e for 2 days. Attached cells were then removed and resorption pits were imaged under a light microscope. Scale bars, 100 mm. (C) The resorption pit area as a percentage of well area were quantified using ImageJ. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig. 2. Vx-11e reduced the expression of osteoclast marker genes. Real-time quantitative PCR was performed on RNA extracted from BMM-derived osteoclasts stimulated with 50 ng/ml RANKL without or with indicated concentrations of Vx-11e for 5 days. (AeD) The expression levels of MMP-9, ACP5/TRAcP, CTSK, and CTR genes were normalized to GAPDH and then expressed as relative fold change against RANKL-only control. *p < 0.05, **p < 0.01, and ***p < 0.001.
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3.4. Vx-11e inhibited the activity of ERK in response to RANKL The MAPK signaling pathway is one of the key signaling cascades activated in response to RANKL during osteoclast differentiation. As a potent and selective ERK inhibitor, we thus examined the RANKL-induced early activation of the ERK MAPK pathway in the absence or presence of Vx-11e. To this end, we performed Western blot analyses of the phosphorylation status of ERK1/2 and it's downstream substrate p90RSK, an established biomarker for evaluating compound effects native ERK1/2 activity and inhibition [15]. Interestingly, the results showed that Vx-11e enhanced the phosphorylation of ERK1/2 but blocked ERK-mediated phosphorylation of p90RSK (Fig. 3A). This finding suggests that Vx-11e does not affect ERK1/2 phosphorylation but rather ERK kinase activity for downstream substrates and is in line with previous reports of contradictory increases in ERK1/2 phosphorylation in various tumor cells treated with Vx-11e [16]. Consistent with its specificity towards ERK, Vx-11e did not affect the activation of the other MAPK members, JNK and p38, or the NF-kB signaling pathway (Fig. 3B). Taken together, these results demonstrate that Vx-11e inhibited osteoclast differentiation by blocking ERK kinase activity which ultimately impairs downstream ERK signaling. 3.5. Vx-11e suppressed titanium particle-induced bone loss in vivo Given the promising in vitro inhibitory effect on osteoclast formation, we next explored the potential therapeutic potential of Vx11e on preventing titanium (Ti) particle-induced bone loss in mice calvarias. Three dimensional mCT reconstructions showed extensive bone loss with numerous surface erosions and resorption pits in the Ti group treated with PBS vehicle as compared to Sham controls where the calvarial surface were smooth with no signs of bone erosion (Fig. 4A). Treatment with Vx-11e on the other hand, attenuated Ti particle-induced osteolysis in a dose-dependent manner. Resorption pits and surface erosion were mildly reduced with low-dose of Vx-11e, whereas the calvarial surface in mice treated with high-dose Vx-11e was comparable to Sham controls. Morphometric quantification of bone volume/total volume (BV/ TV), number of porosity, and the percentage of total porosity in the ROI confirmed the dose-dependent attenuation of Ti particleinduced bone loss (Fig. 4CeE). Again bone morphometric
parameters showed that high-dose Vx-11e treatment was comparable to Sham controls. Histological and histomorphometric analysis further confirmed the protective effect of Vx-11e treatment against Ti particleinduced bone erosion in vivo. TRAcP and H&E stained tissue sections showed extensive osteolytic bone destruction in the Ti group treated with PBS vehicle group, with abundant TRAcP positive osteoclasts along the eroded bone surface (Fig. 4B and F). Treatment with low- and high-dose Vx-11e treatment group significantly reduced osteolytic bone loss and reduced the number of osteoclasts on the bone surface (Fig. 4B and F). Collectively, the in vivo data provides evidence for the potential therapeutic application of Vx11e against osteolytic bone loss. 4. Discussion Aseptic loosening due to titanium wear particle-induced osteolysis around artificial prosthesis remains one of the most common causes of total joint arthroplasty failure despite advances in implant design and biomaterials used in making the prostheses [17]. Despite the complex molecular and cellular interplays of the osteolytic bone destruction, wear particle-induced inflammation, enhanced osteoclast formation and activation, leading to excessive bone resorption remains the most important cause [18]. As such, pharmacological interventions with bisphosphonates, estrogens, denosumab, and/or teriparatide have been widely used after arthroplasty to inhibit the wear particle-induced osteoclast-mediated osteolysis [19]. However, due to side effects from mild fevers, throat and stomach ulcers to more serious malignant tumor formation and osteonecrosis of the jaw, limit the long-term and systemic use of these drugs. Hence, current intervention therapies are far from ideal and thus the identification of new and safer options are warranted. Given the important role the osteoclasts play in the bone destruction process, agents that can inhibit osteoclast formation and/or bone resorption are ideal therapeutic agents for preventing and ameliorating wear particle-induced osteolysis [20]. In this study, we demonstrated that Vx-11e has a protective effect against Ti particle-induced calvarial osteolysis in mice in vivo. This effect was mediated by the inhibition of RANKL-induced ERK activity leading to suppression of osteoclast formation from BMM precursors and attenuation of mature osteoclast bone resorption.
Fig. 3. Vx-11e specifically inhibited the activity of ERK in response to RANKL. Total cellular proteins (TCPs) extracted from BMM pre-treated with 150 nM Vx-11e for 4 h s and then stimulated with 50 ng/ml RANKL for the indicated times were subjected to immunoblot analyses using specific antibodies against components of the ERK, RSK, JNK, P38 and NF-kB signaling cascade, b-actin was used as internal loading control.signaling cascade. (A-B)The ratio of phosphorylated proteins relative to unphosphorylated proteins was determined.
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Fig. 4. Vx-11e suppressed against titanium particle-induced bone loss in vivo as assessed by mCT and histology. (A) Representative 3-dimensional mCT reconstructed images of mice calvaria from sham-operated (with PBS injection), Ti particles with PBS injection (vehicle), and Ti particles with low dose (2 mg/kg body weight) or high dose (5 mg/kg body weight) Vx-11e 14 days after treatment. (CeE) Quantitative morphometric analyses of bone volume to tissue volume (BV/TV, %), the number of porosity, and the percentage of porosity were conducted. (B) Representative images of H&E (magnifications of 100 and 200 ) and TRAcP (magnifications of 50 and 100 ) stained calvarial tissue sections from sham-operated (with PBS injection), Ti particles with PBS injection (vehicle), and Ti particles with low dose (2 mg/kg body weight) or high dose (5 mg/kg body weight) Vx-11e groups. Black arrows indicate TRAcP-positive osteoclasts. (F) The number of TRAcP-positive osteoclasts in the calvarial bone sections was quantified.*p < 0.05, **p < 0.01, and ***p < 0.001.
These results provide evidence for the potential therapeutic application of Vx-11e to treat wear particle-induced osteolysis. Vx-11e (Vertex-11e) was originally defined as a potent, selective, and orally bioavailable ERK2 inhibitor [21]. However, further studies have shown it to be a type-I kinase inhibitor that potently inhibits both ERK1 and ERK2 kinase activity [22] and exhibits unique kinetic properties defined by slow dissociation rates [23]. In this study, we found that Vx-11e enhanced ERK1/2 phosphorylation in response to RANKL, a paradoxical phenomena that has been previously documented in various tumor cells treated with VX-11e [24]. It has been reported that the expression of phosphorylated ERK does not necessarily correlate with ERK pathway activation and inhibitor sensitivity [25]. Thus this enhanced phosphorylation of ERK1/2 could be due to feedback activation pathways or crosstalk with parallel signaling pathways as previously described [26]. It could also be due to compensatory effect to the inhibition of kinase activity by increasing the levels of activated ERK1/2. The exact mechanism will require further investigations. However, despite the enhanced ERK1/2 phosphorylation, the phosphorylation of the
downstream ERK substrate p90RSK was blocked by Vx-11e, confirming the inhibition of ERK kinase activity. Furthermore, confirming the specificity for ERK1/2 signaling, Vx-11e did not have any discernible effects on the other MAPK members, p38 or JNK, nor the NF-kB signaling pathway. The early activation of the NF-kB and MAPK signaling cascades by RANKL are crucial prerequisite for osteoclast precursor differentiation and fusion, and subsequent mature osteoclast bone resorption [27]. RANKL-induced activation of ERK is necessary for efficient osteoclast formation, regulation of osteoclast survival and apoptosis, as well as maintenance of cell polarity during bone resorption [28]. Consistent with impaired ERK activity, we found that Vx-11e dose-dependently inhibited osteoclast formation from BMM precursors as well as mature osteoclast bone resorption. The concentration of Vx-11e at which osteoclast formation was inhibited did not induce any cytotoxicity demonstrating that inhibitory effect of Vx-11e was not due to decreased cell viability. Furthermore, BMM cells treated with Vx-11e formed smaller osteoclasts as defined by size of the polysomal actin belt
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circumscribing the periphery of the osteoclasts and exhibited less nuclei per cell suggesting impairment in precursor cell fusion. ERK activation is also required for the induction and activation of key osteoclastic transcription factors c-Fos and NFATc1 [29]. NFATc1 is considered the master transcriptional activator for a number of osteoclast marker genes involved in osteoclast formation and bone resorption [30]. Our real-time PCR results showed that Vx-11e suppressed the expression osteoclast maker genes MMP9, ACP5/TRAcP, CTSK, and CTR during RANKL-induced osteoclast formation. The decrease in the expression of these genes which are associated with bone resorption is in line with decreased resorptive activity of mature osteoclasts following Vx-11e treatment. Our in vitro biochemical and cellular results provided evidence for the inhibitory effect of Vx-11e on osteoclast formation, gene expression and bone resorption. With these promising results we examined the potential effects of Vx-11e on Ti particle-induced osteolysis in vivo. We found that mice treated with Vx-11e group dose-dependently alleviated Ti particle-induced osteolysis by reducing the number of TRAcP-positive osteoclasts thereby ameliorating Ti particle-induced osteoclast-mediated bone erosion. Consequently, Vx-11e may be of therapeutic interest in osteolytic bone diseases. Conflicts of interest The authors declare no conflicts of financial or other interests. Acknowledgements The authors acknowledged all the members of our laboratory for their assistance. An Qin, and Qian Liu designed the study; Chen Li, Zuoxing Wu, Guixin Yuan, Ruoyu Pu, Siyuan Shao, Yanbin Kang, Jiaxin Ding, Li, Zhanfei Fang and Xixi Lin participated in the experiments, analyzed the data, and prepared the figures. The manuscript was wrote by Chen Li. All authors have reviewed the manuscript. This project is supported by National Natural Science Foundation of China (81501910) (81772373) (81572167), Guangxi Natural Science Foundation under Grant (2015GXNSFCA414001), and Guangxi Collaborative Innovation Center for Biomedicine Talent Cultivation (GCICB-TC-2017001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.054. References [1] A.D. Van Citters, C. Fahlman, D.A. Goldmann, J.R. Lieberman, K.M. Koenig, D.G.A. Rd, B. O'Donnell, J. Martin, F.A. Federico, R.A. Bankowitz, Developing a pathway for high-value, patient-centered total joint arthroplasty, Clin. Orthop. Relat. Res. 472 (2014) 1619e1635. [2] S. Kurtz, K. Ong, E. Lau, F. Mowat, M. Halpern, Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030, J. Bone Joint Surg. Am. Vol. ume (2007). €rrholm, Highly crosslinked [3] P.E. Johanson, G. Digas, P. Herberts, J. Thanner, J. Ka polyethylene does not reduce aseptic loosening in cemented THA 10-year findings of a randomized study, Clin. Orthop. Relat. Res. 470 (2012) 3083e3093. [4] S. Jin, J.Y. Park, J.M. Hong, T.H. Kim, H.I. Shin, E.K. Park, S.Y. Kim, Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-a release and in vivo osteolysis, Exp. Mol. Med. 43 (2011) 411e418. [5] X. Jiang, T. Sato, Z. Yao, M. Keeney, J. Pajarinen, T.H. Lin, F. Loi, K. Egashira, S. Goodman, F. Yang, Local delivery of mutant CCL2 protein-reduced orthopaedic implant wear particle-induced osteolysis and inflammation in vivo, J. Orthop. Res. 34 (2016) 58e64.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Vx-11e protects against titanium-particle-induced osteolysis and osteoclastogenesis by supressing ERK activity Chen Li a, 1, Zuoxing Wu a, 1, Guixin Yuan d, Zhanfei Fang d, Xixi Lin a, Ruoyu Pu f, Yanbin Kang b, Li Li e, Siyuan Shao a, Jiaxin Ding a, Jinmin Zhao b, Qian Liu b, **, An Qin a, c, * a
Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Nanning, Guangxi, 530021, China Research Centre for Regenerative Medicine, Department of Trauma Orthopedic and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Guangxi, 530021, China c Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China d Department of Orthopedics, The Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, 515041, China e Pharmaceutic College, Guangxi Medical University, Nanning, Guangxi, 530021, China f Department of Obstetrics and Gynaecology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2019 Accepted 6 May 2019 Available online 17 May 2019
Wear particle-induced osteolysis around the prosthesis is the most common long-term complication after total joint replacement surgery which often leads to aseptic loosening of the prosthesis. Osteoclasts play key roles in the osteolytic process. Currently there is a lack of clinically effective measures to prevent or treat peri-prosthetic osteolysis and thus identification of new agents that can inhibit the enhanced osteoclastic bone resorption is warranted. Through this study, we discovered that the specific and potent ERK1/2 inhibitor, Vx-11e, can protect against calvarial osteolysis caused by titanium (Ti) particles in vivo. Low doses of Vx-11e mildly reduced osteoclast resorption whilst no calvarial osteolysis was observed with high dose Vx-11e treatment. Histological examination showed fewer osteoclasts and reduced bone erosion in the Vx-11e treated groups. In vitro cellular analyses showed that Vx-11e inhibited osteoclast formation from BMM precursors in response to RANKL, as well as bone resorption by mature osteoclasts. Mechanistically, Vx-11e impaired RANKL-induced ERK1/2 signaling by inhibiting its kinase activity thereby blocking the phosphorylation of downstream substrates. Moreover, Vx-11e significantly reduced the expression of RANKL-mediated genes such as ACP5/TRAcP, CTR, MMP-9, CTSK. Collectively, our data provides evidence for the potential therapeutic use of Vx-11e for the treatment of osteolysis diseases caused by extremely actived osteoclastogenesis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Osteolytic Vx-11e Osteoclast RANKL
1. Introduction Total joint replacement is widely used in arthritis, arthrosis, femoral head necrosis and other diseases [1]. In the United States, the number of arthroplasty cases reached 70 million and showed an increasing trend [2]. With the number of patients receiving this
* Corresponding author. Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China. ** Corresponding author. Research Centre for Regenerative Medicine, Guangxi Medical University, Guangxi, China. E-mail addresses: [email protected] (Q. Liu), [email protected] (A. Qin). 1 Chen Li and Zuoxing Wu contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.05.054 0006-291X/© 2019 Elsevier Inc. All rights reserved.
treatment constantly increasing, so too has the number of patients with failed arthroplasty requiring revision surgery. Although there are substantial advances in biomaterials and prosthesis design, loosening of the prosthesis caused by osteolysis remain the most difficult problem to overcome [3]. Wear particles such as titanium particles generated from prosthesis is considered one of the main causes of TJA (total joint arthroplasty) failure [4]. The pathophysiology of wear particle-induced osteolysis is complex but can be generalized as the local induction of inflammation near and around the prosthesis leading to release of proinflammatory signals that result in the recruitment of immune cells, lymphocytes and osteoclast precursors such as monocytes and macrophages [5]. Subsequently, various pro-inflammatory
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cytokines are released by these cells into the tissues surrounding the loose prosthesis [5,6]. These factors such as TNF-a, IL-6 and IL17 stimulate osteoblasts to release receptor activator of nuclear factor-kB ligand (RANKL) which then promotes the osteoclastogenesis of BMMs and bone resorption leading to peri-prosthetic bone loss [7,8]. Osteoclasts are therefore key players in mediating the osteolytic bone destruction induced by wear particles, and are prime targets for pharmacological intervention. Stimulation with RANKL, monocytic precursors of the monocyte/macrophage lineage fuse into osteoclasts [9]. Binding of RANKL to its cognate receptor RANK on the membranes of monocytic precursor cells induces the rapid activation of a myriad of signaling pathways, of which the NF-kB and MAPK pathways are the most important [10]. Together, these pathways induce the expression and activation of NFATc1, the master transcriptional factor necessary for osteoclastogenesis [11]. Thus, the targeted suppression of NF-kB or MAPK signaling pathways might alleviate enhanced osteoclast bone resorption providing a new strategy for the prevention of osteoclast-mediated bone diseases. Vx-11e is a potent, selective, and orally bioavailable ERK1/2 inhibitor and ERK pathway plays an important role in osteoclast differentiation, thus we hypothesize that Vx-11e could effectively inhibit osteoclastogenesis. According to our results, we found that Vx-11e has a significant effect on inhibiting osteoclastogenesis in vitro. Futhermore, we explored that Vx-11e can protect mice against titanium particle-induced calvarial osteolysis. To sum up, we suggest that Vx-11e has the potential to be exploited as an effective drug in the treatment of osteoclast-mediated osteolysis diseases. 2. Materials and methods 2.1. Media and reagents ERK inhibitor, Vx-11e, was obtained from Selleckchem (Houston, Texas, USA) and dissolved in DMSO. a-MEM and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA). TRAcP Staining Kit and Cell Counting Kit-8 (CCK8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant mouse MCSF and RANKL were obtained from R&D Systems (Minneapolis, MN, USA). Specific antibodies against phosphorylated and total forms of ERK1/2, RSK (p90) p38 and JNK were obtained from Cell Signaling Technology (Danvers, MA, USA), IkBa, and b-actin were obtained from Abcam (China).
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2.3. mCT, histology and histomorphometric analysis Prior to histological assessment, 3D images of whole calvaria from each group were acquired using a high resolution mCT scanner (SkyScan 1076) and associated analysis software (SkyScan, Aartselaar, Belgium). All calvaria were scanned with the following parameters: pixel size, 14.4 mm; X-ray voltage, 50 kV; electric current, 800 mA. A square region of interest (ROI) around the midline suture was chosen for qualitative and quantitative analysis after reconstruction. The percentage of bone volume/tissue volume (BV/TV, %), number of porosities, and percentage of total porosity of each sample were measured in the ROI as previously reported [13]. After micro-CT scanning, the PFA-fixed calvarias were decalcified and embedded in paraffin for H&E and TRAcP staining. The histological sections were examined under a microscope at 50 , 100 , and 200 magnifications. The number of TRAcP-positive osteoclasts was quantified using ImageJ software (NIH, USA). 2.4. Cell culture, osteoclast differentration, and bone resorption in vitro BMMs (bone marrow macrophage) were isolated from femurs and tibias of 4-week-old C57BL/6 J mice by marrow flushing as previously described [14]. The extracted cells were cultured in complete a-MEM (a-MEM with 10% FBS and 100U/mL penicillin/ streptomycin) containing 30 ng/mL M-CSF. For osteoclast differentiation, M-CSF depend BMMs were seeded in 96-well plates at a density of 6 103 cells/well. After sticked, cells were treated without or with serial dilutions of Vx-11e (150 nMe37.5 nM) in the stimulation of RANKL (50 ng/mL) until multinucleated ‘pancake’ shaped osteoclasts were formed in RANKL-treated only control groups. Cells were stained with TRAcP Staining Kit after fixed with 4% paraformaldehyde for 10 min. The number of TRAcP-positive osteoclasts with 3 or more nuclei was quantified by ImageJ software. For bone resorption assay, small pre-osteoclasts cultured with RANKL for at least 3e4 days were dissociated from culture plates and re-seeded onto hydroxyapatite-coated 96-well plates (Corning Inc, NY, USA). Cell was allowed to adhere to the wells for a few hours to overnight after which they were treated with 37.5 or 75 nM Vx-11e for 2 days. Adherent cells were removed using 5% sodium hypochlorite solution and the plates were rinsed with water and air-dried. The results of resorption were observed under microscope and the pit area normalized to total well area was quantified by ImageJ software.
2.2. Murine model of Ti particle-induced calvarial osteolysis 2.5. Cell viability assay To evaluate the potential therapeutic efficacy of Vx-11e in vivo, we generated a mouse calvarial osteolysis model. The experimental protocol was approved by the Animal Ethics Committee of Guangxi Medical University. Thirty two healthy male C57BL/6 J mice were randomly divided into four groups (n ¼ 8 per group): Shamoperated (with PBS injection), Vehicle (Ti particles with PBS injection), low-dose Vx-11e (Ti particles treated with 2 mg/kg Vx-11e), and high-dose Vx-11e (Ti particles treated with 5 mg/kg Vx-11e). Titanium particles were soaked in 75% alcohol for two days and then dried at 37 C [12]. To induce calvarial osteolysis 30 mg of sterilized Ti particles were uniformly coated under the periosteal membrane around the suture of the calvaria. To minimize infections, mice were intraperitoneally injected with 1:100 dilution of penicillin/streptomycin in PBS (100 mL final volume). Two days after Ti particles implantation, PBS (Vehicle group) or Vx-11e (2 mg/kg and 5 mg/kg groups) were injected under the scalp every two days for 2 weeks. After fourteen days, mice were euthanized, and the calvarias were harvested for micro-CT (mCT) and histological analysis.
CCK-8 Cell Proliferation/Cytotoxicity kit was assessed to explore the effect of Vx-11e to the BMMs activity. MeCSFedependent BMMs seeded in 96-well plates at a density of 6 103 cells/well in complete a-MEM were treated with serial dilutions of Vx-11e (from 600 nM to 18.6 nM) for 48 h s, and then 10 mL of CCK-8 reagent was added to each well and incubated at 37 C for 2 h s, the absorbance was measured at 490 nm on an ELX800 Absorbance Microplate Reader (BioTek; Winooski, VT, USA). 2.6. Podosomal actin belt immunofluorescence BMM-derived osteoclasts cultured as described above were fixed with 4% paraformaldehyde and then permeabilized with 0.1% (v/v) Triton X-100 for 5 min s. After extensive washed with PBS, cells were incubated with Alexa-Fluor 647 phalloidin (Invitrogen; San Diego, CA, USA) diluted in 0.2% (w/v) BSA-PBS for 1 h at room temperature. Cells were counterstained with DAPI to visualize cell
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nuclei. Fluorescence images were acquired using the LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany) and processed using the associated Zeiss ZEN software. The number of podosomal actin belts was quantified using ImageJ software. 2.7. RNA extraction and quantitative PCR assay Total RNA were extracted from BMM-derived osteoclasts cultured with RANKL in the absence or presence of serial dilutions of Vx-11e (from 150 nM to 37.5 nM) for 5 days using TRIzol reagent. Complementary DNAs (cDNAs) were synthesized using M-MLV reverse transcriptase with 1 mg extracted RNA template and oligo-dT primers (Promega, USA). The resultant cDNA was then used as template in the SYBR Premix Ex Tag Kit (TaKaRa, Shiga Prefecture, Japan) master mix according to manufacturer's instuctions and qPCR reactions performed on the ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA, USA) using the following cycling conditions: initial denaturation at 93 C for 2 min s; followed by 40 repeated cycles of 93 C for 60 s s, 55 C for 60 s s, and 72 C for 60 s s; and a final extension at 72 C for 7 min s. The following primer sets based on mouse sequences were used: matrix metalloproteinase-9 (MMP-9)Forward:50 -CGTGTCTGGAGATTCGACTTGA-3'; Reverse:50 -TTGGAAACTCACACGCCAGA-30 ), 0 ACP5/TRAcP(Forward:5 -TGTGGCCATCTTTATGCT-3'; Reverse:50 GTCATTTCTTTGGGGCTT-30 ), cathepsinK(CTSK)(Forward:forward:50 GGGAGAAAAACCTGAAGC-3'; reverse: 50 -ATTCTGGGGACTCAGAGC30 ), and calcitonin receptor (CTR)(Forward:50 -TGCAGACAACTCTTGGTTGG; Reverse:50 -TCGGTTTCTTCTCCTCTGGA).
differentiation and therefore inhibition of ERK activity will negatively impact osteoclastogenesis. Thus, we first examined the in vitro effect of the ERK inhibitor, Vx-11e, on RANKL-induced osteoclast differentiation from BMM precursors. To this end, stimulated with RANKL, BMMs were treated with or without indicated concentrations of Vx-11e for osteoclastogenesis, after which cells were fixed and TRAcP stained. As shown in graphical Highlight, BMMs stimulated with RANKL only differentiated efficiently into abundant typical ‘pancake’-shaped multinucleated osteoclasts which are highly TRAcP positive. In contrast, BMMs treated with Vx-11e showed a dose-dependent decrease in the formation of large TRAcP-positive osteoclasts (graphical Highlight). To ensure that the anti-osteoclastogenic effect of Vx-11e was not the result of cell cytotoxicity, we evaluated the viability of the BMM precursor cells in the presence of various concentrations of Vx-11e using the CCK-8 cell proliferation/cytotoxicity assay. As demonstrated in the graphical Highlight, no cytotoxic effects were observed on BMM precursor cells 48 h s after treatment with Vx-11e, even at concentrations of up to 600 nM. Thus it can be concluded that the inhibitory effect of Vx-11e on osteoclast formation at concentrations of up to 150 nM is not a result of cellular cytotoxicity. 3.2. Vx-11e impaired BMM precursor cell fusion and osteoclast bone resorption in vitro
To examine early RANKL-induced signaling pathways, total cellular proteins (TCPs) were extracted from BMMs pre-treated with Vx-11e for 4 h s followed by RANKL stimulation for0, 5, 10, 20, 30, or 60 min. Cells were lysed in a NP-40 lysis buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 1 mM EDTA; and 10% glycerol) supplemented with protease and phosphatase inhibitor cocktail. Protein concentrations were assessed using the BCA protein assay reagent (Beyotime, China) in accordance with manufacturer's protocol, and then 25 mg of TCPs were resolved on 8% SDS-PAGE gels and electrotransferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA) overnight at 4 C. Membranes were blocked with 5% (w/v) BSA in TBST (0.1% Tween 20 in Tris-buffered saline) for 1hr and then incubated with primary antibodies (diluted 1:1000 in 1% (w/v) BSATBST) overnight at 4 C. Membranes were then washed and subsequently incubated with the appropriate secondary fluorescence antibodies conjugated to IRDye 800CW (diluted 1:5000 in 1% (w/v) BSA-TBST) (LI-COR Biociences; Lincoln, NE, USA) for 2 h s. Antibody immunoreactivity was detected by exposing the membrane blots in an Odyssey Infrared Imaging System (LI-COR).
As noted, Vx-11e inhibited the formation of typical large multinucleated osteoclasts suggesting that the precursor cell fusion process is impaired. To further show an impairment of precursor cell fusion, we examined the actin cytoskeleton and nuclei number in BMM-derived osteoclasts treated without or with Vx-11e. Osteoclasts cultured on plastic plates have a belt-like structure of podosomes called the polysomal actin belt at the periphery and circumscribe the cells. Osteoclasts treated with Vx-11e also displayed the polysomal actin belt but were much smaller and less nucleated compared to RANKL-only controls, further suggesting that the fusogenic process is impaired (Fig. 1A). The actin cytoskeleton is also crucial for the bone resorptive function of the osteoclasts. When polarized towards bone resorption, the polysomal actin belt condenses into a tight F-actin ring surrounding the sealing zone from which resorption will take place. Thus based on the previous observations, it was inferred that the bone resorptive activity of mature osteoclasts would also be affected by Vx-11e. As expected, mature osteoclasts exposed to Vx11e exhibited significantly reduced bone resorptive ability (Fig. 1B). When compared with control untreated osteoclasts which almost completely absorbed the total area of the HA-coated well, osteoclasts treated with 75 or 150 nM absorbed only ~60% and 20% of the total well area, respectively (Fig. 1C). These findings demonstrated that Vx-11e impaired osteoclast bone resorption and F-actin ring formation in vitro.
2.9. Statistical analysis
3.3. Vx-11e reduced the expression level of RANKL-mediated genes
All data presented are representative of at least three independent experiments, and are expressed as mean ± standard deviation. The data were analyzed by SPSS 16.0, and Student's t-test was used for comparison between groups with p < 0.05, **p < 0.01 and ***p < 0.001 considered statistically significant.
Osteoclast differentiation and function are related to the expression level of RANKL-mediated genes. Real-time PCR was conducted on RNA extracted from osteoclasts treated with different doses of Vx-11e. As shown in Fig. 2, the expression of MMP-9, ACP5/ TRAcP, CTSK, and CTR was significantly reduced at 150 nM. The expression of MMP-9 and CTSK was dose-dependently reduced whereas TRAcP was only significantly reduced at 150 nM (Fig. 2AeD). The impaired expression of these genes in the presence of Vx-11e coincided with the decreased osteoclast formation and bone resorption observed earlier. Thus our real-time PCR results confirmed the inhibitory effect of Vx-11e on osteoclast differentiation and bone resorption in vitro.
2.8. Western blotting analysis
3. Results 3.1. Vx-11e attenuated RANKL-induced osteoclastogenesis in vitro ERK is one of the key early signaling cascades activated following RANKL stimulation and is required for efficient osteoclast
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Fig. 1. Vx-11e impaired precursor cell fusion and mature osteoclast bone resorption in vitro. (A) Effects of Vx-11e on BMM precursor fusion. MeCSFedependent BMMs stimulated with RANKL in the absence or presence of indicated concentrations of Vx-11e were fixed and immuno-stained for actin (Alexa-Fluor 647 phalloidin) and nuclei (DAPI). (B) Vx-11e inhibits mature osteoclast bone resorption. Equal numbers of BMM-derived osteoclasts stimulated with 50 ng/mL RANKL for at least 3e4 days were seeded onto hydroxyapatite-coated wells and then treated with indicated concentrations of Vx-11e for 2 days. Attached cells were then removed and resorption pits were imaged under a light microscope. Scale bars, 100 mm. (C) The resorption pit area as a percentage of well area were quantified using ImageJ. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig. 2. Vx-11e reduced the expression of osteoclast marker genes. Real-time quantitative PCR was performed on RNA extracted from BMM-derived osteoclasts stimulated with 50 ng/ml RANKL without or with indicated concentrations of Vx-11e for 5 days. (AeD) The expression levels of MMP-9, ACP5/TRAcP, CTSK, and CTR genes were normalized to GAPDH and then expressed as relative fold change against RANKL-only control. *p < 0.05, **p < 0.01, and ***p < 0.001.
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3.4. Vx-11e inhibited the activity of ERK in response to RANKL The MAPK signaling pathway is one of the key signaling cascades activated in response to RANKL during osteoclast differentiation. As a potent and selective ERK inhibitor, we thus examined the RANKL-induced early activation of the ERK MAPK pathway in the absence or presence of Vx-11e. To this end, we performed Western blot analyses of the phosphorylation status of ERK1/2 and it's downstream substrate p90RSK, an established biomarker for evaluating compound effects native ERK1/2 activity and inhibition [15]. Interestingly, the results showed that Vx-11e enhanced the phosphorylation of ERK1/2 but blocked ERK-mediated phosphorylation of p90RSK (Fig. 3A). This finding suggests that Vx-11e does not affect ERK1/2 phosphorylation but rather ERK kinase activity for downstream substrates and is in line with previous reports of contradictory increases in ERK1/2 phosphorylation in various tumor cells treated with Vx-11e [16]. Consistent with its specificity towards ERK, Vx-11e did not affect the activation of the other MAPK members, JNK and p38, or the NF-kB signaling pathway (Fig. 3B). Taken together, these results demonstrate that Vx-11e inhibited osteoclast differentiation by blocking ERK kinase activity which ultimately impairs downstream ERK signaling. 3.5. Vx-11e suppressed titanium particle-induced bone loss in vivo Given the promising in vitro inhibitory effect on osteoclast formation, we next explored the potential therapeutic potential of Vx11e on preventing titanium (Ti) particle-induced bone loss in mice calvarias. Three dimensional mCT reconstructions showed extensive bone loss with numerous surface erosions and resorption pits in the Ti group treated with PBS vehicle as compared to Sham controls where the calvarial surface were smooth with no signs of bone erosion (Fig. 4A). Treatment with Vx-11e on the other hand, attenuated Ti particle-induced osteolysis in a dose-dependent manner. Resorption pits and surface erosion were mildly reduced with low-dose of Vx-11e, whereas the calvarial surface in mice treated with high-dose Vx-11e was comparable to Sham controls. Morphometric quantification of bone volume/total volume (BV/ TV), number of porosity, and the percentage of total porosity in the ROI confirmed the dose-dependent attenuation of Ti particleinduced bone loss (Fig. 4CeE). Again bone morphometric
parameters showed that high-dose Vx-11e treatment was comparable to Sham controls. Histological and histomorphometric analysis further confirmed the protective effect of Vx-11e treatment against Ti particleinduced bone erosion in vivo. TRAcP and H&E stained tissue sections showed extensive osteolytic bone destruction in the Ti group treated with PBS vehicle group, with abundant TRAcP positive osteoclasts along the eroded bone surface (Fig. 4B and F). Treatment with low- and high-dose Vx-11e treatment group significantly reduced osteolytic bone loss and reduced the number of osteoclasts on the bone surface (Fig. 4B and F). Collectively, the in vivo data provides evidence for the potential therapeutic application of Vx11e against osteolytic bone loss. 4. Discussion Aseptic loosening due to titanium wear particle-induced osteolysis around artificial prosthesis remains one of the most common causes of total joint arthroplasty failure despite advances in implant design and biomaterials used in making the prostheses [17]. Despite the complex molecular and cellular interplays of the osteolytic bone destruction, wear particle-induced inflammation, enhanced osteoclast formation and activation, leading to excessive bone resorption remains the most important cause [18]. As such, pharmacological interventions with bisphosphonates, estrogens, denosumab, and/or teriparatide have been widely used after arthroplasty to inhibit the wear particle-induced osteoclast-mediated osteolysis [19]. However, due to side effects from mild fevers, throat and stomach ulcers to more serious malignant tumor formation and osteonecrosis of the jaw, limit the long-term and systemic use of these drugs. Hence, current intervention therapies are far from ideal and thus the identification of new and safer options are warranted. Given the important role the osteoclasts play in the bone destruction process, agents that can inhibit osteoclast formation and/or bone resorption are ideal therapeutic agents for preventing and ameliorating wear particle-induced osteolysis [20]. In this study, we demonstrated that Vx-11e has a protective effect against Ti particle-induced calvarial osteolysis in mice in vivo. This effect was mediated by the inhibition of RANKL-induced ERK activity leading to suppression of osteoclast formation from BMM precursors and attenuation of mature osteoclast bone resorption.
Fig. 3. Vx-11e specifically inhibited the activity of ERK in response to RANKL. Total cellular proteins (TCPs) extracted from BMM pre-treated with 150 nM Vx-11e for 4 h s and then stimulated with 50 ng/ml RANKL for the indicated times were subjected to immunoblot analyses using specific antibodies against components of the ERK, RSK, JNK, P38 and NF-kB signaling cascade, b-actin was used as internal loading control.signaling cascade. (A-B)The ratio of phosphorylated proteins relative to unphosphorylated proteins was determined.
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Fig. 4. Vx-11e suppressed against titanium particle-induced bone loss in vivo as assessed by mCT and histology. (A) Representative 3-dimensional mCT reconstructed images of mice calvaria from sham-operated (with PBS injection), Ti particles with PBS injection (vehicle), and Ti particles with low dose (2 mg/kg body weight) or high dose (5 mg/kg body weight) Vx-11e 14 days after treatment. (CeE) Quantitative morphometric analyses of bone volume to tissue volume (BV/TV, %), the number of porosity, and the percentage of porosity were conducted. (B) Representative images of H&E (magnifications of 100 and 200 ) and TRAcP (magnifications of 50 and 100 ) stained calvarial tissue sections from sham-operated (with PBS injection), Ti particles with PBS injection (vehicle), and Ti particles with low dose (2 mg/kg body weight) or high dose (5 mg/kg body weight) Vx-11e groups. Black arrows indicate TRAcP-positive osteoclasts. (F) The number of TRAcP-positive osteoclasts in the calvarial bone sections was quantified.*p < 0.05, **p < 0.01, and ***p < 0.001.
These results provide evidence for the potential therapeutic application of Vx-11e to treat wear particle-induced osteolysis. Vx-11e (Vertex-11e) was originally defined as a potent, selective, and orally bioavailable ERK2 inhibitor [21]. However, further studies have shown it to be a type-I kinase inhibitor that potently inhibits both ERK1 and ERK2 kinase activity [22] and exhibits unique kinetic properties defined by slow dissociation rates [23]. In this study, we found that Vx-11e enhanced ERK1/2 phosphorylation in response to RANKL, a paradoxical phenomena that has been previously documented in various tumor cells treated with VX-11e [24]. It has been reported that the expression of phosphorylated ERK does not necessarily correlate with ERK pathway activation and inhibitor sensitivity [25]. Thus this enhanced phosphorylation of ERK1/2 could be due to feedback activation pathways or crosstalk with parallel signaling pathways as previously described [26]. It could also be due to compensatory effect to the inhibition of kinase activity by increasing the levels of activated ERK1/2. The exact mechanism will require further investigations. However, despite the enhanced ERK1/2 phosphorylation, the phosphorylation of the
downstream ERK substrate p90RSK was blocked by Vx-11e, confirming the inhibition of ERK kinase activity. Furthermore, confirming the specificity for ERK1/2 signaling, Vx-11e did not have any discernible effects on the other MAPK members, p38 or JNK, nor the NF-kB signaling pathway. The early activation of the NF-kB and MAPK signaling cascades by RANKL are crucial prerequisite for osteoclast precursor differentiation and fusion, and subsequent mature osteoclast bone resorption [27]. RANKL-induced activation of ERK is necessary for efficient osteoclast formation, regulation of osteoclast survival and apoptosis, as well as maintenance of cell polarity during bone resorption [28]. Consistent with impaired ERK activity, we found that Vx-11e dose-dependently inhibited osteoclast formation from BMM precursors as well as mature osteoclast bone resorption. The concentration of Vx-11e at which osteoclast formation was inhibited did not induce any cytotoxicity demonstrating that inhibitory effect of Vx-11e was not due to decreased cell viability. Furthermore, BMM cells treated with Vx-11e formed smaller osteoclasts as defined by size of the polysomal actin belt
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circumscribing the periphery of the osteoclasts and exhibited less nuclei per cell suggesting impairment in precursor cell fusion. ERK activation is also required for the induction and activation of key osteoclastic transcription factors c-Fos and NFATc1 [29]. NFATc1 is considered the master transcriptional activator for a number of osteoclast marker genes involved in osteoclast formation and bone resorption [30]. Our real-time PCR results showed that Vx-11e suppressed the expression osteoclast maker genes MMP9, ACP5/TRAcP, CTSK, and CTR during RANKL-induced osteoclast formation. The decrease in the expression of these genes which are associated with bone resorption is in line with decreased resorptive activity of mature osteoclasts following Vx-11e treatment. Our in vitro biochemical and cellular results provided evidence for the inhibitory effect of Vx-11e on osteoclast formation, gene expression and bone resorption. With these promising results we examined the potential effects of Vx-11e on Ti particle-induced osteolysis in vivo. We found that mice treated with Vx-11e group dose-dependently alleviated Ti particle-induced osteolysis by reducing the number of TRAcP-positive osteoclasts thereby ameliorating Ti particle-induced osteoclast-mediated bone erosion. Consequently, Vx-11e may be of therapeutic interest in osteolytic bone diseases. Conflicts of interest The authors declare no conflicts of financial or other interests. Acknowledgements The authors acknowledged all the members of our laboratory for their assistance. An Qin, and Qian Liu designed the study; Chen Li, Zuoxing Wu, Guixin Yuan, Ruoyu Pu, Siyuan Shao, Yanbin Kang, Jiaxin Ding, Li, Zhanfei Fang and Xixi Lin participated in the experiments, analyzed the data, and prepared the figures. The manuscript was wrote by Chen Li. All authors have reviewed the manuscript. This project is supported by National Natural Science Foundation of China (81501910) (81772373) (81572167), Guangxi Natural Science Foundation under Grant (2015GXNSFCA414001), and Guangxi Collaborative Innovation Center for Biomedicine Talent Cultivation (GCICB-TC-2017001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.054. References [1] A.D. Van Citters, C. Fahlman, D.A. Goldmann, J.R. Lieberman, K.M. Koenig, D.G.A. Rd, B. O'Donnell, J. Martin, F.A. Federico, R.A. Bankowitz, Developing a pathway for high-value, patient-centered total joint arthroplasty, Clin. Orthop. Relat. Res. 472 (2014) 1619e1635. [2] S. Kurtz, K. Ong, E. Lau, F. Mowat, M. Halpern, Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030, J. Bone Joint Surg. Am. Vol. ume (2007). €rrholm, Highly crosslinked [3] P.E. Johanson, G. Digas, P. Herberts, J. Thanner, J. Ka polyethylene does not reduce aseptic loosening in cemented THA 10-year findings of a randomized study, Clin. Orthop. Relat. Res. 470 (2012) 3083e3093. [4] S. Jin, J.Y. Park, J.M. Hong, T.H. Kim, H.I. Shin, E.K. Park, S.Y. Kim, Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-a release and in vivo osteolysis, Exp. Mol. Med. 43 (2011) 411e418. [5] X. Jiang, T. Sato, Z. Yao, M. Keeney, J. Pajarinen, T.H. Lin, F. Loi, K. Egashira, S. Goodman, F. Yang, Local delivery of mutant CCL2 protein-reduced orthopaedic implant wear particle-induced osteolysis and inflammation in vivo, J. Orthop. Res. 34 (2016) 58e64.
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