Kaempferide Prevents Titanium Particle Induced Osteolysis by Suppressing JNK Activation during Osteoclast Formation

Kaempferide (KF) is an O-methylated flavonol, a natural plant extract, which is often found in Kaempferia galanga. It has a variety of effects including anti-carcinogenic, anti-inflammatory, anti- oxidant, anti-bacterial and anti-viral properties. In this study, we aimed to investigate whether KF effectively inhibits titanium particle induced calvarial bone loss via down regulation of the JNK signaling pathway. In the mice with titanium particle induced calvarial osteolysis, the Low dose of KF mildly reduced the resorption pits while in the high dose group, fewer scattered pits were observed on the surface of calvarium. Histological examination showed fewer osteoclasts formation in the KF group. In mouse bone marrow macrophages (BMMs) and RAW264.7 cells, KF significantly inhibited the osteoclast formation and bone resorption at 12.5 μM. However, KF does not affect the mature osteoclast F-actin ring formation. But when being co-treated with KF and anisomycin, BMMs differentiated into mature osteoclasts. At the molecular levels, the JNK phosphorylation was inhibited and the osteoclastogenesis- related specific gene expression including V-ATPase d2, TRAP, calcitonin receptor (CTR), c-Fos and NFATc1 was markedly suppressed. In conclusion, these results indicated that KF is a promising agent in the treatment of osteoclast-related diseases.

End-stage temporomandibular joint (TMJ) diseases such as osteoarthritis, severe inflammatory condylar resorp- tion, idiopathic condylar resorption and TMJ ankylosis usually result in loss of the posterior vertical height of the mandible and therefore need TMJ reconstruction. At present, the commonly used treatment modalities for such diseases include autogenous bone grafts1 such as costochondral graft, sternoclavicular graft, and coronoid graft or total joint replacement (TJR) with artificial prosthesis. TJR of the TMJ is an effective treatment for an intractable pain and impaired TMJ function2. However, those patients are often relatively young (30 to 35 years of age) and need long term use of prosthesis3. Wear particles such as titanium particles generated from prosthesis can cause long term complication such as aseptic peri-prosthesis loosening. Indeed, the loosening and instability of the condylar component and the fixation screws of the TJR are one of the most widely reported complications associated with TMJ prosthetic replacement4. Therefore, the prevention of aseptic loosening of TMJ prosthesis takes on added importance.Theoretically, the long term use of the prosthesis will cause the release of small wear particles between the bone and the implant interface. The released titanium particles can thus recruit and activate macrophages, result- ing in the release of different inflammatory mediators such as IL-1β, IL-6, IL-17 and TNF-alpha5,6, which in turn can enhance the expression of RANKL from the surrounding osteocytes and stromal cells7. The increased RANKL levels can subsequently activate osteoclast formation and bone resorption, leading to periprostheic bone loss and therefore causing prosthetic loosening and instability8.

Accordingly, there are two ways for improving the clinical outcome of the total joint prosthesis: 1) Synthesis of more biocompatible prosthesis materials that can reduce the release of wear particles. 2) Searching for com- pounds that can inhibit macrophage and/or osteoclast activation. During the screening of such compounds that can inhibit osteoclast formation and function, we identified a natural compound derived from the roots of kaempferia galanga, kaempferide (KF) and found KF is capable of suppressing osteoclast function. Previous studies showed that KF has a series of biological activities including antioxidant9,10 and antibacterial11 properties. However, to our knowledge, there are no reports discussing the role of KF on bone metabolism and titanium particles induced osteolysis. Furthermore, the possible use of KF in preventing osteolysis in vivo remains unclear. Therefore, this study aimed to investigate whether KF has an inhibitory effect on titanium particle induced oste- olysis in vivo and to unveil its mode of action in vitro.

Results
KF inhibited osteoclast differentiation in vitro. First, we investigated the effect of KF on osteoclast differentiation in vitro. As shown in Fig. 1A, a large number of TRAP-positive multinucleated osteoclasts formed in the control group, while the presence of KF inhibited osteoclast formation in a dose dependent manner. Treatment of osteoclasts with KF at 3.125 μM mildly inhibited osteoclast formation, with approximately 30% reduction in the number of osteoclast formation. Compared with the control group, the addition of KF at 12.5 μM significantly suppressed osteoclast formation, with almost no round osteoclast formed in this group. There are only about 10.16 ± 4.22 osteoclasts formed in the 12.5 μM group. (Fig. 1B). Collectively, KF inhibited osteoclast differentiation in a dose dependent manner.KF had no cytotoxicity at low concentrations. In order to exclude the possibily of KF on osteoclast differentiation is not due to the cytotoxicity of KF on osteoclasts, the effect of KF on cell viability was evaluated. As shown in Fig. 1C, KF exhibited cytotoxic effect on the osteoclast precursor cells at 50 μM or higher concentrations after the incubation for 48 h, 72 h and 96 h respectively. The IC50 of KF was 159.8 ± 15.6 μM, 90.72 ± 10.3 μM, and 43.13 ± 8.7 μM at 48 h, 72 h and 96 h respectively (Fig. 1D). No cytotoxic effects were observed at 25 μM or lower concentrations. Thus, KF has cytotoxic effect on osteoclast precursor cells at concentrations ≥50 μM. Since we observed KF at 12.5 μM had dramatic inhibitory effect on osteoclast differentiation. Therefore, the inhibitory effect of KF on osteoclastogenesis is
not due to its cytotoxic effect.KF suppressed RANKL-induced gene expression in vitro. The suppression of osteoclast differentia- tion is further evaluated by examination the osteoclast specific gene expression profile. Osteoclasts treated with different doses of KF were harvested for RNA extraction and real-time PCR. As shown in Fig. 1E, the expression of osteoclast specific TRAP gene was expressed in the control group. However, its expression was significantly inhibited after being treated with KF. Approximately 50% reduction was noticed. Similarly, another osteoclast specific marker CTR demonstrated similar expression trend, suggesting a reduced osteoclast number after KF treatment. In addition to these mature osteoclast markers, the expression of key transcription factors NFATc1 and c-fos were also inhibited after KF treatment, which result in the attenuated expression of their downstream gene expression such as V-ATPase d2 (Fig. 1E). Taken together, our realtime PCR results confirmed the inhibitory effect of KF on osteoclast differentiation in vitro.

KF inhibited the function of osteoclasts: bone resorption assay. Since osteoclast differentiation was inhibited, we assumed that KF can subsequently inhibit the osteoclast function. Thus, the osteoclast precursors were seeded on the surface of bone slices with the absence or presence of KF at different concentrations. Scanning electron microscope (SEM) showed that large areas of bone resorption pits were observed in the control group. The percentage of the resorption area in the control group was significantly higher than in the KF group. A dose dependent suppressive effect of KF on osteoclast bone resorption was noticed, while nearly no resorption pits were observed at 12.5 μM (Fig. 2A). Further analysis of the bone resorption area using Image J software also con- firmed the inhibitory effect of KF on bone resorption in vitro. About 60% and 90% reduction of bone resorption area were observed in the groups treated with KF at 6.25 μm and 12.5 μM respectively (Fig. 2B). Collectively, these data indicated that KF inhibited osteoclast bone resorption in vitro.KF does not affect the RANKL-induced F-actin ring formation. Prior to osteoclast induced bone resorption, the differentiated osteoclasts need to reconstruct their cyto-skeletal structure, known as F-actin ring formation. Thus, we then investigated whether KF can affect F-actin ring formation in vitro. As shown in Fig. 2C, under the circumstances of M-CSF (30 ng/ml) and RANKL (50 ng/ml) that induced the mature osteoclast forma- tion, a well-structured F-actin ring was observed in the control group. In agreement with the suppressed osteo- clast differentiation as mentioned above, the number of F-actin ring was indeed reduced after being treated with different doses of KF. However, we can still observe the well-preserved ring-like structures in the drug treated groups at 3.125 μM and 6.25 μM. When KF concentration was increased to 12.5 μM, almost no mature large oste- oclasts were detectable. However, small ring-like structures still existed. Thus, our results suggested KF does not affect the mature osteoclast F-actin ring formation.

KF depressed the osteoclastogenesis via down regulating the JNK and ERK signaling path- way. All the aforementioned results indicate that KF can suppress the osteoclast differentiation and thus inhibiting bone resorption in vitro. However, the underlying molecular mechanisms on how KF affects the oste- oclastogenesis require further investigations. MAPK signaling pathways are known to play pivotal roles in the osteoclast differentiation. Thus, we further checked the activation of these signaling pathways with the presence or absence of KF. As shown in Fig. 3A, the RANKL-induced JNK phosphorylation was observed in the control group, peaking at 20 min and 30 min. However, the activation of JNK phosphorylation was significantly sup- pressed after KF treatment. The expression of p-JNK in the KF group was significantly lower than that in the control group as reflected by the statistical analysis (Fig. 3B). We also examined the effect of KF on other MAPK signaling pathways including p38 and ERK phosphorylation. As shown in Fig. 3A, the phosphorylation of ERK was also observed in the control group. Similarly, KF partially attenuated the ERK phosphorylation as compared to the control group (Fig. 3C). Different from JNK and ERK, the phosphorylation of p38 was not affected after KF treatment (Fig. 3D). However, the NF-kB pathway, which is another downstream signaling pathway, was not affected (data not shown). The above results showed that KF inhibited the phosphorylation and degradation of JNK and ERK, without affecting the p38 signaling pathway.

The inhibitory role of KF on osteoclastogenesis was rescued by anisomycin. In order to further confirm that KF suppressed the osteoclast formation by affecting the JNK activation, we further employed a res- cue assay to validate this. As shown in Fig. 3E, the osteoclast formation was significantly restrained when the cells were treated with KF only. In contrast, the addition of the JNK agonist, anisomycin, rescued osteoclastogenesis, where mature osteoclasts were formed (Fig. 3E). Statistical analysis showed that when the BMMs treated with KF and anisomycin, both at 6.25 μM and 12.5 μM concentrations, the number of osteoclasts was significantly higher than that of the KF treated group (Fig. 3F). Western bolt showed that the JNK phosphorylation was rescued, as shown in Fig. 3H, when treated with RANKL+KF+Anisomycin, the p-JNK expression was observed compared to the RANKL+KF group, which means the JNK phosphorylation was rescued. At the same time, the administra- tion of anisomycin didn’t affect JNK protein synthesis (Fig. 3H), the JNK expression increased gradually from 0 to 120 mins (Fig. 3G). Together, these data implicated KF suppressed osteoclast formation via inhibiting the JNK signaling pathway.

Interaction between KF and the JNK/ERK protein. As the phosphorylation of JNK is an important molecular step in the process of osteoclast differentiation, we next examined whether KF can bind to JNK protein based on a computational calculation. As shown in the molecular docking, KF could form stable connections with JNK1 (Fig. 4A) and JNK2 (Fig. 4B) ATP binding sites. Specifically, KF interacted with MET-111, LEU-110 and GLU-109 of JNK1 and JNK2. However, we failed to find the potential binding sites of KF onto ERK protein (Fig. 4C). Together with the western blotting, the molecular docking indicated that KF suppressed the phospho- rylation of JNK. In order to testify that the other JNK inhibitor might express the similar effects as that of KF, we also examined the possible interaction of 1,9-Pyrazoloanthrone, which is a specific JNK inhibitor, with JNK. As shown in the Fig. 4D, 1,9-Pyrazoloanthrone could form stable connections with JNK ATP binding sites at LEU- 110 and MET-111, which expressed the similar effect as that of KF.KF suppressed Ti-particle induced osteolysis in vivo. Our in vitro experiments suggested KF is effective in preventing the formation and function of osteoclasts. To validate the potential therapeutic effect of KF on prevent- ing titanium particles induced bone loss, we then administered KF in titanium induced osteolysis model in vivo. As shown in Fig. 5A, reconstruction of micro-CT scanning showed an extensive bone loss after titanium stimulation in the vehicle group, with numerous large and deep resorption pits on the calvarial surface.

In contrast, the calvarial surface was relatively smooth in the sham group. Interestingly, the administration of KF attenuated particle induced osteolysis. Low dose of KF (4 mg/kg/day) mildly reduced the resorption pits, with fewer and smaller pits on the cal- varial surface. More obvious attenuation of titanium induced osteolysis was observed in the high dose group (8 mg/ kg/day), with fewer scattered pits observed on the surface, especially along the suture line. Statistical analysis of bone volume/total volume (BV/TV), number of porosity and the percentage of total porosity in the region of interest (ROI) also confirmed the Micro-CT scanning results. On the sham group, BV/TV was significantly higher than that of the vehicle group, while the value of BV/TV was increased with the presence of KF (Fig. 5C). On the vehicle group, both the number of porosity and the percentage of porosity were greatly higher than the other 3 groups. With the increasing concentration of KF, the value of the KF-L and the KF-H group were significantly decreased.Furthermore, the histological examination confirmed the protective effect of KF on Ti-particle induced oste- olysis. Extensive bone destruction was observed in the vehicle group, with a lot of TRAP positive osteoclasts on the surface of the dissolved bone tissue (Fig. 5B). However, the number of mature osteoclasts in KF-L and KF-H group was significantly reduced (Fig. 5D). In addition, no fatalities were recorded and all mice remained with normal activity during the whole experiment. Together, these data indicated KF is capable of preventing titanium induced osteolysis in vivo.

Discussion
For many end-stage diseases of the TMJ such as severe osteoarthritis, ankylosis, idiopathic condylar resorption and tumors involving the mandibular condyle, TJR with an artificial prosthesis is the most effective way to treat these diseases. However, aseptic loosening of the prosthesis is one of the most important causes of failure for hip and knee joint prostheses12, and this complication most likely will be the same for TMJ total joint prostheses. Because TMJ patients are often relatively young (30 to 35 years of age), a total TMJ prosthesis must have a very long lifetime3. The prevention of aseptic loosening of the TMJ prosthesis gains much more attention recently. Generally, the titanium particle induced loosening is due to the periprosthesis osteolysis induced by an activation of a large number of osteoclasts. Therefore, osteoclasts inhibitors are thought to be potential drugs for the tita- nium particle induced osteolysis.Flavonoids are natural phenolic compounds present in fruits and vegetables with antioxidant13, anti-carcinogenic14 and other biological functions15,16 including osteogenic and anti-osteoclastogenic effects. Eerduna et al.17 found that flavonoids reduce the myocardial infarction size after an artery ligation in rats, through regulating the antioxidative enzymes activity and the endothelial nitric oxide synthase activity. Flavonoids also have neuroprotective actions, having the potential as multi-targeted therapeutic tools for protecting the brain18. Recently, being a kind of Flavonoids, kaempferol, an analogue of KF, has been shown to have an inhibitory role on the bone loss in mice long bones by preventing the osteoclast formation19. However, whether KF has the same role on osteoclast formation and bone resorption both in vivo and in vitro has not been discussed before.

In our study, KF was shown to have an inhibitory effect on osteoclast formation and function in a dose dependent manner. At the concentration of 12.5 μM, osteoclastogenesis was inhibited obviously and almost no resorption pits were observed on bone slices. The inhibited bone resorption is most likely due to suppressed osteoclast formation, as supported by the fact that KF suppressed TRAP positive ostgeoclast formation and gene expression in a dose dependent manner. These findings can further reflect the in vivo findings that the admin- istration of KF can prevent osteoclastic bone loss by reducing TRAP positive osteoclast number in our titanium particle induced osteolysis model.Furthermore, the molecular mechanisms underlying this inhibitory effect of KF were elucidated. Western blotting revealed that KF inhibited the RANKL-induced JNK and ERK signaling pathways without affecting the p38 signaling pathway. During the osteoclast metabolism process, JNK plays a very important role20. Johnson et al. found that c-Jun–deficient mice are embryonic lethal21. Ikeda also found that the body size of the transgenic mice in which the domain-negative c-Jun lacking the transcriptional activation domain was much smaller than that of the wild type mice. Also the transgenic mice showed an increased radio density of the long bones, jaw bones, and vertebrae compared with the control mice. An activated JNK subsequently leads to the activation of the transcription factor c-Jun22. C-Jun together with c-Fos, an essential transcription factor for osteoclast forma- tion, can form the activator protein-1 (AP-1) complexes. AP-1 can bind to the NFATc1 promotor and regulate its expression. NFATc1 is a master regulator in osteoclastogenesis process23, and the coupling of c-Jun signaling with NFAT family is crucial for the transcriptional events during osteoclastogenesis24. NFATc1 can regulate the expression of several genes associated with osteoclast differentiation and function. In vitro promotor analyses identified the nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 (TRAP) and Calcr (CTR) promotors.In addition, the transcriptional induction of NFATc1 is considered a major function of c-Fos in osteoclast differentiation25. In this study, KF inhibited JNK phosphorylation as demonstrated by WB results. This is further evidenced by our molecular docking assay that KF can interact with the ATP binding site of MET-111, LEU-110 and GLU-109 of JNK1 and JNK2. Again, the addition of JNK agonist anisomycin reversed the inhibitory effect of KF on osteoclast formation. All the results point to the possible mode of action that KF suppressed JNK activation and lead to impaired osteoclast formation.

In summary, our study demonstrated that KF is able to prevent titanium particle induced osteolysis in vivo and inhibit osteoclastogenesis in vitro, and the inhibitory effect can be played at a lower concentrations. KF could be considered as a potential agent for the prevention of particle induced osteolysis in future. Surface modification or local injection of this natural compound is of potential in the treatment of peri-prosthesis loosening. Also, KF is also used in Chinese cooking and traditional Chinese medicine, so it can be intake from the daily diet. Admittedly, there are some limits of this study. The effect of KF on osteoblast and osteocyte biology needs further investiga- tion. For future translation of this finding into clinic, surface coating of this natural compound onto prosthesis need further validation. Another classical animal model for studying osteoclasts is the collagen-induced arthritis in mice, which will be performed in the further study of elucidating inflammatory lesions of TMJ such as TMJ osteoarthritis. Generally, we demonstrated natural compound KF is of value in Ti-particle induced osteolysis.Media and reagents. KF was obtained from Meilun (Dalian, Liaoning, China) and then it was dissolved in Dimethylsulfoxide (DMSO) with a concentration of 100 mM stock solution. Alpha-MEM, fetal bovine serum (FBS), and penicillin were purchased from Gibco BRL (Gaithersburg, MD, USA). Soluble mouse recombinant M-CSF and RANKL were purchased from R&D Systems (USA). Tartrate-resistant acid phosphatase (TRAP) staining solution was from Sigma–Aldrich. The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Japan). Primary antibodies targeting GADPH, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, NF-kB and IkB-a were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). The Prime Script RT reagent Kit Anisomycin and SYBR® Premix Ex Taq™ II were obtained from TaKaRa Biotechnology (Otsu, Shiga, Japan).