Netrin‐1 regulates ERK1/2 signaling pathway and autophagy activation in wear particle‐induced osteoclastogenesis

Abstract Background Artificial joint replacement surgery is often accompanied by osteolysis induced aseptic loosening around the prosthesis. Wear particles from joint replacement are thought to be one of the main factors leading to local inflammation and osteolysis at the prosthesis site. The aim of this study was to investigate the molecular mechanism of osteoclast formation and dissolution induced by wear particles and the potential roles of Netrin‐1, the ERK1/2 pathway and autophagy activation in this process. Methods The messenger RNA levels in cells and tissues were detected with real‐time quantitative PCR. The western blotting was used to detect the expression of proteins. A CCK‐8 kit was used to detect the viability of RAW 264.7 cells. Moreover, an air pouch model of bone resorption was established. Immunohistochemistry was used to detect the expression of TRAP and Netrin‐1 in rat bone tissue. Cell culture supernatants were collected in the rat air pouch model of bone resorption, and the levels of RANKL and OPG were detected with enzyme‐linked immunosorbent assay. The protein levels of TRAP and Netrin‐1 in bone tissue were examined by immunohistochemistry. Results Titanium wear particles induced osteoclast formation and autophagy activation. Moreover, blocking autophagy suppressed the osteoclastogenesis after exposure to wear particles in vitro. The activation of the ERK1/2 pathway and the overexpression of Netrin‐1 were both found to play important roles in osteoclastogenesis mediated by autophagy. Moreover, 3‐MA effectively decreased the secretion of proinflammatory cytokines mediated by wear particles. Conclusion Blockade of autophagy inhibits the osteoclastogenesis and inflammation induced by wear particles, thus potentially providing novel treatment strategies for abnormal osteoclastogenesis and aseptic prosthesis loosening induced by wear particles.


| INTRODUCTION
As an important treatment for arthropathy, artificial joint replacement surgery is used to relieve joint pain and restore joint function, by using materials such as metal, polymeric polyethylene, or ceramic (Bouhidel et al., 2015;Bozic et al., 2009). With progress in technology and the advancement of biological material applications, the success rate of joint replacement surgery has continually increased.
However, this surgery is often accompanied by complications, including aseptic prosthetic loosening and periprosthetic osteolysis (Bai et al., 2017;Brinkmann et al., 2012). Implanted joint replacement derived wear particles are believed to be the major inducer of local inflammation and periprosthetic bone loss, thus ultimately resulting in failure of the prostheses (Czaja, 2011). During this process, inflammatory cells (such as macrophages and lymphocytes) are recruited to the surface of the prosthesis and local bone tissues (Cody et al., 2011). These cells are activated to release proinflammatory cytokines (such as interleukin [IL]-1, IL-6, tumor necrosis factor alpha [TNF-α], and PGE2), which promote the expression of RANKL, which, in turn, contributes to osteoclastogenesis (Castillo et al., 2012;Cuetara et al., 2006). Additionally, the binding of RANKL and RANK activates the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways (Feng, 2005;Goldring et al., 1983), which have been demonstrated to be crucial for osteoclast formation and bone absorption function. In summary, wear particle mediated osteoclastogenesis and inflammation are the key factors initiating osteolysis and aseptic loosening (Harris & is, 2001). Osteoclasts are differentiated from monocytes and macrophages, and have been demonstrated to be resorptive cells in bone metabolism (Liu et al., 2013). In osteoclastogenesis, hematopoietic osteoclast precursors differentiate into mature osteoclasts; this process is central to bone destruction and resorption (osteolysis).
Autophagy is traditionally identified as a dynamic catabolic process, in which the damaged proteins or organelles enter autophagosomes and are delivered to lysosomes for degradation (Levine & Kroemer, 2008;Li et al., 2014;Liao et al., 2012;Liu et al., 2015).
Autophagy occurs when cells are exposed to adverse conditions, including nutrient deprivation, hypoxia, radiation, and pathogenic infection (Mizushima, 2007). During this process, increased expression of ATG proteins is the main contributor to the formation of double-membrane autophagosome vesicles, which eventually fuse with lysosomes. Acid proteases and hydrolases in the autolysosomes degrade the engulfed autophagosomes. The digested products of autophagosomes, amino acids and fatty acids, are then transported into the cytoplasm to be used again for nutrition (Mediero et al., 2015;Mizushima & Levine, 2010). Studies increasingly indicate links between autophagy and various physiological processes and diseases. Beyond the previously described functions in homeostasis maintenance, autophagy is involved in the development and differentiation of various cell types, such as erythrocytes, lymphocytes, adipocytes, and macrophages (Mazière et al., 2009;Nakahira et al., 2011;Purdue et al., 2006). Netrin-1, a member of the axonal guidance protein family, is also involved in the differentiation and formation of osteoclasts. Antibody blockade of Netrin-1 or Unc5b inhibits osteoclast differentiation (Purdue et al., 2007). Thus, Netrin-1 is likely to play an important role in the osteoclastogenesis induced by wear particles and the resultant bone resorption. Although studies have shown that Netrin-1 ultimately influences osteoclast differentiation through RhoA and FAK, the specific molecular signaling pathway mechanisms have not been clearly elucidated. Studies have shown that in a model of myocardial infarction, Netrin-1 improves cardiac function through signaling by its receptors, DCC and ERK1/2, thus decreasing the occurrence of infarcts (Qin et al., 2011). In addition, Netrin-1 increases the expression of the autophagy marker Beclin-1 and the LC3-II/LC3-I ratio by increasing MAPK phosphorylation and decreasing mTOR phosphorylation (Razani et al., 2012).
Other studies have shown that blockade of the NF-κB and MAPK pathways attenuates wear particle-induced osteoclast differentiation (Rawi et al., 2011). Together, these findings indicate that Netrin-1 is closely associated with activation of the MAPK signaling pathway and cellular autophagy.
Although osteoclastogenesis plays an important role in bone destruction and resorption, the mechanisms of differentiation from osteoclast progenitor cells into osteoclasts has not yet been elucidated. The effects of autophagy on the osteoclastogenesis induced by wear particles also remain unclear. We speculate that Netrin-1 promotes autophagy caused by wear particles and promotes the formation of osteoclasts through the ERK1/2 signaling pathway.

| Western blotting
The cells and pouch wall tissues were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche) to prepare the protein samples.

| Immunohistochemical and immunofluorescence staining
Cells and implanted bones were fixed with 4% paraformaldehyde, then incubated with primary antibody against TRAP; this was followed by washing and incubation with FITC-conjugated goat antirabbit secondary antibodies (1:250; Earthox) for 2 h at 30°C. The nuclei were stained with 4',6-diamidino-2'-phenylindole. Fluorescence images were visualized and captured with an inverted fluorescence microscope (Olympus). The bones were embedded in paraffin and sliced into 4-μm sections, and this was followed by dewaxing and rehydration. The sections were incubated with primary antibody against TRAP, then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:250; Earthox) for 2 h at room temperature. Images were visualized and captured with a phase contrast microscope (Olympus).

| Establishment of the bone resorption air pouch model and naringin intervention
The animal experiments were performed in accordance with the guidelines of the Chinese Council on Animal Care, and the study was approved by the Xi'an Jiaotong University Committee (Xi'an, China).
The air pouch model of bone resorption was established in our laboratories as described previously (Ravikumar et al., 2010;Ru et al., 2016). Briefly, air pouches were generated by injection of sterile air into the backs of female BALB/c mice (8 weeks of age). At Day 7 after air pouch formation, a proximal or distal section of the femur, or a section of calvarium from congeneric littermate donors, was surgically implanted into the established air pouches. Ti particles with or without 3-MA were injected into each pouch, and the mice were killed 7 days later to harvest implanted bones, pouch wall tissues and blood for further measurements.

| Enzyme-linked immunosorbent assay
Cell culture supernatants and serum were analyzed with a Mouse RANKL ELISA Kit (Abcam) and Mouse Osteoprotegerin ELISA Kit Abbreviations: IL, interleukin; TNF, tumor necrosis factor. C Cell ell B Biology iology I International nternational (Abcam). All experiments were performed according to the manufacturer's protocols.

| Pit resorption assay
After the coculture, the lower chamber bone ground sections were removed, washed with phosphate buffered saline three times and treated with 2.5% ethylenediaminetetraacetic acid and trypsin for 10 min to digest the cells on the bone ground sections. After fixation with 2.5% glutaraldehyde for 30 min, the impurities in the bone resorption pits were removed by ultrasonic washing. Then, alcohol gradient dehydration (30%-100% ethanol solution) was performed, and finally isoamyl acetate was added to replace the 100% ethanol. After vacuum drying, the bone resorption pits were observed under a scanning electron microscope.

| Transmission electron microscopy
The bone pieces were quickly cut into sections approximately 1 mm thick and fixed with 2.5% glutaraldehyde at room temperature. After fixation, sections were rinsed with 0.2 M phosphate buffer (pH 7.2).
Then the samples were treated with 1% buffered osmium tetroxide for 1 h, dehydrated in gradient ethanol and embedded in epoxy resin to prepare ultrathin sections, mounted on a copper grid, and stained with acetate and lead acetate with uranyl. Images were collected with a transmission electron microscope.

| Statistical analysis
Data are presented as means ± SEM for each experiment. Statistical comparisons were performed as appropriate, with either Student's two-tailed t test or analysis of variancewith Tukey's multiple comparison posttest. A p value less than .05 was considered significant.

| Wear particles induce osteoclast differentiation by increasing TRAP expression
To investigate the effect of wear particles on osteoclastogenesis in vitro, we cultured RAW 264.7 cells and exposed them to wear debris.
We first observed cell viability and morphology changes. 3.3 | Wear particles regulate autophagy by increasing the expression of Netrin-1 We investigated the role of Netrin-1 in Ti-induced macrophage autophagy models. Ti treatment promoted the expression of Netrin-1 in RAW 264.7 cells (Figure 3a,b). UNC5b and DCC were detected in RAW 264.7 cells (Figure 3c). Further studies demonstrated that neutralizing antibodies against Netrin-1 and its receptor Unc5b effectively decreased the Atg5, Atg7, Atg12, Beclin-1, and LC3-II expression was induced by Ti, whereas neutralizing antibodies to DCC, another receptor of Netrin-1, did not have this effect (Figure 3d,e).
These results indicated that Ti induced autophagy was mediated by Netrin-1 and its receptor Unc5b. Netrin-1 thus plays an important role in the development of autophagy in macrophages.

| Blocking of autophagy inhibits wear particle induced osteoclastogenesis in vitro
We next investigated the effect of autophagy on osteoclast development by suppressing the formation of autophagosomes. The expression of TRAP was upregulated when RAW 264.7 cells were exposed to Ti wear particles; however, the TRAP expression was decreased by the addition of 3-MA (Figures 4a,b,  Therefore, we also detected the expression of RANKL and OPG during osteoclastogenesis induced by wear particles. In agreement with the results above, the expression of RANKL and OPG also clearly increased after stimulation with Ti wear particles; however, the increase was inhibited with 3-MA treatment (Figures 4a and 4c).
We also found that autophagy was induced by Ti but was reversed by addition of 3-MA, on the basis of electron microscopy of bone sections, (Figure 4e). The expression of DCC and UNC5B were tested by the western blotting ( Figure 4f). Together, these findings demonstrated that Ti not only induces osteoclastogenesis by enhancing the expression of TRAP but also upregulates the expression of RANKL and OPG.

| Netrin-1 significantly promotes osteoclastogenesis
After having observed the effects of Netrin-1 on autophagy of macrophages, we further examined its functions in osteoclastogenesis. The results indicated that TRAP was upregulated when RAW 264.7 cells were exposed to Ti wear particles; however, the TRAP expression was decreased with the addition of Netrin-1 neutralizing antibodies and Unc5b (Figure 5a). Furthermore, the expression of TRAP and Beclin-1 was upregulated when RAW 264.7 cells were treated with Netrin-1; however, these effects, particularly Beclin-1 expression, were reversed by 3-MA treatment (Figure 5b). For further elucidation, we explored the roles of ERK1/2 signaling in osteoclastogenesis. The results indicated that the expression of TRAP and p-ERK was upregulated when Netrin-1 was added to RAW 264.7 cells, and these effects were inhibited by U0126, a p-ERK inhibitor ( Figure 5c). Pit resorption assays also demonstrated the role of Netrin-1 in osteoclast differentiation (Figure 5d). In addition to influencing macrophage autophagy, Netrin-1 affects osteoclast formation through the ERK1/2 signaling pathway.

| Blocking of autophagy inhibits wear particle induced osteoclastogenesis in vivo
Next, we established an air pouch model to investigate the role of autophagy in osteoclast development. the expression of TRAP and Netrin-1 increased in implanted bone in response to Ti wear particle treatment, whereas administration of 3-MA weakened the expression of TRAP but not Netrin-1 (Figure 6a,b). In pouch wall tissues, Ti wear particles enhanced RANKL and OPG expression, and this effect was reversed by administration of 3-MA (Figures 6c,d and 6e). Moreover, we measured the proinflammatory factors IL-1β, IL-6, and TNF-α in pouch wall tissues to assess the degree of inflammation due to different treatments. Ti wear particles induced clear inflammation in the bone implanted microenvironment, but this effect was weakened by 3-MA treatment ( Figure 6f). These data demonstrated that Ti wear particles induce Netrin-1 promoted autophagy and osteoclastogenesis through the ERK1/2 signaling pathway. Ti wear particle induced inflammation was additionally found to be regulated by autophagy.

| DISCUSSION
Under normal physiological conditions, autophagy can clear damaged organelles and insoluble protein aggregates in cells, and contribute to cell homeostasis maintenance and cell survival regulation (Rosenfeldt & Ryan, 2009;Rubinsztein et al., 2012). Autophagy occurs when cells are challenged, such as under starvation or pathogen infection. Previous studies have confirmed that autophagy plays an important role in intracellular pathogen clearance, antigen presentation and lymphocyte differentiation (Ren et al., 2004). Roles of autophagy in macrophages, including oxidative and inflammatory stress suppression, and polarization control, have also been reported (Sakiyama et al., 2001;Saitoh et al., 2008;Sato & Sato, 2011;Shi et al., 2012;Shanbhag et al., 1995;Shintani & Klionsky, 2004;Thomas et al., 2012;Tsukamoto et al., 2008).
Artificial joint replacement surgery is an important treatment for arthropathy; however, complications such as septic prosthetic loosening and periprosthetic osteolysis occur very often after this sur- frequently been used in in vitro models to investigate osteoclast differentiation precursors in response to wear debris (Tsao et al., 2008;Udagawa et al., 1990;Wooley & Schwarz, 2004;Xiao et al., 2015). As reported, we observed the presence TRAP expression and multiple nuclei-the characteristic features of mature osteoclasts . We detected the expression of TRAP in RAW 264.7 cells with Ti wear particle treatment. A mouse model based study has used 30 mg of Ti particles embedded under the periosteum at the middle suture of the calvaria, with a Ti particle concentration of 0.1 mg/ml (Zhao et al., 2012). Such particles have been shown to effectively mimic the wear particles retrieved from periprosthetic tissues (Zhou et al., 2011). Nano-sized Ti particles were used in this study at a concentration of approximately 0.0012 mg/ml. The ratio of 100 particles/cell used in the experiment to the ratio of 0.1 mg/ml microparticles delivered to cells was consistent.
As expected, treatment with Ti wear particles enhanced the expression of TRAP and resulted in formation of more cell projec-  In conclusion, our study reveals that disruption of autophagy arrests the differentiation of macrophages into osteoclasts and F I G U R E 6 Blocking of autophagy inhibits wear particle induced osteoclastogenesis in vivo. The air pouch mold was established, and Ti wear particles with or without 3-MA were injected in the air pouch. (a) After 7 days, the implanted bones were harvested to detect the mRNA expression of TRAP and Netrin-1 with real-time PCR. (b) The harvested bones were also subjected to immunohistochemical staining to detect the expression of TRAP and Netrin-1. (c) The mRNA expression of RANKL and OPG in pouch wall tissues was detected by real-time PCR. (d) The protein expression of RANKL and OPG in pouch wall tissues was detected by western blotting. (e) The serum levels of RANKL and OPG were detected by ELISA. The mRNA expression of proinflammatory factors (IL-1β, IL-6, and TNF-α) in pouch wall tissues was measured to assess the degree of inflammation. Data represent mean ± SEM; n = 5 (*p < .05). ELISA, enzyme-linked immunosorbent assay; IL, interleukin; mRNA, messenger RNA; TNF, tumor necrosis factor 620 | C Cell ell B Biology iology I International nternational ameliorates the inflammation induced by wear particles. These data suggest that autophagy may be a novel protective cellular response during osteolysis induced by wear particles. However, the molecular mechanisms linking autophagy and inflammation induced by wear particles remain to be investigated.