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.

Keywords: ERK1/2 signaling pathway; Netrin-1; RAW 264.7 cell morphology; abnormal osteoclastogenesis; the air pouch model of bone resorption; titanium (Ti) wear particles.

Figure 1

Wear particles induce osteoclast differentiation, on the basis of increased expression of TRAP. (a) The cell viability of RAW 264.7 cells was detected with CCK‐8 assays after treatment with Ti wear particles. (b) Phase contrast microscopic images of RAW 264.7 cells with or without treatment with Ti wear particles. (c) The mRNA expression of TRAP in RAW 264.7 cells with or without treatment with Ti wear particles. (d) The immunofluorescence staining of TRAP in RAW 264.7 cells with or without treatment with Ti wear particles. (e) Western blot detection of TARP expression in RAW 264.7 cells with or without treatment with Ti wear particles. (f) Pit resorption assay indicating the effect of Ti treatment on osteoclast differentiation. Data represent mean ± SEM; n = 5 (*p < .05). mRNA, messenger RNA

Figure 2

Wear particles induce autophagy of macrophages. (a) RAW 264.7 cells were transfected with GFP‐LC3 for 24 h and then treated with Ti for 48 h to observe the punctuate GFP‐LC3 distribution by fluorescence microscopy. (b) The mRNA expression of Atg5, Beclin1, Atg7, and Atg12 in RAW 264.7 cells with or without treatment with Ti wear particles. (c) The expression of LC‐3 was detected by western blotting in RAW 264.7 cells with or without treatment with Ti wear particles. Data represent mean ± SEM; n = 5 (*p < .05). mRNA, messenger RNA

Figure 3

Wear particles regulate autophagy by increasing the expression of Netrin‐1. (a) Western blot detection of Netrin‐1 expression in RAW 264.7 cells with or without treatment with Ti wear particles. (b) The mRNA expression of Netrin‐1 in RAW 264.7 cells with or without treatment with Ti wear particles. (c) The protein expression of UNC5b and DCC in RAW 264.7 cells. (d) The mRNA expression of Atg5, Beclin1, Atg7, and Atg12 in RAW 264.7 cells with or without treatment with Ti wear particles and IgG, Netrin‐1, Unc5b, or DCC neutralizing antibodies. (e) The expression of LC‐3 and Beclin1 was detected by western blotting in RAW 264.7 cells with or without treatment with Ti wear particles and IgG, Netrin‐1, Unc5b, or DCC neutralizing antibodies. (f) The mRNA expression of Atg5, Beclin1, Atg7, and Atg12 in RAW 264.7 cells with or without treatment with recombinant Netrin‐1. (g) The expression of LC‐3 and Beclin1 was detected by western blotting in RAW 264.7 cells with or without treatment with recombinant Netrin‐1. mRNA, messenger RNA

Figure 4

Blocking of autophagy inhibits wear particle induced osteoclastogenesis in vitro. (a) The expression of TRAP, RANKL, and OPG was detected by real‐time PCR in RAW 264.7 cells under the indicated treatments. (b) RAW 264.7 cells were transfected with GFP‐LC3 for 24 h and then treated with Ti in the presence or absence of 3‐MA for 48 h to observe the punctuate GFP‐LC3 distribution by fluorescence microscopy; the expression of TRAP was also detected by immunofluorescence staining. (c) The cell culture supernatants in different groups were collected to detect RANKL and OPG with ELISA. (d) The expression of TRAP in RAW 264.7 cells in different groups was detected by western blotting. (e) Ti or 3‐MA treatment induced autophagy was detected by electron microscopy of bone sections. Data represent mean ± SEM; n = 5 (*p < .05). (f) The expression of DCC and UNC5B were tested by the western blotting. ELISA, enzyme‐linked immunosorbent assay; mRNA, messenger RNA

Figure 5

(a) Netrin‐1 significantly promotes osteoclastogenesis. The expression of Trap was detected by western blotting in RAW 264.7 cells with or without treatment with Ti wear particles and IgG, Netrin‐1, or Unc5b neutralizing antibodies. (b) The expression of Trap and Beclin1 was detected by western blotting in RAW 264.7 cells with or without treatment with recombinant Netrin‐1 and 3‐MA. (c) The expression of Trap, p‐ERK and ERK was detected by western blotting in RAW 264.7 cells with or without treatment with recombinant Netrin‐1 and U0126. (d) Pit resorption assay for the effect of Netrin‐1 on osteoclast differentiation. IgG, immunoglobulin G

Figure 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