The USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis in mice by suppressing NF-κB and PI3K/AKT activities

Abstract

Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle-induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)-containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle-induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle-induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle-induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.

Keywords: NF-κB; NLRC5; USP14; cytokine; macrophage; osteoclast; total hip arthroplasty; tumor necrosis factor (TNF); wear particles.

Figure 1.

Expression of TNFα, IL-6, NLRC5, and USP14 in hip synovial membrane of patients with femoral head necrosis and aseptic loosening. A, immunohistochemical staining of TNFα, IL-6, NLRC5, and USP14 from AL and FHN patients' hip synovial membrane. B, immunoreactivity score of TNFα, IL-6, NLRC5, and USP14 from AL and FHN patients' hip synovial membrane. *, p < 0.05.

Figure 2.

mRNA and protein expression levels of TNFα, NLRC5, and USP14 in J774A.1 cells stimulated by Ti particles for different times. A, mRNA expression levels of TNFα, NLRC5, and USP14 at each time point after stimulation of Ti particles (0, 1, 2, 4, 6, and 8 h). B, secreted TNFα protein expression at each time point after stimulation (0, 1, 2, 4, and 6 h), detected by ELISA. *, p < 0.05. C, protein expression of TNFα, NLRC5, and USP14 after stimulation for 24 h and the ubiquitination (Ub) level of NLRC5 by Ti particles and LPS treatment for 1 h. WCL, whole-cell lysate.

Figure 3.

Expression of TNFα and NF-κB pathway in USP14 or NLRC5-knockdown J774A.1 macrophage compares with negative control cells after titanium stimulation. A, higher mRNA expression of TNFα in USP14/NLRC5-knockdown J774A.1 cells compared with negative control cells after titanium stimulation. B, more secreted TNFα protein was detected in USP14/NLRC5-knockdown J774A.1 cells compared with negative control cells at every time point after titanium stimulation. *, p < 0.05. C, P65 was observed with more translocation into the nuclei of USP14-knockdown J774a.1 cells than those of control cells after Ti stimulation. D, protein expression of phospho-IκBα and phospho-P65 in the NF-κB pathway and NLRC5 in USP14-knockdown J774a.1 cells and negative control cells after titanium and LPS stimulation. DAPI, 4,6-diamidino-2-phenylindole.

Figure 4.

Expression of TNFα and NF-κB pathway in NLRC5-overexpressed J774A.1 cells compared with normal cells after titanium stimulation. A, overexpression of NLRC5 significantly suppressed the mRNA expression of TNFα after titanium stimulation. B, lower concentration of secreted TNFα protein was detected in NLRC5-overexpressed J774A.1 cells compared with normal cells at every time point after titanium stimulation. *, p < 0.05. C, P65 was observed with less translocation into the nuclei of NLRC5 overexpressed J774A.1 cells than those of normal cells after titanium stimulation. D, protein expression of phospho-IκBα and phospho-P65 in NLRC5-overexpressed J774a.1 cells and normal cells after titanium and LPS stimulation.

Figure 5.

Expression of TNFα and NF-κB pathway in USP14-overexpressed J774A.1 cells compared with normal cells after titanium stimulation. A, overexpression of USP14 significantly suppressed the mRNA expression of TNFα after titanium stimulation. B, lower concentration of secreted TNFα protein was detected in USP14-overexpressed J774A.1 cells compared with normal cells at every time point after titanium stimulation. *, p < 0.05. C, P65 was observed with less translocation into the nuclei of USP14-overexpressed J774A.1 cells than those of normal cells after titanium stimulation. D, protein expression of phospho-IκBα, phospho-P65, and NLRC5 as well as its ubiquitination level in USP14-overexpressed J774a.1 cells and normal cells after titanium and LPS stimulation.

Figure 6.

Flow cytometry of macrophage polarization and PI3K/Akt pathway in each group of J774A.1 cells after stimulation by Ti particles. A and B, M1 polarization of macrophages detected by flow cytometry in USP14/NLRC5-overexpressed J774A.1 cells compared with those transfected with negative control vector after Ti stimulation. *, p < 0.05. C, protein expression of PI3K/Akt pathway in USP14/NLRC5-overexpressed J774A.1 cells compared with negative control cells.

Figure 7.

Local injection of NLRC5/USP14-overexpressed J774A.1 cells alleviated the cranial osteolysis of mice induced by Ti particles in vivo. A, from day 1 to day 7, bioluminescence was observed focused on the mid-line sagittal suture of the calvaria on the vertical images of mice injected with J774A.1 reporter cells expressing luciferase. B, 3-dimensional reconstruction of the mouse crania micro-CT showed an area of osteolysis around the cranial sutures in the mice treated with titanium particles compared with the sham group, and the osteolysis was alleviated by the overexpression of NLRC5 and USP14. C, BV/TV and BMD of a 1 × 3-mm ROI around the cranial suture evaluated by micro-CT. *, p < 0.05.

Figure 8.

Expression of IL-6, TNFα, and TRAP of mouse calvarial sutures in different treatment groups. A, immunohistochemical staining of IL-6, TNFα, and TRAP of mouse calvarial sutures in different treatment groups. B, Trap⁺ counting of cell numbers in the mouse calvarial sutures in the different treatment groups. *, p < 0.05.