Decoy Receptor 3 Promotes Preosteoclast Cell Death via Reactive Oxygen Species-Induced Fas Ligand Expression and the IL-1 α/IL-1 Receptor Antagonist Pathway

Abstract

Purpose: Interleukin-1α (IL-1α) is a potent cytokine that plays a role in inflammatory arthritis and bone loss. Decoy receptor 3 (DCR3) is an immune modulator of monocytes and macrophages. The aim of this study was to investigate the mechanism of DCR3 in IL-1α-induced osteoclastogenesis.

Methods: We treated murine macrophages with DCR3 during receptor activator of nuclear factor kappa Β ligand- (RANKL-) plus IL-1α-induced osteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed using a pit formation assay. The mechanisms of inhibition were studied by biochemical analyses, including RT-PCR, immunofluorescent staining, flow cytometry, an apoptosis assay, immunoblotting, and ELISA.

Results: DCR3 suppresses IL-1α-induced osteoclastogenesis in both primary murine bone marrow-derived macrophages (BMM) and RAW264.7 cells as it inhibits bone resorption. DCR3 induces RANKL-treated osteoclast precursor cells to express IL-1α, secretory IL-1ra (sIL-1ra), intracellular IL-1ra (icIL-1ra), reactive oxygen species (ROS), and Fas ligand and to activate IL-1α-induced interleukin-1 receptor-associated kinase 4 (IRAK4). The suppression of DCR3 during RANKL- or IL-1α-induced osteoclastogenesis may be due to the abundant secretion of IL-1ra, accumulation of ROS, and expression of Fas ligand in apoptotic osteoclast precursor cells.

Conclusions: We concluded that there is an inhibitory effect of DCR3 on osteoclastogenesis via ROS accumulation and ROS-induced Fas ligand, IL-1α, and IL-1ra expression. Our results suggested that the upregulation of DCR3 in preosteoclasts might be a therapeutic target in inflammatory IL-1α-induced bone resorption.

Figure 1

Effects of DCR3 on RANKL- plus IL-1α-induced osteoclast differentiation and function. (a) RAW264.7 cells were treated with DCR3 (10 μg/ml) in the presence of RANKL (50 ng/ml) or RANKL plus IL-1α (50 ng/ml) for 5 days. After incubation, the cells were fixed and stained for TRAP, and TRAP⁺ multinucleated RAW264.7 cells containing more than five nuclei were counted as multinucleated osteoclasts. (b) RAW264.7 cells were seeded on dentine slices as described in the Materials and Methods. After being incubated for 5 days, the dentine slices were recovered from the culture and were subjected to a pit formation assay to visualize resorption. The percentages of the resorbed areas were determined using the NIH Image software. Data are presented as means ± SD of more than four slices and means ± SD of more than three cultures (N: RAW264.7 cells; R: RANKL; RD: RANKL+DCR3; Rα: RANKL+IL-1α; RαD: RANKL+IL-1α+DCR3; ^∗∗P < 0.01 and ^∗∗∗P < 0.001).

Figure 2

Effects of DCR3 on IL-1α and IL-1ra regulation on RANKL-induced osteoclast differentiation. (a–d) BMM cells were seeded at a density of 2 × 10⁵ cells/well in a 24-well plate and treated with 10 μg/ml of DCR3 or IgG control in the presence of RANKL and M-CSF. After 6 hours of incubation, total RNA was isolated, and 1 μg of total RNA was used to reverse transcribe cDNA. Mouse IL-1α, sIL-1ra, and icIL-1ra were detected by RT-PCR (a) and QPCR (b–d). (e) RAW264.7 cells were treated with 10 μg/ml DCR3 or IgG in the presence of RANKL (50 ng/ml) stimulation for 6, 24, or 48 hours. Cell extracts were analyzed by immunoblotting assay. Equal amounts of protein were loaded in each lane as demonstrated by the level of GAPDH. (f) Supernatants at 24 or 48 hours were analyzed by IL-1α ELISA. Cell extracts (g) and supernatants (h) at 24 or 48 hours were analyzed by IL-1ra ELISA. A representative result of at least three independent experiments is shown (M: BMM cells+MCSF; RM: RANKL+MCSF; RMG: RANKL+MCSF+IgG; RMD: RANKL+MCSF+DCR3; N: RAW264.7 cells; R: RANKL; RG or R+G: RANKL+IgG; RD or R+D: RANKL+DCR3; ^∗∗∗P < 0.001).

Figure 3

Effects of DCR3 on IL-1α protein localization and ROS expression in RANKL-induced osteoclast differentiation. RAW264.7 cells were treated with DCR3 or IgG in the presence of RANKL stimulation for 6 hours. After the time indicated, the cells were stained with an anti-IL-1α antibody (green), endoplasmic reticulum (red), and Hoechst dye (blue) or with a ROX detection kit. (a) The localization of IL-1α was compared in merged images for each group. (b) The ROS levels in each group were evaluated by flow cytometry. A representative result of at least three independent experiments is shown (BF: bright field; ER: endoplasmic reticulum staining; 1α: IL-1α staining; Ho: nuclear staining; M: merge image of ER, 1α, and Ho; N: RAW264.7 cells; R: RANKL; RG: RANKL+IgG; RD: RANKL+DCR3; ^∗∗∗P < 0.001).

Figure 4

Effects of DCR3 on cell apoptosis and interleukin-1-associated kinase activation in osteoclasts. RAW264.7 cells were treated for 48 hours with or without 10 μg/ml of DCR3 in the presence of RANKL or RANKL plus IL-1α. After incubation, cells were harvested for staining with (a) Annexin V and PI to evaluate the percentage of cell apoptosis or with (b) anti-Fas ligand to evaluate death ligand expression. (c) RAW264.7 cells were serum-starved overnight and treated with DCR3 in the presence of RANKL or RANKL plus IL-1α for 15 or 30 minutes. Cell extracts were analyzed by western blot analysis using antibodies specifically directed against the phosphorylated forms of IRAK4, compared to data obtained with antibodies directed against the unphosphorylated states of the kinases. Equal amounts of protein were loaded in each lane as demonstrated by the level of GAPDH. The expression ratio of phosphorylated/nonphosphorylated IRAK4 was quantified and normalized to the GAPDH. A representative result of at least three independent experiments is shown (N: RAW264.7 cells; R: RANKL; Rα: RANKL+IL-1α; RD: RANKL+DCR3; RαD: RANKL+IL-1α+DCR3; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001).