Interleukin-1 induces receptor activator of nuclear factor-κB ligand-independent osteoclast differentiation in RAW264.7 cells

Abstract

Interleukin-1 (IL-1) is a pro-inflammatory cytokine which induces bone destruction in various diseases, such as osteoporosis and rheumatoid arthritis. RAW264.7 cells are frequently used in studies as osteoclast precursors, however it remains unclear whether IL-1 can induce osteoclast differentiation from RAW264.7 cells without the stimulation of receptor activator of nuclear factor-κB ligand (RANKL). Hence, the present study aimed to investigate the effects of IL-1 on the formation of osteoclasts from RAW264.7 cells. The cell viability was determined via the Cell Counting Kit-8 (CCK-8) assay. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR, respectively. Tartrate-resistant acid phosphatase (TRAP) staining and the resorption pit assay were performed to determine the formation and activity of osteoclasts. A significantly increased quantity of osteoclasts were found in the IL-1 group compared with the control group, and also in the RANKL+IL-1 group compared with the RANKL group. In addition IL-1 significantly increased both the protein and mRNA expression of specific genes associated with osteoclastogenesis, including nuclear factor of activated T cells cytoplasmic 1, matrix metalloprotein-9, cathepsin K and TRAP. The findings of the present study suggested that IL-1 can induce osteoclast differentiation and upregulate the quantity of osteoclasts differentiated from RAW264.7 cells. These results may lay a foundation for further study of diseases involving inflammation-associated bone loss. The combined blockade of IL-1 and RANKL may be effective for the prevention of inflammatory bone loss.

Keywords: RAW264.7 cells; interleukin-1; osteoclast differentiation; osteoporosis; receptor activator of nuclear factor-κB ligand-independent.

Figure 1

IL-1/RANKL does not affect the cell viability of RAW264.7 cells. The cells were treated with or without IL-1 (10 ng/ml) and/or RANKL (50 ng/ml). The CCK-8 assay was used to measure the cell viability at indicated time points (days 0, 1, 2, 3 and 4). No statistical difference was found between groups. OD, optical density; IL, interleukin; RANKL, receptor activator of nuclear factor-κB ligand-independent; control, untreated cells.

Figure 2

IL-1 promotes RAW264.7 cells osteoclastogenesis. Following 12 h of incubation, culture medium with IL-1 (10 ng/ml), RANKL (50 ng/ml), or IL-1 (10 ng/ml)+RANKL (50 ng/ml) was added to RAW264.7 cells. (A) RAW264.7 cells were stained 4 days after the stimulation using a TRAP kit according to the manufacturer's instructions and observed under alight microscope. Scale bar=100 µm. Red arrows indicate mature osteoclasts. (B) TRAP-positive osteoclasts with ≥3 nuclei were identified and counted in 5 random fields. (C) TRAP activity was detected at 540 nm and the results were presented as expression related to control. ^**P<0.01 compared with the control group, ^##P<0.01 compared with IL-1-treated or RANKL-treated alone. OD, optical density; IL, interleukin; RANKL, receptor activator of nuclear factor-κB ligand-independent; control, untreated cells; TRAP, tartrate-resistant acid phosphatase.

Figure 3

IL-1 promotes the expression of osteoclastogenesis-specific genes. RAW264.7 cells were treated with IL-1 (10 ng/ml) and/or RANKL (50 ng/ml) for 4 days. (A) Expressions of osteoclastogenesis-specific genes [MMP-9, CTSK, TRAP and NFATc1 (C)] and IL-1RI (B) were determined by western blotting. The exposure time of TRAP, IL-1RI and MMP-9 was 10 sec, the exposure time of NFATc1 was 60 sec and the exposure time of other blots was 5 sec. IL-1RI and NFATc1 were from different gels, TRAP, MMP-9 and CTSK were from the same gel. (D-G) RT-qPCR expression in the 4 groups of RAW 264.7 cells (control, IL-1, RANKL and IL-1+RANKL) of (D) TRAP (E) NFATc1, (F) CTSK and (G) MMP-9. Relative gene expression was normalized to GAPDH. ^**P<0.01 compared with the control group, ^#P<0.05, ^##P<0.01 compared with IL-1-treated or RANKL-treated alone. IL, interleukin; RANKL, receptor activator of nuclear factor-κB ligand-independent; control, untreated cells; RT-q, reverse transcription-quantitative; NFATc1, nuclear factor of activated T cells cytoplasmic 1; MMP-9, matrix metalloprotein-9; CTSK, cathepsin K; TRAP, Tartrate-resistant acid phosphatase; IL-1RI, IL-1 receptor I.

Figure 4

IL-1 promotes the area of bone resorption pit. (A) Images of RAW264.7 cells on Corning Osteo Assay Surface treated with or without IL-1 (10 ng/ml) and/or RANKL (50 ng/ml) for 8 days. Magnification (x40). (B) Relative level of resorption area was measured using Image J Plus software. ^**P<0.01 compared with the control group, ^##P<0.01 compared to IL-1-treated or RANKL-treated alone. IL, interleukin; RANKL, receptor activator of nuclear factor-κB ligand-independent; control, untreated cells.