Receptor activator of NF-κB ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells

Abstract

Interleukin-1 (IL-1) is a proinflammatory cytokine that is a potent stimulator of bone resorption and an inhibitor of bone formation, whereas macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB (RANK) ligand (RANKL) are essential and sufficient for osteoclast differentiation. Recently, we showed that IL-1α affects mineralized nodule formation in vitro and halts bone matrix turnover. We also showed that IL-1α stimulates osteoclast formation via the interaction of RANKL with RANK by increasing M-CSF and prostaglandin E 2 and decreasing osteoprotegerin. Here, we examined the effects of IL-1α or RANKL and/or M-CSF in the presence of IL-1α on the expression of carbonic anhydrase II (CAII), cathepsin K, matrix metalloproteinase-9 (MMP-9), RANK, M-CSF receptor (c- fms), and c- fos transcription factor using RAW264.7 cells as osteoclast precursors. Cells were cultured for up to 14 days in 0 or 100 U/ml IL-1α and either 50 ng/ml RANKL, 10 ng/ml M-CSF, or 50 ng/ml RANKL + 10 ng/ml M-CSF in the presence of 100 U/ml IL-1α. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase staining. Expression of the genes coding for the six proteins of interest was determined using real-time PCR, and the expression of the three enzymes was examined using Western blotting or ELISA. In the presence of IL-1α, expression of CAII, cathepsin K, and MMP-9 was markedly increased in cells cultured with RANKL or M-CSF + RANKL, whereas expression was difficult to detect in cells cultured with IL-1α alone and M-CSF. RANK and c- fos expression was also increased in cells cultured with RANKL or M-CSF + RANKL in the presence of IL-1α, whereas c- fms expression did not change. These results indicate that the expression of CAII, cathepsin K, and MMP-9 in RAW264.7 cells is not induced by M-CSF, but by RANKL in the presence of IL-1α.