Receptor activator of NF-kappaB ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells.
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-kappaB (RANK) ligand (RANKL) are essential and sufficient for osteoclast differentiation. Recently, we showed that IL-1alpha affects mineralized nodule formation in vitro and halts bone matrix turnover. We also showed that IL-1alpha 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-1alpha or RANKL and/or M-CSF in the presence of IL-1alpha 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-1alpha 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-1alpha. 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-1alpha, 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-1alpha 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-1alpha, 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-1alpha.
Fujisaki
Kyosuke
K
Nihon University Graduate School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan.
Tanabe
Natsuko
N
Suzuki
Naoto
N
Kawato
Takayuki
T
Takeichi
Osamu
O
Tsuzukibashi
Osamu
O
Makimura
Masaharu
M
Ito
Koichi
K
Maeno
Masao
M
D000818:Animals; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism*; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004338:Drug Combinations; D015870:Gene Expression / Q000187:drug effects; D053582:Interleukin-1alpha / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000235:genetics / Q000378:metabolism; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004338:Drug Combinations; D053582:Interleukin-1alpha; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor; D002403:Cathepsins; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D020780:Matrix Metalloproteinase 9; D024402:Carbonic Anhydrase II