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
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
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DOI：
10.1016/j.lfs.2006.12.037
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发表时间：
2007-03-13
期刊：
影响因子：
6.1
通讯作者：
Maeno, Masao
中科院分区：
文献类型：
--
作者：
Fujisaki, Kyosuke;Tanabe, Natsuko;Maeno, Masao
白细胞介素-1（IL-1）是一种促炎细胞因子，是骨吸收的有效刺激剂和骨形成的抑制剂，而巨噬细胞集落刺激因子（M-CSF）和NF-κ B受体激活剂（RANK）配体（RANKL）是破骨细胞分化所必需和充分的。最近，我们发现IL-1 α影响体外矿化结节的形成，并阻止骨基质的转换。我们还发现，IL-1 α通过RANKL与RANK的相互作用，通过增加M-CSF和前列腺素E-2以及减少骨保护素，刺激破骨细胞形成。在此，我们使用RAW264.7细胞作为破骨细胞前体，检测了IL-1 α或RANKL和/或M-CSF在存在IL-1 α的情况下对碳酸酐酶H（CAII）、组织蛋白酶K、基质金属蛋白酶-9（NIMP-9）、RANK、M-CSF受体（c-fms）和c-fos转录因子表达的影响。在存在100 U/ml IL-1 α的情况下，将细胞在0或100 U/ml IL-1 α和50 ng/ml RANKL、10 ng/ml M-CSF或50 ng/ml RANKL + 10 ng/ml M-CSF中培养长达14天。用抗酒石酸酸性磷酸酶染色法检测破骨细胞样细胞的形成。使用实时PCR测定编码六种目的蛋白质的基因的表达，并使用Western印迹或ELISA检查三种酶的表达。在存在IL-1 α的情况下，CAR、组织蛋白酶K和N4 MP-9的表达在与RANKL或M-CSF+RANKL一起培养的细胞中显著增加，而在与单独的IL-1 α和M-CSF一起培养的细胞中难以检测到表达。在IL-1 α存在下，RANKL或M-CSF + RANKL培养的细胞中RANK和c-fos表达也增加，而c-fins表达没有变化。这些结果表明，RAW264.7细胞中CAII、组织蛋白酶K和MNW-9的表达不是由M-CSF诱导的，而是在IL-1 α存在下由RANKL诱导的。(c)2007年爱思唯尔公司All rights reserved.
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-kappa B (RANK) ligand (RANKL) are essential and sufficient for osteoclast differentiation. Recently, we showed that IL-1 alpha affects mineralized nodule formation in vitro and halts bone matrix turnover. We also showed that IL-1 alpha 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-1 alpha or RANKL and/or M-CSF in the presence of IL-1 alpha on the expression of carbonic anhydrase H (CAII), cathepsin K, matrix metalloprotemase-9 (NIMP-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-1 alpha 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/mI IL-1 alpha. 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-1 alpha, expression of CAR, cathepsin K, and N4MP-9 was markedly increased in cells cultured with RANKL or M-CSF+RANKL, whereas expression was difficult to detect in cells cultured with IL-1 alpha 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-1 alpha, whereas c-fins expression did not change. These results indicate that the expression of CAII, cathepsin K, and MNW-9 in RAW264.7 cells is not induced by M-CSF, but by RANKL in the presence of IL-1 alpha. (c) 2007 Elsevier Inc. All rights reserved.