Heparin inhibits osteoclastic differentiation and function
Heparin inhibits osteoclastic differentiation and function
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DOI：
10.1002/jcb.21559
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发表时间：
2008-04-15
影响因子：
4
通讯作者：
Nishihara, Tatsuji
中科院分区：
文献类型：
--
作者：
Ariyoshi, Wataru;Takahashi, Tetsu;Nishihara, Tatsuji
我们研究了糖胺多聚糖(GAG)对体外培养的小鼠单核细胞系分化、增殖和功能的影响。RAW 264.7细胞与核因子-kappaB受体激活剂配体(RANKL)和不同的GAG S共同培养，抗酒石酸酸性磷酸酶(TRAP)染色显示破骨细胞，苯磷酸底物法检测破骨细胞。RAW 264.7细胞与BD Biocoat OSTEOLOGIC(TM)试剂盒中含有的刺激物一起培养，通过计数骨吸收坑的数量来评价骨吸收活性。用四甲基偶氮唑盐比色法检测肝素对细胞生长的影响，免疫印迹法检测c-Src蛋白的表达水平。肝素抑制RANKL诱导的TRAP阳性多核细胞的形成和TRAP活性，而其他GAG对破骨细胞分化无影响。肝素也能抑制吸收陷窝的形成，而其他肝素不能。在四甲基偶氮唑盐比色法中，所有受试GAG均不影响RAW 264.7细胞的增殖。然而，肝素降低RAW 264.7细胞在RANKL刺激下的c-src蛋白水平。用HiTrap肝素柱层析法测定肝素与RANKL的亲和力，收集各组分。Western blotting分析显示RANKL在与肝素结合的部分有表达。用石英晶体微天平证实RANKL与肝素的结合。这些结果表明，肝素对RANKL诱导的破骨细胞生成的抑制作用是由于肝素与RANKL的结合所致。
We investigated the effects of Glycosaminoglycans(GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7cells were cultured with receptor activator of NF-kappa B ligand (RANKL) and various GAG s. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGIC(TM) kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.