Karsdal Morten A, Hjorth Pernille, Henriksen Kim, Kirkegaard Tove, Nielsen Karina L, Lou Henriette, Delaissé Jean-Marie, Foged Niels T
Nordic Bioscience A/S, CCBR, Herlev/Ballerup, Herlev DK-2730, Denmark.
J Biol Chem. 2003 Nov 7;278(45):44975-87. doi: 10.1074/jbc.M303905200. Epub 2003 Aug 20.
Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.
尽管核因子κB受体活化因子配体（RANK-L）对于破骨细胞的形成至关重要，但诸如转化生长因子-β（TGF-β）等因子是破骨细胞生成刺激的有效调节剂。为了系统地研究TGF-β在人类破骨细胞生成中的作用，通过三种不同的方法从外周血中分离单核细胞，得到富含淋巴细胞、淋巴细胞较少或纯破骨细胞前体（CD14阳性）细胞群体。在这些破骨细胞前体群体中的每一个中，研究了TGF-β对增殖、抗酒石酸酸性磷酸酶（TRAP）活性和骨吸收的影响，涉及暴露时间和时长。当使用高度纯化的CD14破骨细胞前体细胞群体时，TGF-β的作用强烈依赖于破骨细胞成熟阶段。当单核细胞在初始培养期（第1 - 7天）暴露于TGF-β时，TRAP活性和骨吸收增加了40%，而细胞数量减少了25%。当在整个培养期（第1 - 21天）存在TGF-β时，观察到细胞数量有类似的减少，但直接相反的是，TRAP活性、细胞融合、组织蛋白酶K和基质金属蛋白酶（MMP）-9表达以及骨吸收几乎完全被消除。此外，我们发现潜伏的TGF-β通过与MMP-9孵育而强烈活化，并认为这是调节破骨细胞活性的高度相关机制。为了进一步研究连续与不连续暴露于TGF-β产生不同作用的分子机制，我们检测了RANK表达和p38丝裂原活化蛋白激酶（MAPK）激活。我们发现TGF-β强烈诱导单核细胞中的p38 MAPK，但在成熟破骨细胞中不诱导，并且TGF-β持续暴露于单核细胞会下调RANK表达。目前的结果表明，TGF-β通过刺激p38 MAPK促进单核细胞中的人类破骨细胞生成，而持续暴露于TGF-β则通过下调RANK表达并因此减弱RANK-RANK-L信号传导来消除破骨细胞生成。
