Zhao S, Zhang Y Kato Y, Harris S, Ahuja S S, Bonewald L F
Department of Medicine, University of Texas Health Science Center, San Antonio, USA.
J Bone Miner Res. 2002 Nov;17(11):2068-79. doi: 10.1359/jbmr.2002.17.11.2068.
Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte-like cell line called MLO-Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO-Y4 cells support osteoclast formation in the absence of 1,25-dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO-Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200-1,000 MLO-Y4 cells per 0.75-cm2 well. No osteoclast formation was observed with 2T3, OCT-1, or MC3T3-E1 osteoblast cells (1,000 cells/well). Conditioned media from the MLO-Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2-week-old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO-Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony-stimulating factor (M-CSF), RANKL, and osteoprotegerin (OPG). MLO-Y4 cells express and secrete large amounts of M-CSF. MLO-Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO-Y4 cells compared with the other cell types. RANK-Fc and OPG-Fc blocked the formation of osteoclasts by MLO-Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M-CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes.
骨细胞是成骨细胞谱系的终末分化细胞，已嵌入矿化基质中，并可能发出调节骨塑形和重塑的信号。本研究要检验的假设是骨细胞可以刺激并支持破骨细胞的形成和激活。为了验证这一假设，将一种名为MLO-Y4的类骨细胞系和原代小鼠骨细胞与脾细胞或骨髓细胞进行共培养。在没有1,25-二羟基维生素D3 [1,25(OH)2D3]或任何其他外源性促骨因子的情况下，MLO-Y4细胞支持破骨细胞的形成。这些细胞单独刺激破骨细胞形成的程度与添加1,25(OH)2D3相同或更大。1,25(OH)2D3与MLO-Y4细胞共同添加可协同增加破骨细胞的形成。每0.75平方厘米孔中接种200 - 1000个MLO-Y4细胞时，在牙本质上观察到最佳的破骨细胞形成和陷窝形成。2T3、OCT-1或MC3T3-E1成骨细胞（1000个细胞/孔）未观察到破骨细胞形成。MLO-Y4细胞的条件培养基对破骨细胞形成没有影响，表明细胞接触是必要的。对2周龄小鼠颅骨进行连续消化，得到的细胞群体与1,25(OH)2D3和骨髓共培养时支持破骨细胞形成，但留在骨颗粒中的细胞群体无论有无1,25(OH)2D3都支持最多数量的破骨细胞。为了研究这些细胞支持破骨细胞形成的机制，将MLO-Y4细胞与一系列成骨细胞和基质细胞比较巨噬细胞集落刺激因子（M-CSF）、核因子κB受体活化因子配体（RANKL）和骨保护素（OPG）的表达。MLO-Y4细胞表达并分泌大量的M-CSF。MLO-Y4细胞在其表面及其树突状突起上表达RANKL。与其他细胞类型相比，MLO-Y4细胞中RANKL与OPG mRNA的比例最大。RANK-Fc和OPG-Fc可阻断MLO-Y4细胞形成破骨细胞。这些研究表明RANKL和OPG可能在骨细胞信号传导中起作用，OPG和M-CSF作为可溶性因子，RANKL作为在骨细胞中或沿其暴露的树突状突起起作用的表面分子。
