Kwan Tat S, Pelletier J-P, Lajeunesse D, Fahmi H, Lavigne M, Martel-Pelletier J
Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, Montreal, Quebec.
Clin Exp Rheumatol. 2008 Mar-Apr;26(2):295-304.
We previously reported that human OA subchondral bone osteoblasts could be discriminated into two subpopulations identified by their levels of endogenous production (low [L] or high [H]) of PGE(2). Here, we investigated the OPG and RANKL expression levels, the histologic analysis of the subchondral bone as well as the osteoclast differentiation effect of osteoblasts on normal and both OA subpopulations (L and H), and further examined on the L OA osteoblasts the modulation of bone remodelling factors on the OPG and RANKL levels, as well as on the resorption activity.
Gene expression was determined using real-time PCR, PGE2 and OPG levels by specific ELISA, and membranous RANKL by flow cytometry. Histological observation of the subchondral bone was performed on human knee specimens. Osteoclast differentiation and formation was assayed by using the pre-osteoclastic cell line RAW 264.7. OPG and RANKL modulation on L OA osteoblasts was monitored following treatment with osteotropic factors, and the resorption activity was studied by the co-culture of differentiated PBMC/osteoblasts.
Human OA subchondral bone osteoblasts expressed less OPG than normal. Compared to normal, RANKL gene expression levels were increased in L OA and decreased in H OA cells. The OPG/RANKL mRNA ratio was significantly diminished in L OA compared to normal or H OA (p<0.02, p<0.03), and markedly increased in H OA compared to normal. Inhibition of endogenous PGE(2) levels by indomethacin markedly decreased the ratio of OPG/RANKL on the H OA. In contrast to H OA osteoblasts, L OA cells induced a significantly higher level of osteoclast differentiation and formation (p<0.05). Histological analysis showed a reduced subchondral bone on the L OA and an increased bone mass on the H OA compared to normal. Treatment of L OA osteoblasts with osteotropic factors revealed that the OPG/RANKL mRNA expression ratio was significantly reduced by vitamin D(3) and significantly increased by TNF-alpha, PTH and PGE(2), while IL-1Beta demonstrated no effect. OPG protein levels showed similar profiles. No true effect was noted on membranous RANKL upon treatment with IL-1Beta, PGE(2) and PTH, but a significant increase was observed with vitamin D3 and TNF-alpha. The resorption activity of the L OA cells was significantly inhibited by all treatments except IL-1Beta, with maximum effect observed with vitamin D(3) and PGE(2).
OPG and RANKL levels, and consequently the OPG/RANKL ratio, differed according to human OA subchondral bone osteoblast classification; it is decreased in L and increased in H OA. These findings, in addition to those showing that L OA osteoblasts have a reduced subchondral bone mass and induce a higher level of osteoclast differentiation, strongly suggest that the metabolic state of the L OA osteoblasts favours bone resorption.
我们之前报道过，人骨关节炎（OA）软骨下骨成骨细胞可根据其前列腺素E2（PGE2）内源性产生水平（低[L]或高[H]）分为两个亚群。在此，我们研究了骨保护素（OPG）和核因子κB受体活化因子配体（RANKL）的表达水平、软骨下骨的组织学分析以及成骨细胞对正常及两种OA亚群（L和H）破骨细胞分化的影响，并进一步研究了骨重塑因子对L OA成骨细胞OPG和RANKL水平以及吸收活性的调节作用。
使用实时聚合酶链反应（PCR）测定基因表达，通过特异性酶联免疫吸附测定（ELISA）检测PGE2和OPG水平，采用流式细胞术检测膜结合RANKL。对人膝关节标本进行软骨下骨的组织学观察。使用前破骨细胞系RAW 264.7检测破骨细胞的分化和形成。用促骨生成因子处理后监测L OA成骨细胞上OPG和RANKL的调节情况，并通过分化的外周血单个核细胞/成骨细胞共培养研究吸收活性。
人OA软骨下骨成骨细胞表达的OPG比正常细胞少。与正常相比，L OA中RANKL基因表达水平升高，而H OA细胞中降低。与正常或H OA相比，L OA中OPG/RANKL mRNA比值显著降低（p<0.02，p<0.03），而与正常相比，H OA中该比值显著升高。吲哚美辛抑制内源性PGE2水平可显著降低H OA上OPG/RANKL的比值。与H OA成骨细胞相反，L OA细胞诱导的破骨细胞分化和形成水平显著更高（p<0.05）。组织学分析显示，与正常相比，L OA的软骨下骨减少，而H OA的骨量增加。用促骨生成因子处理L OA成骨细胞发现，维生素D3可显著降低OPG/RANKL mRNA表达比值，而肿瘤坏死因子-α（TNF-α）、甲状旁腺激素（PTH）和PGE2可显著升高该比值，而白细胞介素-1β（IL-1β）无作用。OPG蛋白水平显示出类似的变化情况。用IL-1β、PGE2和PTH处理后，膜结合RANKL无明显变化，但维生素D3和TNF-α处理后显著增加。除IL-1β外，所有处理均显著抑制L OA细胞的吸收活性，维生素D3和PGE2的作用最为明显。
OPG和RANKL水平以及OPG/RANKL比值根据人OA软骨下骨成骨细胞分类而有所不同；L OA中降低，H OA中升高。这些发现，再加上那些表明L OA成骨细胞软骨下骨量减少且诱导更高水平破骨细胞分化的发现，强烈提示L OA成骨细胞的代谢状态有利于骨吸收。
