PLOS ONE ( IF 2.9 ) Pub Date : 2018-03-16 , DOI: 10.1371/journal.pone.0194591 Christelle Sanchez 1 , Gabriel Mazzucchelli 2 , Cécile Lambert 1 , Fanny Comblain 1 , Edwin DePauw 2 , Yves Henrotin 1
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Objective
Osteoarthritis (OA) is characterized by cartilage degradation but also by other joint tissues modifications like subchondral bone sclerosis. In this study, we used a proteomic approach to compare secretome of osteoblast isolated from sclerotic (SC) or non sclerotic (NSC) area of OA subchondral bone.
Design
Secretome was analyzed using differential quantitative and relative label free analysis on nanoUPLC G2 HDMS system. mRNA of the more differentially secreted proteins were quantified by RT-PCR in cell culture from 5 other patients. Finally, osteomodulin and fibulin-3 sequences were quantified by western blot and immunoassays in serum and culture supernatants.
Results
175 proteins were identified in NSC osteoblast secretome. Data are available via ProteomeXchange with identifier PXD008494. Compared to NSC osteoblast secretome, 12 proteins were significantly less secreted (Osteomodulin, IGFBP5, VCAM-1, IGF2, 78 kDa glucose-regulated protein, versican, calumenin, IGFBP2, thrombospondin-4, periostin, reticulocalbin 1 and osteonectin), and 13 proteins were significantly more secreted by SC osteoblasts (CHI3L1, fibulin-3, SERPINE2, IGFBP6, SH3BGRL3, SERPINE1, reticulocalbin3, alpha-2-HS-glycoprotein, TIMP-2, IGFBP3, TIMP-1, SERPINF1, CSF-1). Similar changes in osteomodulin, IGF2, SERPINE1, fibulin-3 and CHI3L1 mRNA levels were observed. ELISAs assays confirm the decrease by half of osteomodulin protein in SC osteoblasts supernatant compared to NSC and in OA patients serum compared to healthy subjects. Fibulin-3 epitopes Fib3-1, Fib3-2 and Fib3-3 were also increased in SC osteoblasts supernatant compared to NSC.
Conclusions
We highlighted some proteins differentially secreted by the osteoblasts coming from OA subchondral bone sclerosis. These changes contribute to explain some features observed in OA subchondral bone, like the increase of bone remodeling or abnormalities in bone matrix mineralization. Among identified proteins, osteomodulin was found decreased and fibulin-3 increased in serum of OA patients. These findings suggest that osteomodulin and fibulin-3 fragments could be biomarkers to monitor early changes in subchondral bone metabolism in OA.
中文翻译：
OA 中硬化与非硬化软骨下骨来源的成骨细胞分泌组的比较：一项初步研究
客观的
骨关节炎 (OA) 的特点是软骨退化，但也有其他关节组织改变，如软骨下骨硬化。在这项研究中，我们使用蛋白质组学方法来比较从 OA 软骨下骨的硬化 (SC) 或非硬化 (NSC) 区域分离的成骨细胞的分泌组。
设计
在nanoUPLC G2 HDMS系统上使用差异定量和相对无标记分析来分析分泌组。通过 RT-PCR 对其他 5 名患者的细胞培养物中差异较大的分泌蛋白的 mRNA 进行定量。最后，通过蛋白质印迹和免疫分析对血清和培养物上清液中的骨调节蛋白和 fibulin-3 序列进行定量。
结果
NSC 成骨细胞分泌组中鉴定出 175 种蛋白质。数据可通过 ProteomeXchange 获得，标识符为 PXD008494。与 NSC 成骨细胞分泌蛋白组相比，12 种蛋白的分泌量显着减少（骨调节蛋白、IGFBP5、VCAM-1、IGF2、78 kDa 葡萄糖调节蛋白、多功能蛋白聚糖、calumenin、IGFBP2、血小板反应蛋白-4、periostin、reticulocalbin 1 和骨粘连蛋白），13 种蛋白的分泌量显着减少。 SC 成骨细胞分泌的蛋白质显着增多（CHI3L1、fibulin-3、SERPINE2、IGFBP6、SH3BGRL3、SERPINE1、reticulocalbin3、α-2-HS-糖蛋白、TIMP-2、IGFBP3、TIMP-1、SERPINF1、CSF-1）。骨调节蛋白、IGF2、SERPINE1、fibulin-3 和 CHI3L1 mRNA 水平也观察到类似的变化。 ELISA 测定证实，与 NSC 相比，SC 成骨细胞上清液中的骨调节蛋白减少了一半，与健康受试者相比，OA 患者血清中的骨调节蛋白减少了一半。与 NSC 相比，SC 成骨细胞上清液中的 Fibulin-3 表位 Fib3-1、Fib3-2 和 Fib3-3 也有所增加。
结论
我们强调了来自 OA 软骨下骨硬化的成骨细胞差异分泌的一些蛋白质。这些变化有助于解释 OA 软骨下骨中观察到的一些特征，例如骨重塑的增加或骨基质矿化的异常。在已鉴定的蛋白质中，OA 患者血清中骨调节蛋白减少，fibulin-3 增加。这些发现表明骨调节蛋白和 fibulin-3 片段可以作为监测 OA 软骨下骨代谢早期变化的生物标志物。
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