Screening of Chondrogenic Factors With a Real-Time Fluorescence-Monitoring Cell Line ATDC5-C2ER Identification of Sorting Nexin 19 as a Novel Factor
Screening of Chondrogenic Factors With a Real-Time Fluorescence-Monitoring Cell Line ATDC5-C2ER Identification of Sorting Nexin 19 as a Novel Factor
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DOI：
10.1002/art.24878
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发表时间：
2009-11-01
影响因子：
--
通讯作者：
Kawaguchi, Hiroshi
中科院分区：
文献类型：
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作者：
Kan, Akinori;Ikeda, Toshiyuki;Kawaguchi, Hiroshi
客观的。建立无创实时监测成软骨分化的细胞培养体系，筛选成软骨因子。方法。通过荧光素酶报告基因测定和胰岛素培养过程中的荧光分析，选择转染到软骨形成 ATDC5 细胞中的最佳报告基因构建体。根据 SOX 蛋白、骨形态发生蛋白 2 (BMP-2) 或转化生长因子 β (TGF β) 刺激后的荧光来验证建立的细胞系，并与 COL2A1 的信使 RNA 水平以及阿尔新蓝染色程度进行比较。使用从人气管软骨制备的逆转录病毒表达文库通过表达克隆进行软骨形成因子的筛选。通过原位杂交和免疫组织化学检查所鉴定分子的表达模式。通过转染已鉴定的基因、小干扰RNA和突变基因进行功能分析。结果。我们建立了一个 ATDC5 细胞系，其中 4 个重复的高度保守的增强子连接到 COL2A1 基础启动子和 DsRed2 报告基因 (ATDC5-C2ER)。在 SOX 蛋白、BMP-2 或 TGF beta 刺激下诱导荧光，显示出与软骨形成标记物良好的对应性。使用 ATDC5-C2ER 系统进行筛选，确定了多种软骨形成因子，包括分选 nexin 19 (SNX19)。 SNX19 在小鼠胚胎的肢体软骨和骨关节炎进展过程中成年小鼠膝关节的退化软骨中表达。功能获得和功能丧失分析揭示了 SNX19 的有效软骨形成活性。结论。我们建立了 ATDC5-C2ER 系统，通过荧光分析有效监测软骨形成分化，并利用该系统鉴定了一种新型软骨形成因子 (SNX19)。该系统将有助于阐明软骨分化的分子网络。
Objective. To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors.Methods. The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor beta (TGF beta) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene.Results. We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGF beta, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19.Conclusion. We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.