Department of Sensory & Motor System Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan.
Osteoarthritis Cartilage. 2009 Aug;17(8):1065-75. doi: 10.1016/j.joca.2009.02.003. Epub 2009 Feb 20.
Although SOX9 is a key molecule for chondrogenic differentiation, little is known about the upstream signal. The present study attempted to identify transcription factors to induce SOX9 expression and examined the mechanism.
Sequences of about 1 kb of 5'-end flanking regions were compared between human and mouse SOX9 genes. In vivo localization was examined by immunohistochemistry in the limb cartilage of fetal mice. Promoter activities of the SOX9, SOX6, and type II collagen (COL2A1) genes were determined in human non-chondrogenic HeLa cells and mouse chondrogenic ATDC5 cells transfected with a luciferase-reporter gene containing the promoter fragments. Protein-DNA binding was examined by electrophoretic mobility shift and chromatin immunoprecipitation assays. The chondrogenic differentiation was assessed by endogenous SOX9, SOX6, and COL2A1 mRNA levels, and by Alcian blue staining and alkaline phosphatase activity.
Among transcription factors whose binding motifs were identified in the highly-conserved regions between human and mouse SOX9 promoters, a nuclear factor kappa B (NF-kappaB) member RelA strongly activated the promoter activity. RelA and SOX9 were co-localized in the limb cartilage. Deletion, mutagenesis, and tandem-repeat analyses identified the core region responsive to RelA at the NF-kappaB binding motif to be around -250bp of the human SOX9 promoter, and this was confirmed to show specific binding to RelA. RelA induced the chondrogenic differentiation parameters in HeLa and ATDC5 cells.
We have identified RelA as a transcriptional factor for SOX9 induction and chondrogenic differentiation via binding to an NF-kappaB binding motif in the SOX9 promoter.
尽管 SOX9 是软骨分化的关键分子，但对于其上游信号知之甚少。本研究试图鉴定诱导 SOX9 表达的转录因子，并研究其机制。
比较了人 SOX9 基因和鼠 SOX9 基因约 1kb 5'端侧翼序列。通过免疫组织化学检测胎鼠肢体软骨中的体内定位。用人非软骨细胞系 HeLa 和转染含启动子片段的荧光素酶报告基因的鼠软骨细胞系 ATDC5 检测 SOX9、SOX6 和 II 型胶原（COL2A1）基因的启动子活性。通过电泳迁移率变动和染色质免疫沉淀实验检测蛋白-DNA 结合。通过内源性 SOX9、SOX6 和 COL2A1mRNA 水平以及阿利新蓝染色和碱性磷酸酶活性评估软骨分化。
在人 SOX9 启动子高度保守区鉴定出的转录因子结合基序中，核因子 kappa B（NF-kappaB）成员 RelA 强烈激活启动子活性。RelA 和 SOX9 在肢体软骨中存在共定位。缺失、突变和串联重复分析确定了响应 RelA 的核心区域，即人类 SOX9 启动子的 NF-kappaB 结合基序周围约-250bp，并且证实该区域与 RelA 具有特异性结合。RelA 诱导 HeLa 和 ATDC5 细胞中的软骨分化参数。
我们已经鉴定出 RelA 作为 SOX9 诱导和软骨分化的转录因子，通过结合 SOX9 启动子中的 NF-kappaB 结合基序。
