Identification of the Core Element Responsive to Runt-Related Transcription Factor 2 in the Promoter of Human Type X Collagen Gene
Identification of the Core Element Responsive to Runt-Related Transcription Factor 2 in the Promoter of Human Type X Collagen Gene
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DOI：
10.1002/art.24243
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发表时间：
2009-01-01
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：
Higashikawa, Akiro;Saito, Taku;Kawaguchi, Hiroshi
目标。已知X型胶原和矮小相关转录因子2(RUNX-2)在小鼠骨骼生长和骨关节炎(OA)修复和发展过程中对软骨细胞肥大起重要作用。本研究以临床应用为目的，研究RUNX-2对人X型胶原基因转录的调节作用。用免疫组织化学方法检测X型胶原和RUNX-2在培养的小鼠成软骨ATDC-5细胞中的定位，并观察它们在功能上的相互作用。在HeLa、Huh7和OUMS27细胞中检测了人X型胶原基因(COL10A1)的启动子活性，该基因含有4.5 kb的启动子和片段。用凝胶电泳法和染色质免疫沉淀法检测其与RUNX-2的结合情况。RUNX-2和X型胶原在小鼠肢体软骨和骨折骨痂中共存。RUNX-2功能的获得和丧失表明，RUNX-2对X型胶原的表达和软骨细胞的终末分化是必不可少的。人COL10A1启动子活性在RUNX-2单独作用下增强，RUNX-2与辅活化子核心结合因子0联合使用时作用更强。缺失、突变和串联重复分析确定核心反应元件位于-和-60bp之间的区域(称为肥大框[HY box])，该区域显示与RUNX-2特异结合。人COL10A1启动子中的其他RUNX-2结合基序在人细胞中不能与RUNX-2反应。我们的发现表明，在人类COL10IA1启动子中，HY盒是响应RUNX-2的核心元件。对RUNX-2和HY box相关分子网络的研究将有助于骨骼生长迟缓、骨折和骨关节炎的治疗。
Objective. Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells.Methods. Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation.Results. RUNX-2 and type X collagen were colocalized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor 0 in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between -89 and -60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells.Conclusion. Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10IA1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.