USE OF A NEW RAT CHONDROSARCOMA CELL-LINE TO DELINEATE A 119-BASE PAIR CHONDROCYTE-SPECIFIC ENHANCER ELEMENT AND TO DEFINE ACTIVE PROMOTER SEGMENTS IN THE MOUSE PRO-ALPHA-1(II) COLLAGEN GENE
USE OF A NEW RAT CHONDROSARCOMA CELL-LINE TO DELINEATE A 119-BASE PAIR CHONDROCYTE-SPECIFIC ENHANCER ELEMENT AND TO DEFINE ACTIVE PROMOTER SEGMENTS IN THE MOUSE PRO-ALPHA-1(II) COLLAGEN GENE
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DOI：
10.1074/jbc.270.46.27711
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发表时间：
1995-11-17
影响因子：
4.8
通讯作者：
DECROMBRUGGHE, B
中科院分区：
文献类型：
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作者：
MUKHOPADHYAY, K;LEFEBVRE, V;DECROMBRUGGHE, B
我们发现，从Swarm肿瘤长期培养中建立的一种新的大鼠软骨肉瘤（RCS）细胞系显示出稳定的分化软骨细胞样表型。事实上，这些细胞产生II型、IX型和XI型胶原蛋白和阿新蓝软骨特异性蛋白聚糖，但不产生I型或III型胶原蛋白。用II型胶原/β Geo嵌合基因稳定转染细胞，该基因在转基因小鼠中赋予基本上完美的软骨细胞特异性表达，RCS细胞表达β-半乳糖苷酶和G418抗性，与类似转染的10 T1/2和NIH/3 T3成纤维细胞相比，然后使用这些细胞进行小鼠II型胶原蛋白基因（Col 2a 1）的第一内含子的系统缺失分析，使用瞬时表达实验来确定哪些片段刺激RCS细胞中而不是10 T1/2成纤维细胞中荧光素酶报告基因的表达，156 bp内含子1片段的两个串联拷贝的克隆（+2188至+2343）在含有314-bp Col 2a 1启动子的构建体中引起RCS细胞中启动子活性增加近200倍，但在10 T1/2细胞中，对该156-bp片段的DNA酶I足迹分析揭示了两个相邻的保护区，FP 1和FP 2，位于该片段的3 '-一半，但在RCS细胞和10 T1/2成纤维细胞的核提取物中没有观察到差异。缺失FP 2以留下119-bp片段使增强子活性降低了几倍，但RCS细胞特异性得以保持，进一步的缺失表明，RCS细胞特异性增强子活性同时需要119-bp片段的5'部分和FP 1中的序列。当在瞬时表达实验中单独测试这些启动子时，小鼠Col 2a 1基因的启动子区域中的一系列缺失逐渐降低活性。在RCS细胞中，这些启动子缺失都被包括156-bp增强子的231-bp内含子1片段激活到相似的水平。即使Col 2a 1启动子被最小的腺病毒主要晚期启动子替代，RCS细胞特异性活性仍然存在，该231-bp内含子1片段在瞬时转染的小鼠原代软骨细胞中也具有强的增强活性。我们的实验建立了RCS细胞作为研究软骨细胞特异性基因调控的实验系统的有用性，提供了一个广泛的描绘节段的Col 2a 1第一内含子参与软骨细胞特异性活动，并显示启动子序列是软骨细胞特异性。
We show that a new rat chondrosarcoma (RCS) cell line established in long-term culture from the Swarm tumor displayed a stable differentiated chondrocyte-like phenotype, Indeed, these cells produced the collagen types II, IX, and XI and alcian blue-stainable cartilage-specific proteoglycans, but no type I or type III collagen, To functionally characterize their chondrocytic nature, the cells were stably transfected with a type II collagen/beta geo chimeric gene which confers essentially perfect chondrocyte-specific expression in transgenic mice, RCS cells expressed both beta-galactosidase and G418 resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3 fibroblasts which did not, These cells were then used to perform a systematic deletion analysis of the first intron of the mouse type II collagen gene (Col2a1) using transient expression experiments to determine which segments stimulated expression of a luciferase reporter gene in RCS cells but not in 10T1/2 fibroblasts, Cloning of two tandem copies of a 156-base pair (bp) intron 1 fragment (+2188 to +2343) in a construction containing a 314-bp Col2a1 promoter caused an almost 200-fold increase in promoter activity in RCS cells but no increase in 10T1/2 cells, DNase I footprint analysis over this 156-bp fragment revealed two adjacent protected regions, FP1 and FP2, located in the 3'-half of this segment, but no differences were seen with nuclear extracts of RCS cells and 10T1/2 fibroblasts, Deletion of FP2 to leave a 119-bp segment decreased enhancer activity by severalfold, but RCS cell specificity was maintained, Further deletions indicated that sequences both in the 5' part of the 119-bp fragment and in FP1 were needed simultaneously for RCS cell-specific enhancer activity, A series of deletions in the promoter region of the mouse Col2a1 gene progressively reduced activity when these promoters were tested by themselves in transient expression experiments, However, these promoter deletions were all activated to a similar level in RCS cells by a 231-bp intron 1 fragment that included the 156-bp enhancer, The RCS cell-specific activity persisted even if the Col2a1 promoter was replaced by a minimal adenovirus major late promoter, This 231-bp intron 1 fragment also had strong enhancing activity in transiently transfected mouse primary chondrocytes, Our experiments establish the usefulness of RCS cells as an experimental system for studies of the control of chondrocyte-specific genes, provide an extensive delineation of segments in the Col2a1 first intron involved in chondrocyte-specific activity, and show that promoter sequences are dispensable for chondrocyte specificity.