DIFFERENTIAL UTILIZATION OF REGULATORY DOMAINS WITHIN THE ALPHA-1(I) COLLAGEN PROMOTER IN OSSEOUS AND FIBROBLASTIC CELLS
DIFFERENTIAL UTILIZATION OF REGULATORY DOMAINS WITHIN THE ALPHA-1(I) COLLAGEN PROMOTER IN OSSEOUS AND FIBROBLASTIC CELLS
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DOI：
10.1083/jcb.116.1.227
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发表时间：
1992-01-01
影响因子：
7.8
通讯作者：
ROWE, DW
中科院分区：
文献类型：
--
作者：
PAVLIN, D;LICHTLER, AC;ROWE, DW
I型胶原蛋白在多种结缔组织细胞中表达，并且由于许多发育、环境和激素因素的影响，其转录调控是高度复杂的。为了研究这种复杂调控的一个方面的分子基础，即骨组织中α-1（I）胶原（COL 1A 1）基因的表达，我们将大鼠COL 1A 1启动子的碱基-3，521和+115之间的3.6-kb DNA片段和三个缺失突变体融合到氯霉素乙酰转移酶（CAT）标记基因上。在稳定转染的成骨细胞系ROS 17/2.8、Py-1a和MC 3 T3-E1以及三个成纤维细胞系NIH-3 T3、Rat-1和EL 2中测量这些ColCAT转基因的表达。全长ColCAT3.6构建体的远端1.2 kb片段的缺失使成骨细胞系中的启动子活性降低7- 30倍，在EL 2中降低2倍，并且在NIH-3 T3和Rat-1细胞中没有影响。为了开始评估完整动物中COL 1A 1上游调控元件的功能，我们建立了转基因小鼠系，并检查了ColCAT 3.6构建体在新生动物的各种组织中的活性。该构建体的表达遵循高和低胶原蛋白产生组织之间的预期分布：颅骨、牙齿和肌腱中的CAT活性水平高，皮肤中的水平低，肝脏和大脑中没有检测到活性。此外，CAT活性在颅骨是三至四倍高于相邻的骨膜层。CAT蛋白在ColCAT3.6小鼠的颅骨和发育中的牙胚中的免疫染色也证实了与骨膜和牙乳头的成纤维细胞样细胞相比，转基因在分化的成骨细胞和成牙本质细胞中的优选表达。该研究表明，3.6-kb DNA片段赋予了COL 1A 1基因在完整动物的高胶原产生组织中的强表达，并且5'侧翼启动子序列在-3，521和-2，295 bp之间包含一个或多个在成骨细胞中优先具有活性的刺激元件。
Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha-1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-1a, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-I cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.