Pavlin D, Lichtler A C, Bedalov A, Kream B E, Harrison J R, Thomas H F, Gronowicz G A, Clark S H, Woody C O, Rowe D W
Department of Pediatrics, University of Connecticut Health Center, Farmington 06032.
J Cell Biol. 1992 Jan;116(1):227-36. doi: 10.1083/jcb.116.1.227.
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-la, 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 ColCAT 3.6 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-1 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.
I型胶原蛋白在多种结缔组织细胞中表达，由于受到众多发育、环境和激素因素的影响，其转录调控高度复杂。为了研究这种复杂调控某一方面的分子基础，即骨组织中α1（I）胶原蛋白（COL1A1）基因的表达，我们将大鼠COL1A1启动子-3521至+115碱基之间的一个3.6 kb DNA片段以及三个缺失突变体与氯霉素乙酰转移酶（CAT）标记基因融合。在稳定转染的成骨细胞系ROS 17/2.8、Py-la和MC3T3-E1以及三个成纤维细胞系NIH-3T3、Rat-1和EL2中检测这些ColCAT转基因的表达。全长3.6 kb的ColCAT构建体远端1.2 kb片段的缺失使成骨细胞系中的启动子活性降低了7至30倍，在EL2中降低了两倍，而在NIH-3T3和Rat-1细胞中没有影响。为了开始评估完整动物中COL1A1上游调控元件的功能，我们建立了转基因小鼠系，并检测了ColCAT3.6构建体在新生动物各种组织中的活性。该构建体的表达遵循了预期的在高胶原蛋白产生组织和低胶原蛋白产生组织之间的分布：在颅骨、牙齿和肌腱中CAT活性水平高，在皮肤中活性低，在肝脏和大脑中未检测到活性。此外，颅骨中的CAT活性比相邻骨膜层中的高3至4倍。对ColCAT3.6小鼠颅骨和发育中的牙胚进行CAT蛋白免疫染色也证实，与骨膜和成牙乳头的成纤维样细胞相比，转基因在分化的成骨细胞和成牙本质细胞中优先表达。这项研究表明，3.6 kb DNA片段赋予了COL1A1基因在完整动物高胶原蛋白产生组织中的强表达，并且-3521至-2295 bp之间的5'侧翼启动子序列包含一个或多个在成骨细胞中优先活跃的刺激元件。
