Pavlin D, Bedalov A, Kronenberg M S, Kream B E, Rowe D W, Smith C L, Pike J W, Lichtler A C
Department of Orthodontics, University of Texas Health Science Center, San Antonio 78284.
J Cell Biochem. 1994 Dec;56(4):490-501. doi: 10.1002/jcb.240560409.
The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
骨细胞中I型胶原蛋白的合成受到钙调节激素1,25 - 二羟基维生素D3的抑制。我们实验室早期的研究表明，基于核转录分析和由3.6 kb COL1A1启动子片段与氯霉素乙酰转移酶报告基因融合而成的杂交基因的功能启动子分析结果，维生素D的调节作用发生在转录水平。在本研究中，我们调查了维生素D介导的COL1A1基因转录抑制的分子基础，并报告了在COL1A1上游启动子内鉴定出一个区域（核苷酸 -2295至 -1670之间的HindIII - Pstl限制性片段），该区域对于成骨细胞中1,25 - 二羟基维生素D3的反应性是必需的。这种激素介导的对标记基因的抑制作用与内源性胶原蛋白基因的抑制作用相似。来自该区域的一个41 bp片段（核苷酸 -2256至 -2216之间）包含一个与骨钙素基因中鉴定出的维生素D反应元件非常相似的序列。通过凝胶迁移分析表明，表达高水平维生素D受体的培养细胞提取物中含有一种激素：受体复合物，该复合物能特异性结合这个41 bp片段。然而，从 -3.5 kb或 -2.3 kb启动子片段中缺失这个维生素D受体结合区域并没有消除维生素D反应性。这些结果表明，与其他维生素D反应基因（骨钙素和骨桥蛋白）中描述的类似的维生素D反应元件并不能单独介导1,25 - 二羟基维生素D3对COL1A1的抑制作用。
