Niessen R W, Rezaee F, Reitsma P H, Peters M, de Vijlder J J, Sturk A
Center for Haemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Centre, Amsterdam, The Netherlands.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):263-70. doi: 10.1042/bj3180263.
We studied potential modulators of antithrombin gene expression. A putative hormone response element (HRE) was identified by sequence similarity analysis of the antithrombin promoter, situated between nucleotides -92 and -54 relative to the transcription start site. This HRE contains three hexa-nucleotide motifs with an AGGTCA consensus, which are potential targets of members of the steroid/thyroid superfamily of nuclear receptors. Stimulation of the hepatoma cell line HepG2 with the receptor ligands L-3,5,3'-tri-iodothyronine, all-trans retinoic acid, or their combination, increased production of antithrombin into the culture medium by 1.3-, 1.6-, and 2.0-fold, respectively. In contrast, the receptor ligand 1,25-dihydroxy-cholecalciferol[1,25-(OH)2VitD3] did not influence antithrombin production. Analysis of promoter chloramphenicol acetyl-transferase (CAT) constructs, showed that the first 86 bp of the antithrombin promoter region are sufficient for basal transcription. The DNA length polymorphism of 32 bp or 108 bp, located upstream of position -276, did not influence anti-thrombin promoter activity. The antithrombin promoter activity dropped to background values when deleting the region -97/-49 of promoter fragment -453/+57. Transactivation of the antithrombin promoter by retinoid X receptor alpha (RXR alpha) (5-7-fold) or thyroid hormone receptor beta (TR beta) (4-5-fold) was only observed when at least -167/+57 bp of the promoter region is present in CAT constructs, and when the appropriate ligand of the nuclear receptor was added. This transactivation was not observed upon deletion of the antithrombin promoter region -97/-49. With three copies of the antithrombin promoter fragment -109/-42 in front of the thymidine kinase minimal promoter, transactivation was only obtained with RXR alpha, and not with TR beta. In conclusion, these results indicate that the ligand-dependent enhancement of antithrombin gene expression is regulated by RXR alpha as well as by TR beta. Transactivation of antithrombin gene expression by RXR alpha and TR beta appears to be dependent upon the presence of promoter region up to nucleotide -167. The HRE segment (-109/-42) only confers RXR alpha responsiveness to a heterologous promoter. Further study is needed to unravel the exact nature of this HRE and its 5'-flanking sequences.
我们研究了抗凝血酶基因表达的潜在调节因子。通过对抗凝血酶启动子进行序列相似性分析，确定了一个假定的激素反应元件（HRE），它位于相对于转录起始位点的核苷酸-92至-54之间。该HRE包含三个具有AGGTCA共有序列的六核苷酸基序，它们是核受体类固醇/甲状腺超家族成员的潜在靶点。用受体配体L-3,5,3'-三碘甲状腺原氨酸、全反式维甲酸或它们的组合刺激肝癌细胞系HepG2，可使培养基中抗凝血酶的产量分别增加1.3倍、1.6倍和2.0倍。相比之下，受体配体1,25-二羟基胆钙化醇[1,25-(OH)2VitD3]不影响抗凝血酶的产生。对启动子氯霉素乙酰转移酶（CAT）构建体的分析表明，抗凝血酶启动子区域的前86 bp足以进行基础转录。位于-276位点上游的32 bp或108 bp的DNA长度多态性不影响抗凝血酶启动子活性。当缺失启动子片段-453/+57的-97/-49区域时，抗凝血酶启动子活性降至背景值。仅当CAT构建体中存在至少-167/+57 bp的启动子区域，并且添加了核受体的适当配体时，才观察到维甲酸X受体α（RXRα）（5 - 7倍）或甲状腺激素受体β（TRβ）（4 - 5倍）对抗凝血酶启动子的反式激活。在缺失抗凝血酶启动子区域-97/-49后未观察到这种反式激活。在胸苷激酶最小启动子前有三个抗凝血酶启动子片段-109/-42的拷贝时，仅用RXRα可获得反式激活，而用TRβ则不能。总之，这些结果表明，抗凝血酶基因表达的配体依赖性增强受RXRα和TRβ调节。RXRα和TRβ对抗凝血酶基因表达的反式激活似乎依赖于直至核苷酸-167的启动子区域的存在。HRE片段（-109/-42）仅赋予异源启动子对RXRα的反应性。需要进一步研究以阐明该HRE及其5'侧翼序列的确切性质。
