Ríus C, Smith J D, Almendro N, Langa C, Botella L M, Marchuk D A, Vary C P, Bernabéu C
Department of Immunology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Blood. 1998 Dec 15;92(12):4677-90.
Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.
内皮糖蛋白（CD105）是转化生长因子-β（TGF-β）受体复合物的一种细胞表面成分，在内皮细胞中高度表达。内皮糖蛋白基因突变是1型遗传性出血性毛细血管扩张症（HHT1）的病因，HHT1也被称为奥斯勒-韦伯-伦杜综合征（OMIM 187300）。这是一种常染色体显性血管疾病，可能由单倍剂量不足机制引起，表现为正常蛋白水平较低。为了了解内皮糖蛋白调控表达的潜在机制，已分离出一个包含人内皮糖蛋白基因5'侧翼序列3.3 kb的基因组DNA克隆。内皮糖蛋白基因的5'侧翼区域缺乏共有TATA盒和CAAT盒，但包含两个富含GC的区域以及Sp1、ets、GATA、AP-2、NFκB和Mad的共有基序，还有TGF-β、糖皮质激素、维生素D和雌激素反应元件。通过引物延伸和5' RACE实验确定，转录起始位点簇位于翻译起始密码子上游350 bp处。为了分析内皮糖蛋白启动子活性，将上游-400/+341片段与荧光素酶基因融合，并在几种细胞类型中进行瞬时转染。该构建体在人和牛内皮细胞中表现出组织特异性活性。对各种缺失构建体的分析表明，-81/+350片段内存在一个基础启动子区域，-400/-141片段内存在主要转录调控元件。电泳迁移率变动分析表明，ets家族的一个成员与位于-68位的共有基序发生特异性相互作用。与野生型构建体相比，在该ets序列处突变的启动子构建体活性大幅降低，这支持了该ets基序参与启动子基础活性的观点。内皮糖蛋白启动子在TGF-β1存在时表现出诱导性，这提示了对HHT1患者可能的治疗方法，在这类患者中，正常内皮糖蛋白等位基因的表达水平可能未达到其功能所需的阈值。人内皮糖蛋白启动子的分离和表征是阐明内皮糖蛋白基因受控表达的第一步。
