Lengyel E, Wang H, Stepp E, Juarez J, Wang Y, Doe W, Pfarr C M, Boyd D
Department of Obstetrics and Gynecology, Technical University of Munich, 81675 Munich, Germany.
J Biol Chem. 1996 Sep 20;271(38):23176-84. doi: 10.1074/jbc.271.38.23176.
The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface. The present study was undertaken to identify elements in the u-PAR promoter required for the elevated expression of this binding site. Toward this end, we used two cultured colon cancer cell lines; one (RKO) has a transcriptionally activated u-PAR gene, and the other (GEO) overexpresses the receptor only after phorbol ester treatment. A chloramphenicol acetyltransferase (CAT) reporter driven by 398 nucleotides of 5' regulatory sequence of the u-PAR gene was strongly activated in the RKO cells, which displays approximately 3 x 10(5) receptors/cell. A region of this promoter between -197 and -8 was required for optimal expression, as indicated using a CAT reporter driven by 5' deleted fragments. DNase I footprinting revealed three protected regions (I, -190 to -171; II, -148 to -124; and III, -99 to -70) in this part of the promoter. Mutation of an AP-1 binding site at -184 within region I reduced activation of the promoter by 85%. Deletion of either region II or III also reduced promoter activity by over 60%. An oligonucleotide spanning the AP-1 motif at -184 bound, specifically, nuclear factors from RKO cells, and antibodies specific for Jun-D, c-Jun, or Fra-1 proteins supershifted the complex indicating the presence of these proteins. The amount of these factors was reduced in GEO cells in which the u-PAR gene is only weakly transcriptionally activated. Expression of a vector encoding a wild-type Jun-D cDNA increased u-PAR promoter activity in GEO cells. Conversely, transfection of RKO cells with a transactivation domain-lacking Jun-D expression construct resulted in a dose-dependent decrease in u-PAR promoter activity. Treatment of GEO cells with phorbol ester increased u-PAR mRNA and the activity of a CAT reporter driven by the wild-type but not the AP-1 (-184)-mutated u-PAR promoter, and this was associated with a strong induction in the amount of Jun-D, c-Jun, and c-Fos. Methylation interference studies using a fragment of the u-PAR promoter (spanning -201 to -150) bound with nuclear extracted proteins from RKO cells, and phorbol 12-myristate 13-acetate-treated and -untreated GEO cells showed that the contact points corresponded to the AP-1 binding site at -184. Thus, the elevated expression of u-PAR in RKO cells, which constitutively produces this binding site, as well as in phorbol 12-myristate 13-acetate-stimulated GEO cells requires an AP-1 motif located 184 bp upstream of the transcriptional start site.
尿激酶型纤溶酶原激活物受体（u-PAR）通过加速细胞表面纤溶酶的形成促进细胞外基质蛋白水解。本研究旨在确定u-PAR启动子中该结合位点表达升高所需的元件。为此，我们使用了两种培养的结肠癌细胞系；一种（RKO）具有转录激活的u-PAR基因，另一种（GEO）仅在佛波酯处理后过表达该受体。由u-PAR基因5'调控序列的398个核苷酸驱动的氯霉素乙酰转移酶（CAT）报告基因在RKO细胞中被强烈激活，RKO细胞显示约3×10⁵个受体/细胞。如使用由5'缺失片段驱动的CAT报告基因所示，该启动子-197至-8之间的区域是最佳表达所必需的。DNase I足迹分析揭示了该启动子这一部分的三个受保护区域（区域I，-190至-171；区域II，-148至-124；区域III，-99至-70）。区域I中-184处AP-1结合位点的突变使启动子的激活降低了85%。缺失区域II或区域III也使启动子活性降低了60%以上。一个跨越-184处AP-1基序的寡核苷酸特异性结合RKO细胞核因子，针对Jun-D、c-Jun或Fra-1蛋白的特异性抗体使复合物发生超迁移，表明这些蛋白的存在。在u-PAR基因仅被弱转录激活的GEO细胞中，这些因子的量减少。编码野生型Jun-D cDNA的载体的表达增加了GEO细胞中u-PAR启动子的活性。相反，用缺乏反式激活结构域的Jun-D表达构建体转染RKO细胞导致u-PAR启动子活性呈剂量依赖性降低。用佛波酯处理GEO细胞增加了u-PAR mRNA以及由野生型而非AP-1（-184）突变的u-PAR启动子驱动的CAT报告基因的活性，这与Jun-D、c-Jun和c-Fos量的强烈诱导相关。使用与来自RKO细胞、经佛波醇12-肉豆蔻酸酯13-乙酸酯处理和未处理的GEO细胞的核提取物蛋白结合的u-PAR启动子片段（跨越-201至-150）进行的甲基化干扰研究表明，接触点对应于-184处的AP-1结合位点。因此，在组成性产生该结合位点的RKO细胞以及佛波醇12-肉豆蔻酸酯13-乙酸酯刺激的GEO细胞中u-PAR的表达升高需要位于转录起始位点上游184 bp处的AP-1基序。
