Ma J X, Chao J, Chao L
Department of Biochemistry and Molecular Biology Medical University of South Carolina, Charleston 29425, USA.
Biochim Biophys Acta. 1996 Jul 17;1307(3):285-93. doi: 10.1016/0167-4781(96)81374-1.
Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor (serpin) which binds to and inhibits tissue kallikrein activity [1,2]. In this study, we have sequenced and identified two promoter regions of the RKBP gene (RKBP). One promoter is located in the 5' flanking region (P1) of the gene and the other is located in the first intron (P2). Both promoters contain a consensus TATA and CAAT box. These RKBP promoters were fused with a chloramphenicol acetyltransferase (CAT) reporter gene and their promoter activities were determined by measuring CAT levels using a specific ELISA. The P1 promoter exhibited high promoter activities in Hep3B hepatoma cells but not in La-fibroblastoma cells, indicating its tissue-specificity. By deletion analysis, we have identified a negative regulatory element of the P1 promoter between -739 and -472, and defined a minimal sequence between -183 and -2 for maintaining the intact promoter activity. The P2 promoter showed a strong activity only when linked to an SV40 enhancer. Activity of the P1 promoter can be induced by growth hormone in Hep3B cells. Gel retardation assay has identified 5 DNA fragments which were bound by nuclear proteins from rat liver. Two DNA fragments are in the 5' flanking region, one contains a putative glucocorticoid and growth hormone response element and the other one contains a CAAT box and two putative AP-1 binding sites. The remaining three are in the first intron and contain a putative thyroid hormone response element, a putative GATA site and three consensus CAAT boxes, respectively. Nuclear proteins from the kidney showed that spontaneously hypertensive rats (SHR) have a distinct trans-acting factor which binds with the DNA fragment containing the glucocorticoid and growth hormone response elements, as compared with normotensive rats. This result indicates that different trans-acting factors in the kidney of SHR may contribute to the decreased RKBP expression in these hypertensive rats.
大鼠激肽释放酶结合蛋白（RKBP）是一种丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），它能结合并抑制组织激肽释放酶的活性[1,2]。在本研究中，我们对RKBP基因（RKBP）的两个启动子区域进行了测序和鉴定。一个启动子位于基因的5'侧翼区域（P1），另一个位于第一个内含子（P2）。两个启动子都含有共有TATA盒和CAAT盒。这些RKBP启动子与氯霉素乙酰转移酶（CAT）报告基因融合，并通过使用特异性ELISA测量CAT水平来确定它们的启动子活性。P1启动子在Hep3B肝癌细胞中表现出高启动子活性，但在La-成纤维细胞瘤细胞中则没有，表明其组织特异性。通过缺失分析，我们在-739至-472之间鉴定出P1启动子的一个负调控元件，并确定了-183至-2之间维持完整启动子活性的最小序列。P2启动子仅在与SV40增强子连接时才显示出强活性。生长激素可诱导Hep3B细胞中P1启动子的活性。凝胶阻滞试验鉴定出5个与大鼠肝脏核蛋白结合的DNA片段。两个DNA片段位于5'侧翼区域，一个含有假定的糖皮质激素和生长激素反应元件，另一个含有CAAT盒和两个假定的AP-1结合位点。其余三个位于第一个内含子中，分别含有一个假定的甲状腺激素反应元件、一个假定的GATA位点和三个共有CAAT盒。来自肾脏的核蛋白显示，与正常血压大鼠相比，自发性高血压大鼠（SHR）具有一种独特的反式作用因子，它与含有糖皮质激素和生长激素反应元件的DNA片段结合。这一结果表明，SHR肾脏中不同的反式作用因子可能导致这些高血压大鼠中RKBP表达降低。
