Raychowdhury R, Zhang Z, Höcker M, Wang T C
Gastrointestinal Unit and the Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
J Biol Chem. 1999 Jul 23;274(30):20961-9. doi: 10.1074/jbc.274.30.20961.
The human histidine decarboxylase gene is regulated by gastrin through a cis-acting element known as the gastrin response element (GAS-RE) that was initially localized to a site (+2 to +24) downstream of the transcriptional start site. Electrophoretic mobility shift assays using sequentially deleted DNA probes and nuclear extracts from AGS-B gastric cancer cells showed that the GAS-RE is actually composed of two overlapping binding sites (GAS-RE1, +1 to +19; and GAS-RE2, +11 to +27) that bind distinct nuclear factors. Reporter gene assays demonstrated that each element alone could confer gastrin responsiveness, but the presence of both elements was required for complete gastrin response. Stimulation of AGS-B cells with gastrin for 10-20 min resulted in a >2-fold increase in factor binding. The binding was inhibited by pretreatment of AGS-B cells with cycloheximide and the MEK1 inhibitor PD98059, indicating a requirement for protein synthesis and also indicating that activation occurs through the MEK/mitogen-activated protein kinase pathway. UV cross-linking and Southwestern blot analysis showed that GAS-RE1 bound a 52-kDa protein, whereas GAS-RE2 bound a 35-kDa protein. Hence, activation of histidine decarboxylase gene promoter activity by gastrin is most likely mediated by two separate nuclear factors.
人组氨酸脱羧酶基因受胃泌素通过一种称为胃泌素反应元件（GAS-RE）的顺式作用元件调控，该元件最初定位于转录起始位点下游的一个位点（+2至+24）。使用来自AGS-B胃癌细胞的连续缺失DNA探针和核提取物进行的电泳迁移率变动分析表明，GAS-RE实际上由两个重叠的结合位点（GAS-RE1，+1至+19；和GAS-RE2，+11至+27）组成，它们结合不同的核因子。报告基因分析表明，单独的每个元件都可以赋予胃泌素反应性，但完整的胃泌素反应需要两个元件都存在。用胃泌素刺激AGS-B细胞10至20分钟导致因子结合增加2倍以上。用放线菌酮和MEK1抑制剂PD98059预处理AGS-B细胞可抑制这种结合，这表明需要蛋白质合成，也表明激活是通过MEK/丝裂原活化蛋白激酶途径发生的。紫外线交联和蛋白质印迹分析表明，GAS-RE1结合一种52 kDa的蛋白质，而GAS-RE2结合一种35 kDa的蛋白质。因此，胃泌素对组氨酸脱羧酶基因启动子活性的激活很可能是由两个独立的核因子介导的。
