Waeber G, Thompson N, Nicod P, Bonny C
Department of Internal Medicine B, University Hospital, Lausanne, Switzerland.
Mol Endocrinol. 1996 Nov;10(11):1327-34. doi: 10.1210/mend.10.11.8923459.
The homeodomain protein PDX-1, referred as IPF-1/STF-1/IDX-1, is a transcriptional factor that plays a critical role in the control of several genes expressed in the pancreatic islet. PDX-1 gene expression has been previously shown to be reduced in cultured beta-cell lines chronically exposed to high glucose concentrations. As the glucose transporter type 2 (GLUT2) gene expression is selectively decreased in the beta-pancreatic cells of experimental models of diabetes, we postulated that the loss of GLUT2 gene expression in the pancreatic islets of diabetic animals may be due to the loss of PDX-1 transacting function on the GLUT2 gene. We, therefore, investigated the potential role of PDX-1 in the transcriptional control of GLUT2. We have identified a repeat of a TAAT motif (5'-TAATA-ATAACA-3') conserved in the sequence of the human and murine GLUT2 promoters. Recombinant PDX-1 binds to this GLUT2TAAT motif in electrophoretic mobility shift experiments. PDX-1 antiserum detects the formation of the complex of PDX-1 with the GLUT2TAAT motif in nuclear extracts from the pancreatic insulin-secreting cell line, beta TC3. The GLUT2TAAT motif was mutated in the murine GLUT2 promoter (-1308/+49 bp) linked to a luciferase reporter gene and transfected into beta TC3 cells. Compared with the transcriptional activity of the wild type promoter, that of the mutated promoter decreases by 41%. Multiple copies of the GLUT2TAAT motif were ligated 5' to a heterologous promoter and transfected into a PDX-1-expressing cell line (beta TC3) and into cell lines lacking the homeobox factor (InR1-G9 and JEG-3). The GLUT2TAAT motif mediates the activation of the heterologous promoter in the PDX-1-expressing cell line but not in InR1-G9 or JEG-3 cell lines. Furthermore, cotransfection in a PDX-1-deficient cell line with the expression vector encoding PDX-1 transactivates specifically the heterologous promoter containing the multimerized GLUT2TAAT motif. These data demonstrate that the murine GLUT2 promoter is controlled by the PDX-1 homeobox factor through the identified GLUT2TAAT motif.
同源结构域蛋白PDX-1，也被称为IPF-1/STF-1/IDX-1，是一种转录因子，在调控胰岛中表达的多个基因方面发挥着关键作用。先前的研究表明，在长期暴露于高葡萄糖浓度的培养β细胞系中，PDX-1基因的表达会降低。由于在糖尿病实验模型的胰腺β细胞中，葡萄糖转运蛋白2（GLUT2）基因的表达选择性降低，我们推测糖尿病动物胰岛中GLUT2基因表达的丧失可能是由于PDX-1对GLUT2基因的反式作用功能丧失所致。因此，我们研究了PDX-1在GLUT2转录调控中的潜在作用。我们在人和小鼠GLUT2启动子序列中鉴定出一个保守的TAAT基序重复序列（5'-TAATA-ATAACA-3'）。在电泳迁移率变动实验中，重组PDX-1与这个GLUT2TAAT基序结合。PDX-1抗血清可检测到在胰腺胰岛素分泌细胞系β TC3的核提取物中，PDX-1与GLUT2TAAT基序形成复合物。在与荧光素酶报告基因相连的小鼠GLUT2启动子（-1308/+49 bp）中，GLUT2TAAT基序发生突变，并转染到β TC3细胞中。与野生型启动子的转录活性相比，突变型启动子的转录活性降低了41%。将多个GLUT2TAAT基序拷贝连接到异源启动子的5'端，并转染到表达PDX-1的细胞系（β TC3）以及缺乏同源框因子的细胞系（InR1-G9和JEG-3）中。GLUT2TAAT基序在表达PDX-1的细胞系中介导异源启动子的激活，但在InR1-G9或JEG-3细胞系中则不然。此外，在缺乏PDX-1的细胞系中，与编码PDX-1的表达载体共转染可特异性激活含有多聚化GLUT2TAAT基序的异源启动子。这些数据表明，小鼠GLUT2启动子受PDX-1同源框因子通过已鉴定的GLUT2TAAT基序调控。
