Olsen Line, Bressendorff Simon, Troelsen Jesper T, Olsen Jorgen
Dept. of Medical Biochemistry and Genetics, University of Copenhagen, The Panum Institute Bldg. 6.4. Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Am J Physiol Gastrointest Liver Physiol. 2005 Aug;289(2):G220-6. doi: 10.1152/ajpgi.00449.2004. Epub 2005 Apr 14.
The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.
肠碱性磷酸酶基因（ALPI）编码一种消化性刷状缘酶，在小肠上皮细胞分化过程中高度上调。为了确定在肠上皮细胞分化过程中负责调控ALPI表达的新的假定启动子基序，我们对人ALPI近端启动子序列进行了计算机辅助顺式元件搜索。相对于翻译起始位点，在-94至-82位置预测到转录因子肝细胞核因子（HNF）-4的一个假定识别位点。在非肠道的Hela细胞系中研究了HNF-4α刺激ALPI启动子表达的能力。与HNF-4α表达载体共转染证明通过这个-94至-82元件直接激活了ALPI启动子。电泳迁移率变动分析表明，来自分化的肠上皮细胞（Caco-2）核提取物中的HNF-4α与预测的HNF-4结合位点具有高亲和力结合。含有HNF-4结合位点的521 bp启动子片段在Caco-2细胞中显示出启动子活性的分化依赖性增加。这种增加的发生需要HNF-4结合位点的存在。
