Gevrey J-C, Malapel M, Philippe J, Mithieux G, Chayvialle J-A, Abello J, Cordier-Bussat M
INSERM Unité 45, IFR 62, Faculté de Médecine RTH Laënnec, Lyon, France.
Diabetologia. 2004 May;47(5):926-36. doi: 10.1007/s00125-004-1380-0. Epub 2004 Apr 14.
AIMS/HYPOTHESIS: Protein hydrolysates (peptones) increase not only glucagon-like peptide-1 (GLP-1) secretion but also transcription of the proglucagon ( PG) gene in the intestine. The critical physiological roles of gut-derived GLPs raised hope for their therapeutic use in several disorders, especially GLP-1 in diabetes. We aimed to investigate the molecular mechanisms involved in this nutrient- PG gene interaction.
Wild-type and mutated PG promoter fragments fused to the luciferase reporter gene were transfected into enteroendocrine STC-1 cells, which were then either treated or not with peptones. Co-transfection with expression vectors of dominant-negative forms of cAMP response element binding protein (CREB) and protein kinase A (PKA) proteins were performed, as well as electrophoresis mobility shift assays.
Deletion analysis showed that the promoter region spanning between -350 and -292 bp was crucial for the transcriptional stimulation induced by peptones. Site-directed mutagenesis of the canonical cAMP response element (CRE(PG)) and of the adjacent putative CRE site (CRE-like1) led to a dramatic inhibition of the promoter responsiveness to peptones. Over expression of a dominant-negative mutant of CREB or of PKA produced a comparable and selective inhibitory effect on the activity of transfected promoter fragment containing the -350/-292 sequence. EMSA showed that CREB and fra2 transcription factors bound to CRE(PG) and CRE-like1 elements respectively, independently of peptone treatment.
CONCLUSIONS/INTERPRETATION: Our report identified cis- and trans-regulatory elements implicated in the transcriptional control of PG gene by nutrients in enteroendocrine cells. It highlights the role of a previously unsuspected CRE-like1 element, and emphasises the importance of CRE-related sequences in the regulation of PG gene transcription in the intestine.
目的/假设：蛋白水解产物（蛋白胨）不仅能增加胰高血糖素样肽-1（GLP-1）的分泌，还能促进肠道中胰高血糖素原（PG）基因的转录。肠道源性GLP的关键生理作用为其在多种疾病中的治疗应用带来了希望，尤其是在糖尿病治疗中使用GLP-1。我们旨在研究这种营养素与PG基因相互作用的分子机制。
将与荧光素酶报告基因融合的野生型和突变型PG启动子片段转染至肠内分泌STC-1细胞，然后用蛋白胨处理或不处理这些细胞。进行了与环磷酸腺苷反应元件结合蛋白（CREB）和蛋白激酶A（PKA）蛋白的显性负性形式的表达载体的共转染，以及电泳迁移率变动分析。
缺失分析表明，-350至-292 bp之间的启动子区域对于蛋白胨诱导的转录刺激至关重要。对典型的环磷酸腺苷反应元件（CRE(PG)）和相邻的假定CRE位点（CRE-like1）进行定点诱变，导致启动子对蛋白胨的反应性显著抑制而戏剧性下降。CREB或PKA的显性负性突变体的过表达对含有-350/-292序列的转染启动子片段的活性产生了类似且选择性的抑制作用。电泳迁移率变动分析表明，CREB和fra2转录因子分别与CRE(PG)和CRE-like1元件结合，与蛋白胨处理无关。
结论/解读：我们的报告确定了肠内分泌细胞中营养素对PG基因转录控制所涉及的顺式和反式调节元件。它突出了一个先前未被怀疑的CRE-like1元件的作用，并强调了CRE相关序列在肠道中PG基因转录调控中的重要性。
