Identification and Synergism of cis-acting Elements Essential for Basal Promoter Activity of the Human Type 1 Angiotensin II Receptor Gene in PLC-PRF-5 Cells

摘要

The basal promoter activity of the human AT_1 receptor gene was characterized using a human hepatoma cell line with a considerably high expression of AT_1, PLC-PRF-5. Four cis-acting, positively regulating elements termed AT_1PRE1 (-113 to -102 bp), AT_1PRE2 (-49 to -43 bp), AT_1PRE3 (-5 to -2 bp) and AT_1PRE4 (+44 to +50 bp) were identified. AT_1PRE2 contained a GC-box-like sequence and bound to Sp1. AT_1PRE1 contained two tandem GC-boxes and was bound to several nuclear proteins in addition to Sp1. Nuclear proteins that were bound sequence-specifically to AT_1PRE1, AT_1PRE2 and AT_1PRE4 were found in both PLC-PRF-5 cells and 8505C cells, while those bound to AT_1PRES were not found in 8505C cells, which showed no expression of AT_1 and almost no promoter activity for the AT_1 gene. Significant promoter activity was still observed even when AT_1PRE1, AT_1PRE2 and AT_1PRE4 were all mutated. Mutagenesis of AT_1PRE3, however, substantially inactivated promoter activity. AT_1PRE1, AT_1PRE2 and AT_1PRE4 synergistically enhanced AT_1 gene transcription promoted by AT_1PRE3. These results suggested that AT_1PRE3 is responsible for the tissue-specific expression of the human AT_1 gene, and that AT_1PRE1, AT_1PRE2 and AT_1PRE4 function as a general enhancer in liver-derived cells.