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

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(1)PRE1 (-113 to -102 bp), AT(1)PRE2 (-49 to -43 bp), AT(1)PRE3 (-5 to -2 bp) and AT(1)PRE4 (+44 to +50 bp) were identified. AT(1)PRE2 contained a GC-box-like sequence and bound to Spl. AT(1)PRE1 contained two tandem GC-boxes and was bound to several nuclear proteins in addition to Spl. Nuclear proteins that were bound sequence-specifically to AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 were found in both PLC-PRF-5 cells and 8505C cells, while those bound to AT(1)PRE3 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(1)PRE1, AT(1)PRE2 and AT(1)PRE4 were all mutated. Mutagenesis of AT(1)PRE3, however, substantially inactivated promoter activity. AT(1)PRE(1), AT(1)PRE2 and AT(1)PRE4 synergistically enhanced AT(1) gene transcription promoted by AT(1)PRE3. These results suggested that AT(1)PRE3 is responsible for the tissue-specific expression of the human AT(1) gene, and that AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 function as a general enhancer in liver-derived cells.