TRANSCRIPTIONAL REGULATION OF HUMAN ADRENOMEDULLIN GENE IN VASCULAR ENDOTHELIAL-CELLS

Adrenomedullin (AM) is a recently identified vasodilator peptide produ ced in cardiovascular organs such as the heart, vascular wall, kidney and lung. Because plasma levels of AM are not different between variou s portions of the cardiovascular system, vascular cells are supposed t o be the main source of circulating AM. To elucidate the regulatory me chanism of human AM gene expression, functional elements of 5'-flankin g region of AM gene were studied in human aortic endothelial cells (HA EC). Northern blot analysis revealed considerable AM mRNA expression i n cultured HAEC, and 2 transcription start sites were recognized at 21 and 25 bp downstream from the TATA box. The 1534 bp 5'-flanking regio n DNA inserted in the luciferase vector showed significant promoter ac tivity when transfected into HAEC using liposome. Serial deletion of t he inserted 5'-flanking region DNA length resulted in reduction in pro moter activity by 41% between 110 and 90 bp and further reduction by 4 2% between 66 and 29 bp. The 19 bp DNA fragment without TATA box had a lmost no promoter activity. Gel shift assay revealed presence of HAEC nuclear proteins specifically bound to nuclear factor for interleukin- 6 expression (NF-IL6) consensus sequence existing from -85 to -93 bp o f the AM gene 5'-flanking region and activator protein 2 (AP-2) consen sus sequence clustered from -33 to -68 bp. Furthermore, mutation of NF -IL6 consensus sequence in the 5'-flanking region resulted in 42% redu ction in the expression of luciferase activity. These findings suggest that NF-IL6 and AP-2 sites in the promoter region are the functional elements in the transcriptional regulation of human AM gene in vascula r endothelial cells. (C) 1998 Academic Press.