cis-regulatory elements and trans-acting factors directing basal and cAMP-stimulated human renin gene expression in...

Much knowledge was accumulated in the regulation of plasma renin activity and renin secretion during recent years. However, the mechanisms of renin gene transcription, especially for the human gene, have been poorly studied because of the lack of cell lines expressing renin. Cells derived from chorion tissue were used to study renin gene transcription because these cells express renin and regulate renin secretion in a similar way to JG cells. The present study was performed to determine the cis-regulatory elements and the trans-acting factors involved in human renin gene expression using chorionic cells. Transient DNA transfections were performed with various constructs containing the 5'-flanking region of the human renin gene. 5'-Deletion analysis of the human renin promoter (from -2616 to -67 bp) revealed the presence of two proximal negative cis-regulatory elements between -374 and -273 bp and between -273 and -137 bp. These elements were not present in a non-renin-producing cell line, JEG-3 cells. DNase I footprinting revealed that two sequences located within these regions bind trans-factors present in chorionic cellular nuclear extract: AGE3-like sequence (-293/-273) and apolipoprotein A1 regulatory protein-1-like sequence (-259/-245). The first 110 bp of the renin promoter were sufficient to direct specific expression in chorionic cells and contained two footprints sharing homology with ets (-29/-6) and pituitary-specific factor (Pit-1) (-70/-62) sequences. Furthermore, one footprint (-234/-214) contained the sequence TAGCGTCA, which shares strong homology to the cAMP-responsive element (CRE) binding site. Gel shift analysis showed specific DNA/protein complexes within this region, which were displaced by the somatostatin consensus CRE. Finally, luciferase analysis of 5'-deletion mutant revealed that -273 to +16 bp of the renin promoter was sufficient to confer complete forskolin stimulation, whereas deletion to -130 (deletion of the CRE) decreased cAMP responsiveness by 50% and those to -67 bp (deletion of the CRE and Pit-1-like sequences) suppressed it. Thus, these latter two sequences probably act together to confer complete cAMP responsiveness.