CIS-REGULATORY ELEMENTS AND TRANS-ACTING FACTORS DIRECTING BASAL AND CAMP-STIMULATED HUMAN RENIN GENE-EXPRESSION IN CHORIONIC CELLS

Much knowledge was accumulated in the regulation of plasma renin activ ity and renin secretion during recent years. However, the mechanisms o f renin gene transcription, especially for the human gene, have been p oorly studied because of the lack of cell lines expressing renin. Cell s derived from chorion tissue were used to study renin gene transcript ion because these cells express renin and regulate renin secretion in a similar way to JG cells. The present study was performed to determin e the cis-regulatory elements and the trans-acting factors involved in human renin gene expression using chorionic cells. Transient DNA tran sfections were performed with various constructs containing the 5'-fla nking region of the human renin gene. 5'-Deletion analysis of the huma n renin promoter (from -2616 to -67 bp) revealed the presence of two p roximal negative cis-regulatory elements between -374 and -273 bp and between -273 and -137 bp. These elements were not present in a non-ren in-producing cell line, JEG-3 cells. DNase I footprinting revealed tha t two sequences located within these regions bind trans-factors presen t 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 spe cific expression in chorionic cells and contained two footprints shari ng homology with ets (-29/-6) and pituitary-specific factor (Pit-1) (- 70/-62) sequences. Furthermore, one footprint (-234/-214) contained th e sequence TAGCGTCA, which shares strong homology to the cAMP-responsi ve element (CRE) binding site. Gel shift analysis showed specific DNA/ protein complexes within this region, which were displaced by the soma tostatin consensus CRE. Finally, luciferase analysis of 5'-deletion mu tant revealed that -273 to +16 bp of the renin promoter was sufficient to confer complete forskolin stimulation, whereas deletion to -130 (d eletion of the CRE) decreased cAMP responsiveness by 50% and those to -67 bp (deletion of the CRE and Pit-1-like sequences) suppressed it. T hus, these latter two sequences probably act together to confer comple te cAMP responsiveness.