Prorenin and gene activation

Abstract

After the discovery of an inactive, putative renin precursor that could be proteolytically activated, and the proteases involved in vivo, Morris and co-workers directly demonstrated that renin is indeed synthesized as a "pro" form, and from genetic coding sequences they provided the structure of human prorenin. The gene is inactive and must be activated in prorenin-synthesizing tissues. To study the mechanism involved, we have performed transient expression analyses of putative regulatory DNA of the human gene (REN). 5′-Flanking DNA, extending from residue −144 to −2400, was linked to a reporter gene, viz. that for chloramphenicol acetyl transferase (CAT), and its ability to drive a heterologous (thymidine kinase, tk) promoter was examined by transfecting plasmid constructs into ceils in culture and measuring CAT activity 48 h later. Because suitable renin-synthesizing cells were not available, choriocarcinoma (JEG-3) and cervical carcinoma (HeLa) cells were used. Although this DNA caused a reduction in CAT activity relative to the positive control, examination of a range of subfragments suggested that the −2400 to −144 region did not contain negative regulatory elements. In contrast, all fragments containing the −149 to +13 DNA segment gave CAT activities that were lower than the promoterless control. Together, the data were consistent with the presence of negative regulatory element(s) in that fragment of DNA that contained the REN promoter. On the basis of mouse gene studies, it has been suggested that the inactivity of the renin gene in tissues that do not synthesize prorenin is not due to repression, but rather, cells that do express the gene may possess unique trans-acting factor(s) that stimulate enhancer(s) in the renin DNA, therefore activating the renin promoter. Nuclear extracts of JEG-3, but not HeLa, cells contained a binding activity for the −340 to −192 segment, but the relationship, if any, of this to the model proposed is unclear. Thus, having demonstrated the synthesis, structure, and activation of prorenin, the mechanism of activation of the gene represents the next challenge.Key words: gene regulation, transient expression assay, JEG-3 ceils, HeLa cells, prorenin.