Structure of the 5′-Flanking Region of the Rat Prostaglandin F2α Receptor Gene and Its Transcriptional Control Functions in Hepatocytes

Abstract

Prostaglandin F2α (PGF2α), modulates hepatocyte functions via a heptahelical Gq-coupled PGF2α-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5′-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5′-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1.4 kb intron containing the exons 1b and 1c coding for the 5′-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5′-RACE. A DNA-fragment covering the 5′-flanking region of the rFP-R gene from −1 of the transcription initiation site to −2590 bp was cloned and sequenced. Its 3′-two thirds had a 65% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3β, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5′-flanking region were transfected into rat hepatocytes. Luciferase expression ranked −181 ≥ −608 < −1418 > −1821 ≥ −2590. The strongest transcriptional activity was conferred by the region between −608 and −1418 containing a cluster of potential HNF-1 and HNF-3β binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the −2590 promoter fragment were reduced by IL-6 in hepatocytes.