Structure of the 5'-flanking region of the rat prostaglandin F(2alpha) receptor gene and its transcriptional control functions in hepatocytes.
Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-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-3beta, 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-3beta 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.
Neuschäfer-Rube
F
F
Institut für Ernährungswissenschaft, Abteilung Biochemie der Ernährung, Universität Potsdam, Arthur-Scheunert-Allee 114-116, Bergholz-Rehbrücke, D-14558, Germany. fneusch@rz.uni-potsdam.de
Möller
U
U
Püschel
G P
GP
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D005091:Exons; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism*; D015850:Interleukin-6 / Q000502:physiology; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D011982:Receptors, Prostaglandin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics* / Q000502:physiology
D020121:5' Untranslated Regions; D015850:Interleukin-6; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D004247:DNA