title: Promoter function of different lengths of the murine luteinizing hormone receptor gene 5'-flanking region in transfected gonadal cells and in transgenic mice.
abstract: The purpose of these studies was to explore the sex- and tissue-specific expression of the LH receptor (LHR) gene. Fusion genes containing three different lengths of the 5'-flanking region of the mouse LHR gene (7.4 kb, 2.1 kb, and 173 bp), beta-globin intron, and the beta-galactosidase (beta-GAL) reporter gene were constructed. Function of these fusion genes [LHR (7.4 kb)/beta-GAL, LHR (2.1 kb)/beta-GAL, and LHR (173 bp)/beta-GAL] was studied in vitro and in vivo. beta-GAL expression was higher in transfected mouse Leydig (mLTC-1) than in granulosa (KK-1) tumor cells with all three constructs. The shortest LHR (173 bp)/beta-GAL construct showed the highest level of beta-GAL expression in both cell types. beta-GAL expression was clearly suppressed with the 2.1-kb promoter and was nearly undetectable with the 7.4-kb construct. In transgenic mice, all three constructs directed beta-GAL expression to adult Leydig cells, displaying decreasing intensity with increasing promoter length. Unexpectedly, beta-GAL expression was also found in elongating spermatids, but not in fetal Leydig cells. There was no expression in any ovarian cell type with the three constructs used, except that one of five mouse lines with the LHR (7.4 kb)/beta-GAL construct expressed beta-GAL in their thecal cells. Two lines transgenic for the 7.4- and 2.1-kb promoter constructs each directed high beta-GAL expression to the brain, with higher intensity in 7.4-kb lines. All promoters directed expression to the pituitary gland, some faintly to the adrenal gland. Northern hybridization analysis of the beta-GAL transcripts in Leydig cells revealed that the 173-bp promoter mainly gave rise to the full-length beta-GAL messenger RNA, whereas the 2.1- and 7.4-kb promoters mainly induced transcription of truncated beta-GAL messages. This suggests that the 5'-flanking region, upstream of -173, determines the formation of splice variants of the structural gene to be transcribed. The present findings in transgenic mice provide in vivo evidence for basal transcriptional activity of the first 173 bp upstream of the LHR translation initiation codon. In conclusion, the promoter function of the mouse LHR 5'-flanking region is tissue, age, and sex specific. The sequence upstream of the basal promoter determines extragonadal LHR expression as well as the alternate splicing of its message. The promoter sequences directing LHR expression to fetal Leydig cells and ovary reside outside the 7.4-kb 5'-flanking region and remain to be identified.
authors.affiliation: Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Hämäläinen
authors.forename: M
authors.initials: M
authors.lastname: Poutanen
authors.forename: I
authors.initials: I
authors.lastname: Huhtaniemi
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D006106:Granulosa Cell Tumor; D007984:Leydig Cell Tumor; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010053:Ovary / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D011974:Receptors, LH / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013087:Spermatids / Q000378:metabolism; D013799:Theca Cells / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D005914:Globins; D001616:beta-Galactosidase