The INSL3 gene is a direct target for the orphan nuclear receptor, COUP-TFII, in Leydig cells.
Insulin-like 3 (INSL3), a hormone produced by Leydig cells, regulates testicular descent during foetal life and bone metabolism in adults. Despite its importance, little is known about the molecular mechanisms controlling INSL3 expression. Reduced Insl3 mRNA levels were reported in the testis of mice deficient for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), an orphan nuclear receptor known to play critical roles in cell differentiation and lineage determination in several tissues. Although COUP-TFII-deficient mice had Leydig cell dysfunction and impaired fertility, it remained unknown whether Insl3 expression was directly regulated by COUP-TFII. In this study, we observed a significant decrease in Insl3 mRNA levels in MA-10 Leydig cells depleted of COUP-TFII. Furthermore, a -1087 bp mouse Insl3 promoter was activated fourfold by COUP-TFII in MA-10 Leydig cells. Using 5' progressive deletions, the COUP-TFII-responsive element was located between -186 and -79 bp, a region containing previously uncharacterised direct repeat 0-like (DR0-like) and DR3 elements. The recruitment and direct binding of COUP-TFII to the DR0-like element were confirmed by chromatin immunoprecipitation and DNA precipitation assay respectively. Mutation of the DR0-like element, which prevented COUP-TFII binding, significantly decreased COUP-TFII-mediated activation of the -1087 bp Insl3 reporter in CV-1 fibroblast cells but not in MA-10 Leydig cells. Finally, we found that COUP-TFII cooperates with the nuclear receptor steroidogenic factor 1 (SF1) to further enhance Insl3 promoter activity. Our results identify Insl3 as a target for COUP-TFII in Leydig cells and revealed that COUP-TFII acts through protein-protein interactions with other DNA-bound transcription factors, including SF1, to activate Insl3 transcription in these cells.
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6.
Raifish E
RE
Mendoza-Villarroel
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6.
Mickaël
M
Di-Luoffo
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6.
Etienne
E
Camiré
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6.
Xavier C
XC
Giner
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6.
Catherine
C
Brousseau
ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6ReproductionMother and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec, CHUL Room T3-67, 2705 Laurier Boulevard, Québec, City, Québec, Canada G1V 4G2Department of ObstetricsGynecology, and Reproduction, Faculty of Medicine, Centre for Research in Biology of Reproduction, Université Laval, Québec City, Québec, Canada G1V 0A6 Jacques-J.Tremblay@crchul.ulaval.ca.
Jacques J
JJ
Tremblay
D000818:Animals; D051837:COUP Transcription Factor II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D007328:Insulin / Q000235:genetics*; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017384:Sequence Deletion; D054339:Steroidogenic Factor 1 / Q000378:metabolism
D051837:COUP Transcription Factor II; D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D054339:Steroidogenic Factor 1; C515058:steroidogenic factor 1, mouse
Leydig cells; NR2F2; cooperation; insulin-like 3; nuclear receptor