Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site

Reproductive Biology and Endocrinology

Research

Chanho Lee¹  Brett R White¹  Rebecca A Cederberg¹  Amy R Perkins²  Emily A McDonald³  Jacqueline E Smith⁴ 

[1] Laboratory of Reproductive Biology, Department of Animal Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, USA;Laboratory of Reproductive Biology, Department of Animal Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, USA;Current address: Arizona Andrology Laboratory and Cryobank, Tuscon, AZ, USA;Laboratory of Reproductive Biology, Department of Animal Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, USA;Current address: Center for International Health Research, Rhode Island Hospital, Providence, RI, USA;Laboratory of Reproductive Biology, Department of Animal Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, USA;Current address: Stowers Institute for Medical Research, Kansas City, MO, USA;

关键词: GnRH;    GnRH receptor;    Transcriptional regulation;    Gene expression;    Gonadotrope specific element;    Steroidogenic factor 1;    Retinoid X receptor;    Anterior pituitary;    Porcine;   

DOI  :  10.1186/s12958-015-0033-0

 received in 2014-11-21, accepted in 2015-04-16,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundRegulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Binding of GnRH to its receptor regulates follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and levels of this G-protein coupled receptor on the surface of gonadotropes determines their sensitivity to GnRH pulses. While transcriptional regulation of this gene has been studied in mice, rats, humans and sheep, little is known about its regulation in the pig, an important agricultural species and human research model.MethodsWe isolated 5118 bp of 5′ flanking sequence for the porcine GnRHR gene and generated luciferase reporter vectors. Deletion and mutation constructs were evaluated in gonadotrope-derived alphaT3-1 cells to determine regions important for gene transcription. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to identify transcription factors binding to the GnRHR promoter.ResultsTransient transfections revealed that the GnRHR promoter was functional in alphaT3-1 cells but not in cells of non-gonadotrope origin. Mutation of the highly conserved gonadotrope specific element (GSE) located at -179/-171 of proximal promoter completely ablated luciferase activity, whereas mutation of another GSE at -315/-310 reduced activity by 34%. Consistent with this, EMSAs using alphaT3-1 nuclear extracts and a steroidogenic factor (SF)1 antibody confirmed SF1 binding to both GSEs. EMSAs also demonstrated that a retinoid X receptor (RXR) binding site at -279/-274 binds RXRalpha and RXRbeta and mutation of this site eliminated promoter activity. Transient transfection of alphaT3-1 cells with reporter vectors containing selective removal of 5′ flanking region for the porcine GnRHR gene indicated that the -1915/-1431 segment was important for promoter activity. Definition of this region via transfection assays and EMSAs revealed an upstream enhancing region located at -1779/-1667 that increases porcine GnRHR gene expression in alphaT3-1 cells and includes a SF1 binding site at -1760/-1753.ConclusionsPorcine GnRHR promoter activity in alphaT3-1 cells is partially conferred by a distal GSE, two proximal GSEs and a RXR binding site. Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity.

【 授权许可】

Unknown   
© Cederberg et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.