The role of steroidogenic factor-1 (SF-1) in transcriptional regulation of the gonadotropin-releasing hormone (GnRH) receptor gene

Styger, Gustav (2001-03)

Thesis (MSc)--Stellenbosch University, 2001.

Thesis

ENGLISH ABSTRACT: The GnRH receptor is a G-protein-coupled receptor in pituitary gonadotrope cells. Binding of its ligand, GnRH, results in synthesis and release of gonadotropin hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH). Steroidogenic factor 1 (SF-1), a transcription factor, binds to specific sites in the promoter region of gonadotropin genes, and thus regulates transcription of these genes. The promoter region of the GnRHreceptor gene contains two SF-1-like binding sites, one at -14 to -8 (site 1) and another at -247 to -239 (site 2), relative to the methionine start codon. The role played by these two SF-1-like sites in basal transcription of the mouse GnRH receptor (mGnRH-R) gene in a pituitary precursor gonadotrope cell line, aT3 cells, was the first area of investigation during this study. Luciferase reporter constructs containing 580 bp of mGnRH-R gene promoter were prepared, where SF-1-like sites were either wildtype or mutated. Four such constructs were made, i.e. wildtype (LG), site 1 mutant (LGM1), site 2 mutant (LGM2) and mutated site 1 plus site 2 (LGM1/2). These constructs were transfected into aT3 cells to determine the effect of mutations of sites 1 and/or 2 on the basal expression of the mGnRH-R gene. Mutation of either site 1 or site 2 had no effect on basal expression of the mGnRH-R gene. It was found that only upon simultaneous mutation of both sites 1 and 2, a 50% reduction in basal transcription took place. The implications of this is that SF-1 protein seems to only require one intact DNA-binding site, to mediate basal transcription of the mGnRH-R gene, suggesting that these two sites lie in close proximity during basal transcription. The effect of the protein kinase A (PKA) pathway on the endogenous mGnRH-R gene was also investigated by incubating non- , transfected aT3 cells with the PKA activators, forskolin and 8-Br-cAMP. Similar incubations were also performed on the wild type and mutated site 1 constructs transfected into pituitary gonadotrope aT3 cells. It was found that forskolin and 8-Br-cAMP were able to increase endogenous mGnRH-R mRNA levels in a concentration-dependent fashion, showing that endogenous GnRH receptor gene expression is stimulated via a protein kinase A pathway. Similar results were obtained with the wildtype promoter construct, showing that the protein kinase A pathway stimulates transcription of the promoter. This effect was only seen with wild type and not with the mutated site 1. These results are consistent with a role for a SF-1-like transcription factor in mediating the protein kinase A effect via binding to the site 1 at position -14 in the GnRH receptor gene. A separate investigation was performed to determine whether 25-hydroxycholesterol (25-0HC) is a ligand for SF-1, by incubating aT3 cells transfected with the various constructs with 25-0HC. Results show a dose-dependant response, with an increase in gene expression at 1 μM and a decrease at higher concentrations, for both mutant and wild type constructs. This suggests that, if SF-1 is indeed the protein binding to sites 1 and 2, then 25-0HC is not a ligand for SF-1 protein in aT3 cells and that the effect of 25-0HC on the mGnRH-R gene is not mediated via site 1. The results indicate that these decreases of expression at the higher concentrations may be due to cytotoxic effects. Towards the end of the study the laboratory obtained a luminoskan instrument with automatic dispensing features. Optimisation studies on the luciferase and β-Gal assays were performed on the luminoskan in a bid to decrease experimental error. It was found that automation of these assays resulted in a decrease in experimental error, showing that future researchers could benefit substantially from these optimisation studies.

AFRIKAANSE OPSOMMING: Die GnRH reseptor is 'n G proteïen-gekoppelde reseptor in pituitêre gonadotroopselle. Binding van die ligand, GnRH, lei tot die sintese en vrystelling van die gonadotropien hormone, luteïniserende hormoon (LH) en follikel stimulerende hormoon (FSH). Steroidogeniese faktor-t (SF-1) is 'n transkripsie faktor wat aan spesifieke areas in die promotergebied van die gonadotropien hormone bind, en dus transkripsie van hierdie gene reguleer. Die promotergebied van die GnRH reseptor geen bevat twee SF-1 bindings areas, een by -14 to -8 (area 1) asook by -247 to -239 (area 2), relatief to die metionien beginkodon. Die rol wat hierdie twee SF-1 areas speel in basale transkripsie van die muis GnRH reseptor (mGnRH-R) geen in 'n pituïtêre voorloper gonadotroop sellyn, aT3 selle, was die eerste gebied van ondersoek gedurende hierdie studie. Plasmiede bestaande uit die 580 basispaar mGnRH-R promoter verbind aan 'n lusiferase geen is vervaardig, waar SF-1-soortige areas enersyds onveranderd gelaat is, of gemuteer is. Vier sulke plasmiede is vervaardig, nl. onveranderd (LG), area 1 mutant (LGM1), area 2 mutant (LGM2) en gemuteerde area 1 plus area 2 (LGM1/2). Hierdie plasmiede is gebruik om aT3 selle te transfekteer om die effek van mutasies van areas 1 en/of 2 op die basale ekspressie van die mGnRH-R geen te ondersoek. Daar is gevind dat mutasies van areas 1 of 2 geen effek op basale ekspressie op die bogenoemde geen gehad het nie. Slegs tydens gelyktydige mutasie van areas 1 en 2 het 'n 50% vermindering in basale transkripsie plaasgevind. Die implikasies hiervan is dat die SF-1 proteïen blykbaar slegs een volledige DNA-bindingsarea benodig om basale transkripsie van die mGnRH-R geen te reguleer. Dit wil dus voorkom of hierdie twee areas baie na aan mekaar geposisioneer is tydens basale transkripsie. Die effek van die proteïen kinase A (PKA) roete op die natuurlike mGnRH-R geen is ook ondersoek tydens inkubasie van nie-getransfekteerde aT3 selle met die PKA akiveerders, forskolin en 8-Br-cAMP. Soortgelyke inkubasie is ook gedoen op die onveranderde en gemuteerde area 1 plasmiede wat in aT3 selle getransfekteer is. Daar is gevind dat forskolin en 8-Br-cAMP daarin geslaag het om die natuurlike mGnRH-R geen mRNA vlakke op 'n konsentrasie-afhanklike wyse te vermeerder. Hierdie resultaat dui daarop aan dat die natuurlike mGnRH-R geen se ekspressie gestimuleer kan word via 'n proteïen kinase A roete. Soortgelyke resultate is verkry met die onveranderde promoter plasmied en dit wys ook daarop dat proteïen kinase A transkripsie deur die promoter kan stimuleer. Hierdie effek was slegs aanwesig met die onveranderde en nie met die gemuteerde area 1 plasmied nie. Die resultate stem ooreen met 'n rol vir SF-1 transkripsie faktor in die regulering van proteren kinase A effek deur middel van binding aan die area 1 by posisie -14 in die GnRH-R geen. 'n Afsonderlike ondersoek is gedoen om vas te stel of 25-hidroksiecholesterol (25-0HC) 'n ligand vir SF-1 is deur getransfekteerde aT3 selle met 25-0HC te inkubeer. Resultate toon 'n dosis-afhanklike respons met 'n verhoging in geen ekspressie by 1 μM en 'n verlaging met hoër konsentrasies vir beide onveranderde en gemuteerde plasmiede. Dit impliseer dat, indien SF-1 wel die faktor is wat aan areas 1 en 2 bind, 25-0HC nie die ligand vir SF-1 proteren in aT3 selle is nie en dat die effek van 25-0HC op die mGnRH-R geen nie gereguleer word via area 1 nie. Die verlaging in ekspressie gevind by die hoër konsentrasies is dalk die gevolg van sitotoksiese effekte. Teen die einde van die studie het die laboratorium luminoskan toerusting met outomatiese pipettering verkry. Optimiseringstudies van die lusifirase en β-Galtoetse is met die luminoskan gedoen in 'n poging om eksperimentele foute te minimaliseer. Daar is gevind dat outomatisering van hierdie toetse wel gelei het tot 'n verlaging in eksperimentele foute. Toekomstige navorsers kan dus grootliks voordeel trek uit hierdie optimiseringstudies.

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