Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the
pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the
GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone
(FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly
correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements
containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/-
236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence
(TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been
previously shown to be involved in regulation of transcription of the mouse GnRHR
(mGnRHR) promoter in some cell lines, the role of site 3 has not been previously
investigated. This study investigated whether transcription of the mGnRHR gene is regulated
by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein
of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein-
DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1
site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of
investigation during this study. Luciferase reporter plasmids containing 600 bp of the
mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or
mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site
3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into
LβT2 cells was carried out to determine the effect of these mutations on the basal expression
of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional
activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of
basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site
resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2
cells. This suggests that the AP-1 site is involved in positively mediating the basal
transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the
understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR
promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins
involved and the effect of varying doses for varying times of GnRH, as well as the overexpression
of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not
involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA
response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic
mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings
were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was
found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it
could be the c-Jun protein that binds to the AP-1 site under basal conditions.
Another area that was investigated was whether the mGnRHR promoter can be regulated by
dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1
site, as shown by the complete loss of response when these sites were individually mutated,
consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells. / AFRIKAANSE OPSOMMING: Die gonadotropienvrystellings hormoon (GnRH) reseptor is ‘n G-proteïen-gekoppelde
reseptor in die pituitêre gonadotrope en is ’n belangrike beheerpunt vir reproduksie. GnRH
bind aan die GnRH reseptor (GnRHR) met die gevolg dat follikel stimulerende hormoon
(FSH) en luteïeniserende (LH) gesintetiseer en vrygestel word. Die sensitiwiteit van die
pituitêre klier vir GnRH kan direk met GnRHR vlakke gekorreleer word. Die muis GnRHR
promotor bevat drie cis elemente met bindingssetels vir steroïedogeniese faktor 1 (SF1),
naamlik setel 1 (-15/-7), setel 2 (-244/-236) en setel 3 (-304/-296) sowel as ’n aktiveerder
proteïen 1 (AP-1) tipe konsensus sekwens (TGAGTCA) in posisie -336/-330. Terwyl setels 1
en 2 en die AP-1 setel voorheen getoon is om by die regulering van transkripsie van die muis
GnRHR (mGnRHR) promotor in party sellyne betrokke te wees, is die rol van setel 3 nog nie
vantevore bestudeer nie. In hierdie studie is ondersoek of die transkripsie van die mGnRHR
geen deur GnRH en glukokortikoïede in die LβT2 gonadotroop pituitêre sellyn gereguleer
word, en die rol van setel 3 en die AP-1 setel en hulle binders, deur gebruik te maak van in
vitro proteïen-DNA bindings studies en promotor-verslaggewer essais. Die rol wat setel 3 en
die AP-1 setel in basale transkripsie van die mGnRHR gene in LβT2 selle gespeel het, was
die eerste onderwerp wat in hierdie studie bestudeer is. Lusiferase verslaggewer plasmiede
wat die eerste 600 bp van die mGnRHR promotor bevat het en waarin setel 3 en die AP-1
setels óf wilde tipe óf gemuteer was, is gebruik. Two konstrukte is vanaf die wilde tipe
konstruk berei, naamlik wilde tipe (LG), ’n setel 3 mutant (m3) en ’n AP-1 mutant (mAP-1).
Transfeksie van LG, m3 en mAP-1 plasmiede in LβT2 selle is deurgevoer om te bepaal wat
die effek van hierdie mutasies op die basale ekspressie van die mGnRHR gene was. Mutasie
van setel 3 het ’n 1.5-voudige toename in die transkripsionele aktiwiteit van die mGnRHR
promotor tot gevolg gehad. Dit suggereer dat setel 3 ’n rol in die inhibisie van die basale
transkripsievlakke van die mGnRHR promotor in LβT2 selle speel. Mutasie van die AP-1 setel
het tot ‘n 50% verlaging in basale transkripsievlakke van die mGnRHR promotor in LβT2 selle
gelei. Dit suggereer dat die AP-1 setel betrokke is in die positiewe bemiddeling van die basale
transkriptionele respons van die GnRHR promotor in LβT2 selle. Eksperimente wat gemik
was om die meganisme van die cis-elemente (setel 3 en die AP-1 setel) op die mGnRHR
promotor te verklaar, asook om die rol van proteïen kinase A (PKA) paaie, proteïene daarby
betrokke en die effek van varieende dosisse vir verskillende tye van GnRH, sowel as die
oorekspressie van PKA en die SF-1 proteïen, is deurgevoer. Dit is gevind dat setel 3 en die
AP-1 setel nie betrokke by die GnRH respons is nie. Die resultate suggereer dat setel 3
gedeeltelik betrokke is by die PKA respons van LβT2 selle. Setel 3 kan SF-1 proteïen bind
soos getoon deur kompeterence elektroforetiese mobiliteits verskuiwings essais (EMSA). As
EMSA’s deurgevoer is op die AP-1 setel is bevind dat die c-Fos proteïen nie betrokke is in die
aktivering van die AP-1 setel nie. ’n Faktor is gevind om aan die AP-1 setel te bind wat nie ’n
intakte AP-1 setel vereis het nie, wat gesuggereer het dat dit die c-Jun proteïen kan wees wat
aan die AP-1 setel onder basale omstandighede bind. ’n Ander area wat ondersoek is, is of die GnRHR promotor gereguleer kan word deur
deksametasoon (dex) óf via die AP-1 setel óf via setel 3. ’n Dosis en tyds-afhanklike toename
in promotor aktiwiteit is waargeneem met dex. ’n Vereiste vir hierdie effek blyk om die
teenwoordigheid van setel 3 en die AP-1 setel te wees, soos aangetoon deur die totale verlies
aan response as hierdie twee setels individueel gemuteer is, en wat weereens in
ooreenstemming met die funksionele interaksie tussen setel 3 en die AP-1 setel in LβT2 selle
is.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19595 |
| Date | 03 1900 |
| Creators | Fernandes, S. M. (Sandra Maria) |
| Contributors | Hapgood, J.P., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 97 leaves : ill. |
| Rights | Stellenbosch University |
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