Thesis (PhD)--Stellenbosch University, 2011. / See also the post-print version of the article that was published from the PhD - http://hdl.handle.net/10019.1/19557 / ENGLISH ABSTRACT: Glucocorticoid receptor (GR) levels, which modulate the response to glucocorticoids (GCs), vary
between tissues and individuals and are altered by physiological and pharmacological effectors. In
this study we set out to investigate the effects and implications of differences in GR concentration.
Firstly, we established conditions that resulted in three statistically different GR populations in
transiently transfected COS-1 cells. We demonstrated, using whole cell saturation ligand binding
experiments, that high levels of wild type GR, but not of dimerization deficient GR, exhibited positive
cooperative ligand binding with a concomitant increased ligand binding affinity. Furthermore, we
established, through co-immunoprecipitation and fluorescent resonance energy transfer, that ligand
independent dimerization correlates with positive cooperative ligand binding. This is the first time that
positive cooperative ligand binding and increased ligand binding affinity have been explicitly correlated
and linked to increased ligand independent dimerization of the GR. The downstream consequences of
variation in GR concentration and dimerization included modulation of GR import and export rates, as
investigated through live cell as well as immunofluorescent analysis. Furthermore, the nuclear
distribution of GR was also influenced by GR dimerization. The major function of the GR is as a
transcription factor, which mediates the response to GCs via activation or repression of genes. We
have revealed direct influences of GR concentration and dimerization in a number of promoter reporter
assays as well as in the transactivation of an endogenous gene. Specifically, cooperative ligand
binding was found to be responsible for the GR level dependent potency shift in transrepression of an
NF B containing promoter reporter construct via dexamethasone and the shift in the bio-character of
Compound A, a dissociative GR agonist. Transactivation potency of dexamethasone as well as the
partial agonist bio-character of medroxyprogesterone and mifepristone via a multiple GRE containing
promoter reporter construct were influenced directly by cooperative ligand binding. Dimerization of the
GR was shown to be crucial for ligand dependent transactivation of a single GRE containing promoter
reporter construct, while ligand independent transactivation of both single and multiple GRE containing
constructs was significantly increased due to an increase in GR concentration. The endogenous GC
responsive glucocorticoid induced leucine zipper (GILZ) gene demonstrated significant ligand
independent transactivation at GR levels, which displayed ligand independent dimerization. An
increase in GR concentration resulted in an increase in efficacy through all promoter reporter
constructs as well as the endogenous GILZ gene. Positive cooperative binding and the concomitant
increase in ligand binding affinity to the GR at high levels may be a crucial factor in determining both
the efficacy and potency of the GC response. Considering the significant differences in GR
concentrations expressed by different tissues and by individuals within the same tissue, our findings
may explain the interindividual as well as tissue specific responses to GC treatment and suggest an
important mechanism of action through which the GR is primed to responsed to subsaturating GC
concentrations and displays a significant level of ligand independent activity. / AFRIKAANSE OPSOMMING: Glukokortikoïed reseptor (GR) vlakke, wat die gedrag van glukokortikoïede (GCs) moduleer, wissel
tussen weefsels en onder individue en word verander deur fisiologiese en farmakologiese effektore. In
hierdie studie ondersoek ons die gevolge en implikasies van verskille in GR konsentrasie. Eerstens
het ons die kondisies vasgestel wat benodig word om drie statisties verskillende GR populasies te
vestig in kortstondige getransfekteerde COS-1 selle. Ons het getoon, met behulp van die heel sel
versadigings ligand bindings eksperimente, dat hoë vlakke van wilde-tipe GR, maar nie van
dimeriserings gebrekkige GR, positiewe koöperatiewe ligand binding, met 'n gepaardgaande toename
in ligand bindings affiniteit, toon. Verder het ons bevestig, deur ko-immunopresipitasie en fluoressente
resonansie energie-oordrag, dat ligand onafhanklike dimerisering korreleer met positiewe
koöperatiewe ligand binding. Dit is die eerste keer dat positiewe koöperatiewe ligand binding en
verhoogde ligand bindings affiniteit uitdruklik gekorreleer en gekoppel word aan verhoogde ligand
onafhanklike dimerisering van die GR. Die daarop nagevolge van variasie in GR konsentrasie en
dimerisering sluit in modulasie van die invoer en uitvoer tempo van die GR, soos ondersoek deur
lewendige sel sowel as immunofluorescente analise. Verder is die verspreiding van die GR in die kern
ook beïnvloed deur GR dimerisering. Die belangrikste funksie van die GR is as 'n transkripsie faktor,
wat die respons van GCS bemiddel via aktivering of onderdrukking van gene. Ons het die direkte
invloed van GR konsentrasie en dimerisering in 'n aantal promotor verslaggewer essais sowel as in
die transaktivering van endogene gene onthul. Spesifiek, is gevind dat koöperatiewe ligand binding
verantwoordelik is vir die GR vlak afhanklike verskuiwing in transrepressie potensie van 'n NF B
bevattende promotor verslaggewer konstruk via deksametasoon en die verskuiwing van die biokarakter
van verbinding A,' dissosiatiewe GR agonis. Transaktiverings potensie van
deksametasoon, asook die gedeeltelike agonis bio-karakter van medroksie-progesteroon en
mifepristoon, via 'n veelvoudige GRE bevattende promotor verslaggewer konstruk is direk beïnvloed
deur koöperatiewe ligand binding. Dimerisering van die GR is getoon om deurslaggewend vir ligand
afhanklike transaktivering van 'n enkele GRE bevattende promotor verslaggewer konstruk te wees,
terwyl ligand onafhanklike transaktivering van beide enkel-en veelvoudige GRE bevattende konstrukte
aansienlik toegeneem het as gevolg van toename in GR konsentrasie. Die endogene GC
responsiewe glukokortikoïed geïnduseerde leusien rits (GILZ) gene het beduidende ligand
onafhanklike transaktivering gedemonstreer op GR vlakke wat ligand onafhanklike dimerisering toon.
'n toename in GR konsentrasie het gelei tot toename in die effektiwiteit van al die promotor
verslaggewer konstrukte, sowel as die endogene GILZ gene. Positiewe koöperatiewe ligand binding
en die gepaardgaande toename in ligand bindings affiniteit van die GR by hoë vlakke kan 'n
belangrike faktor wees in die bepaling van sowel die effektiwiteit as die potensie van die GC respons. As die aansienlike verskille in GR konsentrasies van verskillende weefsels en tussen verskillende
individue in dieselfde weefsel in ag geneem word, kan ons bevindings die inter-individuele sowel as
weefsel spesifieke response op GC behandeling verduidelik en stel dit 'n belangrike meganisme van
aksie voor waardeur die GR voorberei word om op sub-versadigings konsentrasies van GC te reageer
deur 'n beduidende vlak van ligand onafhanklike aktiwiteit te toon.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6885 |
| Date | 03 1900 |
| Creators | Robertson, Steven Ernest |
| Contributors | Louw, Ann, Hapgood, Janet, University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xv, 200 p. : ill. (some col.) |
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