Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Although the progestins medroxyprogesterone acetate (MPA) and
norethisterone acetate (NET-A) are widely used in reproductive therapy, the steroid
receptors and their target genes involved in the actions of MPA and NET-A are not
well understood. Surprisingly, it had not yet been investigated whether doses of MPA
and NET-A used for contraception and HRT cause significant side effects through
various target genes via the glucocorticoid receptor (GR).
In this thesis results of in vitro studies showed that, MPA, like dexamethasone
(dex) and prog, significantly repressed tumour necrosis factor (TN F)-stimulated IL-6
protein production, and IL-6 and IL-8 promoter reporter constructs at the
transcriptional level in L929sA cells, via interference with nuclear factor KB (NFKB)
and activator protein-1 (AP-1) transcription factors. Like dex and prog, MPA did not
affect NFKB DNA-binding activity. Furthermore, unlike dex and prog, MPA did not
inhibit mitogen-activated protein kinase (MAPK) activity. The antagonistic effects of
the GR and progesterone receptor (PR) antagonist, RU486, as well as the MPAinduced
nuclear translocation of the GR, strongly suggest that the actions of MPA in
these cells are mediated at least in part via the GR. Although the mechanism was not
investigated as extensively as for MPA, NET-A was shown to repress IL-8 promoter
reporter activity very weakly relative to dex, MPA and prog in Hek293 cells stably
transfected with the rat GR. Furthermore, NET-A, like MPA, dex and prog did not
interfere with the DNA-binding activity of NFKB. Significant transactivation of a GRE-driven promoter reporter construct by
MPA and dex in L929sA via endogenous GR and COS-1 cells via expressed rat GR,
and by MPA, dex and prog in Hek293 cells via expressed rat GR was also observed.
In contrast, NET-A, unlike MPA, dex and prog showed no transactivation in Hek293
cells.
MPA, NET-A and prog were shown to compete with dex for binding to the
endogenous human GR in human lung carcinoma A549 cells. Similarly, MPA and
NET-A were shown to compete with dex for binding to expressed rat GR in COS-1
cells. MPA displayed a higher relative binding affinity than NET-A for the GR in both
systems, and a higher relative binding affinity than prog in A549 cells. Equilibrium
dissociation constants (Ki values) for MPA (Ki = 10.8 ± 1.1 nM), NET-A (Ki = 270 ±
1.3 nM) and prog (Ki = 215 ± 1.1 nM) towards the human GR in A549 cells were also
established. Furthermore, dose-response curves showed that MPA displays
significantly greater GC agonist potency and efficacy than NET-A and prog for both
transactivation of a synthetic GRE-reporter construct and transrepression of a
synthetic IL-8 reporter construct via expressed rat GR in Hek293 cells, as NET-A
showed no transactivation and very weak partial agonist activity for transrepression.
Based on these observations, MPA behaves as a GR agonist whereas NET-A is
proposed to be a weak antagonist. These results show that MPA and NET-A are not
alike and not the same as prog in their mechanism of action via the GR, which may
have serious health implications in vivo. Such insights may provide women and their
clinicians with more information to facilitate the selection of contraception or
reproductive therapy regimes with fewer side effects. / AFRIKAANSE OPSOMMING: Alhoewel MPA en NET-A algemeen gebruik word in hormoontherapie, is dit
nie duidelik watter steroïedreseptore en teikengene betrokke is by die werking van
MPA en NET-A nie. Verrassend is dat geen studie nog gedoen is om te bepaal of die
dosisse van MPA en NET-A wat gebruik word in voorbehoeding en
hormoonvervangingsterapie (HVT), newe-effekte veroorsaak deur die
glukokortikoïedreseptor (GR) en verskeie teikengene nie.
In hierdie tesis is in L929sA selle aangetoon dat MPA, net soos
deksametasoon (dex) en prog, TNF-gestimuleerde IL-6 produksie onderdruk, en dat
IL-6 en IL-8 promoter-rapporteerderkonstrukte op transkripsionele vlak onderdruk
word deur middel van inmenging met NF-KB en AP-1 transkripsie-faktore. Net soos
dex en prog het MPA nie die DNA-bindingsaktiwiteit van NF-KB beïnvloed nie.
Anders as dex en prog het MPA egter nie MAPK aktiwiteit onderdruk nie. Die
antagonistiese effekte van RU486, asook die MPA-geïnduseerde translokasie van
die GR na die selkern, dui sterk daarop dat die effekte van MPA in hierdie selle ten
minste gedeeltelik deur die GR geskied. Alhoewel die meganisme vir NET -A nie so
breedvoerig bestudeer is as dié van MPA nie, is tog aangetoon dat, in Hek293 selle
wat stabiel getransfekteer is met die rot GR, die onderdrukking van die IL-8 promoter
deur NET-A baie swakker is as met dex, prog en MPA. Verder is daar ook gevind dat
NET-A, net soos MPA, dex en prog, nie kon inmeng met die DNA-bindingsaktiwiteit
van NF-KB nie. Beduidende transaktivering van 'n GRE-bevattende promoterrapporteerderkonstruk
deur MPA en dex in L929sA en COS-1 selle, en deur MPA,
dex en prog in Hek293 selle, is ook gevind. Daarteenoor het NET-A, anders as MPA,
dex en prog, geen transaktivering in Hek293 selle getoon nie.
Verder moes die relatiewe bindingsaffiniteit (ewewigs-dissosiasiekonstantes)
van MPA, NET-A en prog vir die GR, asook die relatiewe sterkte en effektiwiteit vir
transaktivering en transonderdrukking van verskeie teikengene deur die GR, ook
bepaal word. Daar is gevind dat MPA, NET-A en prog meeding met dex vir binding
aan die endogene GR in mens longkarsinoom A549 selle. Soortgelyk hieraan is ook
gevind dat MPA en NET-A meeding met dex vir binding aan rot GR wat in COS-1
selle uitgedruk is. MPA het in beide sisteme 'n hoër relatiewe bindingsaffiniteit vir die
GR getoon as NET-A, asook 'n hoër relatiewe bindingsaffiniteit as prog in A549 selle.
Ewewigs-dissosiasiekonstantes (Ki waardes) vir MPA (Ki = 10.8 ± 1.1 nM), NET- A
(Ki = 270 ± 1.3 nM) en prog (Ki = 215 ± 1.1 nM) vir die mens GR in A549 selle is ook
bereken. Dosisrespons-grafieke het ook aangedui dat MPA 'n beduidend beter GC
sterkte en effektiwiteit as NET-A en prog het, vir beide transaktivering van 'n
sintetiese GRE-rapporteerderkonstruk en transonderdrukking van 'n sintetiese IL-8
rapporteerderkonstruk via rot GR wat uitgedruk is in Hek293 selle. Dit kon afgelei
word aangesien NET-A geen transaktivering en slegs baie swak gedeeltelike agonisaktiwiteit
vir transonderdrukking getoon het. Op grond van hierdie waarnemings tree
MPA op as 'n GR agonis, terwyl dit lyk asof NET-A 'n swak antagonis is. Hierdie
resultate dui aan dat MPA en NET-A nie dieselfde is nie, en ook nie dieselfde
meganisme van werking deur die GR het as prog nie. Dit kan ernstige
gesondheidsimplikasies inhou in vivo. Hierdie insigte kan dus meer inligting aan
vroue en kliniese personeel verskaf om sodoende die keuse van voorbehoeding of
voortplantingsterapie met minder newe-effekte te vergemaklik.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/70116 |
| Date | 04 1900 |
| Creators | Koubovec, Dominique J. B. M. |
| 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 | Unknown |
| Type | Thesis |
| Format | 199 p. : ill. |
| Rights | Stellenbosch University |
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