Department of Biochemistry

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Browsing Department of Biochemistry by Subject "Acetates"

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    An investigation into the molecular mechanism of action of the progestins, medroxyprogesterone acetate and norethisterone acetate
    (Stellenbosch : Stellenbosch University, 2004-04) Koubovec, Dominique J. B. M.; Hapgood, J. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    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.