Donita Africander

Molecular mechanisms of steroid receptor-mediated actions by synthetic progestins used in HRT and contraception

Synthetic progestins are used by millions of women as contraceptives and in hormone replacement therapy (HRT), although their molecular mechanisms of action are not well understood. The importance of investigating these mechanisms, as compared to those of progesterone, has been highlighted by clinical evidence showing that medroxyprogesterone acetate (MPA), a first generation progestin, increases the risk of breast cancer and coronary heart disease in HRT users. A diverse range of later generation progestins with varying structures and pharmacological properties is available for therapeutic use and it is becoming clear that different progestins elicit beneficial and adverse effects to different extents. These differences in biological activity are likely to be due to many factors including variations in dose, metabolism, pharmacokinetics, bioavailability, and regulation of, and/or binding, to serum-binding proteins and steroidogenic enzymes. Since the intracellular effects on gene expression and cell signaling of steroids are mediated via intracellular steroid receptors, differential actions via the progesterone and other steroid receptors and their isoforms, are likely to be the major cause of differential intracellular actions of progestins. Since many progestins bind not only to the progesterone receptor, but also to the glucocorticoid, androgen, mineralocorticoid, and possibly the estrogen receptors, it is plausible that synthetic progestins exert therapeutic actions as well as side-effects via some of these receptors. Here we review the molecular mechanisms of intracellular actions of old (MPA, norethisterone, levonorgestrel, gestodene) vs. new (drospirenone, dienogest, trimegestone) generation progestins, via steroid receptors.

Abrogation of Glucocorticoid Receptor Dimerization Correlates with Dissociated Glucocorticoid Behavior of Compound A

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [3H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GRdim) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.

11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

Differential regulation of endogenous pro-inflammatory cytokine genes by medroxyprogesterone acetate and norethisterone acetate in cell lines of the female genital tract

BACKGROUND Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives are widely used in female reproductive therapy, but little is known about their mechanisms of action via steroid receptors in the female genital tract. MPA used as a contraceptive has been implicated in effects on local immune function. However, the relative effects of progesterone (Prog), MPA and norethisterone acetate (NET-A) on cytokine gene expression in the female genital tract are unknown. STUDY DESIGN Using two epithelial cell lines generated from normal human vaginal (Vk2/E6E7) and ectocervical (Ect1/E6E7) cells as in vitro cell culture model systems for mucosal immunity of the female cervicovaginal environment, we investigated steroid receptor expression and activity as well as regulation of cytokine/chemokine genes by MPA and NET-A, as compared to the endogenous hormone Prog. RESULTS We show that the Prog, androgen, glucocorticoid and estrogen receptors (PR, AR, GR and ER, respectively) are expressed in both the Vk2/E6E7 and Ect1/E6E7 cell lines, and that the GR and AR are transcriptionally active. This study is the first to show ligand-, promoter- and cell-specific regulation of IL-6, IL-8 and RANTES (regulated-upon-activation, normal T cell expressed and secreted) gene expression by Prog, MPA and NET-A in these cell lines. Moreover, we show that the repression of the TNF-α-induced RANTES gene by MPA in the Ect1/E6E7 cell line is mediated by the AR. CONCLUSION Collectively, these data demonstrate that cell lines from different anatomical sites of the female genital tract respond differently to Prog and the synthetic progestins, most likely due to differential actions via different steroid receptors. The results highlight the importance of choice of progestins for immune function in the cervicovaginal environment. They further suggest that choice of progestins in endocrine therapy may have implications for womens risk of susceptibility to infections due to differential actions on genes involved in inflammation and immune function.

The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor

Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ∼24 nM for transactivation of the anti-inflammatory GILZ gene and ∼4–20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells.

11-ketotestosterone and 11-ketodihydrotestosterone in castration resistant prostate cancer : potent androgens which can no longer be ignored

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). Under castrate conditions, DHT is produced from the metabolism of the adrenal androgen precursors, DHEA and androstenedione. Recent studies have shown that the adrenal steroid 11β-hydroxyandrostenedione (11OHA4) serves as the precursor to the androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). In this study we comprehensively assess the androgenic activity of 11KT and 11KDHT. This is the first study, to our knowledge, to show that 11KT and 11KDHT, like T and DHT, are potent and efficacious agonists of the human androgen receptor (AR) and induced both the expression of representative AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines, LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells, while in vitro conversion assays showed that 11KT and 11KDHT are metabolized at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT, respectively. Our findings show that 11KT and 11KDHT are bona fide androgens capable of inducing androgen-dependant gene expression and cell growth, and that these steroids have the potential to remain active longer than T and DHT due to the decreased rate at which they are metabolised. Collectively, our data demonstrates that 11KT and 11KDHT likely play a vital, but overlooked, role in the development and progression of CRPC.

A comparative study of the androgenic properties of progesterone and the progestins, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A).

The importance of investigating the molecular mechanism of action of medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A), two clinically important progestins used in hormone therapy (HT), has been highlighted by clinical evidence showing that MPA and norethisterone (NET) increase the risk of the development of breast cancer in HRT users, and that MPA may increase susceptibility to- and transmission of HIV-1. The aim of this study was to compare the molecular mechanisms of action of MPA, NET-A and progesterone (Prog) via the androgen receptor (AR) in a cell line model that can minimize confounding factors such as the presence of other steroid receptors. This study is the first to determine accurate apparent Ki values for Prog, MPA and NET-A toward the human AR in COS-1 cells. The results reveal that these ligands have a similar binding affinity for the AR to that of the natural androgen 5α-dihydrotestosterone (DHT) (Kis for DHT, Prog, MPA and NET-A are 29.4, 36.6, 19.4 and 21.9 nM, respectively). Moreover, in both transactivation and transrepression transcriptional assays we demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. One of the most novel findings of our study is that NET-A, like DHT, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does not induce the N/C interaction on a classical ARE and does so only weakly on an AR-selective ARE. This suggests that MPA and NET-A may exert differential promoter-specific actions via the AR in vivo. Consistent with this, molecular modeling suggests that MPA and NET-A induce subtle differences in the structure of the AR ligand binding domain. Taken together, the results from this study suggest that unlike Prog, both MPA and NET-A used in hormonal therapy are likely to compete with DHT and exert significant and promoter-specific off-target transcriptional effects via the AR, possibly contributing to some of the observed side-effects with the clinical use of MPA and NET-A.

Investigating the anti-mineralocorticoid properties of synthetic progestins used in hormone therapy

A more detailed understanding of the affinities and efficacies for transcriptional regulation by the synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) via the mineralocorticoid receptor (MR) is required, to better understand their relative risk profiles. Both MPA and NET-A bind to the MR, although with about 100-fold lower affinities than that of Prog. MPA and NET-A exhibit no agonist activity, but NET-A, unlike MPA, has similar antagonistic efficacy to Prog on the endogenous mineralocorticoid/glucocorticoid response element (MRE/GRE)-containing genes, α-glycolytic protein or orosomucoid-1 (Orm-1) and plasminogen activator inhibitor-1 (PAI-1). This study is the first to show that NET-A, but not MPA, can dissociate between transrepression and transactivation via the MR. Given the relatively low affinity and potency of MPA and NET-A for the MR, our results suggest that these progestins are unlikeley to exert significant effects via the MR at doses used in hormonal therapy. However, considering their relative free concentrations compared to endogenous hormones, the possibility that NET-A may exhibit significant MR antagonist activity, with some possible cardiovascular protective benefits, should not be excluded.

16α-Hydroxyprogesterone: Origin, biosynthesis and receptor interaction

The metabolism of progesterone (PROG) by cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) results in the formation of both 17α-hydroxyprogesterone (17-OHPROG) and 16α-hydroxyprogesterone (16-OHPROG) in humans. Unlike 17-OHPROG, 16-OHPROG is not metabolised further in steroidogenic tissue. While this metabolite can be readily detected in serum and urine, its physiological role remains unclear. This paper reviews the production of 16-OHPROG by human CYP17A1 by providing insight into the catalysis of PROG by CYP17A1 and highlights the role of Ala105 in the 16α-hydroxylation reaction. As 16-OHPROG has been putatively linked to reproductive function, we investigated the interaction of this steroid metabolite with both isoforms of the human progesterone receptor (hPR). We show for the first time that 16-OHPROG can bind to both hPR-A and hPR-B and act as an agonist for both receptors.

Medroxyprogesterone Acetate Differentially Regulates Interleukin (IL)-12 and IL-10 in a Human Ectocervical Epithelial Cell Line in a Glucocorticoid Receptor (GR)-dependent Manner

Background: Little is known about the mechanism of action of MPA in the female genital tract. Results: GR mediates MPA-induced up-regulation of IL-12 and down-regulation of IL-10 mRNA and protein levels. Conclusion: MPA favors a pro-inflammatory milieu in ectocervical epithelial cells. Significance: MPA used in hormonal therapy may modulate inflammation in the ectocervical environment via this genomic mechanism. Medroxyprogesterone acetate (MPA), designed to mimic the actions of the endogenous hormone progesterone (P4), is extensively used by women as a contraceptive and in hormone replacement therapy. However, little is known about the steroid receptor-mediated molecular mechanisms of action of MPA in the female genital tract. In this study, we investigated the regulation of the pro-inflammatory cytokine, interleukin (IL)-12, and the anti-inflammatory cytokine IL-10, by MPA versus P4, in an in vitro cell culture model of the female ectocervical environment. This study shows that P4 and MPA significantly increase the expression of the IL-12p40 and IL-12p35 genes, whereas IL-10 gene expression is suppressed in a dose-dependent manner. Moreover, these effects were abrogated when reducing the glucocorticoid receptor (GR) levels with siRNA. Using a combination of chromatin immunoprecipitation (ChIP), siRNA, and re-ChIP assays, we show that recruitment of the P4- and MPA-bound GR to the IL-12p40 promoter requires CCAAT enhancer-binding protein (C/EBP)-β and nuclear factor κB (NFκB), although recruitment to the IL-10 promoter requires signal transducer and activator of transcription (STAT)-3. These results suggest that both P4 and MPA may modulate inflammation in the ectocervix via this genomic mechanism.