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

CITATION: Pretorius, E., et al. 2016. 11-ketotestosterone and 11-ketodihydrotestosterone in castration resistant prostate cancer : potent androgens which can no longer be ignored. PLoS ONE, 11(7):1-17, doi:10.1371/journal.pone.0159867.The original publication is available at http://journals.plos.org/plosonePublication of this article was funded by the Stellenbosch University Open Access Fund.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.Publisher's versio

Induction of cell proliferation in LNCaP and VCaP cells by DHT, 11KDHT, T and 11KT.

<p>Cells were incubated with media supplemented with CS-FCS for 24 hours prior to treatment with 0.1, 1 or 10 nM steroids. Resazurin assays were carried out on day 7 (LNCaP) or day 10 (VCaP) after treatment. Results are shown as means ± SEM of three independent experiments with eight replicates each.</p

Application of an analysis technique to characterise impulse response of grounding systems

Transient response plays a key role in the evaluation of the performance of grounding systems and for the protection of electrical installations under lightning strikes. The frequency spectrum of the lightning impulse contains harmonics components up to the megahertz range. The measured transient response of grounding systems under test may be distorted by spurious high frequency interference in the acquired signals, which presents challenges for the accurate analysis of high frequency performance of such systems. In this paper, the high frequency performance of a rod electrode is investigated based on measurements of its transient response under impulse energisation. A practical method is implemented to eliminate the high frequency noise in the measured voltage and current shapes, which allows a frequency domain analysis based Fast Fourier Transforms

Biosynthesis of 11KT and 11KDHT from the adrenal androgen precursor 11OHA4.

<p>Enzymes: 11βHSD2, 11β-hydroxysteroid dehydrogenase; 17βHSD2, 17β-hydroxysteroid dehydrogenase; SRD5A1, steroid 5α-reductase type 1; 3αHSD2, 3α-hydroxysteroid dehydrogenase. Steroids: 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OH-5α-dione, 11OH-5α-androstanedione; 11K-5α-dione, 11-keto-5α-androstanedione; 11KDHT, 11-ketodihydrotestosterone; 11OHAST, 11β-hydroxyandrosterone; 11KAST, 11-ketoadrenosterone; 11K-3α-adiol, 11-keto-5α-androstane-3α,17β-diol.</p

Regulation of AR-regulated proteins by DHT, 11KDHT, T and 11KT in VCaP cells.

<p>Cells were incubated with CS-FCS supplemented media for 48 hours prior to treatment with 1 nM steroid. Proteins were subsequently identified using mass spectrometry. Fold changes were calculated relative to the vehicle control. Statistically significant changes are indicated (P<0.05). Results are representative of three independent experiments.</p

Metabolism of DHT, 11KDHT, T and 11KT by LNCaP and VCaP cells.

<p>Steroids were analysed by ultra-performance convergence chromatography-mass spectrometry (UPC²-MS/MS). Results are representative of two independent experiments performed in triplicate.</p

Binding of DHT, T, 11KDHT and 11KT to the human AR (A) and transactivation via an ARE (B and C).

<p>Binding affinities, agonist potencies and efficacies of DHT, 11KDHT, T and 11KT relative to the synthetic AR agonist mibolerone are summarised in (D). Whole cell binding assays (A) were conducted in COS-1 cells transiently transfected with pSVARo. Cells were incubated with 0.2 nM [³H]-Mib in the absence and presence of increasing concentrations of either unlabelled Mib, DHT, 11KDHT, T and 11KT for 16 hours. Results are plotted as % specific binding where the total specific binding of [³H]-Mib only is set to 100% and binding of unlabelled steroid is set as a % binding relative to that. Whole cell binding results are shown as means ± SEM of three independent experiments performed in triplicate. Transactivation assays (B and C) where performed in COS-1 cells transiently transfected with the pSVARo expression vector and the 4xSC ARE1.2-luc reporter. Agonist activity was measured by incubating cells in the presence of increasing concentrations of either Mib, DHT, T, 11KDHT or 11KT for 24 h. Induction is shown as % luciferase activity expressed in relative light units (rlu’s), with the maximal response of Mib (10⁻⁵ M) set to 100%. Luciferase assays are shown as means ± SEM of six independent experiments performed in quadruplicate.</p

Only GR protein is detected in primary cervical epithelial cells (VEN-100).

<p>(<b>A</b>) Upon arrival the VEN-100 cells were rested overnight before being washed once with PBS and harvested with TRIzol®. Total RNA was isolated and 500 ng RNA was reverse-transcribed. Steroid receptor gene expression was measured by qRT-PCR with receptor-specific primers, followed by gel electrophoresis to confirm the PCR products. (<b>B</b>) VEN-100 cells were rested overnight before harvesting in 2X SDS sample buffer. COS-1 cells were transiently transfected with 1 µg/well pcDNA3 (empty vector) which served as negative control (−CTRL) or with 1 µg/well steroid receptor expression vectors (GR, PR-B, AR, MR and ERα) which served as positive controls (+CTRL). Twenty fourhrs later, the COS-1 cells were washed once and lysed with 2X SDS sample buffer. Equal volumes of cell lysate (VEN-100 and COS-1 ctrls) were analysed by Western blotting with antibodies specific for the GR and Flotillin-1 (loading control), respectively.</p

The GR at least in part directly regulates mRNA levels of the inflammatory genes.

<p>End1/E6E7 cells were pretreated with 1 µg/ml cycloheximide (CHX) then treated for 24 hrs with 100 nM DEX, MPA, P4, NET-A or vehicle (ethanol) (CTRL), in the absence or presence of CHX. Total RNA was isolated and reverse-transcribed. Relative (<b>A</b>) GILZ (<b>B</b>) IκBα, (<b>C</b>) RANTES, (<b>D</b>) IL-6 and (<b>E</b>) IL-8 gene expressions was measured by real-time qRT-PCR and normalised to GAPDH mRNA expression. In addition, relative gene expressions were normalized to basal activity (CTRL) in order to obtain relative fold expression. Graphs represent pooled results of at least three independent experiments and are plotted as mean ± SEM. To verify that the CHX inhibited <i>de novo</i> protein synthesis, End1/E6E7 cells were pretreated with CHX then treated with 100 nM DEX or vehicle (ethanol) (CTRL) for 24 hrs. (<b>F</b>) Cells were harvested and equal volumes of lysate were analysed by Western blotting with an antibody specific for IκBα and a GAPDH specific antibody as loading control. (<b>G</b>) Western blots of four independent experiments were quantified to determine the relative GR protein expression. Statistical analysis was carried out using GraphPad Prism software (version 5) using a one-way ANOVA with a Dunnett post-test followed by a student’s t-test to compare specific conditions to each other. Statistical significance is denoted by *, ** or *** to indicate P<0.05, P<0.001 or P<0.0001, respectively.</p