Progestogens exhibit progestogen-, promoter- and isoform-specific effects via the progesterone receptor.

Hormonal contraceptives (HCs) and hormone replacement therapy (HRT) are therapies designed to target the progesterone receptor (PR) to prevent unwanted pregnancy and to alleviate the symptoms of menopause, respectively, in women. Although these therapies are widely used globally, few studies have investigated in parallel how the transcriptional responses of the progestogens used in these therapies compare to each other via the PR isoforms (PR-A and PR-B). Using dose-response promoter-reporter and endogenous gene expression assays, we compared the transcriptional responses of six widely-used progestogens via each PR isoform. The present study shows that progestogens exhibit progestogen-specific potencies and efficacies via both PR isoforms. In addition, the endogenous gene expression data reveals that progestogens exhibit promoter-specific effects. Furthermore, this study reveals that progestogen responses via PR-A are significantly more potent and less efficacious than those observed via PR-B, and that this is unlikely due to differences in PR protein levels. Correlation analysis revealed that there is no detectable correlation between potency or efficacy of progestogens for PR-B or PR-A versus reported relative binding affinity (RBA) of progestogens for the PR, consistent with complex mechanisms of PR regulation. Taken together, our data show that it cannot be assumed that all progestogens have similar transcriptional responses on all genes. Since the PR plays a role in cognition, regulation of inflammation, mitochondrial function, neurogenesis, female reproduction and disease, the data suggest that these important physiological functions could be differentially affected depending on progestogen, promoter, and ratios of PR isoforms.

Characterisation of progestins used in hormonal contraception and progesterone via the progesterone receptor.

Different progestogens are widely used in hormonal therapy and mediate their therapeutic actions via the progesterone receptor (PR). Little published data exist on their relative efficacies and potencies via the PR, while those available may be confounded by off-target receptors, different methodologies and model systems. We performed dose-response analysis to investigate the efficacies and potencies for transcription of progesterone and several progestins widely used in contraception via the B isoform of human PR (PR-B). We compared responses using three different cell lines and two different transient transfection conditions. Results show that in vitro biological responses via PR-B for the select progestogens can vary significantly in biocharacter, rank order and absolute values for efficacies and potencies, depending on the cell line and transfection condition. Progestogen rank orders for published relative binding affinities are mostly different to those for relative efficacies and potencies. These in vitro differences suggest that rank orders and absolute values of the efficacies and potencies of the progestogens are likely to vary in vivo in a cell-specific and progestogen-specific manner, and cannot easily be extrapolated from in vitro data, as is usually the practice. While obtaining such data in vivo is not possible, these in vitro data show proof of concept for likely significant cell- and progestogen-specific PR-B effects.

Maraviroc, tenofovir disoproxil fumarate and dapivirine, activate progesterone receptor B in the absence of progestogens.

Antiretroviral therapy has slowed the HIV/AIDS pandemic and is currently being used as a prophylactic measure for individuals at high risk of infection. However, concerns over adverse effects of long-term use need to be explored. We hypothesize that this may occur, at least in part, through off-target effects via select steroid receptors (SRs) that broadly regulate multiple physiological processes. We investigated the effects of maraviroc (MVC), tenofovir disoproxil fumarate (TDF), and dapivirine (DPV) on progesterone receptor B (PR-B) transcriptional activity. We found that MVC and TDF activate PR-B transcription in the absence of progestogens on a PR-regulated promoter reporter construct and on endogenous PR-regulated genes. MVC and TDF exhibited no direct binding to PR-B; however, increased PR-B phosphorylation was detected with TDF but not MVC. DPV transactivated gilz and ptgs2 in the absence of progestogens and exhibited PR-B binding while showing no effects on phosphorylation, suggesting that it may activate PR-B through a direct mechanism. Our study shows that potential off-target immunomodulatory effects of MVC, TDF and DPV occur in vitro and these are most likely mediated by different mechanisms of PR-B activation.

Reciprocal Modulation of Antiretroviral Drug and Steroid Receptor Function In Vitro.

Millions of women are exposed simultaneously to antiretroviral drugs (ARVs) and progestin-based hormonal contraceptives. Yet the reciprocal modulation by ARVs and progestins of their intracellular functions is relatively unexplored. We investigated the effects of tenofovir disoproxil fumarate (TDF) and dapivirine (DPV), alone and in the presence of select steroids and progestins, on cell viability, steroid-regulated immunomodulatory gene expression, activation of steroid receptors, and anti-HIV-1 activity in vitro Both TDF and DPV modulated the transcriptional efficacy of a glucocorticoid agonist via the glucocorticoid receptor (GR) in the U2OS cell line. In TZM-bl cells, DPV induced the expression of the proinflammatory interleukin 8 (IL-8) gene while TDF significantly increased medroxyprogesterone acetate (MPA)-induced expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. However, peripheral blood mononuclear cell (PBMC) and ectocervical explant tissue viability and gene expression results, along with TZM-bl HIV-1 infection data, are reassuring and suggest that TDF and DPV, in combination with dexamethasone (DEX) or MPA, do not reciprocally modulate key biological effects in primary cells and tissue. We show for the first time that TDF induces progestogen-independent activation of the progesterone receptor (PR) in a cell line. The ability of TDF and DPV to influence GR and PR activity suggests that their use may be associated with steroid receptor-mediated off-target effects. This, together with cell line and individual donor gene expression responses in the primary models, raises concerns that reciprocal modulation may cause side effects in a cell- and donor-specific manner in vivo.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms.

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50-100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.