Progesterone receptor isoform ratios influence the transcriptional activity of progestins via the progesterone receptor.

Progestins (synthetic progestogens) are progesterone receptor (PR) ligands used globally by women in both hormonal contraception and menopausal hormone therapy. Although four generations of unique progestins have been developed, studies seldom distinguish between the activities of progestins via the two functionally distinct PR isoforms, PR-A and PR-B. Moreover, not much is known about the action of progestins in breast cancer tumors where PR-A is mostly overexpressed relative to PR-B. Understanding progestin action in breast cancer is crucial since the clinical use of some progestins has been associated with an increased risk of developing breast cancer. This study directly compared the agonist activities of selected progestins from all four generations for transactivation and transrepression via either PR-A or PR-B, and when PR-A and PR-B were co-expressed at ratios comparable to those detected in breast cancer tumors. Comparative dose-response analysis showed that earlier generation progestins mostly displayed similar efficacies for transactivation on a minimal progesterone response element via the PR isoforms, while most of the 4th generation progestins, similar to the natural progestogen, progesterone (P4), were more efficacious via PR-B. Most of the progestogens were however more potent via PR-A. We are the first to show that the efficacies of the selected progestogens via the individual PR isoforms were generally decreased when PR-A and PR-B were co-expressed, irrespective of the ratio of PR-A:PR-B. While the potencies of most progestogens via PR-B were enhanced when the ratio of PR-A relative to PR-B was increased, those via PR-A were minimally influenced. This study is also the first to report that all progestogens evaluated, except 1st generation medroxyprogesterone acetate and 4th generation drospirenone, displayed similar agonist activity for transrepression via PR-A and PR-B on a minimal nuclear factor kappa B containing promoter. Moreover, we showed that the progestogen activity for transrepression was significantly increased when PR-A and PR-B were co-expressed. Taken together, our results highlight that PR agonists (progestogens) do not always display the same activity via PR-A and PR-B, or when PR-A and PR-B are co-expressed at ratios mimicking those found in breast cancer tumors. These results suggest that biological responses are progestogen- and PR isoform-dependent and may differ in target tissues expressing varying PR-A:PR-B ratios.

The transcriptional activity of progestins used in contraception and menopausal hormone therapy via progesterone receptor A is dependent on the density of the receptor.

Studies directly comparing the efficacies and potencies of multiple progestins used in contraception and menopausal hormone therapy (MHT) in parallel via human progesterone receptor isoform A (PR-A) in the same model system are limited, and how these parameters are influenced by the density of PR-A are unclear. This is surprising as it is known that the expression levels of PR-A vary in different tissues and diseases. We thus determined for the first time the relative efficacies and potencies for transactivation of the natural PR ligand, progesterone (P4), the PR-specific agonist promegestone (R5020), and selected progestins from all four generations in parallel via different densities of PR-A overexpressed in the MDA-MB-231 breast cancer cell line. Comparative dose-response analysis showed that P4, R5020, the 1st generation progestins medroxyprogesterone acetate and norethisterone, 2nd generation progestin levonorgestrel, 3rd generation progestin gestodene, as well as 4th generation progestins nesterone, nomegestrol acetate and drospirenone display differential agonist efficacies and potencies via PR-A. Moreover, we showed that the agonist efficacies and potencies of the progestins via PR-A were modulated in a density- and progestin-specific manner. Our finding that the potencies of the progestins via PR-A, at all densities, do not exceed reported progestin serum concentrations in women, suggest that these progestins are likely to elicit similar effects in vivo. We are the first to report that P4 and the selected progestins display similar agonist activity for transrepression via PR-A, and that the density of PR-A enhances the transrepression activity of some, but not all progestogens. Collectively, our findings provide proof of concept that the effects of the selected progestins via PR-A is progestin-specific and dependent on the density of the receptor, suggesting differential progestin responses in women using these progestins in contraception and MHT.

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.

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.

Progestins used in endocrine therapy and the implications for the biosynthesis and metabolism of endogenous steroid hormones.

Steroidogenesis refers to the de novo synthesis of steroid hormones from cholesterol by a number of sequential enzyme catalysed reactions in the adrenal and the gonads. In addition, circulating steroid hormone precursors are further metabolised in selected peripheral tissues. It has been suggested that the biosynthesis of endogenous steroid hormones can be modulated by progestins, used widely by women in female reproductive medicine. However, as a number of structurally diverse progestins with different pharmacological properties are available, it is possible that these synthetic compounds may vary in their effects on steroidogenesis. This review summarises the evidence indicating that progestins influence the biosynthesis of steroid hormones in the adrenal and gonads, as well as the metabolism of these endogenous hormones in the breast, highlighting the limitations to the current knowledge and directions for future research.