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.     

Hormones and cancer in humans

Hormones play a major role in the aetiology of several of the commonest cancers worldwide, including cancers of the endometrium, breast and ovary in women and cancer of the prostate in men. It is likely that the main mechanisms by which hormones affect cancer risk are by controlling the rate of cell division, the differentiation of cells and the number of susceptible cells. Hormones have very marked effects on cell division in the endometrium; oestrogens stimulate mitosis whereas progestins oppose this effect. The risk for endometrial cancer increases with late menopause, oestrogen replacement therapy and obesity, and decreases with parity and oral contraceptive use; thus risk increases in proportion to the duration of exposure to oestrogens unopposed by progestins, probably because unopposed oestrogens stimulate endometrial cell division. The effects of hormones on breast epithelial cell division in non-pregnant women are much less clear-cut than their effects on the endometrium, but both oestrogens and progestins appear to stimulate mitosis. Breast cancer risk increases with early menarche, late menopause and oestrogen replacement therapy, probably due to increased exposure of the breasts to oestrogen and/or progesterone. Early first pregnancy and multiparity reduce the risk for breast cancer, probably due to the hormonally-induced differentiation of breast cells and the corresponding reduction in the number of susceptible cells. Hormones do not have marked direct effects on the epithelial cells covering the ovaries, but hormones stimulate ovulation which is followed by cell division during repair of the epithelium. Risk for ovarian cancer increases with late menopause and decreases with parity and oral contraceptive use, suggesting that the lifetime number of ovulations may be a determinant of risk. For all three of these cancers risk changes within a few years of changes in exposure to sex hormones and some of the changes in risk persist for many years, indicating that hormones can affect both early and late stages of carcinogenesis. Understanding of the role of sex hormones in the aetiology of prostate cancer and of some rarer cancers is less complete.     

Mechanisms of growth inhibition by antiestrogens and progestins in human breast and endometrial cancer cells.

Marked changes in both growth factor and proto-oncogene expression occur due to treatment of hormonally-responsive human cancers with progestins and antiestrogens. In human endometrial cancer cell lines the antiproliferative effects of progestins and antiestrogens in a particular cell line appear to be associated with similar effects on growth factor and/or proto-oncogene expression. This suggests that although these compounds initially interact with different steroid hormone receptors, the molecular mechanisms of their growth inhibition may be essentially similar. In the case of human breast cancer cell lines, however, the effects of progestins and antiestrogens on gene regulation are often different, suggesting that the molecular mechanisms of progestin and antiestrogen growth inhibition may be essentially dissimilar.     

Progesterone and levonorgestrel regulate expression of 17βHSD-enzymes in progesterone receptor positive breast cancer cell line T47D

The use of combined hormone replacement therapy (HRT) with oestrogens and progestins in postmenopausal women has been associated with an increased risk for developing breast cancer. The reasons are not fully understood, but influence of HRT on endogenous conversion of female sex hormones may be involved. The expression of 17β hydroxysteroid dehydrogenases (17βHSD), which are enzymes catalysing the conversion between more or less potent oestrogens, may partly be regulated by progestins. The breast cancer cell lines T47D, MCF7 and ZR75-1 were treated with progesterone, medroxyprogesterone acetate (MPA) or levonorgestrel for 48 and 72 h at 10(-7) and 10(-9)M to investigate influence on 17βHSD1, 17βHSD2 and 17βHSD5 mRNA expression measured by real time PCR. The expression of 17βHSD1 increased in progesterone and levonorgestrel treated T47D cells (48 h 10(-7)M P=0.002; P<0.001) and 17βHSD5 increased after progesterone treatment (48 h 10(-7)M P=0.003), whereas the expression of 17βHSD2 decreased after the (48 h 10(-7)M P=0.003; P<0.001). Similar, but less prominent effects were seen in MCF7 and ZR75-1. The progestin effects on 17βHSD-expression were lost when T47D cells were co-treated with progestins and the progesterone receptor (PgR) inhibitor mifprestone. We show that both reductive (17βHSD1 and 17βHSD5) and oxidative (17βHSD2) members of the 17βHSD-family are under control of progesterone and progestins in breast cancer cell lines. This is most clear in T47D cells which have high PgR expression. 17βHSD-enzymes are important players in the regulation of sex steroids locally in breast tumours and tumoural expression of various 17βHSD-enzymes have prognostic and treatment predictive relevance. We propose a mechanism for increased breast cancer risk after HRT in which hormone replacement affects the expression of 17βHSD-enzymes, favouring the expression of reductive enzymes, which in turn could increase levels of bioactive and mitogenic estrogens in local tissue, e.g. breast tissue.     

The additive effects of progestins on testosterone-stimulated hepatic ethylmorphine metabolism and cytochrome P-450 content

The actions of several progestins on mouse liver were studied in terms of their inherent potency and for their ability to modify the biologic activity of testosterone. When hepatic ethylmorphine demethylase activity and cytochrome P-450 content were used as end points, the biological potency of progestins was ranked as follows: cyproterone acetate>progesterone>medroxyprogesterone acetate>hydroxyprogesterone caproate controls. The induced alterations of these parameters were, therefore, unrelated to reported progestational (cyproterone acetate medroxyprogesterone acetate>>hydroxyprogesterone caproate>progesterone) or androgenic (medroxyprogesterone acetate>cyproterone acetate = hydroxyprogesterone caproate = progesterone) actions of these steroids. A similar conclusion was reached when hepatic weight and microsomal protein content were used as end points.

When progestins (0.1–10 mg/day) were administered with testosterone (0.1 mg/day), the effect of both steroids were additive. This is in contrast to their actions on other tissues such as kidney and sub-maxillary gland where progestins potentiate and inhibit androgen action. We conclude from these studies that the mechanism of action of progestins on the liver differs from that on other tissues.     

Progesterone and progestins: effects on brain, allopregnanolone and beta-endorphin

The increased use of hormonal therapies over the last years has led to improve the knowledge of pharmacological, biochemical and metabolic properties of several progestins and their effects in target tissues, such as the central nervous system. Progesterone and synthetic progestational agents are able to modulate the synthesis and release of several neurotransmitters and neuropeptides in response to specific physiological and pathological stimuli. While these actions may relay on differential activation of progesterone receptor or recruitment of intracellular pathways, some of the differences found between synthetic progestins may depend on the specific conversion to neuroactive steroids, such as the 3-, 5- reduced metabolite, allopregnanolone. This is a potent endogenous steroid that rapidly affects the excitability of neurons and glia cells through direct modulation of the GABA-A receptors activity exerting hypnotic/sedative, anxiolytic, anaesthetic and anticonvulsive properties. Estrogens increase the CNS and serum levels of allopregnanolone and the addition of certain but not all synthetic progestins determines a further increase in allopregnanolone levels, suggesting that the metabolism into this reduced product is related to the chemical structure of progestin molecule used. In addition, depending on specific progestin molecule used, different interaction are found with the estradiol-induced beta-endorphin synthesis and release, showing that diverse progestins have specific and divergent actions on the opiatergic system. These results highlight the concept that natural and synthetic progesterone receptor agonists may systematically induce different biological actions in CNS. This may have far-reaching implications for the clinical effects and related indications of each compound.     

Membrane actions of progestins at dopamine type 1-like and GABAA receptors involve downstream signal transduction pathways

In the ventral tegmental area (VTA), progestins facilitate lordosis via rapid actions at membrane dopamine Type 1-like (D(1)) and/or GABA(A) receptors (GBRs), rather than via cognate, intracellular progestin receptors (PRs). Downstream signal transduction pathways involved in these effects were investigated using lordosis as a bioassay. If progestins' actions at D(1) and/or GBRs in the VTA require activation of G-proteins, adenylyl cyclase, cyclic AMP-dependent protein kinase A (PKA), phospholipase C (PLC), and/or PKC, then pharmacologically blocking these pathways would be expected to attenuate progestin-facilitated lordosis and its enhancement by D(1) and GBR activity. Ovariectomized, estradiol-primed rats were infused first with vehicle or signal transduction inhibitor, and second with vehicle, a D(1) or GBR agonist, and then with vehicle or progestins to the VTA. Rats were tested for lordosis following infusions. Results indicated that initiation of G-proteins, adenylyl cyclase, PKA, PLC, or PKC in the VTA is required for rapid effects of progestins through D(1) and/or GBRs to facilitate lordosis. As well, progestins' actions at n-methyl-d-aspartate receptors (NMDARs) may modulate activity at D(1) and/or GBRs and mitogen activated protein kinase (MAPK) may be a common signaling pathway. Findings from a microarray study demonstrated that there was upregulation of genes associated with steroid metabolism, GBRs, D(1), NMDARs and signal transduction factors in the midbrain VTA of naturally receptive mated compared to non-mated rats. Thus, in the VTA, progestins have rapid membrane-mediated actions via D(1), GBRs, NMDARs and their downstream signal transduction pathways.     

Aging-related changes in ovarian hormones, their receptors, and neuroendocrine function

Ovarian steroid hormones exert a broad range of effects on the body and brain. In the nervous system, estrogen and progesterone have crucial feedback actions on the hypothalamic neurons that drive the reproductive axis. In addition, hormones exert a variety of actions on other traditionally nonreproductive functions such as cognition, learning and memory, neuroprotection, mood and affective behavior, and locomotor activity. The actions of hormones on the hypothalamus are largely mediated by their nuclear hormone receptors, the two estrogen receptors, ERalpha and ERbeta, and the two progesterone receptor isoforms, PR-A and PR-B. Thus, changes in the circulating concentrations of estrogens and progestins during the life cycle can result in differential activation of their receptors. Furthermore, changes in the numbers, activity, and distribution of hypothalamic ERs and PRs can occur as a function of developmental age. The purpose of this article is to review the literature on the causes and consequences of alterations in steroid hormones, their neural receptors, and their interactions on reproductive senescence. We have also discussed several important experimental design considerations, focusing on rodent models in current use for understanding the mechanisms of menopause in women.     

Risk of breast cancer with progestins: critical assessment of current data

Whether progestins protect against the risk of breast cancer or enhance that risk has been a major area of controversy over the past several years. Observational studies have reported conflicting results and experimental studies examining whether progestins exert mitogenic or anti-mitogenic actions on breast tissue report divergent results. Based upon a wide range of animal, epidemiologic and clinical data, most investigators agree that estrogens contribute to the development of breast neoplasms. However, the additional effect of progestins on this risk has been the subject of substantial discussion and controversy. A variety of experiments have been carried out using human breast cancer cells grown in vitro and as xenografts in nude mice. These studies demonstrated both mitogenic and anti-mitogenic effects depending upon the precise experimental conditions. Two potential reasons for these differences include differential metabolism of progestins into inhibitory pregnenes or stimulatory 5-alpha-reduced pregnanes or the presence of a protein (GPR 30) which allows the anti-mitogenic effects of progestins to be manifest. Based upon the conflicting nature of the results in experimental studies, we believe that only data in patients provide substantial insight into the actions of progestins on the intact human breast. Studies have now demonstrated that cell proliferation and breast density is higher during the luteal than during the follicular phase of the menstrual cycle. In postmenopausal women, long-term exposure to estrogen plus a progestin results in a marked enhancement of proliferation of the terminal duct lobular units as well as in breast density. These data, taken together, provide substantial evidence that progestins are mitogenic on the human breast when given long term to postmenopausal women. To critically evaluate the observational studies regarding breast cancer risk from progestins, we developed a set of stringent criteria for acceptance of individual studies. Four of the five studies meeting these criteria reported a greater risk of breast cancer with combination estrogen/progestin regimens than with estrogen alone. More importantly, the first randomized, prospective, controlled trial of the risk of breast cancer with an estrogen/progestin combination (the Women's Health Initiative Study) has now been published. This study reported a 26% increased relative risk of breast cancer with the estrogen/progestin combination. Based upon these data, we believe that progestins do add to the risk of breast cancer over and above that imparted by estrogen alone. The attributable risk during use for 5 years or less is small but increases logarithmically during long-term use. The majority of data regarding progestins are derived from regimens using MPA. However, we conclude from our analysis that the burden of proof regarding progestins has now shifted. One must now prove that an estrogen/progestin combination is safe with respect to breast cancer rather than having to prove it harmful.     

Mutagenicity of some contraceptive drugs in Drosophila melanogaster

Combinations of 3 progestins, ethynodiol diacetate, norethynodrel and norgestrel, and 2 oestrogens, ethinyloestradiol and mestranol, were fed to larval Oregon-R fruit flies. None of the steroids studied induced X-linked recessive lethal mutations above the control level in Drosophila melanogaster.     

Sex steroid effects at target tissues: mechanisms of action

Our understanding of the mechanisms of sex hormone action has changed dramatically over the last 10 years. Estrogens, progestins, and androgens are the steroid hormones that modulate reproductive function. Recent data have shown that many other tissues are targets of sex hormones in addition to classical reproductive organs. This review outlines new advances in our understanding of the spectrum of steroid hormone ligands, newly recognized target tissues, structure-function relationships of steroid receptors, and, finally, their genomic and nongenomic actions. Sex-based specific effects are often related to the different steroid hormone mileu in men compared with women. Understanding the mechanisms of sex steroid action gives insight into the differences in normal physiology and disease states.     

Indomethacin inhibits lordosis induced by ring A-reduced progestins: possible role of 3alpha-oxoreduction in progestin-facilitated lordosis

Progestins with a delta-4-3-keto configuration bind to the progestin receptor (PR) and facilitate estrous behavior in estrogen-primed rats. Some ring A-reduced progestins [5alpha-dihydroprogesterone (alphaDHP), allopregnanolone, and epipregnanolone] are more potent estrus-inducing agents than progesterone when iv injected despite their lower affinity for the PR. Yet the estrus-inducing action of such progestins is reduced by the antiprogestin RU486, suggesting that binding to the PR is required for this effect. Because allo- and epi-pregnanolone are oxidized to alpha- and betaDHP, respectively, by 3alpha-hydroxysteroid oxo-reductase (3alphaHSOR), part of their estrus-inducing action may occur through the binding of such DHPs to the PR. Conversely, because 3alphaHSOR reduces alpha- and betaDHP to allo- or epi-pregnanolone, both of which exert membrane effects, the estrus-inducing effect of DHPs may involve actions independent of the PR. To test these possibilities we assessed the effect of indomethacin, a blocker of 3alphaHSOR, on the estrus-inducing action of such progestins. Because indomethacin also inhibits cyclooxygenases, we selected a dose and treatment schedule that does not interfere with prostaglandin-mediated brain processes (e.g., LHRH release). Indomethacin did not significantly modify the effect of progesterone or megestrol acetate on lordosis. Yet, it significantly reduced the action of all ring A-reduced progestins. Results suggest that: (a) oxidation is essential for lordosis facilitation by 3alpha-pregnanolones and (b) reduction of 3-keto progestins generates 3alpha-hydroxy metabolites which synergize with processes triggered by occupation of the PR by 3-keto progestins. The possible participation in this response of other events influenced by indomethacin (e.g., prostaglandin or melatonin synthesis) is discussed.