Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites

doi:10.1016/j.jsbmb.2009.03.008

The Journal of Steroid Biochemistry and Molecular Biology

Volume 116, Issues 1–2, August 2009, Pages 8-14

https://doi.org/10.1016/j.jsbmb.2009.03.008 Get rights and content

Abstract

This work was undertaken (i) to study deeply the estrogen, androgen and progestative activities of tibolone and its metabolites (ii) to determine whether tibolone and its metabolites present glucocorticoid or mineralocorticoid activity. For this purpose, we used human cell lines bearing a luciferase gene with a responsive element under the control of human estrogen receptor α (ERα) or estrogen receptor β (ERβ) or androgen receptor (AR) or chimeric Gal4 fusion with progesterone receptor (PR), glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). The major tibolone metabolites, the two hydroxymetabolites, bind and activate ER with a preference for ERα. Tibolone and the Δ⁴-tibolone are agonists for AR and PR and surprisingly 3α- and 3β-OH-tibolone are antagonists for them. Moreover we showed for the first time that tibolone and its primary metabolites are GR and MR antagonists with a stronger affinity for MR than for GR. In conclusion, tibolone by these actions on different receptors and by this capacity to transform in different metabolites, has more complex effects than initially supposed.

Introduction

Tibolone (Org OD14), a synthetic 19-norsteroid related to norethynodrel (NED) is an interesting alternative for hormonal treatment of menopause. In randomized studies, tibolone has been shown to improve menopausal symptoms to a similar degree of combined estrogen/progestogen hormonal therapy [1], [2], [3]. Moreover, clinical trials showed its interest on bone density [4], [5], since tibolone acts as a bone resorption inhibitor in a similar way to estrogens. In two cohort studies in the UK a small increase in endometrial cancer was observed [6], [7], but the results of a randomized controlled trial (the THEBES trial) does not show any stimulation of endometrial proliferation [8]. In this study, tibolone shows less breast pain than conjugated equine estrogen (CEE) plus medroxyprogesterone acetate (MPA). Tibolone acts as a pro-drug and is rapidly converted into 3 main metabolites. In plasma, the 3α-OH-tibolone is the main metabolite [9] followed by the 3β-OH-tibolone. Tibolone and the Δ⁴ isomer are only present during the first 6 h after oral intake.

The Δ⁴ isomer has progestin and androgen properties while the 3α- and the 3β-OH-tibolone have estrogenic properties [10]. The tissue selective action of tibolone is supposed to be a combination of receptor activation and steroid metabolism [11].

Using in vitro transactivation measurements with Chinese Hamster Ovary (CHO) cells, it has been demonstrated that tibolone and Δ⁴-tibolone were agonistic ligands of progesterone receptor (PR), androgen receptor (AR) and estrogen receptor (ERs) whereas 3α- and 3β-hydroxytibolone were only active on ERs [10]. CHO cells were chosen because the metabolism of the major sex steroids was very limited and the intrinsic hormonal properties of synthetic steroids could be accurately determined [12]. Potential disadvantages of the CHO cells are that they contain a different pallet of co-activators and corepressors than the human target cells. Ideally the receptor–reporter systems should be transfected in a cell line derived from the target tissue, but unfortunately such cell lines are not always accessible. Here we have used the Hela human cell line transfected with human ERs, PR, GR or MR and a reporter system. ERα and ERβ reporter cells were obtained by transfecting HeLa cells (ER negative) successively by the ERE-βGlob-Luc-SVNeo plasmid (HELN cell line) and pSG5puro plasmids expressing ERα or ERβ (HELN ERα and HELN ERβ cells). Since the ERs cell lines are derived from the same HELN cell line, the effect of both ERs is determined by the same ERE. PR, GR and MR recognize the same hormone responsive element. As HeLa cells express GR, we constructed chimeric receptors in which each part ensures to obtain the required hormonal specificity: one part is the hormone binding domain of PR, GR or MR, the other part is the DNA binding domain of yeast Gal4 protein. After binding with the steroid hormone these chimeric receptors are able to interact with the Gal4 responsive element placed upstream the luciferase gene. Thus, PR, GR and MR reporter cells were obtained by transfecting HeLa cells successively by the Gal4RE5-bGlob-Luc-SVNeo plasmid (HG5LN cell line) and the pSG5puro plasmids fused with the Gal4 DBD and the LBD of PR, MR or GR (HG5LN Gal4-PR, -MR and -GR cells). This assay format eliminated background activity from endogenous receptors allowing quantification of relative activity for the three steroid receptors with the same reporter gene. This strategy could not be used for AR since the A/B domain deleted receptor does not transactivate. Thus we developed a PC3 cell line expressing the complete human AR and the same responsive reporter gene as used for GR, MR and PR.

In the present study, we used these different bioluminescent reporter cell lines to confirm that tibolone and its primary metabolites are ligands for human PR, AR and/or ER. Interestingly, our data indicate that these compounds are also able to bind glucocorticoid receptor (GR) and mineralocorticoid (MR) and exhibit low antiglucocorticoid and moderate antimineralocorticoid activities. Altogether, our study point out that tibolone and its primary metabolites exert multiple and complex effects on all steroid hormone receptors.

Section snippets

Chemicals and materials

Materials for cell culture came from Life Technologies (Cergy-Pontoise, France) except 96-well Cellstar plates, which were obtained from Greiner labortechnic (Poitiers, France). Luciferin (sodium salt) and geneticin were purchased from Promega (Charbonnières, France). R1881 was purchased from NEN Life Science Products (Paris, France). [³H]-aldosterone (39 Ci/mmol specific activity) and [³H]-dexamethasone (84 Ci/mmol) were purchased from Amersham-GE Healthcare Europe GmbH (Orsay, France). R5020

In vitro estrogen receptors studies

We used HELN ERα and HELN ERβ cell lines [13], [14] to evaluate the estrogenic activities of tibolone and its primary metabolites. In HELN parental cells, neither E2 nor tibolone and its metabolites induced luciferase expression. On the contrary, a small decrease was observed for tibolone and its metabolites at concentrations higher than 1 μM. This is probably due to toxicity (data not shown). In HELN ERα and HELN ERβ cell lines, the EC₅₀ value of E2 was respectively 0.018 and 0.08 nM (Fig. 1A

Discussion

In this study, we have assessed the potential hormonal profile of tibolone and its primary metabolites on all human steroid receptors (PR, AR, GR, MR, ERα and ERβ) using HeLa or PC3 cells stably transfected with a given receptor and a luciferase reporter gene. We show that tibolone and its Δ⁴ isomer predominantly bind and activate PR and AR whereas 3α and 3β-OH-tibolone predominantly bind and activate ERα (Table 1). The hydroxyl metabolites are full agonists for both ERs and bind preferentially

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Traumatic brain injury (TBI) is a pathology of great social impact, affecting millions of people worldwide. Despite the scientific advances to improve the management of TBI in recent years, we still do not have a specific treatment that controls the inflammatory process after mechanical trauma. The discovery and implementation of new treatments is a long and expensive process, making the repurpose of approved drugs for other pathologies a clinical interest. Tibolone is a drug in use for the treatment of symptoms associated with menopause and has been shown to have a broad spectrum of actions by regulating estrogen, androgen and progesterone receptors, whose activation exerts potent anti-inflammatory and antioxidant effects. In the present study, we aimed to investigate the therapeutic potential of the tibolone metabolites 3α-Hydroxytibolone, 3β-Hydroxytibolone, and Δ4-Tibolone as a possible therapy in TBI using network pharmacology and network topology analysis. Our results demonstrate that the estrogenic component mediated by the α and β metabolites can regulate synaptic transmission and cell metabolism, while the Δ metabolite may be involved in modulating the post-TBI inflammatory process. We identified several molecular targets, including KDR, ESR2, AR, NR3C1, PPARD, and PPARA, which are known to play critical roles in the pathogenesis of TBI. Tibolone metabolites were predicted to regulate the expression of key genes involved in oxidative stress, inflammation, and apoptosis. Overall, the repurposing of tibolone as a neuroprotective treatment for TBI holds promise for future clinical trials. However, further studies are needed to confirm its efficacy and safety in TBI patients.
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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.
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It is formed predominantly by the enzyme 3β-hydroxysteroid dehydrogenase-isomerase from 3β-OH-T and to a lesser extent from TIB [16,21]. Both TIB and Δ4-isomer exhibit affinity for progesterone receptor (PR) A and B and for androgen receptors (ARs), with no effect on ERs [16]. It is noteworthy that this metabolite is formed locally in the endometrium on TIB administration; therefore, it can prevent estrogenic action in this tissue by stimulating local progestagenic and androgenic activity [22], which protects against endometrial cancer [23].
Tibolone (TIB), a selective tissue estrogenic activity regulator (STEAR) in clinical use by postmenopausal women, activates hormonal receptors in a tissue-specific manner. Estrogenic activity is present mostly in the brain, vagina, and bone, while the inactive forms predominate in the endometrium and breast. Conflicting literature on TIB’s actions has been observed. While it has benefits for vasomotor symptoms, bone demineralization, and sexual health, a higher relative risk of hormone-sensitive cancer has been reported. In the brain, TIB can improve mood and cognition, neuroinflammation, and reactive gliosis. This review aims to discuss the systemic effects of TIB on peri- and post-menopausal women and its role in the brain. We suggest that TIB is a hormonal therapy with promising neuroprotective properties.
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This could be related to the fact that this steroid did not have effects over proteins involved in these processes that are overexpressed in glioblastomas, such as VEGF, EGFR or IGFR (44–46). TIB and its metabolites have affinity for ERs and PRs (47); estradiol and progesterone treatments induce proliferation of glioblastoma cells and the inhibition of their receptors blocked this effect (11,18,48). Our results showed that the inhibition of ERs and PRs blocked the proliferative effect of TIB in globlastoma cells, suggesting that TIB exerts its effect on cell proliferation through these steroid receptors.
Ovarian steroid hormones are involved in modulating the growth of glioblastomas (the most common, aggressive, and lethal brain tumor) through the interaction with their intracellular receptors. Activation of sex hormone receptors is involved in glioblastomas progression. Tibolone (TIB) is a selective tissue estrogenic activity regulator widely prescribed to treat menopausal symptoms and to prevent bone lost. The effects of TIB on the growth of glioblastoma are unknown.
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Our results suggest that TIB increases cell number and proliferation of human glioblastoma cells through the regulation of ERs and PRs actions and content.
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Traumatic brain injury (TBI) is the leading cause of death and disability in young and adult people. Since TBI is a lesion characterized by its heterogeneity and complexity, many clinical and pharmacological approaches have been unsuccessful. The current understanding of the physiopathology of TBI indicates that it is imperative to focus on a multifactorial therapy. Endogenous hormones have pleiotropic neuroprotective effects that can be used for a therapeutic approach in TBI. However, these neurosteroids have secondary effects depending on the target tissue. In this concern, the development of more specific estrogenic-progestogenic-androgenic like compounds and the advances in elucidating their protective role in TBI has led to the use of SERMs and STEARs. In vitro and in vivo models have shown promising results in elucidating the role of neurosteroids and how they may be considered as a potential therapy aimed at improving the outcome in TBI.
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Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites

Abstract

Introduction

Section snippets

Chemicals and materials

In vitro estrogen receptors studies

Discussion

BJOG

Maturitas

Fertil. Steril.

Steroids

J. Steroid Biochem. Mol. Biol.

J. Steroid Biochem. Mol. Biol.

Sci. Total Environ.

Biochem. Pharmacol.

Mol. Cell Endocrinol.

Toxicol. Appl. Pharmacol.

Steroids

Maturitas

Fertil. Steril.

J. Biol. Chem.