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. Author manuscript; available in PMC: 2023 Feb 20.
Published in final edited form as: Steroids. 2022 Jul 26;187:109094. doi: 10.1016/j.steroids.2022.109094

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

Kim Enfield 1, Chanel Avenant 1, Janet P Hapgood 1,2,*
PMCID: PMC9939308  NIHMSID: NIHMS1867184  PMID: 35905833

Abstract

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.

Keywords: Progestins, progesterone receptor, receptor isoforms, dose-response, efficacy, potency

1. Introduction

The PR exists as two isoforms, namely PR-A and PR-B, which are transcribed from the same gene, although from distinct promoters [1]. PR-B differs from PR-A by the presence of an additional transcriptional activation domain [2, 3]. The PR-A and -B isoforms regulate a suite of genes that are unique to each isoform, but they also share regulation of a subset of genes [4–6]. In general, the PR regulates functions relating to reproduction, cognition, inflammation and neurogenesis [7]. Understanding how different progestogens act via the PR isoforms is crucial to understanding the effect different progestogens are likely to have on these important physiological functions.

HCs and HRT are hormonal therapies designed to target the PR to prevent unwanted pregnancy and to alleviate the symptoms of menopause, respectively, in women. All HCs and some HRT treatments contain a progestin, a synthetic PR agonist designed to mimic the progestogenic actions of progesterone (P4). In sub-Saharan Africa, the most commonly used method of HC is the 3-monthly, intramuscular (IM) injectable contraceptive depot-medroxyprogesterone acetate (depo-MPA-IM or DMPA-IM), followed by the levonorgestrel-containing implant, and then the oral pill [8]. P4 and promegestone (R5020) are often used as reference agonists for experimental comparison and determination of progestogen activity [9–12]. Norethisterone (NET) is administered as a 2-monthly injectable (containing 200 mg NET-enanthate (NET-EN)) [13] or as an oral pill [14]. NET-EN is administered in some forms of HC which is then converted to the metabolically active form, NET [15, 16]. Etonogestrel (ETG) and levonorgestrel (LNG) are both administered as an implant as well as an oral pill [17–19]. Nestorone (NES) is currently under investigation in a clinical trial for use in a vaginal ring at different concentrations in combination with ethyl estradiol (EE) [20].

Given the multifunctional roles of the PR and the widespread use of HCs, surprisingly few published studies directly compare the activity of different progestogens via the PR isoforms. Limited studies have characterised the biological effects of the PR in the presence of different progestogens using the dose-response approach, allowing for accurate determination of maximal ligand response (efficacy) and potency. Potency or EC50, is defined as the concentration of ligand required to generate half the maximal response [21, 22]. Dose-response analysis also allows for determination of ligand biocharacter.

We previously showed by dose-response analysis on reporter genes that biological responses via transiently-expressed PR-B for P4, R5020, MPA, NET, LNG and ETG vary significantly in biocharacter, rank order and absolute values for efficacies and potencies [11]. Others have investigated the dose-response effects of P4, LNG, MPA and NET on mRNA levels of PR-regulated genes in T47D cells (human breast cancer cell line) and found these to be gene-specific, but they did not discriminate between PR-A and PR-B effects, or determine potencies or efficacies [5]. A promoter-reporter study in 1471.1 cells (mouse mammary tumour cell line) using only R5020 found that the activity of PR-A was much lower than that of PR-B; however, the response was more potent via PR-B than PR-A [23]. PR-B was determined to be more transcriptionally active than PR-A in response to P4 in CV-1 cells (monkey kidney fibroblasts) and HepG2 cells (human hepatocellular carcinoma) [6]. In addition, PR-A demonstrated an inhibitory effect on PR-B responses in the presence of P4 [6]. However, in this study potencies, efficacies and biocharacters were not determined [6]. In both these studies, endogenous genes were not investigated [6, 23]. Much of the limited available data is potentially confounded by low power; insufficient data points for robust determination of potencies and efficacies; uncertainty in relative PR isoform levels and a dearth of information on multiple progestogens relevant to hormonal therapy [5, 6, 9, 10, 12, 23–25], while very little data is available on comparative progestogen effects on endogenous genes.

In the present study, MDA-MB-231 PR-A (MDA-PR-A+) or -B (MDA-PR-B+) stably-expressing cell lines were used to investigate in parallel the potencies, efficacies and biocharacters for gene expression of a panel of widely-used progestogens via the PR-A and PR-B isoforms with a view to establishing proof-of-concept progestogen effects via the PR in this model system.

2. Experimental

2.1. Cell culture and materials

The MDA-MB-231 PR-A+ and -B+ cell lines were obtained from V. Lin (Nanyang Technological University, Singapore) [26]. Cells were cultured in humidified 37°C incubators containing 5% CO2 in the BSL2 Mammalian Tissue Culture Facility. The cells were maintained in 75 cm2 flasks (Greiner Bio-one International, Austria) in Dulbecco’s modified Eagle’s medium (catalogue no. D0819, DMEM, Sigma Aldrich, RSA) supplemented with 1 mM sodium pyruvate (catalogue no. P5280, Sigma Aldrich, RSA), 44 mM sodium bicarbonate (catalogue no. 144-55-8, Sigma Aldrich, RSA), 7.5% (v/v) foetal calf serum (FCS, catalogue no 26140, Thermo Scientific, RSA) and 500 μg/mL geneticin (G418, catalogue no. 11811023, Thermo Scientific, USA). The following compounds were purchased from Sigma Aldrich, RSA: MPA, P4, NET, ETG, LNG, NES. R5020 was purchased form PerkinElmer Life and Analytical Sciences, USA. All steroids were prepared in absolute ethanol (EtOH) and added to cells such that final [EtOH] was 0.1% (v/v). The luciferase reporter gene plasmid, pTAT-GRE-E1b-LUC (TAT-GRE-LUC), containing two glucocorticoid response elements (GREs) from the rat tyrosine amino transferase (TAT) gene under the control of the E1b promoter, was a gift from G. Jenster (Erasmus University of Rotterdam, Rotterdam, Netherlands) [27]. Cells were routinely checked for mycoplasma contamination by Hoescht staining [28] and fluorescence microscopy and only mycoplasma-negative cells were used for experiments.

2.2. Promoter-reporter assays

Promoter-reporter assays using the luciferase reporter gene were carried out as described previously for MDA-MB-231 cells [11] with a few modifications. After seeding, the cells were transiently transfected with 9000 ng pTAT-GRE-LUC only using XtremeGENE-9 transfection reagent and added dropwise onto cells. After 24 hours, the cells were treated as previously described [11].

2.3. RNA isolation and real-time qPCR

RNA was isolated as previously described [29] with a few modifications. MDA-MB-231 cells were seeded at 1 X 105 cells per well in 12-well plates. After 24 hours, the cells were treated with the indicated ligands or vehicle (0.1% v/v ethanol) in phenol red-free DMEM supplemented with 5% (v/v) charcoal stripped-fetal calf serum (cs-FCS) for a further 24 hours, after which the cells were harvested in Tri Reagent (Sigma-Aldrich, South Africa). RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, South Africa) according to the manufacturer’s instructions. The primer sets were as follows: Glucocorticoid-induced leucine zipper (GILZ) (cat#QIA249900-QT00091035, Qiagen, RSA), Prostaglandin-Endoperoxide Synthase 2 (PTGS2) (forward) 5’-CATATTTACGGTGAAACTCTGGCT-3’and (reverse) 5’-CATTCAGGATGCTCCTGTTTAAG-3’ [30], ATPase Na+/K+ Transporting Subunit Alpha 1 (ATP1A1) (forward) 5’-CACACAGCCTTCTTCGTCAGTATC-3’ and (reverse) 5’-CGAATTCCTCCTGGTCTTACAGA-3’ [5] and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (forward) 5’TGAACGGGAAGCTCACTGG-3’ and (reverse) 5’CCACCACCCTGTTGCTGTA-3’ [31, 32]

2.4. Protein isolation and western blotting

Protein was isolated and western blots were performed as previously described [33] and separated on a 8% SDS-polyacrylamide gel before transfer to nitrocellulose membranes (Amersham) and blocking in 10% fat-free milk powder. Membranes were probed with anti-PR (PGR-312-L-CE, Leica Biosystems, UK) or anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH; 0411, Santa Cruz Biotechnology, USA) (loading control) followed with the HRP-conjugated secondary goat anti-mouse antibody (Santa Cruz Biotechnology, USA). Proteins were visualised using enhanced chemiluminescence (Bio-Rad Laboratories, Inc. USA) and expression levels quantified using ImageJ Software (Version 1.80).

2.5. Data and Statistical Analysis

All statistical tests were performed using GraphPad Prism software (version 9) and are indicated, as well as n values indicating number of independent experiments, in the figure legends. All data were first analysed for normal distribution using a Shapiro Wilk normality test. Normally distributed data were analysed using either one-way or two-way ANOVA with a Tukey post-test for multiple comparisons or two-tailed paired t-tests for comparison between two samples. Non-normally distributed data were analysed using a non-parametric Kruskal-Wallis test (non-parametric one-way ANOVA) with Dunn’s post-test (compared to control sample) for multiple comparisons or a Wilcoxon signed-rank test for comparisons between two samples. Graphs are plotted as mean ± SEM and the variation between independent experiments is represented by the spread of the black dots, indicating individual experiments.

3. Results

3.1. The progestins are agonists for transactivation but have some significant differences in potency via PR-B

To investigate the efficacy and potency of the progestogens, promoter-reporter assays were performed in MDA-PR-B+ cells. For transactivation, we define here a PR agonist as a ligand which induces a maximal response (efficacy) of at least 70% relative to the chosen synthetic reference agonist (R5020). For the purposes of this study, the term “partial agonist” will be used to define a ligand that induces maximal activity that is less than 70% of that of the reference agonist, whether or not antagonism has been established.

Promoter-reporter assays revealed that, compared to R5020, all the progestogens are agonists for transactivation via PR-B, except for NES. However, the efficacy of NES was not significantly less than that of the other progestins or P4 (Fig. 1 ac and Table A.1). Unexpectedly, R5020 efficacy was significantly higher compared to that of the other progestogens (Fig. 1a, c and Table A.1). R5020 was significantly more potent than the other progestogens (Fig. 1b and Table A.1). Additionally, NET, ETG and MPA were similar in potency while LNG and NES had similar potencies (Fig. 1b). The absolute −logEC50 values suggested that R5020 is the most potent (13.80) followed by NES (12.51) ≥ LNG (12.05) > ETG (11.02) ≥ MPA (10.71) ≥ NET (10.46) > P4 (9.56) (Fig. 1b and Table A.1), although significant differences were detected between some, but not all, of these progestogen potencies.

Figure 1: The progestogens are agonists for transactivation and show some significant differences in potency via PR-B on a reporter gene in MDA-PR-B+ cells.

Figure 1:

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a) Shows the % PR-B activity at increasing concentrations of each progestogen relative to R5020 efficacy = 100%. b) Shows the mean −logEC50 values relative to R5020 ± SEM of each ligand while c) shows the mean efficacies ± SEM of each progestogen relative to R5020. One-way ANOVA with a Tukey post-test was performed to determine statistical differences between progestogens where different letters denote statistically significant differences while the same letters denote no significant difference. The SEM is calculated based on the following number of biological repeats: R5020, MPA = 6; P4, NET, LNG, NES = 4, ETG = 5, each containing three technical repeats of each condition.

3.2. Progestogens act differently via PR-B on select endogenous genes

Having shown that the progestogens have similar efficacies (except R5020) but vary in potency via PR-B, we next sought to investigate the effects of the progestogens on endogenous gene expression. The mRNA expression levels of three PR-regulated genes were investigated, namely ptgs2, gilz and atp1a1 in MDA-PR-B+ cells. These genes were selected because the promoter sequences either contain P4response elements (PREs) [34–37] or their expression has previously been shown to be regulated by P4 [38–40]. For the ptgs2 gene, R5020, P4 and NET acted as agonists via PR-B; however, MPA appeared to act as a partial agonist (Fig. 2a). All the progestogens acted as agonists for gilz expression via PR-B (Fig. 2b) while for the atp1a1 gene, MPA generated the most efficacious response (Fig. 2c).

Figure 2: Select progestogens act differently via PR-B on endogenous genes, ptgs2, gilz and atp1a1 in MDA-PR-B+ cells.

Figure 2:

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Relative mRNA levels of a) ptgs2, b) gilz and c) atp1a1 were normalized to gapdh mRNA levels. Relative expression was determined by normalising to control (ctrl) set to 1. Thereafter, data were set relative to R5020 efficacy = 100%. The SEM of above data is calculated based on three biological repeats.

There were some significant differences in potencies and efficacies for the progestogens via PR-B on the expression of all three genes (Table A.2 and Fig. A.1). For the ptgs2 gene, NET generated the most potent response which was significantly more potent than R5020 and MPA (Fig. A.1a). For efficacy, MPA generated a significantly less efficacious response compared to the other progestogens for ptgs2 expression (Fig. A.1b). For gilz expression, there were no significant differences in −logEC50 values (Fig. A.1c) nor efficacies (Fig. A.1d) between progestogens. Similar to the gilz gene expression, there were no significant differences in potency for atp1a1 gene expression (Fig. A.1e). In terms of efficacy, however, the atp1a1 gene was expressed significantly more in the presence of MPA compared to P4 (Fig. A.1f).

When comparing the potencies and efficacies for each progestogen between the three genes, there were no significant differences in potencies for all except MPA (Fig. A.2a). However, MPA generated a significantly higher efficacy for the atp1a1 gene compared to the ptgs2 gene while it approached a significant difference between ptgs2 and gilz expression (p-value = 0.08) (Fig. A.2b). These results suggest that R5020, P4 and NET act similarly on the three genes investigated while MPA may elicit more differential efficacies via different promoters in the same cell.

3.3. Most progestogen responses via PR-A are significantly more potent and less efficacious than PR-B

After establishing the activity of progestogens via PR-B, we next investigated the activity of the progestogens via PR-A. Relative to the PR-A R5020 efficacy set to 100%, all progestogens were significantly less efficacious than the reference progestin R5020 (Fig. A.3a and A.3c). When assessing the absolute values, R5020 was the most potent progestogen; however, the absolute potency was not significantly different to that of NET, ETG, MPA and NES (Fig. A.3b). As shown in Figure A.3, the potency of P4 was significantly less than that of R5020, while LNG was significantly less potent than all the progestogens via PR-A (Fig. A.3b). In terms of efficacy, R5020 was significantly more efficacious than all the progestogens which acted as partial agonists via PR-A (Fig. A.3c and Table A.3). Additionally, no significant differences were detected between the efficacies for ETG, MPA and NES which were all significantly more efficacious than NET and LNG (Fig. A.3c).

The relative progestogen-induced PR isoform responses were next investigated by comparing the progestogen dose responses on a PR-regulated reporter gene in MDA-PR-A+ cells compared to MDA-PR-B+ cells. When comparing progestogen-induced PR-A activity relative to the efficacy of R5020 via PR-B (set to 100%), promoter-reporter assays revealed that the efficacies of the progestogens via PR-A were much lower than the PR-B R5020 response (Fig. 3a and Table A.4). From the dose response curves, all the progestogen responses appeared to be more potent via PR-A than the PR-B R5020 response (Fig. 3a and Table A.4). When assessing the progestogen responses via PR-A relative to the PR-B R5020 efficacy, both P4 and LNG were significantly less potent than R5020 via PR-A (Fig. 3b). Additionally, all progestogen responses via PR-A, except for ETG, were significantly less efficacious than that of R5020 (Fig. 3c). Relative to the PR-B R5020 efficacy = 100%, when comparing the progestogen-induced PR-A and PR-B responses, all progestogens, except LNG, were significantly more potent via PR-A (Fig. 4a). All progestogen responses were significantly less efficacious via PR-A than PR-B (Fig. 4b).

Figure 3: The progestogens are significantly less efficacious but more potent (except LNG) via PR-A than PR-B relative to PR-B R5020 efficacy = 100%.

Figure 3:

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MDA-PR-A+ cells were seeded and after 24 hours, the cells were transiently transfected with 9 μg 2XPRE-E1b-luc reporter plasmid. Twenty-four hours later, the cells were re-seeded into 96-well tissue culture plates and allowed to adhere overnight. After 24 hours, the cells were treated with increasing concentrations (1X10−13 M–1X10−5 M) of each ligand or vehicle/ctrl (0.1% EtOH) or 24 hours. Thereafter, the cells were harvested and the Luciferase and Bradford assays were performed. a) Shows % PR-A activity at increasing concentrations of each progestogen relative to PR-B R5020 efficacy = 100%. b) Shows the mean −logEC50 values relative to PR-B R5020 ± SEM of each ligand while c) shows the mean efficacies ± SEM of each progestogen relative to PR-B R5020 efficacy = 100%, in MDA-PR-A+ cells. Relative −logEC50 values and efficacies were analysed using one-way ANOVA with a Tukey post-test between progestogens, where different letters denote statistically significant differences while the same letters denote no significant difference. The SEM of the above data is calculated based on the following number of biological repeats: R5020, P4, NES = 5; NET, ETG, MPA = 4 and LNG = 3, each containing three technical repeats of each condition.

Figure 4: Most progestogens are significantly more potent and less efficacious via PR-A relative to PR-B.

Figure 4:

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Mean relative a) −logEC50 values and b) efficacies for PR-B and PR-A are shown from Fig. 1 and Fig. 3, respectively. PR-B R5020 efficacy was set to 100%. Paired t-tests were used to determine statistical differences between cell lines for each progestogen where *, **, *** and **** denote p-value < 0.5, 0.01, 0.001 and 0.0001, respectively.

One possible factor that could contribute to the observed progestogen- and isoform-specific differences is different expression levels of these receptors. We thus investigated the protein expression levels between the two PR stably-expressing MDA cell lines by western blotting. Within each cell line, no significant differences in PR protein levels were detected before and after progestogen treatments (Fig. 5a and b). When the protein levels were set relative to PR-B vehicle (ctrl) = 100%, the PR-A protein levels were significantly higher than PR-B before and after all progestogen treatments (Fig. 5c and d).

Figure 5: MDA-MB-231 PR stable cell line express significantly more PR-A than PR-B proteins.

Figure 5:

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Total a) PR-B and b) PR-A protein levels normalised to GAPDH after progestogen treatment, relative to each respective vehicle control (ctrl) = 100%. c) PR protein levels normalised to GAPDH are plotted relative to the PR-B ctrl set to 100%. d) Shows a representative western blot of the PR-B, PR-A and GAPDH protein levels. For a) and b), one-way ANOVA with a Tukey post-test was performed to determine statistical differences within each cell line where different letters denote statistically significant differences while the same letters denote no significant difference. For c) statistical comparisons were carried out using parametric unpaired t-tests to determine significant differences between cell lines for each progestogen where * and ** denote p-values <0.05 and <0.01 respectively while ns indicates no significant difference. The data are pooled from three independent experiments.

3.5. Neither progestogen potency nor efficacy via PR-A or PR-B correlate with relative binding affinity

We previously reported that there is a correlation between the RBA of glucocorticoid receptor (GR) agonists and their potencies for transactivation via the GR [41]. To determine whether this relationship also holds for the PR, we performed correlations between published RBAs (Table A.5) [42, 43] of the progestogens for the PR and the progestogen-induced PR activity from this study. Figure A.4 shows that there was no significant correlation between RBAs and the progestogen-induced PR-B or PR-A efficacies or potencies for transactivation on the synthetic reporter gene in MDA-PR-B+ cells (Fig. A.4a and b) or MDA-PR-A+ cells (Fig. A.4c and d).

4. Discussion

The PR regulates transcription of specific target genes via multiple mechanisms including direct binding to PREs in the promoter region of these genes or tethering to various DNA-bound transcription factors [44]. We report significant PR-B-mediated dose-dependent and progestogen-specific differences in relative potencies and efficacies via a synthetic PRE-containing promoter-reporter gene, with potencies being more progestogen-specific than efficacies (Fig. 1). In a study by Kumar et al. progestogen-specific responses determined by promoter-reporter assays showed that P4 and LNG exhibited potencies of 98 pM and 5.8 pM via PR-B, respectively [25]. Similarly, PR-B promoter-reporter assays by Sasagawa et al. using P4, MPA and NES showed convincing progestogen-specific responses [12]. Attardi et al. used promoter-reporter assays in the presence of both PR-A and PR-B and the following potency trend was observed: NES > LNG > P4 [9].

We also report significant progestogen-specific dose dependent effects via the PR-B on mRNA levels on the endogenous Ptgs2, gilz and atp1a1 genes. Although significant differences were not detected for all responses, differences may occur but may have been beyond the statistical power of the assays. When comparing our promoter-reporter to our endogenous gene data, some differences were observed. For both the atp1a1 and ptgs2 genes, different relative potencies and efficacies were obtained compared to the promoter-reporter data (Fig. 2). For example, MPA was equally as efficacious as the other progestogens (except R5020) via PR-B for the promoter-reporter assay (Fig. 1); however, MPA generated the least for ptgs2 but the most efficacious response for atp1a1 expression (Fig. 2). However, similar to the promoter-reporter data where NET and MPA generated overlapping dose-response curves, the same trend was seen for gilz expression. NET and MPA generated very similar responses for gilz expression with no significant differences detected between these ligands for both potency and efficacy (Fig. 2 and Fig. A.1). Additionally, as seen for the promoter-reporter data, R5020 was the most potent for gilz expression (Fig. 2 and Fig. A.1). Results suggest that these similarities are due to the presence of multiple PREs in both promoters while the expression of the other genes may be dependent on other mechanisms.

Of particular interest was the effect of MPA on endogenous genes expression which suggested a highly promoter-specific response. The effect of MPA on ptgs2 gene expression was significantly less efficacious than the other progestogens and in contrast, it was the most efficacious on the atp1a1 promoter (Fig. 2 and Table A.2). Since MPA acts via the glucocorticoid receptor (GR) [47] as well as the PR isoforms, this promoter-specific effect may be due to off-target GR activation on some genes in addition to PR-mediated effects. Consistent with this hypothesis, both the atp1a1 and gilz genes contain GREs [34, 37] and the MDA-PR-B+ cells express the GR (Figure A.5). Additionally, others have suggested that expression of the ptgs2 gene may be inhibited by activated GR [48], consistent with the significantly lower efficacy we report in response to MPA for the ptgs2 gene (Fig. 2). Physiologically, the variable MPA response suggests that MPA activity via PR-B may also be highly variable compared to the other progestogens, in vivo. This may be especially relevant in regions with high DMPA-IM use [8].

Some of the progestogen- and promoter-specific effects on mRNA levels may be due to different epigenetic changes such as the degree of DNA methylation, histone modification and RNA silencing. Ligand-induced epigenetic changes to DNA [49, 50] may contribute to differential gene expression patterns. While both MPA and P4 have been shown in separate studies to result in epigenetic changes to the genome [49, 51] compared to estrogen, whether all the progestogens investigated in the present study induce differential epigenetic changes to the genome compared to each other on the same gene, or differences between genes for a particular progestogen, remains to be investigated. Progestogen-specific effects may also be explained by progestogen-specific induced PR conformational changes [45, 46]. Alternatively, they may be because of differential cofactor recruitment, chromatin remodeling and accessibility, resulting in ligand-induced differential gene expression patterns [2]. Promoter-specific differences in potencies and efficacies for endogenous gene expression could also be due to differences in the sequence of DNA flanking the PREs and promoter architecture in terms of presence and frequency of different cis-elements and regulatory motifs within these genes [52, 53].

While previous studies have investigated the activity of some progestins via PR-B or in the presence of both PR isoforms, to the best of our knowledge, this is the first study to determine efficacies and potencies of a wide panel of progestogens (R5020, NET, ETG, LNG, NES, MPA) and P4 in parallel via PR-A and PR-B in two cell lines that only differed in the PR isoform that was stably-expressed. Relative to the PR-B R5020 efficacy, all the progestogens were significantly less efficacious via PR-A than PR-B on the reporter gene (Fig. 4), consistent with previous studies for R5020 [54] and P4 [6]. Additionally, relative to the PR-B R5020 efficacy, the progestogen responses via PR-A were significantly more potent via PR-A than PR-B (Fig. 4), consistent with results by others for P4, LNG and NES [9]. Unlike the present study, an investigation by Lim et al. showed that PR-B responses were more potent than PR-A. Differences in relative potency of PR-A vs PR-B in this study and others, on promoter-reporter genes could be due to differences in the sequences and context of the synthetic promoters or differences in methodology.

Our data highlight the potential importance of PR ratios. They suggest that in cells where more PR-B is expressed than PR-A, the resultant progestogen response will be more efficacious and in cases where PR-A expression is greater, the progestogen response may be more potent. A limitation, however, of ourstudy is that the higher PR-A signal compared to PR-B (Fig. 5) may be due differences in protein expression or the antibody’s enhanced sensitivity to PR-A protein compared to PR-B [57].

Taken together, our findings showing that select and widely-used progestogens exhibit progestogen-, promoter- and isoform-specific dose-response effects via the PR in our model system provide valuable proof of concept insights into differential gene expression responses and highlight the potential importance of choice of progestogen. Although we used a breast cancer cell line, these experiments were not designed to investigate physiologically relevant effects of progestogens on breast cancer. They do, however, offer insights into differential effects of progestogens specifically via the PR isoforms, but within the limits of a cell line with stable PR expression. The extent to which the results obtained are cell-specific remains to be investigated, but this was beyond the scope of the present study. Our data suggest that the PR isoforms act differently in the presence of the same progestogen and the varying PR ratios could have profound effects on biological outcomes.

Supplementary Material

Enfield et al, Steroids, supp

Funding

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant R01HD83026, to J. P. H.).

Abbreviations:

HCs

Hormonal contraceptives

HRT

hormone replacement therapy

PR

progesterone receptor

RBA

relative binding affinity

PRE

progesterone response element

IM

intramuscular

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Enfield et al, Steroids, supp
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