Conceived and designed the experiments: PM ZP KDW. Performed the experiments: ZP EK AT NH CZ EM IC AK. Analyzed the data: PM ZP LN GC KDW. Wrote the paper: PM ZP EK NH LN CZ KDW.
This research was supported by the following commercial sources: Gaea Products SA, Aktina SA, Yiotis SA, Pierre Fabre Hellas, Bee Culturing Co “Attiki” Alex Pittas, Korres SA (EPAN-TP27, PENED-70-3-6340, PAVE-70-3-8966). This support does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Royal jelly (RJ) excreted by honeybees and used as a nutritional and medicinal agent has estrogen-like effects, yet the compounds mediating these effects remain unidentified. The possible effects of three RJ fatty acids (FAs) (10-hydroxy-2-decenoic-10H2DA, 3,10-dihydroxydecanoic-3,10DDA, sebacic acid-SA) on estrogen signaling was investigated in various cellular systems. In MCF-7 cells, FAs, in absence of estradiol (E2), modulated the estrogen receptor (ER) recruitment to the pS2 promoter and pS2 mRNA levels via only ERβ but not ERα, while in presence of E2 FAs modulated both ERβ and ERα. Moreover, in presence of FAs, the E2-induced recruitment of the EAB1 co-activator peptide to ERα is masked and the E2-induced estrogen response element (ERE)-mediated transactivation is inhibited. In HeLa cells, in absence of E2, FAs inhibited the ERE-mediated transactivation by ERβ but not ERα, while in presence of E2, FAs inhibited ERE-activity by both ERβ and ERα. Molecular modeling revealed favorable binding of FAs to ERα at the co-activator-binding site, while binding assays showed that FAs did not bind to the ligand-binding pocket of ERα or ERβ. In KS483 osteoblasts, FAs, like E2, induced mineralization via an ER-dependent way. Our data propose a possible molecular mechanism for the estrogenic activities of RJ's components which, although structurally entirely different from E2, mediate estrogen signaling, at least in part, by modulating the recruitment of ERα, ERβ and co-activators to target genes.
Royal jelly (RJ), a yellowish material excreted by the mandibular and hypopharyngeal glands of worker bees of the genus
Estrogens play pivotal roles in regulating the function of many tissues and organs and estrogen signaling has been associated with a number of diseases, including breast and uterine cancers, disorders of lipid metabolism, cardiovascular diseases, autoimmune inflammatory diseases, osteoporosis, menstrual abnormalities and infertility
In the present study, we investigated the possible estrogenic/antiestrogenic effects of the RJ-derived fatty acids, 10H2DA, 3,10DDA and SA, in various cellular systems in vitro. We examined the ability of FAs, at physiologically achievable levels, to modulate 1) the recruitment of ERα and ERβ to the E2 responsive region of the pS2 promoter in the MCF-7 cell line, 2) the regulation of pS2 mRNA levels in the MCF-7 cell line, 3) the activity of ERα and ERβ on an ERE-driven Luc-reporter gene in MCF-7 and HeLa cells and 4) the E2-induced recruitment of the EAB1 co-activator peptide to ERα. Furthermore, we examined the potential of FAs to induce mineralization in KS483 osteoblasts, which is an ER regulated process in bone remodeling. Finally, we assessed the capacity of FAs to bind to ERs and we also modeled the interaction of FAs with ERα to reveal potentional sites of interaction.
The 10-hydroxydec-2-enoic (10H2DA), 3,10-dihydroxydecanoic (3,10DDA) and sebacic (SA) fatty acids were isolated from RJ by chromatographic separation (Liquid Chromatography, LC and Medium Pressure Liquid Chromatography, MPLC) and identified by means of spectroscopic data analysis, mainly via the concerted application of 1D and 2D Nuclear Magnetic Resonance (NMR) techniques (Heteronuclear Multiple Quantum Coherence, HMQC and Heteronuclear Multiple Bond Coherence, HMBC) and mass spectrometry, as described previously
A cervical adenocarcinoma ER negative cell line (HeLa, ATCC Cell Bank), an endometrial ER positive cancer cell line (Ishikawa ECACC Cell Bank, No 99040201), an ERα positive breast carcinoma cell line (MCF-7, ATCC Cell Bank) and a human hepatoma ER negative cell line (Huh7, ATCC Cell Bank) were used. For chromatin immunoprecipitation (ChIP) experiments, a stable cell line, MCF-7 tet-off Flag-ERβ that expresses an inducible version of ERβ fused to a Flag-tag, was used. This cell line expresses endogenous ERα. The KS483 bone cell line is a non-transformed stable subclone of a parental mouse cell line KS4 that has the ability to form mineralized nodules in vitro. All cell lines were maintained as previously described
Cells were seeded in 150-mm dishes and grown in the presence (ERα+/ERβ−) or in the absence of tetracycline (ERα+/ERβ+) for 4 days in phenol red (PR) free DMEM supplemented with 10% dextran-coated charcoal (DCC)-treated fetal bovine serum (FBS). Cells were treated with 10−8 M E2 or 10−6 M FAs for 45 min. Co-incubation was performed with 10−8 M E2 and 10−6 M FAs. ChIP was performed as previously described
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Cells were seeded in 6-well plates and grown in the presence (ERα+/ERβ−) or in the absence of tetracycline (ERα+/ERβ+) for 4 days in PR free DMEM 10% DCC-FBS. Cells were treated with 10−8 M E2 or 10−10–10−5 M FAs for 24 hrs. Co-incubation was performed with 10−8 M E2 and 10−9, 10−7 or 10−6 M FAs. Total RNA were purified using the RNeasy Mini Kit. Two µg of total RNA was reverse transcribed into cDNA using TaqMan Reverse Transcription Reagents with random hexamer primers. Real time PCR assays were conducted using SYBR green master mix RT-PCR reagent. Acidic ribosomal phosphoprotein PO (36B4) was used as an internal control gene
Before each transfection experiment cells were maintained for 2 days in PR free DMEM containing 10% DCC-FBS. For transfection assays, cells were plated in 6-well or 24-well plates in PR free DMEM with 10% DCC-treated FBS and transfected using reagents and plasmids as stated in
Plasmids | DNA quantity/well | Reagents | |
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ERE (2xERE-TATA-Luc) | 0.2 µg | Lippofectamine (Invitrogen) |
pRL-TK (Renilla-Promega) | 0.01 µg | ||
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GRE (MMTV-Luc) | 0.2 µg | Effectene Tranfection Reagent (Qiagen) |
β-gal (pCMVβ) | 0.2 µg | ||
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ERα (HO-hERα) | 0.5 µg | Polyfect Transfection Reagent (Qiagen) |
ERE (3xERE-TATA-Luc) | 0.5 µg | ||
β-gal (pCMVβ-Clontech) | 0.5 µg | ||
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ERβ (pSG5-hERα) | 0.5 µg | Polyfect Transfection Reagent (Qiagen) |
ERE (3xERE-TATA-Luc) | 0.5 µg | ||
β-gal (pCMVβ-Clontech) | 0.5 µg |
The day before the transfection, Huh7 cells were seeded into 24-well plates in PR free medium 10% DCC-FBS and 2 mM L-glutamine. Cells were transfected with Genejuice as instructed by the manufacturer. After transfection, cells were treated with E2 (1 µM), 4OH-TMX (500 nM), FAs (5 µM) or FAs in combination with E2 for 16 h. C. Luciferase and β-galactosidase activity was assayed as earlier described
For the assays, cells were seeded in 12-well plates in a-MEM 10% DCC-FBS. Three days after plating, cells reached confluence and were subsequently induced to differentiate by the addition to the culture medium of 50 µg/ml ascorbic acid in the absence or presence of FAs in a concentration range 10−10–10−7 M. E2 (10−9–10−6 M) was used as positive control. Co-incubation with ICI182780 (10−7 M) was also performed. B-glycerophosphate was added after day 10. The medium with the reagents was refreshed every 3–4 days for 24 days in total. After 24 days, cells were rinsed with PBS. The number of mineralized bone nodules was identified with Alizarin Red-S. For Alizarin Red- S (sodium alizarin sulphonate) staining, 2% Alizarin Red- S (Sigma) was prepared in distilled water and the pH was adjusted to 5.5. Cultures were fixed with 5% formalin (10 min), washed, and stained with Alizarin Red- S for 5 min. After removal of unincorporated excess dye with distilled water, the mineralized nodules were labeled as red spots. Mineralized nodules were counted by light microscopy at a 10-fold magnification as described previously
Ishikawa cells and MCF-7 were cultured and the effect of FAs (1.6×10−7–4×10−4 M) on cell viability was estimated by a modification of the MTT assay, as previously described
The ligand binding domain of the human ERα (hERα-LBD) and human ERβ (hERβ-LBD) were produced individually in
Three-dimensional models of the FAs (10H2DA, 3,10 DDA, and SA), as well as of the co-factor peptide EAB1, were built using PyMol. The FAs were docked to the ligand pocket and to the co-activator binding site and then the complexes were minimized using 100 steps of Steepest Descent followed by 500 steps of Adopted Basis Newton-Raphson minimization in CHARMM
The RJ's FAs may modulate estrogen signaling by various mechanisms, involving binding to the ligand binding pocket of the receptor, influencing the abundance/distribution of ER subtypes and their recruitment to E2 responsive genes, modulating co-activators and/or co-repressors, physically blocking co-activator and co-repressor recruitment, or alternatively by inducing proteins which may disrupt ER dimerization. Estrogenic effects of RJ FAs could also involve GPR30-mediated signaling
We examined the estrogenic/antiestrogenic activity of 10H2DA, 3,10DDA and SA, which were isolated and identified previously
Effects of FAs on pS2 mRNA levels in the presence of ERα (A) or ERα and ERβ (B) together (II). MCF-7 tet-off Flag-ERβ cells were treated as mentioned in
In the presence of ERα, FAs at all concentrations tested did not change pS2 mRNA levels, while pS2 mRNA levels were increased after E2 treatment (
The addition of 10H2DA, 3,10DDA or SA (10−10–10−5 M), in the presence of E2 (10−8 M), inhibited the E2-mediated induction of an ERE-driven luciferase reporter gene in MCF-7 cells in a dose-dependent manner (
Effects of FAs, on ERE-mediated transactivation in MCF-7cells (II). Cells were transfected under conditions as shown in
The ability of E2, ICI182780, 4OH-TMX and FAs to modulate ERE-driven luciferase activity in HeLa cells transfected with either ERα (A) or ERβ (B) is shown in
The molecular basis for ER agonism is dependent on formation of a hydrophobic surface within the LBD, which represents the docking surface for α-helical leucine-rich peptide motifs in co-activators
As shown in
Cells were treated as mentioned in
To examine a possible binding of FAs to the ligand pocket of the receptor, we used a competition binding assay. Using ERα (PPT) and ERβ (DPN) selective agonists, we confirmed the expression and specificity of the receptors in the cell extracts used in this assay. PPT exhibited 1000-fold higher relative binding affinity in ERα- than in ERβ-expressing cell extracts (10−9 M and 10−6 M respectively), while DPN had 200-fold higher relative binding affinity in ERβ-expressing cell extracts compared to ERα-expressing cell extracts (10−8 M). E2 had equal Relative Binding Affinity (RBA) in both cell extracts (10−9). The assays revealed that SA and 3,10DDA did not bind to ERα or ERβ at all concentrations tested (data not shown). However, 10H2DA exhibited binding to both receptors, but only at extreme concentrations (10−4 M).
The FAs were docked in the ERα ligand binding pocket, with the EAB1 peptide present at the co-activator binding site, and interaction energies between FAs and ERα were obtained in the range of −44 to −63 kcal/mol. For comparison, the interaction energy between the receptor molecule and E2 obtained by the same computational procedure is -70 kcal/mol (
A. The fatty acid 10H2DA in the ligand pocket of ERα. The protein molecule is represented by its contact surface, whereas the fatty acid is represented by spheres (oxygen atoms in red, carbon atoms in grey). B. The pair Glu353-Arg394 (residue numeration follows that of PDB entry 1GWR) and the carboxyl group of 10H2DA (van der Waals spheres) in the ligand pocket of the estrogen receptor. The orientation of the protein molecule is identical to that in A. The co-activator EAB1 is represented by ribbon in blue.
In this study, we determined the possible estrogenic/antiestrogenic properties of 10H2DA, 3,10DDA and SA, isolated from RJ and identified by spectroscopic methods
Using a ChIP assay in MCF-7 breast cancer cells, which are stably transfected with an inducible version of ERβ and express endogenous ERα, we examined the ligand-dependent recruitment of ERα and ERβ to chromatin. None of the tested FAs could modulate ERα recruitment to the pS2 promoter, whilst they increased ERβ recruitment to this promoter. All FAs inhibited the effect of E2 on ERα and ERβ recruitment. Consistent with the effects on receptor recruitment to DNA, experiments revealed that in the presence of ERβ, FAs could decrease pS2 mRNA levels, when added alone, and that they decreased E2's effect in the presence and absence of ERβ. However, since in this cell system endogenous ERα is always present, effects on pS2 expression cannot easily be determined for ERβ alone. We further assessed the effects of FAs on ERα alone and ERβ alone in HeLa cells. This cell line, in contrast to MCF-7 cells, lacks endogenous ER. In HeLa cells, we demonstrated that all FAs, when assayed alone, were weak enhancers of ERα-mediated activity, while they antagonized ERβ-mediated effects. In the presence of E2 they antagonized the E2-mediated effects via ERα and ERβ. The well characterized selective estrogen receptor modulator (SERM) 4OH-TMX also exhibited agonistic effects on ERα-mediated activity, while it was a complete antagonist of ERβ-mediated action. This is in agreement with a previous study reporting that 4OH-TMX induced ERE-mediated reporter gene activity in a stably transformed ERα expressing cell line, but exhibited pure antagonism in the corresponding ERβ expressing system
Recruitment of co-factors is an essential component of ER signaling. The best defined structure-function of a co-regulator interaction is with co-activators that interact through a conserved LxxLL motif, termed an NR box. Interestingly, in MCF-7 cells we show that the recruitment of the EAB1 co-activator peptide upon E2 binding is reduced when FAs are present. This suggests that FAs are preventing proper ER activity, possibly by inducing a conformational response at the co-activator binding site, leading to masking of the co-activator site.
In the ERE-driven luciferase reporter gene assay in MCF-7 cells, all 3 FAs inhibited the E2-mediated increase in luciferase activity, suggesting an ER-mediated effect and a common signal transduction pathway for E2 and FAs at the level of ERE-containing promoters. Additionally, all 3 FAs showed a trend towards increasing the ERE-driven luciferase activity when tested alone. This is consistent with results from Suzuki et al. showing that 10H2DA increased the ERE-driven luciferase activity in MCF-7 cells at the same concentration range. However, co-incubation of FAs with E2 was not investigated in their study
The specificity of FAs with regard to steroid receptor activation was explored by assaying the effects of FAs in MCF-7 cells on GRE-mediated transactivation. The FAs did not alter the basal nor the Dex-induced GRE-mediated transcriptional activity, indicating that the inhibition by FAs has specificity with respect to modulation of NR-mediated functions. In line with our findings, Thurmond et al.
We have explored possible mechanism(s) for the effects of FAs on ER signaling by molecular modeling. As mentioned above, it is possible that the FAs compete with the LXXLL- containing co-activator for the activation function domain 2 (AF2) binding site of the receptor. Of note, docking experiments showed significant favorable interaction energy between the FAs and ERs. However, similar interaction energies were also observed for other locations on the protein's surface, distant from the co-activator binding site. Among the locations showing substantially more favorable intermolecular interactions (−211 kcal/mol) is a region including the loop around Tyr459. This loop is part of the subunit interface in the dimeric ER. Hence, binding of FAs may interfere with the dimerization of ERs and in this way influence co-activator binding (
FAs may bind to the ligand pocket, thus competing with E2. The computational fitting showed very good compatibility of the ligand pocket for all three FAs (
In line with our findings, a recent study on 3,3′-diindolylmethane, a selective activator of ERβ that does not bind to ERβ, proposes a possible mechanism of activation through recruitment of co-activators (i.e. SRC-2)
On the basis of the findings by Narita et al.
RJ is used extensively in commercial nutritional supplements, medical products, and cosmetics in many countries, while SA, one of its major components, is widely employed in medical practice, e.g. parenteral nutrition, orthopedic applications, drug delivery systems, vaccine development
Conclusions are highlighted in lined text boxes (I). Possible molecular mechanism for how FAs modulate E2 signaling through ERs (II). A. Classical E2 regulation of gene transcription through recruitment of ERα or ERβ to the promoter region B. In the presence of E2, FAs seem to block the effect of E2 on ERα and ERβ recruitment to DNA and gene expression (pS2 and ERE-Luc). FAs could bind to a distinct region away from ligand binding pocket either to the co-activator binding pocket or to the dimerization region. This is consistent with the lack of competition by FAs for E2 binding to the ligand binding pocket and with the interference of FAs with E2 induced binding of a co-activator peptide.
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We specially thank M. Bengtsson (KaroBio AB, Sweden) for binding assays, M.Putnik for offering MCF-7 tet-off Flag ERβ stable cell line, JÅ Gustafsson for pSG5-hERβ, K Tomoshige for HO-hERα and MMTV-Luc, KS Korach and D McDonnell for 3xERE-TATA-Luc, T Yamashita and M Karperien for KS483cells.