Fatty Acids Derived from Royal Jelly Are Modulators of Estrogen Receptor Functions

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.


Introduction
Royal jelly (RJ), a yellowish material excreted by the mandibular and hypopharyngeal glands of worker bees of the genus Apis mellifera, is a food essential for the longevity of the queen bee. RJ exerts estrogen effects in vitro and in vivo, similar to those evoked by 17b-estradiol (E 2 ) [1,2,3]. However, the mediators of RJ's estrogenic effects remain unknown. While RJ contains a considerable amount of proteins, free amino acids, sugars, vitamins and sterols, the medium chain fatty acids (FAs) 10hydroxy-2-decenoic (10H2DA), 3,10-dihydroxydecanoic (3,10 DDA) and sebacic (SA) acids ( Fig. 1) are major and unique RJ components [4,5,6].
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 [7]. Estrogens exert their effects via intracellular receptors, estrogen receptors alpha (ERa) and beta (ERb) [8,9,10]. In the presence of ligands, both ERa and ERb are activated and as dimers interact with specific DNA sequences. Activated ERs interact with other nuclear proteins, such as steroid receptor coregulators, altering the transcription rates of responsive genes. The activated ERa and ERb can also bind to other transcription factors, such as activator protein 1 (AP-1) and nuclear factor kappa B (NF-kB), affecting their binding to their cognate DNA sequences and their transcriptional effects [11]. More recently, the G proteincoupled receptor, GPR30/GPER, has been shown to mediate rapid estrogen effects as well as to regulate transcriptional activation. Possible synergism and antagonism with classical estrogen receptors has been suggested [12].
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 ERa and ERb 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 ERa and ERb on an ERE-driven Luc-reporter gene in MCF-7 and HeLa cells and 4) the E 2 -induced recruitment of the EAB1 co-activator peptide to ERa. 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 ERa to reveal potentional sites of interaction.

Cell cultures
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 ERa 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-ERb that expresses an inducible version of ERb fused to a Flag-tag, was used. This cell line expresses endogenous ERa. 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 [13,14,15].

Determination of mRNA and protein levels
Cells were seeded in 6-well plates and grown in the presence (ERa+/ERb2) or in the absence of tetracycline (ERa+/ERb+) for 4 days in PR free DMEM 10% DCC-FBS. Cells were treated with 10 28 M E 2 or 10 210 -10 25 M FAs for 24 hrs. Co-incubation was performed with 10 28 M E 2 and 10 29 , 10 27 or 10 26 M FAs. Total RNA were purified using the RNeasy Mini Kit. Two mg 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 [18]. The sequences of the primers are listed in Table 1. For detecting ERa protein levels, cells were incubated as mention above. Western blot analysis was carried out as previously described [19] using the following antibodies: anti-ERa (HC-20, Santa Cruz Biotechnology) and anti-b actin (A2228, Sigma).

Transfection studies in HeLa cells and MCF-7 cells
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 Table 2, according to the manufacturer's instructions and as previously described [13]. MCF-7 cells transfected with EREs were incubated with E 2 (10 28 M) or FAs (10H2DA, 3,10DDA, SA) in a concentration range of 10 210 -10 25 M. Co-incubation of FAs with E 2 (10 28 M) was also carried out. MCF-7 cells transfected with Glucocorticoid  Table 2.

Mammalian two-hybrid assay
The day before the transfection, Huh7 cells were seeded into 24-well plates in PR free medium 10% DCC-FBS and 2 mM Lglutamine. Cells were transfected with Genejuice as instructed by the manufacturer. After transfection, cells were treated with E 2 (1 mM), 4OH-TMX (500 nM), FAs (5 mM) or FAs in combination with E 2 for 16 h. C. Luciferase and b-galactosidase activity was assayed as earlier described [15].

Mineralization assay in KS483
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 mg/ml ascorbic acid in the absence or presence of FAs in a concentration range 10 210 -10 27 M. E 2 (10 29 -10 26 M) was used as positive control. Co-incubation with ICI182780 (10 27 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 [13,20].

MTT cell viability assay
Ishikawa cells and MCF-7 were cultured and the effect of FAs (1.6610 27 -4610 24 M) on cell viability was estimated by a modification of the MTT assay, as previously described [13]. This assay measures the fraction of active mitochondria of living cells. Thus, since results depend both on the mitochondria activity per cell and on the number of cells present, MTT assay estimates cell proliferation and survival [21].

Ligand binding assay
The ligand binding domain of the human ERa (hERa-LBD) and human ERb (hERb-LBD) were produced individually in Escherichia coli in 2xLB medium supplemented with 50 mM biotin. The cells were harvested by centrifugation and the cell pellet stored frozen at 220uC. The pellets were suspended in Tris buffer and the cell walls were disrupted in a Microfluidizer M-110L. The supernatants with receptor were stored at 270uC. The expression of recombinant ERa and ERb, respectively, in the extracts was confirmed using the ERa selective agonist PPT (propylpyrazol Table 1. Primer pairs for amplification of ChIP enriched regions of pS2 promoter and 18s and mRNA levels of ERa, pS2 and acidic ribosomal phosphoprotein PO (36B4).  triol) and the ERb selective agonist DPN (2,3,-bis(4-hydroxyphenyl) propionitrile) [22,23]. Receptor extracts were thawed on ice from 270uC and mixed with streptavidin coated SPA beads in pH8 buffer (

Modeling of fatty acid interactions with ERa
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 coactivator 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 [24]. The parameters for the FAs were compiled using the CHARMM force field for proteins [25], lipids [26,27] and the CHARMM general force field [28]. The X-ray structure of the ERa receptor with PDB entry code 1GWR [29,30] was used in the calculations. Missing atoms were built and E 2 was parameterized as previously described [31]. The binding of the organic molecules to the receptor was evaluated on the basis of the interaction energy (Coulomb and van der Waals interactions) between receptor and ligand or cofactor peptide.

Results
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 E 2 responsive genes, modulating coactivators 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 [12]. We investigated the RJ FAs with regard to effects on a panel of in vitro bioassays that detect estrogenicity/antiestrogenicity of a test substance [21,32].
We examined the estrogenic/antiestrogenic activity of 10H2DA, 3,10DDA and SA, which were isolated and identified previously [6], in several estrogen-responsive biological systems (Fig. 1). E 2 was used as positive control for agonist activity, whereas ICI182780, a well-known complete estrogen antagonist, served as control for antagonist action. 4OH-TMX served as control for partial estrogen agonism/antagonism activity.
FAs induce ERb recruitment to the pS2 promoter

FAs modulate pS2 mRNA levels
In the presence of ERa, FAs at all concentrations tested did not change pS2 mRNA levels, while pS2 mRNA levels were increased after E 2 treatment (Fig. 2.II.A). However, when co-incubated (10 26 M) with E 2 , FAs decreased E 2 -mediated induction of pS2 mRNA consistent with the results of ChIP assay. When ERb was co-expressed with endogenous ERa, 10H2DA and 3,10DDA significantly decreased pS2 mRNA levels at concentrations of 10 26 M (Fig. 2.II.B). In this system, 10H2DA and 3,10DDA also abolished the induction of pS2 mRNA by E 2 . In MCF-7 cells, with or without ERb expression, FAs alone, at all concentrations tested, do not affect ERa mRNA or nuclear ERa protein levels (Fig. S1).

FAs reduce ERE-mediated transcriptional activity in MCF7 cells
The addition of 10H2DA, 3,10DDA or SA (10 210 -10 25 M), in the presence of E 2 (10 28 M), inhibited the E 2 -mediated induction of an ERE-driven luciferase reporter gene in MCF-7 cells in a dose-dependent manner (Fig. 3.II). When incubated in the absence of E 2, all FAs increased slightly, but not significantly, the basal ERE-driven luciferase activity, in the concentration range of 10 26 -10 25 M (Fig. S2). In MCF-7 cells transfected with GREdriven luciferase reporter, the addition of 10H2DA, 3,10DDA or SA (10 210 -10 25 M) did not alter the GRE-mediated transcriptional activity, when assayed alone or in the presence of DEX (10 26 M) (Fig. S3).

FAs modulate ERa-and ERb-mediated reporter gene activity in HeLa cells
The ability of E 2 , ICI182780, 4OH-TMX and FAs to modulate ERE-driven luciferase activity in HeLa cells transfected with either ERa (A) or ERb (B) is shown in Figure 3.I. The presence of E 2 (10 29 M) increased the ERa-and ERb-mediated luciferase activity, while co-incubation with ICI182780, as expected, diminished the E 2 -enhancing effect in both systems. ICI182780 (10 28 M), when added alone, diminished the basal luciferase activity mediated by ERa and ERb. In agreement with previous reports, 4OG-TMX was a weak agonist of ERa and a potent antagonist of ERb in this system [33]. All FAs enhanced the ERamediated activity, when incubated alone at various concentrations (10 210 -10 25 M) (Fig. S4). Moreover, FAs attenuated the effects of E 2 under co-incubation conditions (Fig. 3.I.A). All FAs diminished ERb-mediated activity when incubated alone at various concentrations (10 210 -10 25 M) (Fig. S4). These FAs also attenuated the effects of E 2 under co-incubation conditions (Fig. 3.I.B). Figure 3.I shows the data for the effects of FAs on ERE-luciferase activity at a FAs concentration of 10 26 M and co-incubation with 10 29 M E 2 (full data in Fig. S4).

FAs alter E 2 -induced co-activator recruitment to ERa
The molecular basis for ER agonism is dependent on formation of a hydrophobic surface within the LBD, which represents the docking surface for a-helical leucine-rich peptide motifs in coactivators [29]. A mammalian two-hybrid assay was used to monitor induction of an agonist conformation in the receptor, which allows recruitment of a peptide containing an a-helical leucine-rich motif (LxxLL) upon ligand binding [15]. The LxxLLcontaining peptide EAB1 is strongly associated with the receptor when E 2 is added, indicating a structural change where the receptor adopts an agonist conformation. The fatty acids, while alone, do not induce a detectable conformational change in ERa. However, when the fatty acids are co-incubated with E 2 , recruitment of the LxxLL peptide is diminished (Fig. 3.III).

FAs induce mineralization in osteoblasts
As shown in Fig. 4, the presence of E 2 (10 29 -10 28 M) induced mineralization in osteoblasts, as expected [20]. Similarly, 10H2DA and SA at 10 29 -10 28 M exhibited an agonistic effect by inducing nodule formation, an effect which was diminished in the presence of ICI182780, thereby suggesting an ER-mediated action.

FAs do not bind to ERa or ERb
To examine a possible binding of FAs to the ligand pocket of the receptor, we used a competition binding assay. Using ERa (PPT) and ERb (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 ERa-than in ERb-expressing cell extracts (10 29 M and 10 26 M respectively), while DPN had 200-fold higher relative binding affinity in ERb-expressing cell extracts compared to ERaexpressing cell extracts (10 28 M). E 2 had equal Relative Binding Affinity (RBA) in both cell extracts (10 29 ). The assays revealed that SA and 3,10DDA did not bind to ERa or ERb at all concentrations tested (data not shown). However, 10H2DA exhibited binding to both receptors, but only at extreme concentrations (10 24 M).

Modeling of FA interactions with ERa
The FAs were docked in the ERa ligand binding pocket, with the EAB1 peptide present at the co-activator binding site, and interaction energies between FAs and ERa were obtained in the range of 244 to 263 kcal/mol. For comparison, the interaction energy between the receptor molecule and E 2 obtained by the same computational procedure is -70 kcal/mol (Fig. 5). We also docked SA at the co-activator binding site, replacing EAB1. In this case also, the interaction energy between the two molecules was favorable (about -140 kcal/mol). However, when SA was docked at other locations on the protein surface, distant from the coactivator binding site, the interaction energy turned out to be similar or even more favorable (data not shown).

Discussion
In this study, we determined the possible estrogenic/antiestrogenic properties of 10H2DA, 3,10DDA and SA, isolated from RJ and identified by spectroscopic methods [6]. In choosing the concentrations we considered 1) the commonly used RJ dietary supplementation (1-3 g daily), 2) the concentration of 10H2DA and the concentration of sebacic acid in RJ (3-6% and 0.5% respectively) [34,35], 3) the concentration of 10H2DA, sebacic acid and 3,10 DDA as well as 10HDA acid in marketed RJ samples in Greece (40-50%, 5%, 4% and 20% respectively), 4) the human blood volume and bioavailability. Based on the above information, we decided to examine the biological effects of FAs in a concentration range of 10 210 M-10 25 M, which are physiologically achievable concentrations.
Using a ChIP assay in MCF-7 breast cancer cells, which are stably transfected with an inducible version of ERb and express endogenous ERa, we examined the ligand-dependent recruitment of ERa and ERb to chromatin. None of the tested FAs could modulate ERa recruitment to the pS2 promoter, whilst they increased ERb recruitment to this promoter. All FAs inhibited the effect of E 2 on ERa and ERb recruitment. Consistent with the effects on receptor recruitment to DNA, experiments revealed that in the presence of ERb, FAs could decrease pS2 mRNA levels, when added alone, and that they decreased E 2 's effect in the presence and absence of ERb. However, since in this cell system endogenous ERa is always present, effects on pS2 expression cannot easily be determined for ERb alone. We further assessed the effects of FAs on ERa alone and ERb 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 ERa-mediated activity, while they antagonized ERb-mediated effects. In the presence of E 2 they antagonized the E 2 -mediated effects via ERa and ERb. The well characterized selective estrogen receptor modulator (SERM) 4OH-TMX also exhibited agonistic effects on ERa-mediated activity, while it was a complete antagonist of ERb-mediated action. This is in agreement with a previous study reporting that 4OH-TMX induced ERE-mediated reporter gene activity in a stably transformed ERa expressing cell line, but exhibited pure antagonism in the corresponding ERb expressing system [33].
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 E 2 binding is reduced when FAs are present. This suggests that  Table 2 and treated as mentioned in Materials and Methods. Results represent the mean 6 SD of three independent experiments. *Significantly different from vehicle (*p,0.05, **p,0.01, ***p,0.001), +significantly different from E 2 (10 28 M) (+p,0.05, ++p,0.01, +++p,0.001). Analysis of ER-co-activator peptide (EAB1) interaction with mammalian two-hybrid assay (III). Huh7 cells were transiently transfected and treated as mentioned in Materials and Methods. Luciferase activity was normalized to b-galactosidase activity. Mean values 6 SE are shown from the results of four independent experiments (* p,0.05 or p,0.01 significantly different from E 2 (10 26 M). doi:10.1371/journal.pone.0015594.g003 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 E 2 -mediated increase in luciferase activity, suggesting an ER-mediated effect and a common signal transduction pathway for E 2 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, coincubation of FAs with E2 was not investigated in their study [36]. In previous reports [2] fresh RJ displays agonistic activity in the  ERE-driven luciferase reporter gene assay in MCF-7 cells similar to that observed for E 2 whereas the isolated FAs in our study show little agonist activity and possess antagonistic activity. RJ contains multiple FA components [6] and data indicate that 10H2DA, sebacic acid and 3,10 DDA (investigated in this study) may not be the only FA determinants that predict estrogen/antiestrogen activity in RJ [36]. Additionally, RJ may exhibit biological effects determined by synergistic and/or antagonistic interactions between its constituents thus showing different biological effects than the biological activity of its isolated components.
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. [37] proposed that medium chain FAs (hexanoate) at high concentrations (mM range) interacted with ERs to inhibit ligand stimulated transcription, while there was no effect on GR-mediated activity. Previous reports have shown that short chain FAs (valproic acid or butyrate and methoxyacetic acid) may act as deacetylase inhibitors at high concentrations (mM range) resulting in the induction of transcriptional silencing of ERa expression, which would imply that they are antiestrogenic in MCF-7 cells [38,39,40,41]. The antiestrogen effects of the above short chain FAs are considered an effect that may be due to their inherent HDAC inhibitory activities, since they have all been shown to reduce endogenous ERa expression and have been characterized as HDAC inhibitors. Interestingly, a recent report showed that methoxyacetic acid (MAA at mM concentrations) modulates ERa and ERb-mediated signaling, lowers endogenous ERa expression and antagonizes E 2 -stimulated expression of ERa target genes, yet it does not compete with E 2 for binding to ERa [41,42]. However, in our study, FAs (at mM concentrations) did not affect ERa mRNA or protein levels.
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 coactivator 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 (2211 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 (Fig.5).
FAs may bind to the ligand pocket, thus competing with E 2 . The computational fitting showed very good compatibility of the ligand pocket for all three FAs (Fig.5). Although the calculated interaction energies between ligands and receptor are only indirectly related to binding affinities, they do indicate that, similarly to E 2 , the three FAs interact favorably with the ER when they are in the ligand pocket. However, our competition binding study did not show any binding of SA and 3,10DDA and binding only at extreme concentrations (10 24 M) of 10H2DA, indicating that an interaction with ERs is not mediated via the ligand binding pocket. In agreement, Suzuki et al Figure 6. Flow chart of assays and summary of findings. Conclusions are highlighted in lined text boxes (I). Possible molecular mechanism for how FAs modulate E2 signaling through ERs (II). A. Classical E 2 regulation of gene transcription through recruitment of ERa or ERb to the promoter region B. In the presence of E 2 , FAs seem to block the effect of E 2 on ERa and ERb 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 E 2 binding to the ligand binding pocket and with the interference of FAs with E 2 induced binding of a co-activator peptide. doi:10.1371/journal.pone.0015594.g006 (2007) showed that 10H2DA had little effect (about 20% inhibition) upon the ability of E 2 to bind to ERa and 50% inhibition of E 2 to bind to ERb at a concentration of approximately 100 mM [36].
In line with our findings, a recent study on 3,39-diindolylmethane, a selective activator of ERb that does not bind to ERb, proposes a possible mechanism of activation through recruitment of co-activators (i.e. SRC-2) [43]. Moreover, it has been shown that the methoxyacetic study which modulates ERa signaling yet does not bind to ERa [41]. Of note, recent findings indicate that ligands, without binding affinity to ERa, activate GPR30 signaling and may act synergistically or may antagonize ERa-mediated gene expression [12]. Future studies should address the potential of FAs to activate GPR30 signaling or phosphorylation pathways in cooperation with ERs.
On the basis of the findings by Narita et al. [3] demonstrating that RJ stimulates bone formation, we used the osteoblastic cell line KS483 followed by the Alizarin Red-S staining as a model system to study the effect of FAs on the mineralization process [20], which is known to be an estrogen induced effect. The murine KS483 cell line is a mesenchymal precursor cell line, which differentiates into mature mineralizing osteoblasts during a three-week culture period, when cultured under osteogenesis inducing conditions. This differentiation process can be divided in a proliferation, matrix formation, matrix maturation and finally a mineralization phase, according to the model of Stein and Lian [44,45]. Thus, the defining characteristic of the mature osteoblast is its ability to produce a mineralized bone matrix. Moreover, KS483 cell model is among few osteoblastic culture systems that can produce discrete, three-dimensionally organized mineralized matrices which are recognizably bone like. These bone nodules consist of woven bone matrix covered by cuboidal osteoblastic cells and containing osteocyte-like cells embedded in the matrix. Characterization of mineralized bone nodules has demonstrated that the processes of nodule formation, matrix deposition and subsequent mineralization follow a well ordered, temporally defined pattern which appears analogous to bone formation and mineralization in vivo. Low concentrations of SA or 10H2DA significantly induced mineralization, which was suppressed by the addition of ICI182780, indicating an ER-mediated effect. As expected, the presence of E 2 significantly stimulated the mineralization of osteoblasts [20]. Our results imply that 10H2DA and SA may be the RJ components that stimulate osteoblasts. None of the FAs stimulated or inhibited cell viability/proliferation of endometrial cancer (Ishikawa) or breast cancer (MFC-7) cells (Fig. S5). The antiestrogenic effect of FAs in breast cancer cells, their favorable effect on osteoblasts and the lack of effect on endometrial cell viability suggest that FAs may be potential natural SERMs.
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 [46,47,48,49,50]. This honey bee-excreted biological fluid possesses estrogen-like activity, yet the compounds mediating its estrogenic effects are largely unknown. The present report investigated the effects of RJ-derived FAs, namely 10hydroxy-2-decenoic, 3,10-dihydroxydecanoic and sebacic acid, on estrogen signaling (Fig.6.I.) and suggests that these RJ-derived medium chain fatty acids, structurally entirely different from E 2 , mediate estrogen signaling, at least in part, by modulating the recruitment of ERa, ERb and co activators to target genes (Fig.6.II.).  Table 2