Identification and characterization of a novel estrogenic ligand actinopolymorphol A
Graphical abstract
Introduction
The Estrogen Receptors (ERs) are hormone dependent transcription factors existing in two forms: ERα and ERβ. The binding of both endogenous (i.e. 17β-estradiol, also known as E2) and exogenous estrogenic ligands to these receptors induces conformational changes leading to dissociation from the Hsp90 molecular chaperone complex, subsequent receptor dimerization, interaction with coactivator proteins, and recognition of Estrogen Response Elements (EREs) in the promoter regions of target genes to activate target gene transcription. Transcriptional activity of ERs is strongly influenced by ligands at each step including (i) the binding of a given ligand for ERα vs. ERβ, (ii) the conformational changes induced upon ligand binding which influence dimer partner preference (i.e. ERα/α and ERβ/β homodimers, or ERα/β heterodimers), (iii) cofactor recruitment, and (iv) interaction with chromatin. The differential regulation of ERα and ERβ by endogenous and exogenous estrogenic compounds has extensive physiological implications, as transcriptional activation of ERα by these ligands is known to stimulate cellular proliferation, while transcriptional activation of ERβ inhibits cell growth [1], [2], [3], [4], [5], [6].
In addition to the genomic transcriptional activities of ERα and ERβ in estrogenic signaling, ERs can also be regulated by growth factors such as epidermal growth factor (EGF) and insulin-like growth factor (IGF) [7], [8], [9], [10]. The effects of many of these growth factor pathways are believed to reflect their abilities to change the phosphorylation state of ERs, as well as that of coregulators and other proteins with which ERs interact to modulate gene expression. Furthermore, in addition to the well-documented synergistic effects of estrogens and growth factors on gene transcription, estrogens also exert rapid membrane-initiated effects that are known to massively impact cell signaling and may also influence gene transcription in the nucleus. Membrane-bound ERs have been shown to mediate estrogenic effects in ER-negative cells via activation of the MAPK pathway [11]. These non-genomic mechanisms of estrogenic signaling should therefore be carefully considered as important mechanisms of global estrogen action.
The cellular functions of ERα and ERβ homodimers are well established. However, the biological role of the ERα/β heterodimer remains a topic for intense study and debate, due largely to the lack of tools to study ERα/β heterodimerization in a physiological context. The co-expression of ERα and ERβ results in a heterogeneous pool of homodimers and heterodimers, and thus the activity of heterodimers cannot be deciphered from that of either homodimer, although evidence strongly suggests that ERβ antagonizes the proliferative action of ERα via formation of growth-inhibitory heterodimers [1], [3], [4], [12], [13]. To elucidate the biological role of these heterodimers, we have developed novel bioluminescence resonance energy transfer (BRET) assays in order to study ERα/β heterodimerization in a cell-based, physiological environment in real time [14]. We have used these assays to study the basic intermolecular mechanism of ERα/β heterodimerization. Another important application of the BRET assay involves the identification of selective ERα/β heterodimer-inducing small molecules. Of particular significance here is the application of the BRET assay to identify new natural products able to activate ERα/β heterodimerization.
Exogenous estrogenic ligands are ubiquitous in the natural environment and include xenoestrogenic industrial by-products and phytoestrogens such as genistein, a principle constituent of soy. Metabolites of xenoestrogenic and phytoestrogenic compounds have been demonstrated to be produced by several bacterial strains including those present in the intestinal flora [15], [16], [17], [18], [19], [20] and soil bacteria [21], [22]. For example, bisphenol A (BPA) can be metabolized by many organisms ranging from microorganisms to animals, and these transformations represent an important pathway for its detoxification [23]. Other studies have shown that many natural products produced by bacteria serve as xenoestrogens [24], [25]. Screening of such compounds for their ability to selectively activate ER homodimers and heterodimers is important in order to determine the physiological effects of these environmental ligands as they may act through pro-proliferative ERα/α homodimers or anti-proliferative ERβ/β heterodimers. Moreover, the identification of such compounds represents an important undertaking as compounds displaying selective ER activation may serve as scaffolds which may be used for the development of novel therapeutics or biochemical tools. Inspired by the realization that the chemical structures of natural products remain either the source of, or the basis for, the majority of drug discovery and synthesis [26], this study sought to identify new natural products able to induce selective ER heterodimer formation leading to subsequent transcriptional activity with the rationale that these structures may be useful as a basis for chemical synthesis of therapeutically-useful ER dimer-selective ligands. Natural products of interest were produced by actinomycetes of terrestrial origin.
The application of our novel ER dimer-specific BRET assay [14], [27] for high throughput screening (HTS) of a microbial library of crude extracts resulted in the identification of actinopolymorphol A from the actinomycete Actinopolymorpha rutilus whose structure has not previously been reported or characterized as an ER ligand. This discovery was enabled by the novelty of the BRET assay with its rapid in-cell format which circumvents the need for tissue-culture grade crude extracts and serves as an excellent assay for activity-guided chemical fractionation of crude extracts containing an assortment of natural products.
Section snippets
High throughput screening BRET of the UWCCC SMSF Discovery Library
HTS BRET was performed at the University of Wisconsin Small Molecule Screening Facility. ERα/α homodimerization was examined using ERα-RLuc and ERα-YFP, ERβ/β homodimerization was examined using RLuc-ERβ and YFP-ERβ, and ERα/β heterodimerization was examined using ERα-RLuc and YFP-ERβ using the optimized conditions described previously [14]. Cells were transfected with these fusion proteins (0.73 μg RLuc fusion + 2.8 μg YFP fusion) in batches on 10 cm plates to reduce well-to-well variation in
High throughput BRET screening of the University of Wisconsin Discovery Library (WDL)
In order to identify ligands capable of differentially inducing ERα/α and ERβ/β homodimerization and ERα/β heterodimerization, a BRET assay was developed and optimized [14]. Distinct from the existing ER reporter assay, the BRET assay is exquisitely sensitive and allows the formation of different dimer types to be detected independently, including ERα/α, ERβ/β, and ERα/β dimers. This is especially important in the case of the ERα/β heterodimer, as the co-expression of ERα and ERβ allows the
Discussion
It is well established that phytochemicals serve as recruitment signals resulting in the symbiotic activation of plant growth signals by soil bacteria, and that the presence of endocrine disrupting compounds (EDCs) contained in pesticides and other industrial by-products can disrupt this process [38], [39], [40]. The direct production of xenoestrogenic compounds by soil bacteria is not as well established and therefore represents an opportunity for discovery of new chemical scaffolds with
Acknowledgements
We thank Erin Shanle for proofreading the manuscript and the Analytical Instrumentation Center of the School of Pharmacy, UW-Madison for support in obtaining MS and NMR data. This work is supported by NIH Grants R01CA125387 and R03MH089442 to W.X., T32 CA009135 to E.P. and CA113297 to B.S.
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