ERR is expressed in several adult and embryonic tissues but
ERRγ is expressed in several adult and embryonic tissues but its biological role is still largely unknown . Overexpression of ERRγ in breast cancer correlates with a favorable outcome . In addition, ERRγ has been reported to inhibit the growth of prostate tumor AZD 3965 . In this study we investigated whether phytoestrogens modulated the transcriptional activity of ERRγ. We analyzed a selection of phytoestrogens for their potential agonistic or antagonistic activities on ERRγ. Using reporter gene assays in transiently transfected U2-OS and PC-3 cells we identified equol as an ERRγ agonist. Based on structural models of the ERRγ LBD we were able to introduce mutations that modulated the agonistic effect of equol. Finally, the growth inhibitory effect of ERRγ on the prostate cancer cell line PC-3 was enhanced by equol. In conclusion, our data suggest that ERRγ is involved in mediating the biological effects of equol.
Materials and methods
Discussion A major step in defining the biological functions of an orphan nuclear receptor is the identification of ligands that can be used to modulate its activity in cell cultures and in vivo. Identification of physiological orphan receptor ligands provides also information about the signaling pathways the receptor is involved in. Recently, several synthetic ERR ligands have been identified. Studies utilizing the synthetic ERRα inverse agonist XCT790 facilitated the elucidation of the role of ERRα as a regulator of oxidative phosphorylation , . Synthetic ligands modulating the activities of ERRβ and ERRγ have been recently identified but the biological functions of these receptors are not as well characterized. The identified ERRγ ligands such as 4-OHT and DES imply, however, that ERRγ potentially impinges on estrogen signaling pathways. In addition, the endocrine disruptor bisphenol A (BPA) which is an estrogenic chemical with only a weak binding ability to ERs was recently reported to bind to ERRγ and prevent 4-OHT from antagonizing the intrinsic transcriptional activity of ERRγ . Most of the identified ERR ligands act as inverse agonists. However, the activities of ERRs can also be stimulated by ligand binding as indicated by the identification of the synthetic ERRβ/γ agonists GSK4716 and DY131 , , . The best-documented biological actions of phytoestrogens are mediated by ERs . Some isoflavonoids modulate also steroid metabolism for example by inhibiting the key steroidogenic enzymes such as 5α-reductase, 17β-hydroxysteroid oxidoreductase, and aromatase , , . Phytoestrogens have been reported to act as ligands for also other nuclear receptors than ERs such as PPARγ and ERRα , . In addition to their hormonal effects, phytoestrogens have many other actions including antioxidant activity and inhibition of tyrosine kinases and topoisomerase , , . We report here that the transcriptional activity of ERRγ is stimulated by the isoflavan equol. In contrast, ERRα and ERRβ failed to transactivate the ERRE3tk-Luc reporter in PC-3 or SaOs-2 cells and equol did not add to their transcriptional activity (Fig. 2A and B). The intrinsic transcriptional activity of ERRα and ERRβ is very low in many cell lines most likely due to differential expression of transcriptional coactivator proteins in different cell lines , . When PC-3 cells were cotransfected with the expression vectors for ERRs and the coactivator PGC-1α, all three ERRs transactivated the ERRE3tk-Luc reporter efficiently. However, equol was able to further enhance the activity ERRγ and ERRβ but not that of ERRα (Fig. 2C). These results suggest that equol is selective for ERRγ and ERRβ as has been reported for GSK4716 . Structural studies and site-directed mutagenesis experiments were performed to examine the binding modes of equol, GSK4716, 4-OHT, and DES to ERRγ. The effect of the different mutants on ligand-responsiveness was studied using reporter gene assays. All the tested mutants maintained constitutive transcriptional activity indicating that they were able to adopt an active conformation and to interact with transcriptional cofactor proteins despite the mutations (Fig. 7B).