Abstract
To characterize human nuclear receptor (NR) specificity of synthetic pharmaceutical chemicals we established stable cell lines expressing the ligand binding domains (LBDs) of human FXR, LXRα, LXRβ, CAR, and RORγ fused to the yeast GAL4 DNA binding domain (DBD). As we have already done for human PXR, a two-step transfection procedure was used. HeLa cells stably expressing a Gal4 responsive gene (HG5LN cell line) were transfected by Gal4-NRs expressing plasmids. At first, using these cell lines as well as the HG5LN PXR cells, we demonstrated that the basal activities varied from weak (FXR and LXRs), intermediate (PXR), to strong (CAR and RORγ), reflecting the recruitment of HeLa co-regulators in absence of ligand. Secondly, we finely characterized the activities of commercially available FXR, LXRα, LXRβ, CAR, RORγ, and PXR agonists/antagonists GW4064, feraxamine, DY268, T0901317, GW3965, WAY252623, SR9238, SR9243, GSK2033, CITCO, CINPA1, PK11195, S07662, SR1078, SR0987, SR1001, SR2211, XY018, clotrimazole, dabrafenib, SR12813, and SPA70, respectively. Among these compounds we revealed both, receptor specific agonists/antagonists, as well as less selective ligands, activating or inhibiting several nuclear receptors. FXR ligands manifested high receptor selectivity. Vice versa, LXR ligands behaved in non-selective manner, all activating at least PXR. CAR was selectively influenced by their ligands, while it also responded to several LXR ligands. Finally, although PXR was quite selectively activated or antagonized by its own ligands, it responded to several NRs ligands as well. Thus, using these reporter cell lines enabled us to precisely characterize the selectivity of pharmaceutical ligands for different nuclear receptors.
Highlights
The superfamily of nuclear receptors (NRs) roofs the essential biological processes implying cell proliferation, differentiation, development and cell death, in both, normal and pathologic states (Aranda and Pascual, 2001; Evans and Mangelsdorf, 2014)
We took of interest the human farnesoid X receptor (FXR), the human liver X receptors (LXRa and LXRb) and the human retinoic-acid-receptor-related orphan nuclear receptor g (RORg), serving as major actors of energy metabolism and circadian rhythm in organisms, transcriptionally regulating bile salt, cholesterol, fatty acid, and glucose homeostasis (Kalaany and Mangelsdorf, 2006; Evans and Mangelsdorf, 2014), as well as promiscuous xenobiotic receptors, the human constitutive androstane receptor (CAR) and the human pregnane X receptor (PXR), sensing to toxic by-products of endogenous metabolism, as well as exogenous chemicals arranging their elimination
Several synthetic NR ligands are commercially available, their selectivity among nuclear receptors has not always been thoroughly evaluated. For this purpose we developed cellular assay for identification and characterization of FXR, LXRs, RORg, PXR, and CAR ligands
Summary
The superfamily of nuclear receptors (NRs) roofs the essential biological processes implying cell proliferation, differentiation, development and cell death, in both, normal and pathologic states (Aranda and Pascual, 2001; Evans and Mangelsdorf, 2014). The remaining NRs called orphan receptors lack their regulatory ligands, so their transcriptional activity is alternatively regulated by post-translational events or expression of coregulatory proteins and eventually, by exogenous chemicals. As we have already done for other NRs (Seimandi et al, 2005; Grimaldi et al, 2015), we have established stable HG5LN-derived reporter cell lines in which FXR, LXRs, RORg and CAR respective ligands induce luciferase activity. These cells stably express a chimeric protein containing the yeast transactivator GAL4 DBD fused to LBD regions of individual human NRs and contain luciferase reporter gene driven by recognition sequence of GAL4 pentamer in front of b-globin promoter. We clearly demonstrated that in vitro cell-based assay enables an easy and rapid identification of nuclear receptor specific ligands with potential therapeutic applications
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