Abstract
Here, we describe several assays to analyze the transcriptional activity of retinoic acid-related orphan receptors (RORs) and the effect of inverse agonists on their activity. One assay measures the effect of an inverse agonist on the transcriptional activation of a luciferase reporter by RORs in a Tet-On cell system. A mammalian two-hybrid assay analyzes the interaction of the ROR ligand binding domain with a coactivator peptide. Two additional assays examine the effect of an inverse agonist on the activation of a luciferase reporter under control of the promoter of the ROR target gene, IL17, and on ROR-mediated activation using a mammalian monohybrid assay.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Jetten AM (2009) Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal 7:e003
Takeda Y, Kang HS, Lih FB, Jiang H, Blaner WS, Jetten AM (2014) Retinoid acid-related orphan receptor gamma, RORγ, participates in diurnal transcriptional regulation of lipid metabolic genes. Nucleic Acids Res 42:10448–10459
Takeda Y, Kang HS, Freudenberg J, DeGraff LM, Jothi R, Jetten AM (2014) Retinoic acid-related orphan receptor γ (RORγ): a novel participant in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. PLoS Genet 10:e1004331
Takeda Y, Jothi R, Birault V, Jetten AM (2012) RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo. Nucleic Acids Res 40:8519–8535
Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ et al (2006) The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17(+) T helper cells. Cell 126:1121–1133
Yang XO, Pappu BP, Nurieva R, Akimzhanov A, Kang HS, Chung Y et al (2008) T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 28:29–39
Kallen JA, Schlaeppi JM, Bitsch F, Geisse S, Geiser M, Delhon I et al (2002) X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORα. Structure 10:1697–1707
Hu X, Wang Y, Hao L-Y, Liu X, Lesch CA, Sanchez BM et al (2015) Sterol metabolism controls T(H)17 differentiation by generating endogenous RORγ agonists. Nat Chem Biol 11:141–147
Jetten AM, Takeda Y, Slominski A, Kang HS (2018) Retinoic acid-related Orphan Receptor (RORγ): connecting sterol metabolism to regulation of the immune system and autoimmune disease. Curr Opion Toxicol 8:66–80
Santori FR, Huang P, van de Pavert SA, Douglass EF Jr, Leaver DJ, Haubrich BA et al (2015) Identification of natural RORγ ligands that regulate the development of lymphoid cells. Cell Metab 21:286–297
Soroosh P, Wu J, Xue X, Song J, Sutton SW, Sablad M et al (2014) Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation. Proc Natl Acad Sci U S A 111:12163–12168
Kojetin DJ, Burris TP (2014) REV-ERB and ROR nuclear receptors as drug targets. Nat Rev Drug Discov 13:197–216
Tuong ZK, Lau P, Du X, Condon ND, Goode JM, Oh TG et al (2016) RORα and 25-hydroxycholesterol crosstalk regulates lipid droplet homeostasis in macrophages. PLoS One 11:e0147179
Slominski AT, Kim TK, Hobrath JV, Oak ASW, Tang EKY, Tieu EW et al (2017) Endogenously produced nonclassical vitamin D hydroxy-metabolites act as "biased" agonists on VDR and inverse agonists on RORα and RORγ. J Steroid Biochem Mol Biol 173:42–56
Fauber BP, Magnuson S (2014) Modulators of the nuclear receptor retinoic acid receptor-related orphan receptor-gamma (RORγ or RORc). J Med Chem 57:5871–5892
Kallen J, Izaac A, Be C, Arista L, Orain D, Kaupmann K et al (2017) Structural states of RORγt: X-ray elucidation of molecular mechanisms and binding interactions for natural and synthetic compounds. Chem Med Chem 12:1014–1021
Xiao S, Yosef N, Yang J, Wang Y, Zhou L, Zhu C et al (2014) Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms. Immunity 40:477–489
Xue X, Soroosh P, De Leon-Tabaldo A, Luna-Roman R, Sablad M, Rozenkrants N et al (2016) Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis. Sci Rep 6:37977
Wang Y, Cai W, Cheng Y, Yang T, Liu Q, Zhang G et al (2015) Discovery of biaryl amides as potent, orally bioavailable, and CNS penetrant RORγt inhibitors. ACS Med Chem Lett 6:787–792
Huh JR, Leung MW, Huang P, Ryan DA, Krout MR, Malapaka RR et al (2011) Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature 472:486–490
Guendisch U, Weiss J, Ecoeur F, Riker JC, Kaupmann K, Kallen J et al (2017) Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo. PLoS One 12:e0188391
Banerjee D, Zhao L, Wu L, Palanichamy A, Ergun A, Peng L et al (2016) Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo. Immunology 147:399–413
Chang MR, Lyda B, Kamenecka TM, Griffin PR (2014) Pharmacologic repression of retinoic acid receptor-related orphan nuclear receptor gamma is therapeutic in the collagen-induced arthritis experimental model. Arthritis Rheumatol 66:579–588
de Wit J, Al-Mossawi MH, Huhn MH, Arancibia-Carcamo CV, Doig K, Kendrick B et al (2016) RORγt inhibitors suppress T(H)17 responses in inflammatory arthritis and inflammatory bowel disease. J Allergy Clin Immunol 137:960–963
Guo Y, MacIsaac KD, Chen Y, Miller RJ, Jain R, Joyce-Shaikh B et al (2016) Inhibition of RORt skews TCRalpha gene rearrangement and limits T cell repertoire diversity. Cell Rep 17:3206–3218
Smith SH, Peredo CE, Takeda Y, Bui T, Neil J, Rickard D et al (2016) Development of a topical treatment for psoriasis targeting RORγ: from bench to skin. PLoS One 11:e0147979
Gege C (2017) RORγt inhibitors as potential back-ups for the phase II candidate VTP-43742 from Vitae Pharmaceuticals: patent evaluation of WO2016061160 and US20160122345. Expert Opin Ther Pat 27:1–6
Kurebayashi S, Nakajima T, Kim SC, Chang CY, McDonnell DP, Renaud JP et al (2004) Selective LXXLL peptides antagonize transcriptional activation by the retinoid-related orphan receptor RORγ. Biochem Biophys Res Commun 315:919–927
Acknowledgments
This research is supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, the National Institutes of Health (Z01-ES-101585).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Takeda, Y., Kang, H.S., Jetten, A.M. (2019). Analysis of the Transcriptional Activity of Retinoic Acid-Related Orphan Receptors (RORs) and Inhibition by Inverse Agonists. In: Badr, M. (eds) Nuclear Receptors. Methods in Molecular Biology, vol 1966. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9195-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9195-2_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9194-5
Online ISBN: 978-1-4939-9195-2
eBook Packages: Springer Protocols