A second class of nuclear receptors for oxysterols: Regulation of RORα and RORγ activity by 24S-hydroxycholesterol (cerebrosterol)

Abstract

The retinoic acid receptor-related orphan receptor α and γ (RORα [NR1F1] and RORγ [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORα and RORγ with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORα and RORγ (K i ∼ 25 nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORα/γ inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORα target genes including BMAL1 and REV-ERBα in a RORdependent manner. We also demonstrate that 24S-OHC decreases the ability of RORα to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORγ. These data indicate that RORα and RORγ may serve as sensors of oxsterols. Thus, RORα and RORγ display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRα [NR1H3] and LXRβ [NR1H2]).

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation

“…7α-OHC failed to show significant agonist activity in enhancing IL-17 production in Cyp27a1 KO CD4 + T cells, and its level was highly increased in Cyp27a1 KO mice, suggesting that it is not an RORγt agonist in vivo. Both 7α-OHC and 24(S)-OHC were previously reported by others (15,16) to function as inverse agonists for RORγ. It is possible that they may play a role in regulating the effect of full endogenous agonists, such as 7β, 27-OHC and 7α, 27-OHC, by competing for binding to RORγ or RORγt.…”

“…For example, certain oxysterols have been shown to serve as endogenous ligands of EBI2 receptor (10,11), activate the liver X receptor (12), function as a selective estrogen receptor modulator (13), or affect Hedgehog signaling by binding to the Smoothened molecule (14). In addition, several oxysterols were previously shown to function as RORγ or RORα inverse agonists (such as 7-hydroxylated oxysterols) or 24(S)-OHC agonists (such as 25-OHC) (4,15,16). However, their role as endogenous RORγ, RORγt, or RORα ligands in vivo was not clearly shown.…”

The Identification of Naturally Occurring Neoruscogenin as a Bioavailable, Potent, and High-Affinity Agonist of the Nuclear Receptor RORα (NR1F1)

“…In contrast, reporter activity was only minimally induced in COS7 cells that expressed the Gal4-DBD construct. The capacity of neoruscogenin to induce RORα target gene expression in the natural chromatin context was then demonstrated in HepG2 cells, where the expression of G6Pase and Bmal1, both well-established RORα target genes, 14,18,22 was significantly induced upon neoruscogenin treatment (Fig. 3C).…”

“…12,13 Subsequently, 7-oxygenated sterols (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol) and (24S)-hydroxycholesterol were identified as novel RORα ligands, with comparable nanomolar binding affinities, 14,15 and recently the structures of synthetic RORα ligands, based on either the benzenesulfonamide or thiourea scaffolds, have been published. [16][17][18] However, no potent and bioavailable ligands are available to date to address important questions about the complex biology of the RORα receptor in vivo.…”

Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

“…Protein complexes are intricately-ordered, dynamic structures that translate biological information into function (11). While some endogenous/synthetic ligands of RORγ were found such as 7-oxygenated sterol, SR1078, T0901317, and 24S-OHC, which have been demonstrated to regulate the transcriptional activity of RORγ (12)(13)(14)(15), only protein Mi-2β has been identified to interact with RORγ to function as a corepressor by using the yeast two hybrid strategy (16). Considering RORγ probably function via forming protein-protein complex, it will be very beneficial to exactly explore the RORγ-interacting proteins by directly capturing and dissecting such complex by using a more suitable strategy, in contrast to the yeast two hybrid method.…”