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

Abstract

The retinoic acid receptor-related orphan receptors alpha and gamma (RORalpha [NR1F1] and RORgamma [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 RORalpha and RORgamma 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 RORalpha and RORgamma (K(i) approximately 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 RORalpha/gamma inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORalpha target genes including BMAL1 and REV-ERBalpha in a ROR-dependent manner. We also demonstrate that 24S-OHC decreases the ability of RORalpha 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 RORgamma. These data indicate that RORalpha and RORgamma may serve as sensors of oxsterols. Thus, RORalpha and RORgamma display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRalpha [NR1H3] and LXRbeta [NR1H2]).

Figure 1

24S-hydroxycholesterol suppresses the transcriptional activity of the RORα and RORγ LBDs. (A) Chemical structure of 24S-hydroxycholesterol. (B) Cotransfection assay in HEK293 cells illustrating the ability of 10 μM 24S-OHC to suppress the transcriptional activation activity of either Gal4-RORα LBD or Gal4-RORγ LBD. T0901317 (T1317) functions as an inverse agonist for both RORs and is used as a positive control. * indicates p<0.05.

Figure 2

24S-hydroxycholesterol suppresses the transcriptional activity of the RORα and RORγ LBDs in a dose-dependent manner. Cotransfection assay in HEK293 cells illustrating the ability 24S-OHC to suppress the transcriptional activation activity of either Gal4-RORα LBD (A) or Gal4-RORγ LBD (B). (C) 24S-hydroxycholesterol is a RORα and RORγ ligand. Results from a competitive radioligand binding assay are shown where [³H]25-OHC was used as the radioligand.* indicates p<0.05.

Figure 3

24S-hydroxycholesterol suppresses the transcriptional activity of full-length RORα and RORγ. Cotransfection assay in HEK293 cells illustrating the ability of 10 μM 24S-OHC to suppress the transcriptional activation activity of either RORα (A) or RORγ (B) driving expression of a BMAL1 promoter – luciferase construct. (C) Cotransfection assay in HepG2 cells illustrating the ability of 10 μM 24S-OHC to suppress the transcriptional activation activity of RORα driving expression of a G6Pase promoter – luciferase construct. * indicates p<0.05.

Figure 4

24S-hydroxycholesterol modulates the expression of RORα target genes. 24S-hydroxycholesterol (10 μM) suppresses the expression of BMAL1 (A) and REV-ERBα (B) mRNA in a RORα dependent manner in HepG2 cells. (C) Expression of RORα following treatment of cells with siRNA targeting RORα. Control cells received scrambled siRNA. (D) ChIP-reChIP assay illustrating the ability of 24S-OHC to decrease SRC-2 recruitment to RORα occupying the BMAL1 promoter. The graph below the gel indicates the average band intensity from three experiments. IgG is used as a negative control and α-RNA pol II antibody is used as a positive control. * indicates p<0.05.

Figure 5

24R-hydroxycholesterol suppresses the transcriptional activity of the RORγ LBD. . (A) Cotransfection assay in HEK293 cells illustrating the inability of 10 μM 24S-OHC to affect the transcriptional activation activity of Gal4-RORα LBD. (B) Cotransfection assay in HEK293 cells illustrating the inability of 10 μM 24S-OHC to suppress the transcriptional activation activity of Gal4-RORγ LBD. (C) Cotransfection assay in HEK293 cells illustrating the ability 24S-OHC to suppress the transcriptional activation activity of Gal4-RORγ LBD dose-dependently. (D) Results from a competitive radioligand binding assay are shown where [³H]25-OHC was used as the radioligand and 24R-OHC was used as a competitor. * indicates p<0.05.

Figure 6

24(S),25-epoxycholesterol suppresses the transcriptional activity of the RORγ LBD.. (A) Cotransfection assay in HEK293 cells illustrating the inability of 10 μM 24S-OHC to affect the transcriptional activation activity of Gal4-RORα LBD. (B) Cotransfection assay in HEK293 cells illustrating the inability of 10 μM 24(S),25-epoxycholesterol to suppress the transcriptional activation activity of Gal4-RORγ LBD. (C) Cotransfection assay in HEK293 cells illustrating the ability 24(S),25-epoxycholesterol to suppress the transcriptional activation activity of Gal4-RORγ LBD dose-dependently. (D) Results from a competitive radioligand binding assay are shown where [³H]25-OHC was used as the radioligand and 24(S),25-epoxycholesterol was used as a competitor. * indicates p<0.05.