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1α-Hydroxy derivatives of 7-dehydrocholesterol are selective liver X receptor modulators

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Highlights

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    1α-Hydroxy derivatives of 7-dehydrocholesterol (7-DHC) activate liver X receptors.

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    1,25-(OH)2-7-DHC induces unique cofactor peptide association.

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    1,25-(OH)2-7-DHC suppresses proinflammatory gene expression in macrophages.

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    1,25-(OH)2-7-DHC interacts with liver X receptor α differently from potent agonists.

Abstract

The nuclear receptors liver X receptor (LXR) α and LXRβ are involved in the regulation of lipid metabolism, inflammation, immunity, cellular proliferation, and apoptosis. Oxysterols are endogenous LXR ligands, and also interact with other nuclear and membrane receptors. We previously reported that a phytosterol derivative with a 1α-hydroxy group acts as a potent LXR agonist with intestine-selective action and that 25-hydroxy and 26/27-hydroxy metabolites of 7-dehydrocholesterol (7-DHC) exhibit partial LXR agonism. In this study, we report that 1α-hydroxy derivatives of 7-DHC, 1α-OH-7-DHC and 1,25-(OH)2-7-DHC, act as LXR modulators. Luciferase reporter gene assays showed that 1α-OH-7-DHC activates LXRα and LXRβ and that 1,25-(OH)2-7-DHC activates both LXRs and vitamin D receptor. Examination of cofactor peptide association showed that the 1α-hydroxy derivatives, specifically 1,25-(OH)2-7-DHC, induce association of coactivator/corepressor peptide in a different manner from the agonist T0901317. Docking modeling and alanine mutational analysis of LXRα demonstrated that 1,25-(OH)2-7-DHC interacts with LXRα residues in a manner distinct from potent agonists, such as T0901317 and 24(S),25-epoxycholesterol. 1α-OH-7-DHC and 1,25-(OH)2-7-DHC induced expression of LXR target genes in a cell type- and gene-selective manner. 1,25-(OH)2-7-DHC effectively suppressed lipopolysaccharide-stimulated proinflammatory gene expression in an LXR-dependent manner. Therefore, 1α-hydroxy derivatives, such as 1,25-(OH)2-7-DHC, are unique LXR modulators with selective agonistic activity and potent transrepression function. These oxysterols have potential as LXR-targeted therapeutics for inflammatory disease.

Introduction

The nuclear receptors liver X receptor (LXR) α and LXRβ are ligand-dependent transcription factors of the nuclear receptor superfamily that regulate several physiological processes, including lipid metabolism and immunity [1], [2]. Oxysterols, such as 24(S),25-epoxycholesterol (24,25-EC) and 22(R)-hydroxycholesterol (22R-HC), activate LXRα and LXRβ [3], [4], while 22(S)-hydroxycholesterol and fatty acids, such as arachidonic acid and linolenic acid, are LXRα/β antagonists [5], [6]. LXRα is expressed in the liver, adipose tissue, small intestine and macrophages, while LXRβ is ubiquitous [1], [2]. Liganded LXR forms a heterodimer with retinoid X receptor (RXR) and binds to an LXR-responsive element that consists of a two-hexanucleotide motif (AGGTCA or a related sequence) on specific target genes, such as genes involved in cholesterol and fatty acid metabolism. LXRα stimulates conversion of cholesterol to bile acids in the rodent liver by inducing cholesterol 7α-hydroxylase. LXRα and LXRβ are involved in intestinal and biliary excretion of cholesterol by increasing expression of the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8, and also in reverse cholesterol transport by inducing ABCA1 and ABCG1. The regulatory effects on cholesterol metabolism lead to anti-atherosclerotic actions of synthetic LXR ligands in mouse models. Hepatic LXRs, specifically LXRα, stimulate lipogenesis by inducing lipogenic genes, such as sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase. In addition, LXR activation modulates immune and inflammatory responses via transrepression of genes, such as proinflammatory genes in macrophages [1], [7]. Furthermore, LXR ligand exhibits anti-cancer effects, which have been characterized in colon cancer, melanoma and glioblastoma [8], [9], [10], [11]. Thus, LXR is a possible drug target in the treatment of cardiovascular disease, inflammatory and autoimmune disease, cancer, and neurodegenerative disease [2], [12].

Recently, we demonstrated that sterol 27-hydroxylase-mediated metabolites of 7-dehydrocholesterol (7-DHC), 25-hydroxy-7-DHC (25-OH-7-DHC) and 26/27-OH-7-DHC, modulate LXR activity [13]. 7-DHC is generated from acetyl CoA by several enzymatic reactions, and is an intermediate precursor in both the vitamin D3 and cholesterol biosynthetic pathways [14]. In vitamin D synthesis, 7-DHC is converted to vitamin D3 (cholecalciferol) by an ultraviolet-induced photo-cleavage reaction in the skin [15]. Vitamin D3 is metabolized to 25-hydroxyvitamin D3 in the liver and further to 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] mainly in the kidney. 1,25(OH)2D3 is the active form of vitamin D and acts as a potent ligand for vitamin D receptor (VDR). In cholesterol synthesis, 7-DHC is converted to cholesterol by 3β-hydroxy-sterol 7-reductase (7-DHC reductase; DHCR7) [14]. Mutations in the DHCR7 gene cause Smith-Lemli-Optiz syndrome, and patients with this disease accumulate 7-DHC, which is converted to 25-OH-7-DHC and 26/27-OH-7-DHC [13]. These 7-DHC metabolites suppress SREBP-1c expression in hepatic HepG2 cells and HaCaT keratinocytes but weakly increase ABCA1 expression in HaCaT cells, indicating that these compounds are selective LXR modulators. We also reported that synthetic derivatives of natural steroids, such as the 1α-hydroxy-sterol (22E)-ergost-22-en-1α,3β-diol (YT-32), act as potent LXR agonists [16]. The 1α-hydroxy group of YT-32 is necessary for activation of LXRα and LXRβ, because its derivative lacking a 1α-hydroxy group (YT-33) loses LXR agonistic activity. In this study, we examined the effect of synthetic 7-DHC derivatives with the 1α-hydroxy group (Fig. 1) on LXR activity and found that 1α-hydroxy derivatives of 7-DHC are unique LXR ligands that bind to LXR in a manner distinct from natural ligands.

Section snippets

Compounds

T0901317 [N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide]] was purchased from Cayman Chemical Company (Ann Arbor, MI), 24,25-EC was from Enzo Life Science (Farmingdale, NY), 22R-HC was from Steraloids (Newport, RI), 1,25(OH)2D3 was from Wako (Osaka, Japan), lithocholic acid (LCA) and chenodeoxycholic acid (CDCA) were from Nacalai Tesque (Kyoto, Japan), and 7-DHC was from Sigma-Aldrich (St. Louis, MO). 25-OH-7-DHC

1α-Hydroxylation of 7-DHC increases LXR transactivation activity

We previously reported that a synthetic sterol, YT-32, which has a 1α-hydroxy group, is a potent LXR agonist [16] and that 7-DHC derivatives, 25-OH-7-DHC and 26/27-OH-7-DHC, are LXR modulators with weak agonistic activity [13]. Here, we examined the effect of 1α-hydroxy derivatives of 7-DHC and 25-OH-7-DHC (Fig. 1) on the oxysterol receptors LXRα and LXRβ. HEK293 cells were transfected with human full-length LXRα or LXRβ expression plasmid and LXR-responsive luciferase reporter, and treated

Discussion

In this study, we report that 1α-hydroxy derivatives of 7-DHC act as selective LXR modulators. 1,25-(OH)2-7-DHC and related compounds, 1,25-(OH)2-tachysterol3 and 1,25-(OH)2-lumisterol3, were previously reported to activate VDR less effectively than 1,25(OH)2D3 [42]. We also observed VDR transactivation activity of 1,25-(OH)2-7-DHC (Fig. 2). Although 1α-OH-7-DHC was not effective on VDR, 1α-OH-7-DHC and 1,25-(OH)2-7-DHC activated LXRα and LXRβ similar to or more effectively than the natural

Conflict of interest

The authors have no conflict of interest.

Funding

This work was supported by MEXT KAKENHI Grant Number JP 18077005 (to M.M.), JSPS KAKENHI Grant Number JP 25860248 (to K.E.-U.), JSPS KAKENHI Grant Number JP 16K19061 (to K.E.-U.), and “Strategic Research Base Development” Program for Private Universities subsidized by MEXT (2008–2012).

Acknowledgments

The authors thank members of Makishima lab for technical assistance and helpful comments, and Dr. Andrew I. Shulman for editorial assistance.

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