Ligand specificity and evolution of liver X receptors

doi:10.1016/j.jsbmb.2008.02.007

The Journal of Steroid Biochemistry and Molecular Biology

Volume 110, Issues 1–2, May 2008, Pages 83-94

https://doi.org/10.1016/j.jsbmb.2008.02.007 Get rights and content

Abstract

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRα and LXRβ from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.

Introduction

Liver X receptor α (LXRα; NR1H3) and β (LXRβ; NR1H2) are members of the nuclear hormone receptor (NR) superfamily of ligand-activated transcription factors. NRs work in concert with co-activators and co-repressors to regulate gene expression [1]. NRs share a modular domain structure, which includes, from N- to C-terminus, a modulatory A/B domain, the DNA-binding domain (DBD; C domain), the hinge D domain, the ligand-binding domain (LBD; E domain) and a variable C-terminal F domain [1]. LXRs, like other members of the NR1 family, function as permissive heterodimers with the retinoid X receptors (NR2B1, 2B2, and 2B3). LXRs were originally classified as ‘orphan’ NRs but are perhaps best referred to now as ‘adopted orphans’ [2], following the identification of endogenous ligands, namely oxysterols such as 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol [3], [4], oxysterol metabolites [5], and some bile acids [6].

LXRs are key regulators of lipid and cholesterol metabolism [2]. More recently, LXRs have been shown to regulate uterine contractility [7]. In all mammals whose genomes have been sequenced so far, two distinct LXR genes are found. LXRα is typically detected at high levels in macrophages, adipose tissues, kidney, lung, and spleen; in contrast, LXRβ is expressed at similar levels in a wide variety of tissues, the basis for an alternative name for this receptor as ‘ubiquitous receptor’ [8]. Based on the sequenced genomes of chicken, pufferfish (fugu; Takifugu rubripes) [9], freshwater pufferfish (Tetraodon nigroviridis), zebrafish (Danio rerio), and Western clawed frog (Silurana or Xenopus tropicalis), non-mammalian species appear to generally have only a single LXR gene.

For non-mammalian species, the pufferfish LXR has been the subject of the most detailed study [9]. Pufferfish LXR is more closely related to mammalian LXRα genes by sequence similarity, although the pattern of tissue expression more closely resembles mammalian LXRβ genes in the ubiquity of expression, including expression in brain, gill, gut, heart, ovary, and liver [9]. The current sequence data suggests that a single LXR gene duplicated before mammalian evolution or early in mammalian evolution [9]. If this hypothesis is correct, then one of the duplicated genes maintained ubiquitous tissue expression (LXRβ) while the other (LXRα) carried out specific roles in cholesterol and lipid metabolism with more restricted expression in adipose tissue, liver, and macrophages.

So far, the ligand specificities of non-mammalian LXRs have not been reported. To this end, we cloned and functionally expressed the LXR genes from three model non-mammalian species, African clawed frog (Xenopus laevis), Western clawed frog (X. tropicalis), and zebrafish (D. rerio). We compared the specificities for ligand activation of these LXRs to human and mouse LXRα and LXRβ. We also sought to probe the evolution of LXR in invertebrates by studying the chordate invertebrate sea squirt (Ciona intestinalis), a member of Urochordata, a subphylum that may contain the closest extant invertebrate relatives to modern vertebrates [10]. The completed genome of C. intestinalis revealed a gene that is an apparent ortholog to vertebrate LXRs [11]. From the Ghost database of C. intestinalis Genomic and cDNA Resources (http://ghost.zool.kyoto-u.ac.jp/indexr1.html), cDNA clone IDs cigd011h11 and cieg096k22 correspond to this putative ‘Ciona LXR’ (ciLXR). This expression profile of these cDNAs based on expressed sequence tag counts shows high expression in gonadal tissue and neural complex, and lower expression in blood cells, eggs, cleaving embryos, gastrulae/neurulae, tailbud embryos, young adult animals, and mature adult animals. We cloned and expressed this ciLXR to determine how similar this receptor is to its vertebrate orthologs with respect to activation by ligands. We also used molecular modeling studies to compare and contrast the ciLXR to human LXRs.

Section snippets

Chemicals

The sources of the chemicals were as follows: fexaramine, GW3965, GW4064, glycocholic acid, taurocholic acid (Sigma–Aldrich, St. Louis, MO, USA); T-0901317 (Axxora, San Diego, CA, USA); 5α-petromyzonol (5α-cholan-3α,7α,12α,24-tetrol), petromyzonol sulfate, 3-ketopetromyzonol sulfate (Toronto Research Chemical, Inc., North York, ON, Canada); Nuclear Receptor Ligand Library (76 compounds known as ligands of various nuclear hormone receptors; BIOMOL International, Plymouth Meeting, PA, USA). Other

Ligand specificity of vertebrate LXRs

To compare ligand activation of mammalian and non-mammalian LXRs, the LBDs of hLXRα, hLXRβ, mLXRα, mLXRβ, xlLXR, xtLXR, and zfLXR were cloned and inserted into the pM2-GAL4 vector to create LBD/GAL4 ‘chimeric’ receptors. The chimeric receptors were then used together with the reporter plasmid tk-UAS-Luc in a luciferase-based reporter assay described previously [15]. An initial screening of possible ligands for these receptors revealed that T-0901317 and GW3965 (see Fig. 1 for chemical

Discussion

LXRs are part of the NR1H subfamily that also includes FXRs (NR1H4 and H5) and the ecdysone receptors (NR1H1; found exclusively in invertebrates). The receptors in the NR1H subfamily all respond to steroidal compounds: FXRs to bile salts, LXRs to oxysterols (and limited numbers of bile acids), and ecdysone receptors to ecdysteroids such as α-ecdysone [7,(5α)-cholesten-2β,3β,14α,22(R),25-pentol-6-one]. The major endogenous ligands identified so far for mammalian LXRs are oxysterols such as 24(S

Acknowledgements

The authors thank the laboratory of Dr. Alan F. Hofmann (University of California, San Diego, CA, United States) for providing 5β-cyprinol. SE acknowledges Dr. David Lawson for initial help providing information on the disorder analysis approach.

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^☆: This study was supported by National Institutes of Health grant K08-GM074238 (to MDK), the Competitive Medical Research Fund from the University of Pittsburgh Medical Center (to MDK), and USEPA-funded Environmental Bioinformatics and Computational Toxicology Center (ebCTC), under STAR Grant number GAD R 832721-010 (to NA and WJW).

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Ligand specificity and evolution of liver X receptors☆

Abstract

Introduction

Section snippets

Chemicals

Ligand specificity of vertebrate LXRs

Discussion

Acknowledgements

J. Biol. Chem.

Steroids

J. Biol. Chem.

J. Lipid Res.

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Lipid Res.

Dev. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Cell

Binding of ligands and activation of transcription by nuclear receptors

Annu. Rev. Biophys. Biomol. Struct.

LXRs and FXR: the Yin and Yang of cholesterol and fat metabolism

Annu. Rev. Physiol.

An oxysterol signalling pathway mediated by the nuclear receptor LXRα

Nature

Cholestenoic acid is a naturally occurring ligand for liver X receptor alpha

Endocrinology

Ubiquitous receptor: a receptor that modulates gene activation by retinoic acid and thyroid hormone receptors

Proc. Natl. Acad. Sci. U.S.A.

The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily

Nucleic Acids Res.

Tunicates and not cephalochordates are the closest living relatives of vertebrates

Nature