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, 23 (3), 864-72

Identification of Farnesoid X Receptor Beta as a Novel Mammalian Nuclear Receptor Sensing Lanosterol

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Identification of Farnesoid X Receptor Beta as a Novel Mammalian Nuclear Receptor Sensing Lanosterol

Kerstin Otte et al. Mol Cell Biol.

Abstract

Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRbeta as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRbeta is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRalpha and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRbeta. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRbeta as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

Figures

FIG. 1.
FIG. 1.
FXRβ genomic structures and molecular cloning. (A) Schematic representation of FXRβ genomic structure. mFXRβ and hFXRβ loci show similar exon-intron structures. Here the murine locus is displayed, consisting of at least 11 exons spanning 26 kb of genomic DNA. Striped boxes represent the DBD, gray boxes represent the LBD, the asterisk represents the stop codon, and the flag represents the start codon. The 5′ untranslated region is encoded by exon 1, while exon 11 encodes the 3′ untranslated region (black boxes). Human stop codons and frame shifts are indicated by vertical arrows. Untranslated regions are not completely sequenced for the human locus. (B) Sequence of mFXRβ. Shown are cDNA and protein sequences of mFXRβ (GenBank accession no. AY094586). Start and stop codons are bold, and the poly(A) signal is underlined.
FIG. 1.
FIG. 1.
FXRβ genomic structures and molecular cloning. (A) Schematic representation of FXRβ genomic structure. mFXRβ and hFXRβ loci show similar exon-intron structures. Here the murine locus is displayed, consisting of at least 11 exons spanning 26 kb of genomic DNA. Striped boxes represent the DBD, gray boxes represent the LBD, the asterisk represents the stop codon, and the flag represents the start codon. The 5′ untranslated region is encoded by exon 1, while exon 11 encodes the 3′ untranslated region (black boxes). Human stop codons and frame shifts are indicated by vertical arrows. Untranslated regions are not completely sequenced for the human locus. (B) Sequence of mFXRβ. Shown are cDNA and protein sequences of mFXRβ (GenBank accession no. AY094586). Start and stop codons are bold, and the poly(A) signal is underlined.
FIG. 2.
FIG. 2.
Phylogenetic tree containing FXR and FXRβ genes from different species. The phylogenetic tree was calculated on the basis of protein alignments by using PHYLIP (phylogeny interference package, 1993; distributed by J. Felsenstein, University of Washington, Seattle, Wash.) on the basis of the following sequences (accession numbers): chicken FXR (AF492497), hFXR (U68233), rat FXR (U18374), mFXR (NM009108), Fugu FXR1 (S001361), mFXRβ (AY094586), frog FOR2 (AF456452), frog FOR1 (AF4564519), Fugu FXR2 (S000248), and drosophila EcR (M74078). Numbers indicate the bootstrap support (out of 1,000) for the respective nodes. Asterisks indicate putatively nonfunctional proteins in primates.
FIG. 3.
FIG. 3.
Transcription of mFXRβ. (A) Splice variants of mFXRβ. Five splice variants were isolated and sequenced (GenBank accession no. AY094586 through AY094590). Striped boxes indicate exons encoding the DBD, and gray boxes indicate exons encoding the LBD. Numbers indicate exons. Shown are only coding regions. (B) Tissue distribution of mFXRs. mFXR and mFXRβ transcripts were determined by RT-PCR amplification of nonhomologous sequences derived from the LBDs of the respective receptors. β-Actin served as an internal control. RNAs from indicated embryonic and adult tissues were used as templates. Lane M, DNA size marker. d, day.
FIG. 4.
FIG. 4.
mFXRβ binds selectively to nuclear REs and forms heterodimers with RXRα. (A) DNA REs recognized by mFXRβ. HEK 293 cells were cotransfected with expression plasmids containing mFXRβ fused to an NF-κB AD (black bars) or an empty vector (white bars) and the indicated plasmid from a panel of luciferase reporter constructs containing synthetic IR, ER, or DR REs with different spacers between the half sites (zero to eight nucleotides). Luciferase activities are means of triplicate experiments, and the SDs are indicated. (B) Interaction of mFXRβ with RXRα is enhanced by 9-cis-RA. HEK 293 cells were cotransfected with the reporter vector pFR-Luc and RXRα fused to an NF-κB AD (pCMV-AD-hRXRα) in combination with either pCMV-BD (white bars), FXR fused to the Gal4 DBD (pCMV-BD-hFXR; gray bars) or an mFXRβ fusion with the Gal4 DBD (pCMV-BD-mFXRβ; black bars). Cells were treated with 1 μM 9-cis-RA (+) or an equivalent volume of DMSO (−). Cells from triplicate experiments were assayed for luciferase activity, and means ± SDs are shown. RLU, relative light units.
FIG. 5.
FIG. 5.
Identification of ligands for mFXRβ. (A) Lanosterol selectively induces SRC1 binding to mFXRβ at physiological concentrations. GST-mFXRβ LBD and biotinylated mSRC1 peptide were mixed, and the effect of lanosterol and the FXR agonist GW4064 on the interaction was determined by HTRF assay. Cofactor interaction is induced by lanosterol with an EC50 of 1 μM, whereas GW4064 only acts as a weak agonist. Signals are plotted as means of triplicates, and error bars indicate SDs. (B) GST-hFXR LBD was mixed with biotinylated, His-tagged hTif2, and the effects of lanosterol and GW4064 on the respective interaction were assayed. The FXR agonist GW4064 induces cofactor interaction with an EC50 of 70 nM, whereas lanosterol does not show any effect. (C) A diverse set of compounds induces cofactor interaction with mFXRβ but not FXR. Shown are EC50s and the relative efficacy of SRC1 recruitment (the efficacy of lanosterol was set to 100%) calculated from dose-response experiments for a selected group of compounds found to be active for FXRβ. (D) Cholates and cholic acids do not induce significant SRC1 binding to mFXRβ, whereas lanosterol does induce SRC1 recruitment. GST-mFXRβ LBD and biotinylated mSRC1 peptide were mixed. The effects of various compounds at a concentration of 40 μM on the interaction was determined by HTRF assay. Ratios of signals versus DMSO controls are plotted as means of triplicates, and SDs are indicated. CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; CA, cholic acid.
FIG. 6.
FIG. 6.
Lanosterol activates mFXRβ. (A) Lanosterol is an intermediate of the cholesterol metabolism pathway. Cholesterol is synthesized from acetyl coenzyme A (acetyl-CoA) via lanosterol and catabolized into bile acids and steroids. Ketoconazole inhibits CYP51, the enzyme catalyzing the demethylation of lanosterol. (B) mFXRβ is activated by 9-cis-RA. HEK 293 cells were cotransfected with an mFXRβ full-length expression vector (black bars) or an empty vector (white bars) and the I-BABP RE luc reporter. Cells were treated with the indicated micromolar concentrations of 9-cis-RA. Mean luciferase activities and SDs determined from triplicate experiments are shown. (C) mFXRβ is activated by lanosterol. HEK 293 cells were cotransfected with an mFXRβ full-length expression vector (black bars) or an empty vector (white bars) and the I-BABP RE luc reporter and treated with the indicated micromolar concentrations of lanosterol, 9-cis-RA, and ketoconazole. Mean luciferase activities and SDs determined from triplicate experiments are shown. RLU, relative light units.

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