Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro and in vivo
Graphical abstract
Introduction
The pregnane X receptor (PXR; NR1I2) is a member of the nuclear receptor (NR) superfamily, expressed predominantly in the liver and gastrointestinal tract. It has been well-characterized as a xenobiotic sensor that binds to structurally diverse chemicals, including numerous clinical drugs and endogenous substances [1]. As a xenobiotic sensor, human PXR (hPXR) functions mainly through its transcriptional activation of target genes encoding proteins involved in xenobiotic detoxification and endobiotic metabolism, such as drug-metabolizing enzymes and transporters [2]. Unwanted activation of hPXR by xenobiotics may lead to adverse drug-drug interactions (DDIs), cancer drug resistance, liver toxicity, and possible liability concerns affecting drug development and clinical therapy [3], [4]. Therefore, a full understanding of hPXR regulation of transcriptional activation is crucial for understanding the metabolism of xenobiotics and circumventing the adverse effects of unwanted hPXR-mediated activation.
The transcriptional regulation of hPXR involves its 4 major structural domains: a sequence-specific DNA-binding domain (DBD), a flexible hinge, a ligand-binding domain (LBD), and an activation function-2 (AF-2) domain located in the LBD [5]. Ligand binding to the hPXR LBD is an important means of control of hPXR transcriptional activity. Upon ligand binding, a conformational change in the LBD and AF-2 domains of hPXR allows dissociation of the corepressor and recruitment of the coactivator; the hPXR-coactivator interaction facilitates DBD binding to site-specific DNA sequences of target genes, resulting in their transcriptional activation. Heterodimerization of hPXR with retinoid X receptor α (RXRα) is another step required for ligand-induced PXR activation [6]. Thus, ligand binding, hPXR-coactivator recruitment, and hPXR-RXRα heterodimerization are the three key steps in hPXR's precise regulation of target gene expression.
Ligand binding of hPXR and coactivator recruitment have long been the main focus of the studies on regulation of transcriptional activity of hPXR. A number of hPXR ligands and hPXR-coactivators have been discovered [7]. However, mechanisms other than ligand binding and co-activator recruitment have been found involved in the regulation of other NR function, including post-translational modifications [7]. As a member of the NR superfamily, hPXR can be phosphorylated in biochemical and cell-based assays, and multiple putative phosphorylation sites have been proposed. Studies by our laboratory and others have shown that hPXR can be phosphorylated by cyclin-dependent kinase 2 (CDK2) and several other kinases including cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), glycogen synthase kinase 3 (GSK3), casein kinase II (CK2), cyclin-dependent kinase 5 (CDK5), 70-kDa ribosomal S6 kinase (p70 S6K) [8], [9], [10], [11]. To elucidate how phosphorylation affects the transcriptional activity of hPXR, phospho-mimetic mutations have been generated at different putative hPXR phosphorylation sites, and their transcriptional activity has been evaluated by a reporter-gene assay using the cytochrome P450 3A4 (CYP3A4) promoter. These assays have revealed increased (e.g., Thr248) or decreased (e.g., Ser8, Thr57, Ser114, Ser208, Ser350, Thr408, and Thr422) transcriptional activity associated with these residues [8], [9], [11], [12].
In addition to post-translational modification, dimerization is also a key regulatory mechanism of NR, such as homodimerization of glucocorticoid receptors and hetero-dimerization of retinoic acid receptors (RARs) with RXRα [7]. The hPXR-RXRα hetero-dimerization has been established as a key step of hPXR activation, but how hPXR-RXRα dimerization regulates hPXR activation has not been well understood. A recent study of hPXR-RXRα LBD heterotetramer crystal structure identified 21 key amino acid residues of hPXR in the hPXR-RXRα interacting surface [13]. Among them, 15 (Lys325, Arg353, His359, Arg360, Asp363, Gln366, Glu367, Ile371, Lys374, Leu391, Met394, Glu399, Arg401, Ser402, Gln409) form either direct or water-meditated interaction with RXRα, while 6 (Lys332, Ser350, Glu378, Thr398, Gln406, Arg413), which are not involved in direct interaction with RXRα, can affect hPXR conformation and intramolecular interactions that potentially strengthen the dimer interface. However, it remains largely unknown whether and how these amino acid residues in hPXR affect the heterodimerization process and consequently hPXR's function.
Ser350 residue of PXR is of particular interest in our study of the regulation of PXR function for a few reasons. First, Ser350 is present in a sequence (350SPDR353) that matches the consensus CDK phosphorylation motif [(S/T)PX(R/K)] [14], thus, it is a putative cyclin-dependent kinase 2 (CDK2) phosphorylation site. Since PXR can be phosphorylated by CDK2, and using a phospho-mimetic hPXR mutation of Ser350, S350D (a mutation of serine to a negatively charged aspartate), we and others have found that this mutation reduced hPXR-activated promoter transactivation of its target genes in cellular promoter-reporter assays, including phase I drug-metabolizing enzyme CYP3A4 [8], [11], [15] and phase II drug-metabolizing enzyme UDP-glucuronosyltransferase 1A1 [16]. Second, Ser350 is one of the 6 residues of PXR that can affect PXR conformation and intramolecular interactions, potentially leading to indirect effects on PXR-RXR dimerization [13]. The current knowledge suggests to us that Ser350 of hPXR could be crucial for hPXR's function. A mechanistic understanding of the Ser350 would give us insights into the transcriptional regulation of hPXR at either post-translational modification or receptor dimerization level.
In the present study, using a S350D mutation in hPXR, which allows constitutive mimic of phosphorylation of hPXR at Ser350 residue, we examined the functional effect of the S350D mutation on the hPXR transcriptional regulation of endogenous CYP3A4 gene expression in human liver-derived HepG2 and intestinal epithelia-derived LS180 cells, including its effects on the ligand binding, co-activator recruitment and hPXR-RXRα dimerization. We identified the molecular mechanism responsible for the impairment of transcriptional activity of the S350D mutant in these cells. Further, we demonstrated that the S350D mutation alone in hPXR is sufficient to impair hPXR activity in mouse liver in vivo by creating and functionally characterizing a novel humanized transgenic mouse model expressing the hPXRS350D transgene.
Section snippets
Chemicals and plasmids
Rifampicin (RIF), SR12813 (SR), T0901317 (TO), hyperforin (Hyp) and 2,2,2-tribromoethanol were purchased from Sigma (St. Louis, MO). The plasmids pcDNA3-hPXR, pcDNA3-hPXRS350D, pcDNA3-hPXRS350A and pGL3-CYP3A4-luc were described previously [8].
Cell culture
The human liver carcinoma cell line HepG2, the human intestinal epithelial cell line LS180 (derived from colorectal adenocarcinoma), and the 293T cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in
Reduced intracellular transactivation of endogenous hPXR target genes by hPXRS350D
We first evaluated the transcriptional activity of the hPXRS350D mutant in human HepG2 cells (liver cells) and LS180 cells (intestine cells), which are commonly used to investigate ligand-induced transactivation of hPXR target genes [25], [26]. After treatment with RIF (a known potent hPXR agonist), endogenous CYP3A4 expression in HepG2 or LS180 cells transfected with empty vector, hPXR, hPXRS350D (phosphomimetic) mutant, or hPXRS350A (phospho-deficient) mutant was compared with that in the
Discussion
To our knowledge, the present study is the first to demonstrate that Ser350 of hPXR is crucial for heterodimerization between hPXR and RXRα and the subsequent transcriptional activation of hPXR target genes. As shown schematically in Fig. 7, this conclusion was proved by establishing, through different in vitro and in vivo approaches, that a phosphomimetic mutation of Ser350 prevents hPXR-RXRα dimerization, which is a necessary step in hPXR-mediated transcriptional activation (Fig. 7).
Although
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgments
We thank the St. Jude Animal Resources Center for technical assistance, Dr. Martin Privalsky for kindly providing the SMRTτ construct, other members of the Chen research laboratory for valuable discussions, and Sharon Naron (St. Jude Department of Scientific Editing) for editing the manuscript. This work was supported by the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's Research Hospital, and the National Institutes of Health [Grants RO1GM086415, RO1GM110034, &
References (48)
- et al.
Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1
Toxicol. Appl. Pharmacol.
(2013) Nuclear receptor drug discovery
Curr. Opin. Chem. Biol.
(2008)- et al.
Cell
(2014) - et al.
Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells
J. Biol. Chem.
(2008) - et al.
Identification and characterization of phosphorylation sites within the pregnane X receptor protein
Biochem. Pharmacol.
(2014) - et al.
Structural and functional analysis of the human nuclear xenobiotic receptor PXR in complex with RXRalpha
J. Mol. Biol.
(2013) - et al.
NSAID sulindac and its analog bind RXRalpha and inhibit RXRalpha-dependent AKT signaling
Cancer Cell
(2010) - et al.
A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay
Anal. Biochem.
(2013) - et al.
Sulforaphane is not an effective antagonist of the human pregnane X-receptor in vivo
Toxicol. Appl. Pharmacol.
(2013) - et al.
Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver
J. Biol. Chem.
(2004)
Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity
J. Biol. Chem.
Overcoming drug resistance by regulating nuclear receptors
Adv. Drug Deliv. Rev.
Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR
J. Biol. Chem.
PXR, CAR and drug metabolism
Nat. Rev. Drug Discov.
Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions
Curr. Drug Metab.
Evaluation of pregnane X receptor (PXR)-mediated CYP3A4 drug-drug interactions in drug development
Drug Metab. Rev.
Overview of nomenclature of nuclear receptors
Pharmacol. Rev.
A systematic analysis of predicted phosphorylation sites within the human pregnane X receptor protein
J. Pharmacol. Exp. Ther.
A phosphomimetic mutation at threonine-57 abolishes transactivation activity and alters nuclear localization pattern of human pregnane x receptor
Drug Metab. Dispos.
The role of residues T248, Y249 and T422 in the function of human pregnane X receptor
Arch. Toxicol.
Mechanisms of specificity in protein phosphorylation
Nat. Rev. Mol. Cell Biol.
Induced CYP3A4 expression in confluent Huh7 hepatoma cells as a result of decreased cell proliferation and subsequent pregnane X receptor activation
Mol. Pharmacol.
Regulation of pregnane X receptor (PXR) function and UGT1A1 gene expression by posttranslational modification of PXR protein
Drug Metab. Dispos.
The PREgnane X receptor gene-humanized mouse: a model for investigating drug-drug interactions mediated by cytochromes P450 3A
Drug Metab. Dispos.
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S.R.P is currently at the Department of Anatomy, Physiology and Pharmacology, Auburn University, Auburn, AL 36849, USA.