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

Abstract

The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR's transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser³⁵⁰) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive. Here we investigated the transactivation of hPXR target genes in vitro and in vivo by hPXR with a phosphomimetic mutation at Ser³⁵⁰ (hPXR^S350D). The S350D phosphomimetic mutation reduced the endogenous expression of cytochrome P450 3A4 (an hPXR target gene) in HepG2 and LS180 cells. Biochemical assays and structural modeling revealed that Ser³⁵⁰ of hPXR is crucial for formation of the hPXR–retinoid X receptor alpha (RXRα) heterodimer. The S350D mutation abrogated heterodimerization in a ligand-independent manner, impairing hPXR-mediated transactivation. Further, in a novel humanized transgenic mouse model expressing the hPXR^S350D transgene, we demonstrated that the S350D mutation alone is sufficient to impair hPXR transcriptional activity in mouse liver. This transgenic mouse model provides a unique tool to investigate the regulation and function of hPXR, including its non-genomic function, in vivo. Our finding that phosphorylation regulates hPXR activity has implications for development of novel hPXR antagonists and for safety evaluation during drug development.

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 hPXR^S350D transgene.