Pregnane X receptor (PXR) plays an important role in the regulation of metabolic homeostasis. Yes-associated protein (YAP) is a critical regulator of liver size and liver regeneration. Recently, we ...reported that PXR-induced liver enlargement and regeneration depend on YAP signalling, but the underlying mechanisms remain unclear. This study aimed to reveal how PXR regulates or interacts with YAP signalling during PXR-induced hepatomegaly and liver regeneration. Immunoprecipitation (IP), Co-IP and GST pull-down assays were performed in vitro to reveal the regulatory mechanisms involved in the PXR-YAP interaction. The roles of YAP-TEAD binding and Sirt2-driven deacetylation and polyubiquitination of YAP were further investigated in vitro and in vivo. The results showed that the ligand-binding domain (LBD) of PXR and the WW domain of YAP were critical for the PXR-YAP interaction. Furthermore, disruption of the YAP-TEAD interaction using the binding inhibitor verteporfin significantly decreased PXR-induced liver enlargement and regeneration after 70 % partial hepatectomy (PHx). Mechanistically, PXR activation significantly decreased YAP acetylation, which was interrupted by the sirtuin inhibitor nicotinamide (NAM). In addition, p300-induced YAP acetylation contributed to K48-linked YAP ubiquitination. Interestingly, PXR activation remarkably inhibited K48-linked YAP ubiquitination while inducing K63-linked YAP polyubiquitination. Sirt2 interference abolished the deacetylation and K63-linked polyubiquitination of YAP, suggesting that the PXR-induced deacetylation and polyubiquitination of YAP are Sirt2 dependent. Taken together, this study demonstrates that PXR induce liver enlargement and regeneration via the regulation of YAP acetylation and ubiquitination and YAP-TEAD binding, providing evidences for using PXR as potential target to promote hepatic development and liver repair.
Display omitted
Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences ...of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.
Abstract
Regulators of biotransformation are of particular interest in pharmacology and toxicology, determining in part the metabolism, disposition, and toxicity of chemicals. The nuclear receptor ...NR1I2 (pregnane X receptor, PXR) is a prominent xenosensor that regulates the expression of biotransformation enzymes governing elimination of many exogenous as well as endogenous compounds. Zebrafish (Danio rerio) has only one gene locus for pxr, but different genetic variants have been identified in zebrafish. However, the prevalence and significance of these variants are unknown. We hypothesize that sequence variation occurring in the Pxr gene of zebrafish may affect the action and fate of many chemicals in this species, a key model organism in various fields of research, including environmental toxicology. Here, we examine variation in Pxr sequences from four different strains of zebrafish and assess the responses of each Pxr to clotrimazole and butyl-4-aminobenzoate. The Pxr variants differed in both their ability to bind these structurally different ligands and to regulate reporter gene expression in vitro. We infer that the observed sequence variations in zebrafish Pxrs likely affect the response to putative Pxr agonists in vivo and potentially cause strain-specific biotransformation of xenobiotics in zebrafish. Thus, the choice of zebrafish strain could affect the outcome of downstream toxicological studies.
Abstract
Pregnane X receptor (PXR; NR1I2) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure, including induction of CYP3A transcription, in many vertebrate ...species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the chemical defensome most relevant when approached in a species-specific manner. Knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Morpholino knockdown of Pxr in zebrafish indicated a similar requirement. Here, we report on the generation of 2 zebrafish lines each carrying a heritable deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise, larvae homozygous for either of the pxr mutant alleles retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone. Thus, zebrafish carrying pxr alleles with deletions in either the DNA binding or the ligand-binding domains did not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Alternative possibilities are that Pxr is not required for the induction of selected genes, or that truncated yet functional mutant Pxr is sufficient for the downstream transcriptional effects. It is crucial that we develop a better understanding for the role of Pxr in this important biomedical test species. This study highlights the potential for compensatory mechanisms to avoid deleterious effects arising from gene mutations.
Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the ...therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.
Nuclear receptors are ligand-activated transcription factors that can often be useful drug targets. Unfortunately, ligand promiscuity leads to two-thirds of receptors remaining clinically untargeted. ...PXR is a nuclear receptor that can be activated by diverse compounds to elevate metabolism, negatively impacting drug efficacy and safety. This presents a barrier to drug development because compounds designed to target other proteins must avoid PXR activation while retaining potency for the desired target. This problem could be avoided by using PXR antagonists, but these compounds are rare, and their molecular mechanisms remain unknown. Here, we report structurally related PXR-selective agonists and antagonists and their corresponding co-crystal structures to describe mechanisms of antagonism and selectivity. Structural and computational approaches show that antagonists induce PXR conformational changes incompatible with transcriptional coactivator recruitment. These results guide the design of compounds with predictable agonist/antagonist activities and bolster efforts to generate antagonists to prevent PXR activation interfering with other drugs.
Display omitted
Previously, we demonstrated that Schisandrol B (SolB) protected against lithocholic acid (LCA)-induced cholestatic liver injury (CLI) through pregnane X receptor (PXR). Additionally, ...growing evidence has revealed that pyroptosis is involved in CLI. Whether the hepatoprotective effect of SolB driven by PXR activation is related to pyroptosis in CLI remains unclear. First, the hepatoprotective effect of SolB was confirmed, as evidenced by the decreased mortality, morphological and histopathological changes, and biochemical parameters. The upregulated serum lactic dehydrogenase (LDH) level, increased number of TUNEL-positive cells, and formation of hepatocyte membrane pores induced by LCA were significantly alleviated after SolB pretreatment, indicating that SolB attenuated LCA-induced hepatocyte damage. Further analysis revealed that both NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and apoptosis protease activating factor-1 (Apaf-1) pyroptosome-induced noncanonical pyroptosis were significantly inhibited after SolB pretreatment, as illustrated by the decreased expression levels of NLRP3, ASC, caspase-1, and GSDMD and the levels of Apaf-1, caspase-11 p20, caspase-3 p20, and GSDME. Furthermore, the activation of the NF-κB and FoxO1 signaling pathways was inhibited after SolB pretreatment. In addition, the activation of PXR via SolB was proven by luciferase reporter gene assays and the upregulation of PXR targets. The results illustrated that SolB could significantly inhibit NLRP3 inflammasome-induced canonical pyroptosis through the PXR/NF-κB/NLRP3 axis and inhibit Apaf-1 pyroptosome-induced noncanonical pyroptosis through the PXR/FoxO1/Apaf-1 axis. Collectively, this study revealed that SolB protected against CLI by inhibiting pyroptosis through PXR, providing new insights for understanding the molecular mechanism of SolB as a promising anti-cholestatic agent.
Display omitted
•Twenty potential hepatic lipid biomarkers were screened and identified by lipidomic analysis in the PXR agonist-treated mice.•PXR-induced lipid deposition is mainly attributed to ...accumulation of TG with lower number of carbon atoms and double bonds.•Lipids such as TG were significantly accumulated during PXR-induced hepatomegaly.
Pregnane X receptor (PXR) is highly expressed in the liver and plays an integral role in the control of xenobiotic and endobiotic metabolism to maintain homeostasis. We previously reported that activation of PXR significantly induced liver enlargement. But the lipid profiling during PXR-induced hepatomegaly remains unclear. This study aimed to characterize the effect of PXR activation on hepatic lipid homeostasis by lipidomics analysis. Mice were intraperitoneally administered with the typical mPXR agonist, pregnenolone 16α-carbonitrile (PCN, 100 mg/kg/d), for 5 days. Liver and serum were collected for further analysis. The results confirmed that PXR activation can significantly induce liver enlargement. An obvious hepatic lipid accumulation was observed in PCN-treated mice, as determined by H&E and Oil Red O staining. Ultra-high performance liquid chromatography-Q Exactive Orbitrap high-resolution mass spectrometer (UHPLC-Q Exactive Orbitrap HRMS)-based lipidomics was performed to characterize the change in lipid species. A total of 20 potential lipid biomarkers were significantly perturbed. The most significant change was found in the triacylglycerol (TG), which constituted with the lower number of carbon atoms and double bonds. Moreover, the mRNA expression levels showed that PCN-induced PXR activation significantly regulated the expression of genes involved in the uptake, synthesis and metabolism of TG, which was consistent with increased TG levels. Collectively, these findings demonstrated that lipids such as TG were significantly accumulated during PXR-induced hepatomegaly.
Pregnane X receptor (PXR) is a xenobiotic‐sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic ...functions of PXR activation, we observed that rifampicin, a well‐established ligand for human PXR, 600 mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow–derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α‐carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.
Display omitted
•Insulin suppresses expression of CESs in an Akt-dependent manner.•PXR mediates insulin-induced suppression of CESs.•PXR regulates CESs transcription by directly binding to its ...promoter.
Insulin is a major therapy for diabetes, and therefore, its role and mechanisms in the regulation of drug-metabolizing enzymes (DMEs), is of clinical importance to facilitate the rational drug use. Carboxylesterases are regarded as one of the major determinants of the metabolism and disposition of various substrates through their actions in the liver and intestine, alterations in the activity of CESs enzymes are often important causes of drug interactions. Therefore, investigation on the mechanism of CESs regulation is significantly important. In this study, we demonstrated that insulin markedly down-regulated CESs expression and suppressed the hydrolytic activity of CESs in an Akt-dependent manner. Moreover, overexpression of PXR abrogated the decrease of CES1 and CES2 expression induced by insulin in HepG2 cells, suggesting PXR was involved in insulin-induced reduction of CESs. Mechanistically, luciferase reporter assay showed that PXR increased the transcriptional activity of CES1 and CES2 gene promoter, and chromatin immunoprecipitation assay verified that PXR bound to the site (−244 to −234) in CES1 gene promoter region and bound to the site (−814 to −804 and −794 to −784) in CES2 gene promoter region for the first time. In summary, our data indicate that down-regulation of PXR mediates insulin-induced suppression of CESs. Accordingly, insulin may impact the therapeutic effects of carboxylesterases substrate drugs and also inhibit expression of other genes targeted by PXR, thus inducing a wide range of potential drug–drug interactions (DDIs) during the treatment of diabetes.
PDF
