Pregnane X Receptor Activation Research Articles

The F-box-only protein 44 regulates pregnane X receptor protein level by ubiquitination and degradation.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug-drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.

Ligand flexibility and binding pocket malleability cooperate to allow selective PXR activation by analogs of a promiscuous nuclear receptor ligand

The human nuclear receptor (NR) family of transcription factors contains 48 proteins that bind lipophilic molecules. Approved NR therapies have had immense success treating various diseases, but lack of selectivity has hindered efforts to therapeutically target the majority of NRs due to unpredictable off-target effects. The synthetic ligand T0901317 was originally discovered as a potent agonist of liver X receptors (LXRα/β) but subsequently found to target additional NRs, with activation of pregnane X receptor (PXR) being as potent as that of LXRs. We previously showed that directed rigidity reduces PXR binding by T0901317 derivatives through unfavorable protein remodeling. Here, we use a similar approach to achieve selectivity for PXR over other T0901317-targeted NRs. One molecule, SJPYT-318, accomplishes selectivity by favorably utilizing PXR's flexible binding pocket and surprisingly binding in a new mode distinct from the parental T0901317. Our work provides a structure-guided framework to achieve NR selectivity from promiscuous compounds.

Resveratrol attenuates efavirenz-induced hepatic steatosis and hypercholesterolemia in mice by inhibiting pregnane X receptor activation and decreasing inflammation

Efavirenz (EFV), a widely prescribed antiviral medication, has been implicated in dyslipidemia and can activate the pregnane X receptor (PXR), leading to hepatic steatosis and hypercholesterolemia in mice. Resveratrol (RES) can ameliorate hepatic steatosis and functions as a partial PXR agonist, capable of mitigating PXR expression induced by other PXR agonists. Therefore, we hypothesized that RES could attenuate EFV-induced hepatic steatosis and hypercholesterolemia by downregulating PXR expression and suppressing inflammatory cytokine production. Here, we conducted an in vivo study involving 6-week-old male mice, which were divided into 4 groups for a 7-day intervention: control (carrier solution), EFV (80 mg/kg), RES (50 mg/kg), and RES + EFV groups. Serum and hepatic tissue samples were collected to assess cholesterol and triglyceride concentrations. Hepatic lipid accumulation was evaluated through hematoxylin-eosin and oil red O staining. Polymerase chain reaction and western blot were performed to quantify hepatic inflammatory factors, lipogenic gene, and PXR expression. Our results indicated that hepatic lipid droplet accumulation was reduced in the RES + EFV group compared with the EFV group. Similarly, the expressions of hepatic inflammatory factors were attenuated in the RES + EFV group relative to the EFV group. Furthermore, RES counteracted the upregulation of hepatic lipid-metabolizing enzymes induced by EFV at both the transcriptional and protein levels. Importantly, PXR expression was downregulated in the RES + EFV group compared with the EFV group. Conclusively, our findings suggest that RES effectively mitigates EFV-induced hepatic steatosis and hypercholesterolemia by inhibiting PXR activation and decreasing inflammation.

Switching on/off aryl hydrocarbon receptor and pregnane X receptor activities by chemically modified tryptamines

Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.

NcBAF enhances PXR-mediated transcriptional activation in the human and mouse liver

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug-drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice

ObjectiveBoth obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. MethodsWildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. ResultsPXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. ConclusionsObesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.

NEAT1_2 and DAZAP1, Paraspeckle Components, Interact with PXR to Negatively Regulate CYP3A4 Induction.

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.

Pregnane X receptor activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signaling.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with various etiologies, which seriously affects the structure and function of the kidney. Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and plays a critical role in regulating the genes related to xenobiotic and endobiotic metabolism in mammals. Previous studies show that PXR is expressed in the kidney and has protective effect against acute kidney injury (AKI). In this study, we investigated the role of PXR in CKD. Adenine diet-induced CKD (AD) model was established in wild-type and PXR humanized (hPXR) mice, respectively, which were treated with pregnenolone-16α-carbonitrile (PCN, 50 mg/kg, twice a week for 4 weeks) or rifampicin (RIF, 10 mg·kg-1·d-1, for 4 weeks). We showed that both PCN and RIF, which activated mouse and human PXR, respectively, improved renal function and attenuated renal fibrosis in the two types of AD mice. In addition, PCN treatment also alleviated renal fibrosis in unilateral ureter obstruction (UUO) mice. On the contrary, PXR gene deficiency exacerbated renal dysfunction and fibrosis in both adenine- and UUO-induced CKD mice. We found that PCN treatment suppressed the expression of the profibrotic Wnt7a and β-catenin in AD mice and in cultured mouse renal tubular epithelial cells treated with TGFβ1 in vitro. We demonstrated that PXR was colocalized and interacted with p53 in the nuclei of tubular epithelial cells. Overexpression of p53 increased the expression of Wnt7a, β-catenin and its downstream gene fibronectin. We further revealed that p53 bound to the promoter of Wnt7a gene to increase its transcription and β-catenin activation, leading to increased expression of the downstream profibrotic genes, which was inhibited by PXR. Taken together, PXR activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signalingpathway.

Effects of perfluorooctanoic acid (PFOA) on gene expression profiles via nuclear receptors in HepaRG cells: Comparative study with in vitro transactivation assays

Perfluorooctanoic acid (PFOA), a synthetic perfluorinated eight-carbon organic chemical, induces hepatotoxicity in rodents, indicated increased liver weight, hepatocellular hypertrophy, necrosis, and peroxisome proliferation. Epidemiological studies have demonstrated the association between serum PFOA levels and various adverse effects. In this study, we investigated the gene expression profiles of human HepaRG cells exposed to 10 and 100 μM PFOA for 24 h. Treatment with 10 and 100 μM PFOA significantly modulated the expression of 190 and 996 genes, respectively. Genes upregulated or downregulated by 100 µM PFOA included peroxisome proliferator-activated receptor (PPAR) signaling genes related to lipid metabolism, adipocyte differentiation, and gluconeogenesis. Moreover, we identified the “Nuclear receptors-meta pathways” following the activation of other nuclear receptors: constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR), as well as the transcription factor nuclear factor E2-related factor 2 (Nrf2). The expression levels of some target genes (CYP4A11, CYP2B6, CYP3A4, CYP7A1, and GPX2) of these nuclear receptors and Nrf2 were confirmed using quantitative reverse transcription polymerase chain reaction. Next, we performed transactivation assays using COS-7 and HEK293 cells to investigate whether these signaling-pathways were activated by the direct effects of PFOA on human PPARα, CAR, PXR, FXR and Nrf2. PFOA concentration-dependently activated PPARα, but not CAR, PXR, FXR, or Nrf2. Taken together, these results suggest that PFOA affects the hepatic transcriptomic responses of HepaRG cells through the direct activation of PPARα and indirect activation of CAR, PXR, FXR, and Nrf2. Our finding indicates that PPARα activation in the “Nuclear receptors-meta pathways” functions as a molecular initiating event for PFOA, and indirect activation of alternative nuclear receptors and Nrf2 also induce important molecular mechanisms in PFOA-induced human hepatotoxicity.

Comprehensive in vitro analysis evaluating the variable drug–drug interaction risk of rifampicin compared to rifabutin

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Pregnane X receptor (PXR) deficiency protects against spinal cord injury by activating NRF2/HO-1 pathway.

As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand-activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI. The clip-compressive SCI model was performed in male wild-type C57BL/6 (PXR+/+ ) and PXR-knockout (PXR-/- ) mice. The N2a H2 O2 -induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α-carbonitrile (PCN), a mouse-specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO-1 pathway in the SCI process. The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase-1 (HO-1) after SCI. We further verified that PXR deficiency activated the NRF2/HO-1 pathway and PXR activation inhibited this pathway in vitro. PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway.

Panaxytriol upregulates CYP3A4 expression through the interaction between nuclear regulators and DNA response elements.

Cytochrome P3A4 (CYP3A4) is a crucial drug-metabolizing enzyme, and its expression is regulated by the pregnane X receptor (PXR), constitutive androstane receptor (CAR), steroid receptor coactivator 1 (SRC-1), and acetyltransferase P300. Panaxytriol is a naturally derived active substance extracted from the roots of Panax ginseng C. A. Mey. which is widely used clinically. Our previous studies have shown that panaxytriol induces CYP3A4 expression through PXR activation, which is antagonized by high CAR expression. However, the underlying mechanism remains unclear. This study aimed to investigate the mechanism of panaxytriol in inducing CYP3A4 expression via interactions between nuclear regulators and DNA response elements. Immunoprecipitation technique was used to assess the binding levels of PXR and CAR with the coactivators SRC-1 and P300 in HepG2 and Huh-7cells. Furthermore, chromatin immunoprecipitation assay was used to investigate the PXR and CAR interaction with the CYP3A4 promoter response element ER-6/DR-3. The binding of PXR to SRC-1, P300, and the response elements ER-6 and DR-3 was improved with an increase in panaxytriol concentration (10-80μM), and the binding affinity was further enhanced upon CAR silencing. The binding of CAR to SRC-1 and the response elements ER-6 and DR-3 was significantly higher at 80μM panaxytriol, whereas no significant binding was observed between CAR and P300. Panaxytriol promoted the recruitment of PXR to SRC-1 and P300, binding to ER-6 and DR-3, and upregulating CYP3A4 expression. Furthermore, an interactive dialogue regulatory mechanism between PXR and CAR was observed.

Machine learning and traditional QSAR modeling methods: a case study of known PXR activators

Pregnane X receptor (PXR), extensively expressed in human tissues related to digestion and metabolism, is responsible for recognizing and detoxifying diverse xenobiotics encountered by humans. To comprehend the promiscuous nature of PXR and its ability to bind a variety of ligands, computational approaches, viz., quantitative structure–activity relationship (QSAR) models, aid in the rapid dereplication of potential toxicological agents and mitigate the number of animals used to establish a meaningful regulatory decision. Recent advancements in machine learning techniques accommodating larger datasets are expected to aid in developing effective predictive models for complex mixtures (viz., dietary supplements) before undertaking in-depth experiments. Five hundred structurally diverse PXR ligands were used to develop traditional two-dimensional (2D) QSAR, machine-learning-based 2D-QSAR, field-based three-dimensional (3D) QSAR, and machine-learning-based 3D-QSAR models to establish the utility of predictive machine learning methods. Additionally, the applicability domain of the agonists was established to ensure the generation of robust QSAR models. A prediction set of dietary PXR agonists was used to externally-validate generated QSAR models. QSAR data analysis revealed that machine-learning 3D-QSAR techniques were more accurate in predicting the activity of external terpenes with an external validation squared correlation coefficient (R 2) of 0.70 versus an R 2 of 0.52 in machine-learning 2D-QSAR. Additionally, a visual summary of the binding pocket of PXR was assembled from the field 3D-QSAR models. By developing multiple QSAR models in this study, a robust groundwork for assessing PXR agonism from various chemical backbones has been established in anticipation of the identification of potential causative agents in complex mixtures. Communicated by Ramaswamy H. Sarma

Hepatic ZBTB22-mediated detoxification ameliorates acetaminophen-induced liver injury by inhibiting pregnane X receptor signaling

Overdose acetaminophen (APAP) can cause acute liver injury (ALI), but the underlying mechanism remains undetermined. This study explored the role of hepatic Zinc Finger And BTB Domain Containing 22 (ZBTB22) in defense against APAP-mediated hepatotoxicity. The results showed that hepatic ZBTB22 expression was significantly reduced in patients with ALI and mice. In mouse primary hepatocytes (MPHs), ZBTB22 deletion aggravated APAP overdose-induced ALI, whereas ZBTB22 overexpression attenuated that pathological progression. The results were further verified in ZBTB22 over-express or knockout mice models. In parallel, hepatocyte-specific ZBTB22 knockout also enhanced ALI. Furthermore, ZBTB22 decreased pregnane X receptor (PXR) expression, and the PXR activator pregnane-16α-carbonitrile suppressed the protective effect of ZBTB22 in APAP-induced ZBTB22-overexpressing mice. Collectively, our findings highlight the protective effect of ZBTB22 against APAP-induced ALI and unravel PXR signaling as the potential mechanism. Strategies to increase hepatic ZBTB22 expression represent a promising therapeutic approach for APAP overdose-induced ALI.

Structure-guided approach to modulate small molecule binding to a promiscuous ligand-activated protein

Ligand-binding promiscuity in detoxification systems protects the body from toxicological harm but is a roadblock to drug development due to the difficulty in optimizing small molecules to both retain target potency and avoid metabolic events. Immense effort is invested in evaluating metabolism of molecules to develop safer, more effective treatments, but engineering specificity into or out of promiscuous proteins and their ligands is a challenging task. To better understand the promiscuous nature of detoxification networks, we have used X-ray crystallography to characterize a structural feature of pregnane X receptor (PXR), a nuclear receptor that is activated by diverse molecules (with different structures and sizes) to up-regulate transcription of drug metabolism genes. Wefound that large ligands expand PXR's ligand-binding pocket, and the ligand-induced expansion occurs through a specific unfavorable compound-protein clash that likely contributes to reduced binding affinity. Removing the clash by compound modification resulted in more favorable binding modes with significantly enhanced binding affinity. We then engineered the unfavorable ligand-protein clash into a potent, small PXR ligand, resulting in marked reduction in PXR binding and activation. Structural analysis showed that PXR is remodeled, and the modified ligands reposition in the binding pocket to avoid clashes, but the conformational changes result in less favorable binding modes. Thus, ligand-induced binding pocket expansion increases ligand-binding potential of PXR but is an unfavorable event; therefore, drug candidates can be engineered to expand PXR's ligand-binding pocket and reduce their safety liability due to PXR binding.

Identification of PXR Activators from Uncaria Rhynchophylla (Gou Teng) and Uncaria Tomentosa (Cat's Claw).

Uncaria rhynchophylla (Gou Teng) and Uncaria tomentosa (cat's claw) are frequently used herbal supplements in Asia and America, respectively. Despite their common usage, information is limited regarding potential herb-drug interactions associated with Gou Teng and cat's claw. The pregnane X receptor (PXR) is a ligand-dependent transcription factor that regulates cytochrome P450 3A4 (CYP3A4) expression and contributes to some known herb-drug interactions. A recent study found that Gou Teng induces CYP3A4 expression, but its mechanism is unknown. Cat's claw has been determined as a PXR-activating herb, but the PXR activators in cat's claw have not been identified. Using a genetically engineered PXR cell line, we found that the extracts of Gou Teng and cat's claw can dose-dependently activate PXR and induce CYP3A4 expression. We next used a metabolomic approach to profile the chemical components in the extracts of Gou Teng and cat's claw followed by screening for PXR activators. Four compounds, isocorynoxeine, rhynchophylline, isorhynchophylline, and corynoxeine, were identified as PXR activators from both Gou Teng and cat's claw extracts. In addition, three more PXR activators were identified from the extracts of cat's claw, including isopteropodine, pteropodine, and mitraphylline. All seven of these compounds showed the half-maximal effective concentration <10 µM for PXR activation. In summary, our work determined Gou Teng as a PXR-activating herb and discovered novel PXR activators from Gou Teng as well as cat's claw. SIGNIFICANCE STATEMENT: This study's data can be used to guide the safe use of Gou Teng and cat's claw by avoiding PXR-mediated herb-drug interactions.

TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs

The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR’s downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein–protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4’s promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.

PXR triggers YAP-TEAD binding and Sirt2-driven YAP deacetylation and polyubiquitination to promote liver enlargement and regeneration in mice

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.

Interaction of pregnane X receptor with hypoxia-inducible factor-1 regulates chemoresistance of prostate cancer cells.

Aim: The nuclear pregnane X receptor (PXR) is a pivotal regulator of steroid and xenobiotics metabolism and plays an important role in shaping tumor cell responses to chemotherapy. Hypoxia within tumor tissue has multifaceted effects, including multiple drug resistance. The goal of this study was to determine whether PXR contributes to hypoxia-induced drug resistance. Methods: Metastatic prostate cancer cells were used to study the interaction of PXR and hypoxia-inducible factor-1 (HIF-1 in drug resistance associated with hypoxia. The activities of PXR and HIF-1 were determined by assays for its reporter gene or target gene expression. Co-immunoprecipitation (Co-IP) was used to determine the interaction of PXR and HIF-1. Ablation or inhibition of PXR or HIF-1 was used to determine their roles in hypoxia-induced chemoresistance. Results: PXR was activated by hypoxia, leading to increased expression of multidrug resistance protein 1 (MDR1). Inhibition of PXR by pharmacological compounds or depletion by shRNAs reduced the hypoxic induction of MDR1 and sensitized prostate cancer cells to chemotherapy under hypoxia. HIF-1 was required for PXR activation under hypoxia. Co-immunoprecipitation results showed that HIF-1 and PXR could physically interact with each other, leading to crosstalk between these two transcription factors. Conclusion: PXR contributes to hypoxia-induced drug resistance in prostate cancer cells through its interaction with HIF-1.

Rifaximin stimulates nitrogen detoxification by PXR-independent mechanisms in human small intestinal organoids.

Recurrent hepatic encephalopathy (HE) is characterized by hyperammonaemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a pregnane X receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability. We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 h with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry. Treatment of wild-type hSI organoids with rifaximin resulted in >twofold differential expression of 131 genes compared to DMSO. These effects were largely PXR independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP binding cassette B1 (ABCB1)-dependent manner. Rifaximin-after uptake into enterocytes-stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium.