Songrong Ren

A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway

The pregnane X receptor (PXR) was isolated as a xenosensor regulating xenobiotic responses. In this study, we show that PXR plays an endobiotic role by impacting lipid homeostasis. Expression of an activated PXR in the livers of transgenic mice resulted in an increased hepatic deposit of triglycerides. This PXR-mediated lipid accumulation was independent of the activation of the lipogenic transcriptional factor SREBP-1c (sterol regulatory element-binding protein 1c) and its primary lipogenic target enzymes, including fatty-acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC-1). Instead, the lipid accumulation in transgenic mice was associated with an increased expression of the free fatty acid transporter CD36 and several accessory lipogenic enzymes, such as stearoyl-CoA desaturase-1 (SCD-1) and long chain free fatty acid elongase. Studies using transgenic and knock-out mice showed that PXR is both necessary and sufficient for Cd36 activation. Promoter analyses revealed a DR-3-type of PXR-response element in the mouse Cd36 promoter, establishing Cd36 as a direct transcriptional target of PXR. The hepatic lipid accumulation and Cd36 induction were also seen in the hPXR “humanized” mice treated with the hPXR agonist rifampicin. The activation of PXR was also associated with an inhibition of pro-β-oxidative genes, such as peroxisome proliferator-activated receptor α (PPARα) and thiolase, and an up-regulation of PPARγ, a positive regulator of CD36. The cross-regulation of CD36 by PXR and PPARγ suggests that this fatty acid transporter may function as a common target of orphan nuclear receptors in their regulation of lipid homeostasis.

Traditional Chinese Medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) Activate Pregnane X Receptor and Increase Warfarin Clearance in Rats

The traditional Chinese medicines (TCMs) are essential components of alternative medicines. Many TCMs are known to alter the expression of hepatic drug-metabolizing enzymes and transporters. The molecular mechanism by which TCMs and/or their constituents regulate enzyme and transporter expression, however, has remained largely unknown. In this report, we show that two TCMs, Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch), and their selected constituents activate the xenobiotic orphan nuclear receptor pregnane X receptor (PXR). Treatment with TCM extracts and the Schisandrol and Schisandrin constituents of Wu Wei Zi induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays and in primary hepatocyte cultures. The affected enzymes and transporters include CYP3A and 2C isozymes and the multidrug resistance-associated protein 2. In transient transfection and reporter gene assays, the Schisandrin constituents of Wu Wei Zi had an estimated EC50 of 2 and 1.25 μM on hPXR and mPXR, respectively. Interestingly, mutations that were intended to alter the pore of the ligand-binding cavity of PXR had species-specific effects on the activities of the individual Schisandrols and Schisandrins. In rats, the administration of Wu Wei Zi and Gan Cao increased the metabolism of the coadministered warfarin, reinforcing concerns involving the safe use of herbal medicines and other nutraceuticals to avoid PXR-mediated drug-drug interactions. Meanwhile, the activation of PXR and induction of detoxifying enzymes provide a molecular mechanism for the hepatoprotective effects of certain TCMs.

Activation of LXRs prevents bile acid toxicity and cholestasis in female mice

Liver X receptors (LXRs) have been identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. In this study, we found that LXRs also affect sensitivity to bile acid toxicity and cholestasis. Activation of LXRα in transgenic mice confers a female‐specific resistance to lithocholic acid (LCA)–induced hepatotoxicity and bile duct ligation (BDL)–induced cholestasis. This resistance was also seen in wild‐type female mice treated with the synthetic LXR ligand TO1317. In contrast, LXR double knockout (DKO) mice deficient in both the α and β isoforms exhibited heightened cholestatic sensitivity. LCA and BDL resistance in transgenic mice was associated with increased expression of bile acid–detoxifying sulfotransferase 2A (Sult2a) and selected bile acid transporters, whereas basal expression of these gene products was reduced in the LXR DKO mice. Promoter analysis showed that the mouse Sult2a9 gene is a transcriptional target of LXRs. Activation of LXRs also suppresses expression of oxysterol 7α‐hydroxylase (Cyp7b1), which may lead to increased levels of LXR‐activating oxysterols. Conclusion: We propose that LXRs have evolved to have the dual functions of maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism and, at the same time, preventing toxicity from bile acid accumulation. (HEPATOLOGY 2007;45:422–432.)

Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice

Efficient detoxification of bile acids is necessary to avoid pathological conditions such as cholestatic liver damage and colon cancer. The orphan nuclear receptors PXR and CAR have been proposed to play an important role in the detoxification of xeno‐ and endo‐biotics by regulating the expression of detoxifying enzymes and transporters. In this report, we showed that the combined loss of PXR and CAR resulted in a significantly heightened sensitivity to bile acid toxicity in a sex‐sensitive manner. A regimen of lithocholic acid treatment, which was tolerated by wild‐type and PXR null mice, caused a marked accumulation of serum bile acids and histological liver damage as well as an increased hepatic lipid deposition in double knockout males. The increased sensitivity in males was associated with genotype‐specific suppression of bile acid transporters and loss of bile acid–mediated downregulation of small heterodimer partner, whereas the transporter suppression was modest or absent in females. The double knockout mice also exhibited gene‐ and tissue‐specific dysregulation of PXR and CAR target genes in response to PXR and CAR agonists. In conclusion, although the cross‐regulation of target genes by PXR and CAR has been proposed, the current study represents in vivo evidence of the combined loss of both receptors causing a unique pattern of gene regulation that can be translated into physiological events such as sensitivity to toxic bile acids. (HEPATOLOGY 2005;41:168–176.)

Identification of Oxysterol 7α-Hydroxylase (Cyp7b1) as a Novel Retinoid-Related Orphan Receptor α (RORα) (NR1F1) Target Gene and a Functional Cross-Talk between RORα and Liver X Receptor (NR1H3)

The retinoid-related orphan receptors (RORs) and liver X receptors (LXRs) were postulated to have distinct functions. RORs play a role in tissue development and circadian rhythm, whereas LXRs are sterol sensors that affect lipid homeostasis. In this study, we revealed a novel function of RORα (NR1F1) in regulating the oxysterol 7α-hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the RORα null (RORαsg/sg) mice, suggesting RORα as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of RORα, and transfection of RORα induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be RORα-specific, because RORγ had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by RORα was suppressed by LXRα (NR1H3), whereas RORα inhibited both the constitutive and ligand-dependent activities of LXRα. The mutual suppression between RORα and LXR was supported by the in vivo observation that loss of RORα increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR α and β isoforms showed activation of selected RORα target genes. Our results have revealed a novel role for RORα and a functional interplay between RORα and LXR in regulating endo- and xenobiotic genes, which may have broad implications in metabolic homeostasis.

Dual role of orphan nuclear receptor pregnane X receptor in bilirubin detoxification in mice

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are implicated in xenobiotic and endobiotic detoxification, including the clearance of toxic bilirubin. Previous studies have suggested both overlapping and preferential regulation of target genes by these receptors, but the mechanism of cross‐talk remains elusive. Here we reveal a dual role of PXR in bilirubin detoxification in that both the loss and activation of PXR led to protection from hyperbilirubinemia induced by bilirubin infusion or hemolysis. The increased bilirubin clearance in PXR‐null mice was associated with selective upregulation of detoxifying enzymes and transporters, and the pattern of regulation is remarkably similar to that of transgenic mice expressing the activated CAR. Interestingly, the increased bilirubin clearance and associated gene regulation were absent in the CAR‐null or double‐knockout mice. In cell cultures, ligand‐free PXR specifically suppressed the ability of CAR to induce the multidrug resistance associated protein 2 (MRP2), a bilirubin‐detoxifying transporter. This suppression was, at least in part, the result of the disruption of ligand‐independent recruitment of coactivator by CAR. In conclusion, PXR plays both positive and negative roles in regulating bilirubin homeostasis, and this provides a novel mechanism that may govern receptor cross‐talk and the hierarchy of xenobiotic and endobiotic regulation. PXR is a potential therapeutic target for clinical treatment of jaundice. (HEPATOLOGY 2005;41:497–505.)

PXR ablation alleviates diet-induced and genetic obesity and insulin resistance in mice

The pregnane X receptor (PXR), along with its sister receptor constitutive androstane receptor (CAR), was initially characterized as a xenobiotic receptor that regulates drug metabolism. In this study, we have uncovered an unexpected endobiotic role of PXR in obesity and type 2 diabetes. PXR ablation inhibited high-fat diet (HFD)–induced obesity, hepatic steatosis, and insulin resistance, which were accounted for by increased oxygen consumption, increased mitochondrial β-oxidation, inhibition of hepatic lipogenesis and inflammation, and sensitization of insulin signaling. In an independent model, introducing the PXR−/− allele into the ob/ob background also improved body composition and relieved the diabetic phenotype. The ob/ob mice deficient of PXR showed increased oxygen consumption and energy expenditure, as well as inhibition of gluconeogenesis and increased rate of glucose disposal during euglycemic clamp. Mechanistically, the metabolic benefits of PXR ablation were associated with the inhibition of c-Jun NH2-terminal kinase activation and downregulation of lipin-1, a novel PXR target gene. The metabolic benefit of PXR ablation was opposite to the reported prodiabetic effect of CAR ablation. Our results may help to establish PXR as a novel therapeutic target, and PXR antagonists may be used for the prevention and treatment of obesity and type 2 diabetes.

Activation of liver X receptor sensitizes mice to gallbladder cholesterol crystallization.

Gallstone disease is a hepatobiliary disorder due to biochemical imbalances in the gallbladder bile. In this report, we show that activation of nuclear receptor liver X receptor (LXR) sensitized mice to lithogenic diet–induced gallbladder cholesterol crystallization, which was associated with dysregulation of several hepatic transporters that efflux cholesterol, phospholipids, and bile salts. The combined effect of increased biliary concentrations of cholesterol and phospholipids and decreased biliary concentrations of bile salts in LXR‐activated mice led to an increased cholesterol saturation index and the formation of cholesterol crystals. Interestingly, the lithogenic effect of LXR was completely abolished in the low‐density lipoprotein receptor (Ldlr) null background or when the mice were treated with Ezetimibe, a cholesterol‐lowering drug that blocks intestinal dietary cholesterol absorption. These results suggest that LDLR‐mediated hepatic cholesterol uptake and intestinal cholesterol absorption play an essential role in LXR‐promoted lithogenesis. Conclusion: The current study has revealed a novel lithogenic role of LXR as well as a functional interplay between LXR and LDLR in gallbladder cholesterol crystallization and possibly cholesterol gallstone disease (CGD). We propose that LXR is a lithogenic factor and that the LXR transgenic mice may offer a convenient CGD model to develop therapeutic interventions for this disease. (HEPATOLOGY 2008.)

A Functional Cross-Talk between Liver X Receptor-α and Constitutive Androstane Receptor Links Lipogenesis and Xenobiotic Responses

The liver X receptor (LXR) and constitutive androstane receptor (CAR) are two nuclear receptors postulated to have distinct functions. LXR is a sterol sensor that promotes lipogenesis, whereas CAR is a xenosensor that controls xenobiotic responses. Here, we show that LXRα and CAR are functionally related in vivo. Loss of CAR increased the expression of lipogenic LXR target genes, leading to increased hepatic triglyceride accumulation, whereas activation of CAR inhibited the expression of LXR target genes and LXR ligand-induced lipogenesis. On the other hand, a combined loss of LXR α and β increased the basal expression of xenobiotic CAR target genes, whereas activation of LXR inhibited the expression of CAR target genes and sensitized mice to xenobiotic toxicants. The mutual suppression between LXRα and CAR was also observed in cell culture and reporter gene assays. LXRα, like CAR, exhibited constitutive activity in the absence of an exogenously added ligand by recruiting nuclear receptor coactivators. Interestingly, although CAR competed with LXRα for coactivators, the constitutive activity and recruitment of coactivators was not required for CAR to suppress the activity of LXRα. In vivo chromatin immunoprecipitation assay showed that cotreatment of a CAR agonist compromised the LXR agonist responsive recruitment of LXRα to Srebp-1c, whereas an LXR agonist inhibited the CAR agonist-responsive recruitment of CAR to Cyp2b10. In conclusion, our results have revealed dual functions of LXRα and CAR in lipogenesis and xenobiotic responses, establishing a unique role of these two receptors in integrating xenobiotic and endobiotic homeostasis.

Pregnane X receptor as a therapeutic target to inhibit androgen activity

The androgen-androgen receptor signaling pathway plays an important role in the pathogenesis of prostate cancer. Accordingly, androgen deprivation has been the most effective endocrine therapy for hormone-dependent prostate cancer. Here, we report a novel pregnane X receptor (PXR)-mediated and metabolism-based mechanism to reduce androgenic tone. PXR is a nuclear receptor previously known as a xenobiotic receptor regulating the expression of drug metabolizing enzymes and transporters. We showed that genetic (using a PXR transgene) or pharmacological (using a PXR agonist) activation of PXR lowered androgenic activity and inhibited androgen-dependent prostate regeneration in castrated male mice that received daily injections of testosterone propionate by inducing the expression of cytochrome P450 (CYP)3As and hydroxysteroid sulfotransferase (SULT)2A1, which are enzymes important for the metabolic deactivation of androgens. In human prostate cancer cells, treatment with the PXR agonist rifampicin (RIF) inhibited androgen-dependent proliferation of LAPC-4 cells but had little effect on the growth of the androgen-independent isogenic LA99 cells. Down-regulation of PXR or SULT2A1 in LAPC-4 cells by short hairpin RNA or small interfering RNA abolished the RIF effect, indicating that the inhibitory effect of RIF on androgens was PXR and SULT2A1 dependent. In summary, we have uncovered a novel function of PXR in androgen homeostasis. PXR may represent a novel therapeutic target to lower androgen activity and may aid in the treatment and prevention of hormone-dependent prostate cancer.