Stacey A. Jones

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.

A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.

Bile acids repress the transcription of cytochrome P450 7A1 (CYP7A1), which catalyzes the rate-limiting step in bile acid biosynthesis. Although bile acids activate the farnesoid X receptor (FXR), the mechanism underlying bile acid-mediated repression of CYP7A1 remained unclear. We have used a potent, nonsteroidal FXR ligand to show that FXR induces expression of small heterodimer partner 1 (SHP-1), an atypical member of the nuclear receptor family that lacks a DNA-binding domain. SHP-1 represses expression of CYP7A1 by inhibiting the activity of liver receptor homolog 1 (LRH-1), an orphan nuclear receptor that is known to regulate CYP7A1 expression positively. This bile acid-activated regulatory cascade provides a molecular basis for the coordinate suppression of CYP7A1 and other genes involved in bile acid biosynthesis.

The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16α-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7α-hydroxylase (Cyp7a1) and the Na+-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

The Pregnane X Receptor: A Promiscuous Xenobiotic Receptor That Has Diverged during Evolution

Transcription of genes encoding cytochrome P450 3A (CYP3A) monooxygenases is induced by a variety of xenobiotics and natural steroids. There are marked differences in the compounds that induce CYP3A gene expression between species. Recently, the mouse and human pregnane X receptor (PXR) were shown to be activated by compounds that induce CYP3A expression. However, most studies of CYP3A regulation have been performed using rabbit and rat hepatocytes. Here, we report the cloning and characterization of PXR from these two species. PXR is remarkably divergent between species, with the rabbit, rat, and human receptors sharing only approximately 80% amino acid identity in their ligand-binding domains. This sequence divergence is reflected by marked pharmacological differences in PXR activation profiles. For example, the macrolide antibiotic rifampicin, the antidiabetic drug troglitazone, and the hypocholesterolemic drug SR12813 are efficacious activators of the human and rabbit PXR but have little activity on the rat and mouse PXR. Conversely, pregnane 16alpha-carbonitrile is a more potent activator of the rat and mouse PXR than the human and rabbit receptor. The activities of xenobiotics in PXR activation assays correlate well with their ability to induce CYP3A expression in primary hepatocytes. Through the use of a novel scintillation proximity binding assay, we demonstrate that many of the compounds that induce CYP3A expression bind directly to human PXR. These data establish PXR as a promiscuous xenobiotic receptor that has diverged during evolution.

Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis

Farnesoid X receptor (FXR) is a bile acid-activated transcription factor that is a member of the nuclear hormone receptor superfamily. Fxr-null mice exhibit a phenotype similar to Byler disease, an inherited cholestatic liver disorder. In the liver, activation of FXR induces transcription of transporter genes involved in promoting bile acid clearance and represses genes involved in bile acid biosynthesis. We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct-ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease.

Identification of a Bile Acid-responsive Element in the Human Ileal Bile Acid-binding Protein Gene INVOLVEMENT OF THE FARNESOID X RECEPTOR/9-cis-RETINOIC ACID RECEPTOR HETERODIMER

Intestinal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acids (BAs) with a high affinity. In the small intestine, its expression is restricted to the ileum where it is involved in the enterohepatic circulation of BAs. Using the human enterocyte-like Caco-2 cell line, we have recently shown that BAs increased I-BABP gene expression. To determine whether this regulation occurs in vivo, the effect of BA depletion or supplementation was studied in mice. A dramatic drop in I-BABP mRNA levels was observed in mice treated with the BA-binding resin cholestyramine, whereas an increase was found in animals fed with taurocholic acid. BAs are physiological ligands for the nuclear farnesoid X receptor (FXR). Both FXR and I-BABP are co-expressed along the small intestine and in Caco-2 cells. To determine the role of FXR in the regulation of I-BABP expression, the promoter of the human I-BABP gene was cloned. In Caco-2 cells, cotransfection of FXR and RXRα is required to obtain the full transactivation of the I-BABP promoter by BAs. Deletion and mutation analyses demonstrate that the FXR/RXRα heterodimer activates transcription through an inverted repeat bile acid responsive element located in position −160/−148 of the human I-BABP promoter. In conclusion, we show that FXR is a physiological BA sensor that is likely to play an essential role in BA homeostasis through the regulation of genes involved in their enterohepatic circulation.

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064.

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.

The Relative Activity of 'Function Sparing' HIV-1 Entry Inhibitors on Viral Entry and CCR5 Internalization: Is Allosteric Functional Selectivity a Valuable Therapeutic Property?

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection ( Science307:1434-1440, 2005 ). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.

Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity

The nuclear receptor, farnesoid X receptor (FXR, NR1H4), is known to regulate cholesterol, bile acid, lipoprotein, and glucose metabolism. In the current study, we provide evidence to support a role for FXR in hepatoprotection from acetaminophen (APAP)-induced toxicity. Pharmacological activation of FXR induces the expression of several genes involved in phase II and phase III xenobiotic metabolism in wild-type, but not Fxr(-/-) mice. We used chromatin immunoprecipitation-based genome-wide response element analyses coupled with luciferase reporter assays to identify functional FXR response elements within promoters, introns, or intragenic regions of these genes. Consistent with the observed transcriptional changes, FXR gene dosage is positively correlated with the degree of protection from APAP-induced hepatotoxicity in vivo. Further, we demonstrate that pretreatment of wild-type mice with an FXR-specific agonist provides significant protection from APAP-induced hepatotoxicity. Based on these findings, we propose that FXR plays a role in hepatic xenobiotic metabolism and, when activated, provides hepatoprotection against toxins such as APAP.

Physiology of FGF15/19

This chapter will review the various biological actions of the mouse fibroblast growth factor 15 (Fgf15) and human fibroblast growth factor 19 (FGF19). Unlike other members of the fibroblast growth factor (FGF) family, the Fgf15 and FGF19 orthologs do not share a high degree of sequence identity. Fgf15 and FGF19 are members of an atypical subfamily of FGFs that function as hormones. Due to subtle changes in tertiary structure, these FGFs have low heparin binding affinity enabling them to diffuse away from their site of secretion and signal to distant cells. FGF signaling through the FGF receptors is also different for this sub-family, requiring klotho protein cofactors rather than heparin sulfate proteoglycan. Mouse Fgf15 and human FGF19 play key roles in enterohepatic signaling, regulation of liver bile acid biosynthesis, gallbladder motility and metabolic homeostasis.