Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, ␤-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

Pregnane x receptor (PXR, NR1I2) was originally characterized as a broad spectrum entero-hepatic xenobiotic ‘sensor’ and master-regulator of drug inducible gene expression. A compelling description of ligand-mediated gene activation has been unveiled in the last decade that firmly establishes this receptor’s central role in the metabolism and transport of xenobiotics in mammals. Interestingly, pharmacotherapy with potent PXR ligands produces several profound side effects including decreased capacities for gluconeogenesis, lipid metabolism, and inflammation; likely due to PXR-mediated repression of gene expression programs underlying these pivotal physiological functions. An integrated model is emerging that reveals a sophisticated interplay between ligand binding and the ubiquitylation, phosphorylation, SUMOylation, and acetylation status of this important nuclear receptor protein. These discoveries point to a key role for the post-translational modification of PXR in the selective suppression of gene expression, and open the door to the study of completely new modes of regulation of the biological activity of PXR.

The pregnane X receptor (PXR, NR1I2) regulates the expression of genes that encode drug-metabolizing enzymes and drug transporter proteins in liver and intestine. Understanding the molecular mechanisms that modulate PXR activity is therefore critical for the development of effective therapeutic strategies. Several recent studies have implicated the activation of kinase signaling pathways in the regulation of PXR biological activity, although direct evidence and molecular mechanisms are currently lacking. We therefore sought to characterize potential phosphorylation sites within the PXR protein by use of a rational, comprehensive, and systematic site-directed mutagenesis approach to generate phosphomimetic mutations (Ser/Thr 3 Asp) and phospho-deficient mutations (Ser/Thr 3 Ala) at 18 predicted consensus kinase recognition sequences in the human PXR protein. Here, we identify amino acid residues Ser8, Thr57, Ser208, Ser305, Ser350, and Thr408 as being critical for biological activity of the PXR protein. Mutations at positions 57 and 408 abolish ligand-inducible PXR activity. Mutations in the extreme N terminus and in the PXR ligand-binding domain at positions Ser8, Ser305, Ser350, and Thr408 decrease the ability of PXR to form heterodimers with retinoid X receptor ␣. Mutations at positions Ser208, Ser305, Ser350, and Thr408 alter PXR-protein cofactor interactions. Finally, the subcellular localization of the PXR protein is profoundly affected by mutations at position Thr408. These data suggest that PXR activity can potentially be regulated by phosphorylation at specific amino acid residues within several predicted consensus kinase recognition sequences to differentially affect PXR biological activity.

Oleanolic acid (OA) is a natural triterpenoid and has been demonstrated to protect against varieties of hepatotoxicants. Recently, however, OA at high doses was reported to produce apparent cholestasis in mice. In this study, we characterized the protective effect of OA at low doses against lithocholic acid (LCA)-induced cholestasis in mice and explored further mechanisms. OA cotreatment (5, 10, and 20 mg/kg, i.p.) significantly improved mouse survival rate, attenuated liver necrosis, and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; more importantly, serum total bile acids and bilirubin, as well as hepatic total bile acids were also remarkably reduced. Gene and protein expression analysis showed that hepatic expression of multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4 was significantly increased by OA cotreatment, whereas other bile acid metabolism-and transport-related genes, including Na+/taurocholate cotransporter, organic anion transporter 1b2, bile salt export pump, multidrug resistance protein 3, Cyp3a11, Cyp2b10, Sulfotransferase 2a1 (Sult2a1), and UDP-glucuronosyltransferase 1a1 (Ugt1a1), were only slightly changed. OA also caused increased nuclear factor-E2-related factor (Nrf2) mRNA expression and nuclear protein accumulation, whereas nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor were not significantly influenced by OA. Luciferase (Luc) assays performed in HepG2 cells illustrated that OA was a strong Nrf2 agonist with moderate PXR and weak FXR agonism. Finally, in mouse primary cultured hepatocytes, OA dose-and time-dependently induced expression of Mrp2, Mrp3, and Mrp4; however, this upregulation was abrogated when Nrf2 was silenced. In conclusion, OA produces a protective effect against LCA-induced hepatotoxicity and cholestasis, possibly due to Nrf2-mediated upregulation of Mrp2, Mrp3, and Mrp4.

Long-term treatment of patients with the macrolide antibiotic and prototypical activator of pregnane X receptor (PXR) rifampicin (Rif) inhibits the inflammatory response in liver. We show here that activation of the inflammatory response in hepatocytes strongly modulates SUMOylation of ligand-bound PXR. We provide evidence that the SUMOylated PXR contains SUMO3 chains, and feedback represses the immune response in hepatocytes. This information represents the first step in developing novel pharmaceutical strategies to treat inflammatory liver disease and prevent adverse drug reactions in patients experiencing acute or systemic inflammation. These studies also provide a molecular rationale for constructing a novel paradigm that uniquely defines the molecular basis of the interface between PXR-mediated gene activation, drug metabolism, and inflammation.