Jessica A. Bonzo

Expression of the human UGT1 locus in transgenic mice by 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643) and implications on drug metabolism through peroxisome proliferator-activated receptor α activation

The UDP-glucuronosyltransferase (UGT) 1A genes in humans have been shown to be differentially regulated in a tissue-specific fashion. Transgenic mice carrying the human UGT1 locus (Tg-UGT1) were recently created, demonstrating that expression of the nine UGT1A genes closely resembles the patterns of expression observed in human tissues. In the present study, UGT1A1, UGT1A3, UGT1A4, and UGT1A6 have been identified as targets of the peroxisome proliferator-activated receptor (PPAR) α in human hepatocytes and Tg-UGT1 mice. Oral administration of the PPARα agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (pirinixic acid, WY-14643) to Tg-UGT1 mice led to induction of these proteins in either the liver, gastrointestinal tract, or kidney. The levels of induced UGT1A3 gene transcripts in liver and UGT1A4 protein in small intestine correlated with induced lamotrigine glucuronidation activity in these tissues. With UGT1A3 previously identified as the major human enzyme involved in human C24-glucuronidation of lithocholic acid (LCA), the dramatic induction of liver UGT1A3 RNA in Tg-UGT1 mice was consistent with the formation of LCA-24G in plasma. Furthermore, PPAR-responsive elements (PPREs) were identified flanking the UGT1A1, UGT1A3, and UGT1A6 genes by a combination of site-directed mutagenesis, specific binding to PPARα and retinoic acid X receptor α, and functional response of the concatenated PPREs in HepG2 cells overexpressing PPARα. In conclusion, these results suggest that oral fibrate treatment in humans will induce the UGT1A family of proteins in the gastrointestinal tract and liver, influencing bile acid glucuronidation and first-pass metabolism of other drugs that are taken concurrently with hypolipidemic therapy.

Suppression of Hepatocyte Proliferation by Hepatocyte Nuclear Factor 4α in Adult Mice

Background: HNF4α is a key factor regulating hepatocyte differentiation and liver-specific functions. Results: Acute disruption of HNF4α in the adult liver causes rapid hepatocyte proliferation through direct and indirect control of multiple pathways. Conclusion: HNF4α is necessary to maintain the differentiated state of hepatocytes in adult liver. Significance: HNF4α expression maintains normal liver function, and its loss stimulates hepatocytes proliferation, possibly leading to cancer. Hepatocyte nuclear factor 4α (HNF4α) regulates genes involved in lipid and bile acid synthesis, gluconeogenesis, amino acid metabolism, and blood coagulation. In addition to its metabolic role, HNF4α is critical for hepatocyte differentiation, and loss of HNF4α is associated with hepatocellular carcinoma. The hepatocyte-specific Hnf4a knock-out mouse develops severe hepatomegaly and steatosis resulting in premature death, thereby limiting studies of the role of this transcription factor in the adult animal. In addition, gene compensation may complicate analysis of the phenotype of these mice. To overcome these issues, an acute Hnf4a knock-out mouse model was generated through use of the tamoxifen-inducible ErT2cre coupled to the serum albumin gene promoter. Microarray expression analysis revealed up-regulation of genes associated with proliferation and cell cycle control only in the acute liver-specific Hnf4α-null mouse. BrdU and ki67 staining confirmed extensive hepatocyte proliferation in this model. Proliferation was associated with induction of the hepatomitogen Bmp7 as well as reduced basal apoptotic activity. The p53/p63 apoptosis effector gene Perp was further identified as a direct HNF4α target gene. These data suggest that HNF4α maintains hepatocyte differentiation in the adult healthy liver, and its loss may directly contribute to hepatocellular carcinoma development, thus indicating this factor as a possible liver tumor suppressor gene.

The stable repression of mesenchymal program is required for hepatocyte identity: A novel role for hepatocyte nuclear factor 4α

The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial‐to‐mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal‐to‐epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real‐time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α‐mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. Conclusion: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;)

The liver X‐receptor alpha controls hepatic expression of the human bile acid–glucuronidating UGT1A3 enzyme in human cells and transgenic mice

Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP‐glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24‐glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol‐activated nuclear receptor liver X‐receptor alpha (LXRα). In human hepatic cells and human UGT1A transgenic mice, LXRα activators induce UGT1A3 mRNA levels and the formation of CDCA‐24glucuronide (24G) and LCA‐24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site‐directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRα is found to interact with the SRC‐1α and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC‐1β. In conclusion, these observations establish LXRα as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively. (HEPATOLOGY 2006;44:368–378.)

Metabolomics Reveals Attenuation of the SLC6A20 Kidney Transporter in Nonhuman Primate and Mouse Models of Type 2 Diabetes Mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na+-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Disruption of the Ugt1 Locus in Mice Resembles Human Crigler-Najjar Type I Disease

The 9 UDP-glucuronosyltranferases (UGTs) encoded by the UGT1 locus in humans are key enzymes in the metabolism of most drugs as well as endogenous substances such as bile acids, fatty acids, steroids, hormones, neurotransmitters, and bilirubin. Severe unconjugated hyperbilirubinemia in humans that suffer from Crigler-Najjar type I disease results from lesions in the UGT1A1 gene and is often fatal. To examine the physiological importance of the Ugt1 locus in mice, this locus was rendered non-functional by interrupting exon 4 to create Ugt1-/- mice. Because UGT1A1 in humans is responsible for 100% of the conjugated bilirubin, it followed that newborn Ugt1-/- mice developed serum levels of unconjugated bilirubin that were 40-60 times higher than Ugt1+/- or wild-type mice. The result of extreme unconjugated bilirubin in Ugt1-/- mice, comparable to the induced levels noted in patients with Crigler-Najjar type 1 disease, is fatal in neonatal Ugt1-/- mice within 2 weeks following birth. The extreme jaundice is present as a phenotype in skin color after 8 h. Neonatal Ugt1-/- mice exhibit no detectable UGT1A-specific RNA, which corresponds to a complete absence of UGT1A proteins in liver microsomes. Conserved glucuronidation activity attributed to the Ugt1 locus can be defined in Ugt1-/- mice, because UGT2-dependent glucuronidation activity is unaffected. Remarkably, the loss of UGT1A functionality in liver results in significant alterations in cellular metabolism as investigated through changes in gene expression. Thus, the loss of UGT1A function in Ugt1-/- mice leads to a metabolic syndrome that can serve as a model to further investigate the toxicities associated with unconjugated bilirubin and the impact of this disease in humans.

Disruption of Thioredoxin Reductase 1 Protects Mice from Acute Acetaminophen-Induced Hepatotoxicity through Enhanced NRF2 Activity

The critical importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen (APAP) is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been extensively studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 (ASK1) and the c-Jun N-terminal kinase (JNK) pathway caused by high doses of APAP. Therefore, a detailed investigation of the role of Txnrd1 in APAP-induced hepatotoxicity was conducted. Liver-specific Txnrd1 knockout mice (Txnrd1(ΔLiv)) were generated and treated with a hepatotoxic dose (400 mg/kg) of APAP for 1 or 6 h. Liver toxicity was assessed by measuring the activities of liver enzymes aspartate aminotransferase and alanine aminotransferase in serum, in addition to histopathological analysis of liver sections and analysis of glutathione levels. At 1 h post-APAP treatment, total and mitochondrial glutathione levels in control and Txnrd1(ΔLiv) mice were similarly depleted. However, at 6 h post-APAP treatment, Txnrd1(ΔLiv) mice were resistant to APAP toxicity as liver enzymes and histology were not significantly different from the corresponding untreated mice. Analyses revealed the compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 (NRF2) target genes and proteins in Txnrd1(ΔLiv) mice with and without APAP treatment. Yet, JNK was phosphorylated to a similar extent in APAP-treated control mice. The results suggest that Txnrd1(ΔLiv) mice are primed for xenobiotic detoxication primarily through NRF2 activation.

Cancer-Produced Metabolites of 5-Lipoxygenase Induce Tumor-Evoked Regulatory B Cells via Peroxisome Proliferator–Activated Receptor α

Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report in this study that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase pathway such as leukotriene B4 to activate the peroxisome proliferator–activated receptor α (PPARα) in B cells. Inactivation of leukotriene B4 signaling or genetic deficiency of PPARα in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARα initiates programs required for differentiation of tBregs. We propose that PPARα in B cells and/or tumor 5-lipoxygenase pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

Diurnal Regulation of the Early Growth Response 1 (Egr-1) Protein Expression by Hepatocyte Nuclear Factor 4α (HNF4α) and Small Heterodimer Partner (SHP) Cross-talk in Liver Fibrosis

Early growth response 1 (Egr-1) protein is a critical regulator of genes contributing to liver fibrosis; however, little is known about the upstream transcriptional factors that control its expression. Here we show that Egr-1 expression is tightly regulated by nuclear receptor signaling. Hepatocyte nuclear factor 4α (HNF4α) activated the Egr-1 promoter through three DR1 response elements as identified by trans-activation assays. Deletion of these response elements or knockdown of HNF4α using siRNA largely abrogated Egr-1 promoter activation. HNF4α activity, as well as its enrichment on the Egr-1 promoter, were markedly repressed by small heterodimer partner (SHP) co-expression. Egr-1 mRNA and protein were transiently induced by HNF4α. On the contrary, HNF4α siRNA reduced Egr-1 expression at both the mRNA and protein levels, and overexpression of SHP reversed these effects. Conversely, knockdown of SHP by siRNA elevated Egr-1 protein. Interestingly, Egr-1 mRNA exhibited diurnal fluctuation, which was synchronized to the cyclic expression of SHP and HNF4α after cells were released from serum shock. Unexpectedly, the levels of Egr-1 mRNA and protein were highly up-regulated in Hnf4α−/− mice. Both HNF4α and Egr-1 expression were dramatically increased in SHP−/− mice with bile duct ligation and in human cirrhotic livers, which was inversely correlated with diminished SHP expression. In conclusion, our study revealed control network for Egr-1 expression through a feedback loop between SHP and HNF4α.

The role of chrysin and the Ah receptor in induction of the human UGT1A1 gene in vitro and in transgenic UGT1 mice

The flavonoid chrysin is an important dietary substance and induces UGT1A1 protein expression in cell culture. As a representative of the class of dietary flavonoids, clinical investigations have been considered as a means of inducing hepatic UGT1A1 expression. We demonstrate the necessity of a xenobiotic response element (XRE) in support of chrysin induction of UGT1A1 in the human hepatoma cell line HepG2. Receptor binding assays confirm that chrysin is a ligand for the Ah receptor by competition with [3H]2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). However, key differences in Ah receptor recognition and activation of UGT1A1 by chrysin exist when compared with classical mechanisms of UGT1A1 induction by TCDD. Ah receptor degradation, an indicator of Ah receptor activation, does not occur after chrysin treatment, and chrysin cannot transactivate the Ah receptor in a TCDD‐dependent fashion. Knock‐down of the Ah receptor by siRNA indicates that chrysin uses the Ah receptor in conjunction with other factors through MAP kinase signaling pathways to maximally induce UGT1A1. Most importantly, oral treatment of chrysin to transgenic mice that express the human UGT1 locus is unable to induce UGT1A1 expression in either the small intestine or liver. Conclusion: Although the implications for chrysin as an atypical agonist of the Ah receptor are intriguing at the molecular level, the relevance of chrysin‐induced transcription for the purpose of clinical therapies or to regulate phase 2–dependent glucuronidation is questionable given the lack of in vivo regulation of human UGT1A1 by chrysin in a transgenic animal model. (HEPATOLOGY 2007;45:349–360.)