COBRE Research

Physiological Function of Nuclear Receptors in Cholestatic Liver Diseases

PI: Bryan Copple, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

The overall goal of this proposal is to determine whether the nuclear receptors farnesoid-X-receptor (FXR), pregnane-X-receptor (PXR), constitutive androstane receptor (CAR), and retinoid-X-receptor alpha (RXRα) protect the liver during cholestatic liver disease.  The main hypothesis is that FXR, PXR, CAR, and RXRα protect the liver during cholestatic liver disease by regulating bile-acid synthesis, transport, and detoxification.  Cholestatic liver disease arises when excretion of bile acids from the liver is interrupted.  This causes toxic bile acids to accumulate in liver, which produces hepatocyte injury.  Recent studies have identified several nuclear receptors expressed by hepatocytes that regulate bile acid homeostasis, including FXR, PXR, CAR, and RXRα.  When activated, these nuclear receptors regulate expression of genes in hepatocytes that encode for proteins that reduce bile-acid uptake and synthesis, as well as increase bile-acid excretion and detoxification.  Studies have shown that some of these nuclear receptors are important for regulating bile-acid toxicity in mice fed toxic quantities of bile acids.  However, it is not known whether these nuclear receptors function similarly and reduce bile acid toxicity during cholestasis.  This forms the basis of this proposal, which will examine the physiological role of each of these nuclear receptors in cholestatic liver disease by systematically determining whether liver injury and bile-acid synthesis, transport, and detoxification are enhanced, reduced, or unaffected in nuclear receptor-null animals with different types of cholestatic liver disease. The studies in this proposal will not only provide important information about the physiological function of each nuclear receptor in cholestasis, but will also provide important insight into whether modulation of these pathways might be beneficial for the treatment of cholestatic liver disease.

Mechanisms by Which Farnesoid-X-receptor Alters Hepatic Lipid Metabolism and Decreases Fatty Liver Development

PI: Grace Liejun Guo, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

The central hypothesis of this proposal is that farnesoid-X-receptor (FXR), in concert with other nuclear receptors, plays a pivotal role in fatty-acid and triglyceride metabolism in liver by regulating their uptake, intracellular transport, lipogenesis, oxidation, and secretion. Fatty acids are crucial for a variety of cellular processes and are stored in the form of triglycerides. The mammalian liver plays a crucial role in regulating fatty-acid and triglyceride metabolism. Abnormal accumulation of lipid in liver causes liver damage, a condition known as fatty liver or steatosis. Recent evidence shows that FXR, a ligand-activated transcriptional factor, is crucial in regulating lipid homeostasis. Deletion of the FXR gene in mice leads to fatty-liver formation. However, the mechanisms by which FXR regulates lipid metabolism in liver remains unclear. I have made hepatocyte-specific FXR-null mice, which will serve as a novel specific tool to study the role of FXR in liver. The goal of this study will be to identify the mechanisms by which FXR regulates hepatic lipid metabolism using hepatocyte-specific FXR-null mice with three specific aims. Aim 1 will determine the role of FXR in suppressing the peroxisome-proliferators-activated receptor g  (PPARg)-regulated pathway of hepatic fatty acid uptake, intracellular transport, and conversion to triglycerides.  Aim 2 is to determine the mechanism by which FXR regulates fatty-acid oxidation in liver by activation of PPARα. Aim 3 will investigate the role of FXR in regulating triglyceride secretion from the liver. Results from this work will elucidate the mechanisms by which FXR regulates hepatic-lipid metabolism, fatty-liver formation, and provide potential therapeutic targets for preventing and treating abnormalities in lipid metabolism.

The Role of Organic Anion Transporting Polypeptides (OATPs) in Nuclear Receptor Activation

PI: Bruno Hagenbuch, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

Organic Anion Transporting Polypeptides (OATPs) are involved in absorption, distribution, and elimination of various endobiotics and numerous drugs. Multispecific OATPs (i.e. OATPs, which accept a broad range of structurally unrelated substrates) like OATP1B1 and OATP1B3, are predominantly expressed in liver where they mediate uptake of numerous bile acids and xenobiotics. The nuclear receptors farnesoid-X-receptor (FXR), pregnane-X-receptor (PXR) and retinoid-X-receptor α (RXRα) are ligand activated transcription factors expressed mainly in liver and small intestine.  When activated by ligands, like bile acids and xenobiotics, they regulate the expression of various enzymes and transporters involved in cholesterol and bile-acid homeostasis, and drug metabolism. The working hypothesis of this project is that OATP1B1 and OATP1B3 mediate hepatocellular uptake of nuclear-receptor ligands, and that malfunction of these OATPs will affect nuclear-receptor activation. In specific aim 1, we will characterize the substrate specificity of OATP1B1 and OATP1B3 with respect to nuclear-receptor ligands using stably transfected cell lines. In specific aim 2, we will perform a detailed quantitative structure activity relationship (QSAR) analysis for OATP1B1 and OATP1B3, which will allow us to predict new OATP substrates and/or inhibitors. In specific aim 3, we will determine the effect of OATP substrates on nuclear-receptor activation using selected ligands in a cell-based reporter system. A detailed characterization of the substrate specificity of OATP1B1 and OATP1B3 is essential for understanding the specificity of OATPs. Furthermore, it will help to understand and predict potential drug-drug interactions of nuclear-receptor ligands and OATP substrates, such as the lipid lowering statins and the antidiabetic glitazones at the transporter level.

Role of SHP in Fatty Liver

PI: Li Wang, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

The current obesity epidemic is resulting in a dramatic increase in the metabolic syndrome, that frequently results in fatty liver, which can cause fibrosis, cirrhosis, and end-stage liver disease. Obesity affects 64% of the U.S. population and results in 300,000 deaths every year. Fatty liver (steatosis) associated with obesity affects more than 50% of people over the age of 50. The goal of these studies is to understand the role that the orphan nuclear receptor small heterodimer partner (SHP) plays in the molecular mechanisms of dyslipidemias associated with fatty liver. Eliminating SHP signaling, in SHP-null mice, protects against a high cholesterol or high-fat diet induced liver steatosis and obesity. Intriguingly, the fatty liver in leptin deficient obese mice (OB/OB) was also prevented by the absence of SHP (OB/SHP-double-null mice). This provides  a unique animal model to explore the function of SHP in fatty liver formation. The hypothesis to be tested is that SHP functions to modulate the expression of critical steps in lipid metabolism, and that SHP regulated pathways are essential for the development of fatty liver. To test this hypothesis, the following specific aims will be addressed: 1) Determine whether decreased serum triglyceride clearance and hepatic lipid uptake or increased hepatic VLDL production occurs in OB/SHP-null mice; 2) Characterize expression of genes involved in lipoprotein transport (e.g., VLDL production, HDL synthesis, triglyceride clearance, lipid uptake, and hepatic lipid synthesis) in OB/OB and OB/SHP-null mice to identify SHP target genes; 3) Identify SHP–regulated-target genes in hepatic lipid metabolic pathways, and examine the transcriptional regulation of these genes by SHP, to determine the mechanism that prevents fatty liver in OB/SHP-null mice. Identifying the specific functions of SHP and the underlying mechanisms that account for the prevention of fatty liver in OB/SHP-null mice, will provide mechanistic insight and novel approaches to understanding and preventing obesity-associated fatty liver in patients.

Identification and Functional Characterization of SNPs in RXRα Gene

PI: Xiaobo Zhong, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

The overall goals of this project are to: identify genetic polymorphisms in the retinoid-X-receptor α (RXRa) gene, determine major haplotypes in American populations, and characterize functional effects of the defined haplotypes on the transcriptional regulation of RXRa-target-gene expression. The main hypothesis is that genetic polymorphisms in the RXRa gene exist in American populations; these polymorphisms are organized in specific haplotypes in different ethnic groups; that the haplotypes, which may contribute to inter-individual variations of RXRa-gene expression, have not yet been defined; and that such variations may cause differences of RXRa-target-gene expression by altering transcriptional regulation. RXRa is a nuclear receptor for 9-cis-retinoic acid and plays a central role for gene transcriptional regulation. RXRa forms heterodimers with the other retinoic-acid receptors (RARa,  b, and g) resulting in activation of various retinoid-mediated-signal pathways for controlling normal cell differentiation and proliferation. RXRa is highly expressed in liver, where different RXRa-containing heterodimers (FXR, PXR, CAR, PPARα, PPARg, etc.) bind to specific DNA- response elements in the promoters of target genes, to regulate their transcription and to control important functions, such as the maintenance of glucose and lipid homeostasis, synthesis of bile acids, and the metabolism of drugs. Three specific aims will be addressed in this project: (1) to identify genetic polymorphisms (mostly single nucleotide polymorphisms, SNPs) in exons, intron/exon boundaries, 3’-untranslated region, and 5’-promoter region of the RXRa gene by sequencing 100 human subjects from the four major American ethnic groups: Caucasian, African-American, East-Asian and Mexican-American; (2) To determine major haplotype patterns by genotyping 12-15 identified SNPs evenly distributed in the RXRa gene in 400 samples from the 4 major American ethnic groups (100 from each); (3) to characterize functional effects of the defined haplotypes on RXRa-target-gene expression by using both in vitro and in vivo approaches. After completion of this project, we expect to learn what genetic polymorphisms exist in our population, how they are organized in different haplotypes, and how they affect RXRa-target-gene expression.

PI: James Luyendyk, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

Joining July, 2007

PI: Partha Krishnamurthy, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

Joining August, 2007

PI: Udayan Apte, Ph.D., Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center

Joining April, 2008

Liver Center Joint Project

PI: Curtis Klaassen, Ph.D., Univ. Distinguished Professor and Chair, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center.