Roger G. Ulrich

Mapping the Genetic Architecture of Gene Expression in Human Liver

Genetic variants that are associated with common human diseases do not lead directly to disease, but instead act on intermediate, molecular phenotypes that in turn induce changes in higher-order disease traits. Therefore, identifying the molecular phenotypes that vary in response to changes in DNA and that also associate with changes in disease traits has the potential to provide the functional information required to not only identify and validate the susceptibility genes that are directly affected by changes in DNA, but also to understand the molecular networks in which such genes operate and how changes in these networks lead to changes in disease traits. Toward that end, we profiled more than 39,000 transcripts and we genotyped 782,476 unique single nucleotide polymorphisms (SNPs) in more than 400 human liver samples to characterize the genetic architecture of gene expression in the human liver, a metabolically active tissue that is important in a number of common human diseases, including obesity, diabetes, and atherosclerosis. This genome-wide association study of gene expression resulted in the detection of more than 6,000 associations between SNP genotypes and liver gene expression traits, where many of the corresponding genes identified have already been implicated in a number of human diseases. The utility of these data for elucidating the causes of common human diseases is demonstrated by integrating them with genotypic and expression data from other human and mouse populations. This provides much-needed functional support for the candidate susceptibility genes being identified at a growing number of genetic loci that have been identified as key drivers of disease from genome-wide association studies of disease. By using an integrative genomics approach, we highlight how the gene RPS26 and not ERBB3 is supported by our data as the most likely susceptibility gene for a novel type 1 diabetes locus recently identified in a large-scale, genome-wide association study. We also identify SORT1 and CELSR2 as candidate susceptibility genes for a locus recently associated with coronary artery disease and plasma low-density lipoprotein cholesterol levels in the process.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity

A rate-limiting step that occurs in the drug discovery process is toxicological evaluation of new compounds. New techniques that use small amounts of the experimental compound and provide a high degree of predictivity would greatly improve this process. The field of microarray technology, which allows one to monitor thousands of gene expression changes simultaneously, is rapidly advancing and is already being applied to numerous areas in toxicology. However, it remains to be determined if compounds with similar toxic mechanisms produce similar changes in transcriptional expression. In addition, it must be determined if gene expression changes caused by an agent in vitro would reflect those produced in vivo. In order to address these questions, we treated rat hepatocytes with 15 known hepatoxins (carbon tetrachloride, allyl alcohol, aroclor 1254, methotrexate, diquat, carbamazepine, methapyrilene, arsenic, diethylnitrosamine, monocrotaline, dimethyl-formamide, amiodarone, indomethacin, etoposide, and 3-methylcholanthrene) and used microarray technology to characterize the compounds based on gene expression changes. Our results showed that gene expressional profiles for compounds with similar toxic mechanisms indeed formed clusters, suggesting a similar effect on transcription. There was not complete identity, however, indicating that each compound produced a unique signature. These results show that large-scale analysis of gene expression using microarray technology has promise as a diagnostic tool for toxicology.

Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.

Identifying toxic mechanisms using DNA microarrays: evidence that an experimental inhibitor of cell adhesion molecule expression signals through the aryl hydrocarbon nuclear receptor.

Microarray analysis of gene expression has become a powerful approach for exploring the biological effects of drugs and other chemicals. In toxicology research, gene expression profiling may help identify hazards by comparing results for an experimental compound with a database, and establish mechanistic hypotheses through examination of discrete gene changes. Here we examine the hepatic effects of a thienopyridine inhibitor of NF-kappa B-mediated expression of cellular adhesion proteins. In a 3-day toxicity study in Sprague-Dawley rats, A-277249 induced hypertrophy of the liver and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). To investigate mechanism, microarray analysis was done on RNA from livers of A-277249-treated rats. Gene expression profiles from A-277249 were compared with a database of profiles from fifteen known hepatotoxins. Agglomerative hierarchical cluster analysis showed A-277249 to have a profile most similar to the aromatic hydrocarbons Aroclor 1254 and 3-methylcholanthrene (3MC), two known activators of the aryl hydrocarbon nuclear receptor (AhR). Several genes regulated by the AhR, including cytochrome P450 1A1, were upregulated by A-277249. In addition, several genes involved in apoptosis and cell cycle were differentially expressed consistent with cell turnover, hypertrophy and hyperplasia observed by histology. Results from this study indicate that A-277249 hepatic toxicity is mediated by the AhR either directly or through effects on NF-kappa B, and demonstrate the utility of microarray analysis for the rapid identification of toxic hazards for new chemical entities.

PTP1B antisense-treated mice show regulation of genes involved in lipogenesis in liver and fat

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.

Profiling the Hepatic Effects of Flutamide in Rats: A Microarray Comparison with Classical AhR Ligands and Atypical CYP1A Inducers

The antiandrogen flutamide (FLU) is used primarily for prostate cancer and is an idiosyncratic hepatotoxicant that sometimes causes severe liver problems. To investigate FLUs overt hepatic effects, especially on inducible drug clearance-related gene networks, FLUs hepatic gene expression profile was examined in female Sprague-Dawley rats using ∼22,500 oligonucleotide microarrays. Rats were dosed daily for 3 days with FLU at 500, 250, 62.5, 31.3, and 15.6 mg/kg/day, and hepatic RNA was isolated. FLU resulted in the dose-dependent regulation of ∼350 genes. Employing a gene-response compendium, FLU was compared with three classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene, benzo[a]pyrene, and β-naphthoflavone, and four atypical CYP1A inducers, indole-3-carbinol (I3C), omeprazole (OME), chlorpromazine (CPZ), and clotrimazole (CLO). The FLU gene response was comparable with classical AhR ligands across a signature AhR ligand gene set that included CYP1A1 and other members of the AhR gene battery. Dose-related responses of CYP1 genes established a maximum response ceiling and discerned potency differences in atypical inducers. FLU had a sharp down-regulation of c-fos that was comparable with all the compounds except CPZ and CLO. FLU absorption, distribution, metabolism, and excretion (ADME) gene expression analysis revealed that FLU, as well as I3C and OME, induced CYP2B and CYP3A, distinguishing them from the classical AhR ligands. By using a compendium of gene expression profiles, FLU was shown to signal in rats similar to an AhR activator with additional CYP2B and CYP3A effects that most resembled the ADME gene expression pattern of the atypical CYP1A inducers I3C and OME.

Isolated human hepatocytes in culture display markedly different gene expression patterns depending on attachment status.

In vitro human hepatocyte cultures are a key tool in the investigation of xenobiotic toxicity and metabolism. In most in vitro hepatocyte studies, the cells are allowed to adhere to an extracellular matrix, such as collagen. Unfortunately, the ability of freshly isolated hepatocytes to adhere to collagen varies from donor to donor. We used microarray analysis to determine what gene expression differences exist between hepatocytes in suspension and hepatocytes attached to collagen. Results from different donors showed a considerable difference in gene expression patterns between the two hepatocyte populations. In addition, we also compared the gene expression profiles of hepatocytes in culture with liver tissue. The results showed that both hepatocytes in suspension and hepatocytes attached to collagen display significant gene expression differences compared with liver tissue. Finally, we show that both populations of hepatocytes are responsive to dexamethasone and regulate some of the same genes. Overall, our results suggest that either significant gene expression changes occur in isolated hepatocytes or that suspended and attached cells represent different populations of hepatocytes found in intact livers.

Microvesicular Steatosis Induced by a Short Chain Fatty Acid: Effects on Mitochondrial Function and Correlation with Gene Expression

Hepatotoxicity characterized by microvesicular steatosis (MVS) is characterized by an abnormal accumulation of numerous small cytoplasmic lipid droplets in hepatocytes. Fulminant or progressive cases of microvesicular steatosis may lead to liver failure and death. Experimentally, short-chain carboxylic acids are known to induce microvesicular steatosis. The identification of gene changes that correlate with MVS concomitant with biochemical and histological indices could provide a better understanding of how this toxicity occurs as well as biomarkers that could be used to avoid this toxicity in the future. Sprague—Dawley rats were dosed days with cyclopropane carboxylic acid (CPCA) a short-chain fatty acid that can induce microvesicular steatosis, and with butyrate, a short chain fatty acid that served as a negative control. CPCA initiated microvesicular steatosis while butyrate did not. In addition, CPCA inhibited beta-oxidation in a concentration-dependent manner in vitro and caused a reduction in mitochondrial respiration ex vivo; no inhibition was evident with butyrate. Microarray results showed that gene expression changes with CPCA resulted in regulation of genes involved in beta-oxidation, as well as other genes associated with mitochondrial function. Overall, these results support altered hepatic mitochondrial function as a mechanism of the toxicity induced by a short-chain fatty acid and may provide potential biomarkers for this toxicity.

Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis

Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.