Expression of uridinediphosphate glucuronosyltransferase genes: focus on alternative splicing and transcriptional regulatory mechanisms that contribute to interindividual differences in drug and carcinogen metabolism
Open Access
- Author:
- Jones, Nathan Richard
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 02, 2012
- Committee Members:
- Philip Lazarus, Ph D, Dissertation Advisor/Co-Advisor
Philip Lazarus, Ph D, Committee Chair/Co-Chair
Jong Kak Yun, Committee Member
John Peter Richie Jr., Committee Member
Thomas E Spratt, Committee Member
Richard Robert Young, Committee Member - Keywords:
- UDP-glucuronosyltransferase
expression
transcriptional regulation
alternative splicing
pharmacogenetics - Abstract:
- There is a complex interplay between genetic and environmental factors that determine inter-individual differences in disease disposition and therapeutic response. Drug metabolism pathways have been a major focus of pharmacogenetic studies because inter-individual differences in the expression and activity of these enzymes may cause clinically significant effects on the kinetic properties of various drugs. Because of their role in the metabolism of chemical toxins and carcinogens, genetic differences in drug metabolizing enzymes are also associated with risk of diseases such as cancer. There are many factors governing inter-individual variation in drug-metabolizing enzymes including SNPs, epigenetics, alternative splicing events, transcriptional regulation, and post-translational modifications. UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and excretion of endogenous and xenobiotic compounds including drugs and carcinogens. UGT enzymes mediate the phase II conjugation of glucuronic acid to their substrates, thereby increasing substrate polarity and facilitating their excretion. Variations in UGT genes are associated with altered drug metabolism and cancer risk. Some of the genetic factors underlying these associations have been discovered, but often there is wide variability in phenotype within a given genotype. The liver is the organ most commonly associated with metabolism, and most UGTs are expressed in the human liver. Expression in extrahepatic tissues has been less well characterized, even though tissues that form a barrier with the environment, such as aerodigestive and gastrointestinal tract tissues represent an important first line of defense against exposures to xenobiotic compounds. A better understanding of the inter-individual variability and relative abundance of UGT gene expression in different tissues is important as this helps determine the physiological relevance of each UGT enzyme. While many previous studies have used qualitative reverse transcription polymerase chain reaction (RT-PCR) for determining which UGT genes are expressed in different tissues, some quantitative analysis of UGT expression has been performed In studies described in this thesis dissertation, real-time PCR was used to quantify the expression of 16 UGT enzymes in multiple specimens of various normal human tissues including lung, liver, larynx, brain, tongue, floor of mouth, tonsil, esophagus, endometrium, and pancreas. Substantial inter-individual variability in expression was observed in both hepatic and extrahepatic tissues. In extrahepatic tissues, unpredictable expression patterns were frequently observed, in which UGT enzymes expressed in some individuals were not expressed in others. In the liver, there was a high degree of correlation between the expression levels of many UGT enzymes within the same individual, suggesting a common mechanism of transcriptional regulation. The hepatic expression of UGTs is known to be transcriptionally regulated by ligand-activated and liver-enriched transcription factors (LETFs). The hepatic transcriptional regulation of several UGTs has been partially described, with UGT2B10 a notable exception. UGT2B10 exhibits glucuronidation activity against pharmacological substrates (olanzapine), toxins (nicotine), and carcinogens (NNAL), suggesting that inter-individual variability in the expression of this gene may affect both drug metabolism and cancer risk. We hypothesized that transcriptional regulatory mechanisms contribute to the observed interindividual variability in UGT2B10 mRNA expression levels. Through luciferase assays and DNaseI footprint analysis, a region between -180 bp and -250 bp upstream from the transcription start site was identified as the UGT2B10 core promoter element. Subsequent gel shift and supershift analyses determined that the Oct-1 and HNF3α proteins were capable of binding to these elements. Mutation of the HNF3α site in the +27bp/-1948bp UGT2B10 promoter construct led to a 49% decrease in promoter activity, and mutation of the Oct-1 site led to a 61% decrease (p=0.0095 and p=0.0004, respectively). The Oct-1/HNF3α double mutant further reduced UGT2B10 promoter activity to 18% of the WT construct (p=0.0002). The HNF3α protein is an LETF, and future studies will be conducted to determine whether variants of HNF3α are associated with UGT2B10 expression levels in human liver, as this may be an important contributor to the observed inter-individual differences in expression. In addition to LETFs, UGTs are also regulated by ligand-activated transcription factors. In particular, UGT genes are known to be regulated by antioxidant response elements (AREs). Phase II enzyme inducers such as L-sulforaphane (SFN) have been shown to induce transcription of UGTs through the nuclear factor-erythroid 2-related factor 2 (nrf2)/ARE pathway. The UGT2B10 enzyme detoxifies carcinogens, making it important to understand how it is being regulated in response to phase II enzyme inducers s as this could influence the carcinogenicity of these compounds. In the current studies, several putative ARE sites were identified in the UGT2B10 promoter and were hypothesized to be regulated by the nrf2/ARE pathway. UGT2B10 expression was found to be significantly decreased in response to SFN treatment (52%, p=0.008). The repression effect was present even after siRNA knockdown of nrf2, indicating that this effect was occurring via an nrf2-independent mechanism. Using luciferase assays, the promoter element responsible for the repression was localized to a 976 bp region between -973 bp and -1948 bp upstream of the transcription start site. Future studies will be needed to elucidate the regulatory factors that are binding and initiating this repression. The repression of UGT2B10 expression via SFN may represent an additional mechanism that is contributing to inter-individual differences in expression. Another potential regulator of inter-individual differences in glucuronidation is alternative splicing. An alternative exon 5 in the common region of the UGT1A gene cluster leads to the expression of 18 additional mRNA species from this locus. The alternative splice isoforms have a dominant-negative effect on the wild type (WT) isoforms in vitro. We hypothesized that inter-individual variation in the relative abundance of WT and splice variant expression affects glucuronidation capacity in human liver, which could carry important pharmacogenetic implications. In these studies, it was determined that UGT1A splice variants, on average, represent less than 7% of the total UGT1A transcript profile in human liver for all hepatic UGT1A species, with relatively low inter-individual variability in expression between different individuals. A consistent pattern was observed in several extrahepatic tissues as well. UGT1A WT and splice variant expression were both correlated with glucuronidation activity. Sequence alignment of the UGT1A alternative exon 5 with the primate-specific alu transposable element revealed that it is a recent evolutionary event, and it displays a low inclusion rate characteristic of similar exons. Alu-derived exons are usually neutral or only slightly deleterious because the novel, alternatively spliced product represents only a small percentage of the total mRNA species. For this reason, the proteins they encode have often been characterized as nonfunctional, evolutionary intermediates. This dissertation research has contributed to the understanding of mechanisms underlying UGT gene expression. Studies examining the inter-individual expression patterns of UGT enzymes in human tissues, an analysis of the importance of UGT1A alternative splice variants and how they affect glucuronidation capacity in human liver, and an assessment of the transcriptional regulation of the UGT2B10 promoter by LETFs and SFN were performed in this work. Together these data have improved our knowledge of how inter-individual differences in the expression of metabolizing enzymes are manifested and how these differences may potentially play a role in drug and carcinogen metabolism, personalized medicine, and cancer risk assessment.