Regulation of uridine diphosphate-glucuronosyltransferase 1A expression by miRNA-214-5p and miRNA-486-3p.

Aim: This study aimed to identify novel miRNAs (miRs) as regulators of UGT1A gene expression and to evaluate them as potential risk factors for the development of liver fibrosis/cirrhosis. Materials & methods: miRNA target sites in UDP-glucuronosyltransferase 1A (UGT1A) 3'-UTR were predicted and confirmed by luciferase assays, quantitative real-time PCR and western blot using HEK293, HepG2 and Huh7 cells. UGT1A and miRNA expression were analyzed in cirrhotic patients and a mouse model of alcoholic liver fibrosis. Results: miR-214-5p and miR-486-3p overexpression reduced UGT1A mRNA, protein levels and enzyme activity in HepG2 and Huh7 cells. miR-486-3p was upregulated in cirrhotic patients and fibrotic mice livers, whereas UGT1A mRNA levels were reduced. Conclusion: In conclusion, we identified two novel miRNAs capable to repress UGT1A expression in vitro and in vivo. Furthermore, miR-486-3p may represent a potential risk factor for the development or progression of liver fibrosis/cirrhosis by means of a reduced UGT1A-mediated detoxification activity.

UDP-glucuronosyltransferase polymorphisms affect diethylnitrosamine-induced carcinogenesis in humanized transgenic mice.

UDP-glucuronosyltransferase (UGT) 1A enzymes detoxify a broad array of exogenous compounds including environmental toxins and carcinogens. Case-control studies identified genetic variations in UGT1A genes leading to reduced glucuronidation activity, which were associated with hepatocellular carcinoma (HCC) formation and progression. The aim of the study was therefore to examine the direct effect of common UGT1A polymorphisms (SNPs) on HCC development and outcome in a diethylnitrosamine (DEN)-induced mouse model. Therefore, a single intraperitoneal DEN injection (20 mg/kg) was administered to 15-day-old htgUGT1A-WT and htgUGT1A-SNP mice (containing a human haplotype of 10 common UGT1A SNPs) either receiving water or coffee cotreatment for the following 39 weeks. After this time, tumor incidence, size (>1 mm), histology, liver-body ratio, serum aminotransferase activities, and UGT1A regulation and activity levels were determined. In DEN-treated htgUGT1A-SNP mice, a markedly higher number of tumors with a bigger cumulative diameter were detected. The relative liver weight and aminotransferase activity levels were also significantly higher in mice carrying UGT1A SNPs. After coffee + DEN cotreatment, susceptibility for tumor development and growth considerably decreased in both mouse lines, but was still higher in htgUGT1A-SNP mice. In conclusion, our study provides experimental evidence for the protective role of UGT1A enzymes in neoplastic transformation. These data confirm case-control studies implicating impaired UGT1A-mediated carcinogen detoxification as a risk factor for individual cancer disposition. Coffee treatment, which is able to activate UGT1A expression and activity, reduced HCC development and provides an explanation for the protective properties of coffee on liver diseases including liver cancer.

The coffee ingredients caffeic acid and caffeic acid phenylethyl ester protect against irinotecan-induced leukopenia and oxidative stress response.

BACKGROUND AND PURPOSE: Irinotecan, used in colorectal cancer therapy, is metabolized by glucuronidation involving different UDP-glucuronosyltransferase (UGT)1A isoforms leading to facilitated elimination from the body. Individuals homozygous for the genetic variants UGT1A1*28 (Gilbert syndrome) and UGT1A7*3 are more susceptible to irinotecan side effects, severe diarrhoea and leukopenia. The aim of this study was to investigate the protective effects and active constituents of coffee during irinotecan therapy using humanized transgenic (htg)UGT1A-WT and htgUGT1A-SNP (carry UGT1A1*28 and UGT1A7*3 polymorphisms) mice. EXPERIMENTAL APPROACH: HtgUGT1A mice were pretreated with coffee or caffeic acid (CA) + caffeic acid phenylethyl ester (CAPE) and injected with irinotecan. The effects of coffee and CA + CAPE were investigated using reporter gene assays, immunoblot, TaqMan-PCR, siRNA analyses and blood counts. KEY RESULTS: Only the combination of the two coffee ingredients, CA and CAPE, mediates protective effects of coffee in a model of irinotecan toxicity by activation of UGT1A genes. Coffee and CA + CAPE significantly increased UGT1A expression and activity along with SN-38 glucuronide excretion in irinotecan-injected htgUGT1A mice, resulting in significant improvement of leukopenia, intestinal oxidative stress and inflammation. CONCLUSION AND IMPLICATIONS: In this study, we identify the compounds responsible for mediating the previously reported coffee-induced activation of UGT1A gene expression. CA and CAPE represent key factors for the protective properties of coffee which are capable of reducing irinotecan toxicity, exerting antioxidant and protective effects. Provided that CA + CAPE do not affect irinotecan efficacy, they might represent a novel strategy for the treatment of irinotecan toxicity.

A Gilbert syndrome-associated haplotype protects against fatty liver disease in humanized transgenic mice.

UDP-glucuronosyltransferases 1 A (UGT1A) enzymes are capable of detoxifying a broad range of endo- and xenobiotic compounds, which contributes to antioxidative effects, modulation of inflammation and cytoprotection. In the presence of low-function genetic UGT1A variants fibrosis development is increased in various diseases. This study aimed to examine the role of common UGT1A polymorphisms in NASH. Therefore, htgUGT1A-WT mice and htgUGT1A-SNP mice (carrying a common human haplotype present in 10% of the white population) were fed a high-fat Paigen diet for 24 weeks. Serum aminotransferase activities, hepatic triglycerides, fibrosis development and UGT1A expression were assessed. Microscopic examination revealed higher hepatic fat deposition and a significant induction of UGT1A gene expression in htgUGT1A-WT mice. In agreement with these observations, lower serum aminotransferase activities and lower expression levels of fibrosis-related genes were measured in htgUGT1A-SNP mice. This was accompanied by reduced PPARα protein levels in htgUGT1A-WT but not in SNP mice. Our data demonstrate a protective effect of a UGT1A SNP haplotype, leading to milder hepatic steatosis and NASH. Higher PPARα protein levels in animals with impaired UGT1A activity are the likely result of reduced glucuronidation of ligands involved in PPARα-mediated fatty acid oxidation and may lead to the observed protection in htgUGT1A-SNP mice.

Differential Regulation of Genes for Cyclic-di-GMP Metabolism Orchestrates Adaptive Changes During Rhizosphere Colonization by Pseudomonas fluorescens.

Bacteria belonging to the Pseudomonas genus are highly successful colonizers of the plant rhizosphere. The ability of different Pseudomonas species to live either commensal lifestyles or to act as agents of plant-growth promotion or disease is reflected in a large, highly flexible accessory genome. Nevertheless, adaptation to the plant environment involves a commonality of phenotypic outputs such as changes to motility, coupled with synthesis of nutrient uptake systems, stress-response molecules and adherence factors including exopolysaccharides. Cyclic-di-GMP (cdG) is a highly important second messenger involved in the integration of environmental signals with appropriate adaptive responses and is known to play a central role in mediating effective rhizosphere colonization. In this study, we examined the transcription of multiple, reportedly plant-upregulated cdG metabolism genes during colonization of the wheat rhizosphere by the plant-growth-promoting strain P. fluorescens SBW25. While transcription of the tested genes generally increased in the rhizosphere environment, we additionally observed a tightly orchestrated response to environmental cues, with a distinct transcriptional pattern seen for each gene throughout the colonization process. Extensive phenotypical analysis of deletion and overexpression strains was then conducted and used to propose cellular functions for individual cdG signaling genes. Finally, in-depth genetic analysis of an important rhizosphere colonization regulator revealed a link between cdG control of growth, motility and stress response, and the carbon sources available in the rhizosphere.