RESUMEN
Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
Asunto(s)
Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Hepatocitos/metabolismo , Propofol/metabolismo , ARN Interferente Pequeño/genética , Regulación hacia Abajo , Glucuronosiltransferasa/genética , Células HEK293 , Humanos , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , UDP Glucuronosiltransferasa 1A9RESUMEN
Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car-/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car-/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucuronosiltransferasa/biosíntesis , Isotiocianatos/farmacología , Hígado/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Bilirrubina/sangre , Receptor de Androstano Constitutivo , Glucuronosiltransferasa/genética , Humanos , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genética , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Humanized UDP-glucuronosyltransferase (UGT)-1 (hUGT1) mice encode the UGT1 locus including the UGT1A1 gene. During neonatal development, delayed expression of the UGT1A1 gene leads to hyperbilirubinemia as determined by elevated levels of total serum bilirubin (TSB). We show in this report that the redox-sensitive NF-κB pathway is crucial for intestinal expression of the UGT1A1 gene and control of TSB levels. Targeted deletion of IKKß in intestinal epithelial cells (hUGT1/Ikkß(ΔIEC) mice) leads to greater neonatal accumulation of TSB than observed in control hUGT1/Ikkß(F/F) mice. The elevation in TSB levels in hUGT1/Ikkß(ΔIEC) mice correlates with a reduction in intestinal UGT1A1 expression. As TSB levels accumulate in hUGT1/Ikkß(ΔIEC) mice during the neonatal period, the increase over that observed in hUGT1/Ikkß(F/F) mice leads to weight loss, seizures and eventually death. Bilirubin accumulates in brain tissue from hUGT1/Ikkß(ΔIEC) mice inducing an inflammatory state as shown by elevated TNFα, IL-1ß and IL-6, all of which can be prevented by neonatal induction of hepatic or intestinal UGT1A1 and lowering of TSB levels. Altering the redox state of the intestines by oral administration of cadmium or arsenic to neonatal hUGT1/Ikkß(F/F) and hUGT1/Ikkß(ΔIEC) mice leads to induction of UGT1A1 and a dramatic reduction in TSB levels. Microarray analysis following arsenic treatment confirms upregulation of oxidation-reduction processes and lipid metabolism, indicative of membrane repair or synthesis. Our findings indicate that the redox state in intestinal epithelial cells during development is important in maintaining UGT1A1 gene expression and control of TSB levels.