Genetic polymorphisms in the 5'-flanking region of human UDP-glucuronosyltransferase 2B7 affect the Nrf2-dependent transcriptional regulation.
OBJECTIVE
Human uridine diphosphate-glucuronosyltransferase 2B7 (UGT2B7) plays important roles in the metabolism of some clinical drugs, carcinogens, and steroid hormones. The molecular mechanisms of the inducible expression of UGT2B7 in response to xenobiotics have not been fully clarified. We sought to investigate whether the UGT2B7 is under the control of NF-E2 p45-related factor 2 (Nrf2), a key transcriptional factor regulating the expression of cytoprotective enzymes.


METHODS
HepG2, HuH7, HLE, and Caco-2 cells were treated with sulforaphane (SFN), and the UGT2B7 mRNA levels were determined by real-time reverse transcriptase PCR. These cells were genotyped for the UGT2B7*2 (H268Y) allele using the PCR-restriction fragment length polymorphism method. Luciferase analyses and gel shift analyses were performed to identify the responsive regions for Nrf2 signaling.


RESULTS
The UGT2B7 mRNA was induced by SFN in HepG2 and HuH7 genotyped as UGT2B7*1/*1, but not in HLE and Caco-2 cells genotyped as UGT2B7*2/*2. In HepG2 cells, the UGT2B7 protein level and morphine glucuronosyltransferase activity were also significantly induced by SFN. The induction was prominently decreased with small interfering RNA for Nrf2. In the 5'-flanking region (-2.5 kb) of the UGT2B7*2 allele, a 324-base pair insertion at -2067 and 12 single nucleotide polymorphisms simultaneously existed. Luciferase analyses and gel shift analyses revealed that an antioxidant responsive element at -1170 was responsible for the transactivation by Nrf2. In addition, a region from -990 to -858 on the UGT2B7*1 allele was also responsible for the transactivation by Nrf2. Abrogation of the Nrf2-dependent transactivation of the UGT2B7*2 allele was owing to the single nucleotide polymorphism -900A>G.


CONCLUSIONS
UGT2B7 is transcriptionally regulated by Nrf2, but the mechanism is hindered by polymorphisms in the promoter region of UGT2B7*2. The allele-specific mechanism may cause variability of the glucuronidation in response to oxidative stress.
Nakamura
Akiko
A
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-Machi, Kanazawa, Japan.
Nakajima
Miki
M
Higashi
Eriko
E
Yamanaka
Hiroyuki
H
Yokoi
Tsuyoshi
T
D024506:5' Flanking Region / Q000235:genetics*; D000483:Alleles; D000975:Antioxidants / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017879:Isothiocyanates; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000187:drug effects; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D017422:Sequence Analysis, DNA; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects
D000975:Antioxidants; D017879:Isothiocyanates; D050984:NF-E2 Transcription Factor, p45 Subunit; D034741:RNA, Small Interfering; D013454:Sulfoxides; D013861:Thiocyanates; D008156:Luciferases; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase; C016766:sulforaphane