The N6-methyladenosine modification posttranscriptionally regulates hepatic UGT2B7 expression.

UDP-glucuronosyltransferases (UGTs) are enzymes catalyzing the glucuronidation of various endogenous and exogenous compounds. In this study, we examined the possibility that N6-methyladenosine (m6A) modification affects hepatic UGT expression. Treatment of HepaRG cells with 3-deazaadenosine, an inhibitor of RNA methylation, significantly increased UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B15 mRNA levels (1.3- to 2.6-fold). Among them, we focused on UGT2B7 because it most highly contributes to glucuronidation of clinically used drugs. Methylated RNA immunoprecipitation assays revealed that UGT2B7 mRNA in HepaRG cells and human livers is subjected to m6A modification mainly at the 5' untranslated region (UTR) and secondarily at the 3'UTR. UGT2B7 mRNA and protein levels in Huh-7 cells were significantly increased by double knockdown of methyltransferase-like 3 (METTL3) and METTL14, whereas those were decreased by knockdown of fat mass and obesity-associated protein (FTO) or alkB homolog 5, RNA demethylase (ALKBH5), suggesting that m6A modification downregulates UGT2B7 expression. By experiments using actinomycin D, an inhibitor of transcription, it was demonstrated that ALKBH5-mediated demethylation would attenuate UGT2B7 mRNA degradation, whereas METTL3/METTL14 or FTO-mediated m6A modification would alter the transactivity of UGT2B7. Luciferase assays revealed that the promoter region at -118 to -106 has a key role in the decrease in transactivity of UGT2B7 by FTO knockdown. We found that hepatocyte nuclear factor 4α (HNF4α) expression was significantly decreased by knockdown of FTO, indicating that this would be the underlying mechanism of the decreased transactivity of UGT2B7 by knockdown of FTO. Interestingly, treatment with entacapone, which is used for the treatment of Parkinson's disease and is an inhibitor of FTO, decreased HNF4α and UGT2B7 expression. In conclusion, this study clarified that RNA methylation posttranscriptionally controls hepatic UGT2B7 expression.

SLC35B1 significantly contributes to the uptake of UDPGA into the endoplasmic reticulum for glucuronidation catalyzed by UDP-glucuronosyltransferases.

The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER.

Methylation of adenosine at the N6 position post-transcriptionally regulates hepatic P450s expression.

The methylation of adenosines at the N6 position (m6A formation) is the most prevalent type of RNA modification in humans. This modification is mediated by methyltransferase like 3 (METTL3)-METTL14 complex, and the methyl group can be removed by RNA demethylases including fat mass and obesity-associated (FTO) and AlkB homolog 5. The formed m6A is recognized by reader proteins such as members of the YT521-B homology (YTH) family, resulting in changes in the splicing, nuclear export, and decay of RNA or translation. In this study, we examined the impact of m6A modification on the expression of drug-metabolizing P450 isoforms. By treatment with 3-deazaadenosine, an inhibitor of RNA methylation, CYP1A2, CYP2B6, and CYP2C8 levels were significantly increased (1.6-fold, 2.2-fold, and 2.7-fold, respectively) in HepaRG cells. In subsequent experiments, we focused on CYP2C8, which showed the largest increase. Consistent with the increase in the mRNA level, CYP2C8 protein level and activity were significantly increased by treatment with 3-deazaadenosine. The CYP2C8 expression levels and activities in HepaRG and Huh-7 cells were increased by knockdown of METTL3/14, whereas they were decreased by knockdown of FTO, suggesting that m6A modification downregulates CYP2C8 expression. With an RNA immunoprecipitation assay using an anti-m6A antibody, it was revealed that the adenosines in the 5'-UTR and the last exon of CYP2C8 are methylated in HepaRG cells and human liver samples. It was demonstrated that YTHDC2, which is known to degrade m6A-containing mRNA, downregulates CYP2C8 expression. In conclusion, we found a novel post-transcriptional regulation mechanism in which the YTHDC2 promotes CYP2C8 mRNA degradation via recognizing the m6A in CYP2C8 mRNA, which is installed by METTL3/14 and removed by FTO.

Quantitative Analysis of UDP-Glucuronosyltransferase Ugt1a and Ugt2b mRNA Expression in the Rat Liver and Small Intestine: Sex and Strain Differences.

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of numerous endogenous and exogenous compounds to facilitate their excretion from the body. Because rats are commonly used in nonclinical studies, information regarding UGT species differences between rats and humans would be helpful for understanding human pharmacokinetics. In this study, we determined the absolute mRNA expressions of Ugt isoforms in the liver and small intestine of male and female Sprague-Dawley, Fischer 344, and Wistar rats. The sum of the mRNA levels of Ugt isoforms expressed in the liver was significantly (P < 0.005) higher than that in the small intestine regardless of the strain and sex. Ugt2b mRNA levels represented approximately 80% of total Ugt mRNA levels in the liver, whereas Ugt1a mRNA levels accounted for almost 90% in the small intestine. Ugt2b2 mRNA was specifically expressed in Wistar rat liver, resulting in 2-fold higher expression of total hepatic Ugt mRNA in Wistar rats than that in the other strains. Wistar rats showed prominently higher Ugt2b3 and Ugt2b8 mRNA levels in the small intestine than the other strains. The difference between sexes was remarkable with regard to hepatic Ugt1a10 in any of the strains, although slight differences between sexes were also observed in multiple Ugt isoforms. Taken together, this study revealed sex and strain differences in mRNA levels of rat Ugts. The data shown here would be useful for the selection of rat strains in nonclinical studies.