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The human UDP-glycosyltransferase ( UGT ) gene superfamily generates 22 canonical transcripts coding for functional enzymes and also produces nearly 150 variant UGT transcripts through alternative splicing and intergenic splicing. In the present study, our analysis of circRNA databases identified backsplicing events that predicted 85 circRNAs from UGT genes, with 33, 11, and 19 circRNAs from UGT1A , UGT2B4 , UGT8 , respectively. Most of these UGT circRNAs were reported by one database and had low abundance in cell- or tissue-specific contexts. Using reverse-transcriptase polymerase chain reaction with divergent primers and cDNA samples from human tissues and cell lines, we found 13 circRNAs from four UGT genes: UGT1A (three), UGT2B7 (one), UGT2B10 (one), and UGT8 (eight). Notably, all eight UGT8 circRNAs contain open reading frames that include the canonical start AUG codon and encode variant proteins that all have the common 274-amino acidN-terminal region of wild-type UGT8 protein. We further showed that one UGT8 circRNA (circ_UGT8-1) was broadly expressed in human tissues and cell lines, resistant to RNase R digestion, and predominately present in the cytoplasm. We cloned five UGT8 circRNAs into the Zinc finger with KRAB and SCAN domains 1 vector and transfected them into HEK293T cells. All these vectors produced both circRNAsand linear transcripts with varying circular/linear ratios (0.17-1.14).Western blotting and mass spectrometry assays revealed that only linear transcripts and not circRNAs were translated. In conclusion, our findings of nearly 100 circRNAs greatly expand the complexity and diversity of the UGT transcriptome; however, UGT circRNAs are expressed at a very low level in specific cellular contexts, and their biologic functions remain to be determined. SIGNIFICANCE STATEMENT: The human UGT gene transcriptome comprises 22 canonical transcripts coding for functional enzymes and approximately 150 alternatively spliced and chimeric variant transcripts. The present study identified nearly 100 circRNAs from UGT genes, thus greatly expanding the complexity and diversity of the UGT transcriptome. UGT circRNAs were expressed broadly in human tissues and cell lines; however, most showed very low abundance in tissue- and cell-specific contexts, and therefore their biological functions remain to be investigated., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Genes involved in drug absorption, distribution, metabolism, and excretion (ADME) are called ADME genes. Currently, 298 genes that encode phase I and II drug metabolizing enzymes, transporters, and modifiers are designated as ADME genes by the PharmaADME Consortium. ADME genes are highly expressed in the liver and their levels can be influenced by liver diseases such as hepatocellular carcinoma (HCC). In this study, we obtained RNA-sequencing and microRNA (miRNA)-sequencing data from 371 HCC patients via The Cancer Genome Atlas liver hepatocellular carcinoma project and performed ADME gene-targeted differential gene expression analysis and expression correlation analysis. Two hundred thirty-three of the 298 ADME genes (78%) were expressed in HCC. Of these genes, almost one-quarter (58 genes) were significantly downregulated, while only 6% (15) were upregulated in HCC relative to healthy liver. Moreover, one-half (14/28) of the core ADME genes ( CYP1A2 , CYP2A6 , CYP2B6 , CYP2C8 , CYP2C9 , CYP2C19 , CYP2E1 , CYP3A4 , NAT1 , NAT2 , UGT2B7 , SLC22A1 , SLCO1B1 , and SLCO1B3 ) were downregulated. In addition, about one-half of the core ADME genes were positively correlated with each other and were also positively ( AHR , ARNT , HNF4A , PXR , CAR , PPARA , and RXRA ) or negatively ( PPARD and PPARG ) correlated with transcription factors known as ADME modifiers. Finally, we show that most miRNAs known to regulate core ADME genes are upregulated in HCC. Collectively, these data reveal 1) an extensive transcription factor-mediated ADME coexpression network in the liver that efficiently coordinates the metabolism and elimination of endogenous and exogenous compounds; and 2) a widespread deregulation of this network in HCC, most likely due to deregulation of both transcriptional and post-transcriptional (miRNA) pathways., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Recent studies have investigated alternative splicing profiles of UDP-glucuronosyltransferase (UGT) genes and identified over 130 different alternatively spliced UGT transcripts. Although UGT genes are highly clustered, the formation of chimeric transcripts by intergenic splicing between two or more UGT genes has not yet been reported. This study identified 12 chimeric transcripts (chimeras A-L) containing exons from two or three genes of the four neighboring UGT genes ( UGT2B15 , UGT2B29P2 , UGT2B17 , and UGT2B29P1 ) in human liver and prostate cancer cells. These chimeras typically contain the first five exons of UGT2B15 or UGT2B17 (exons 1-5) spliced to a terminal exon (exon 6) from a downstream UGT gene. Hence they encode truncated UGTs with novel C-terminal peptides. Functional assays of representative chimeric UGT proteins (termed chimeric UGT2B15 and chimeric UGT2B17) showed that they are inactive and can repress the activity of wild-type UGTs. Coimmunoprecipitation assays demonstrated heterotypic interactions between chimeric UGT2B15 (or chimeric UGT2B17) and the UGT2B7 protein. Thus oligomerization of the chimeric UGTs with wild-type UGTs may explain their inhibitory activity. Studies in breast and prostate cancer cells showed that both wild-type and chimeric UGT2B15 and UGT2B17 transcripts are regulated in a similar way at the transcriptional level by sex hormones through their canonical promoters but are differentially regulated at the post-transcriptional level by micro-RNA 376c via their unique 3'-untranslated regions. In conclusion, the formation of chimeric transcripts by intergenic splicing among UGT genes represents a novel mechanism contributing to the diversity of the human UGT transcriptome and proteome. The differential post-transcriptional regulation of wild-type and variant transcripts by micro-RNAs may contribute to their deregulated expression in cancer., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The gastrointestinal tract expresses several UDP-glucuronosyltransferases (UGTs) that act as a first line of defense against dietary toxins and contribute to the metabolism of orally administered drugs. The expression of UGT1A8 , UGT1A9 , and UGT1A10 in gastrointestinal tissues is known to be at least partly directed by the caudal homeodomain transcription factor, CDX2. We sought to further define the factors involved in regulation of the UGT1A8-1A10 genes and identified a novel composite element located within the proximal promoters of these three genes that binds to both CDX2 and the hepatocyte nuclear factor (HNF) 4 α , and mediates synergistic activation by these factors. We also show that HNF4 α and CDX2 are required for the expression of these UGT genes in colon cancer cell lines, and show robust correlation of UGT expression with CDX2 and HNF4 α levels in normal human colon. Finally, we show that these factors are involved in the differential expression pattern of UGT1A8 and UGT1A10, which are intestinal specific, and that of UGT1A9, which is expressed in both intestine and liver. These studies lead to a model for the developmental patterning of UGT1A8, UGT1A9, and UGT1A10 in hepatic and/or extrahepatic tissues involving discrete regulatory modules that may function (independently and cooperatively) in a context-dependent manner., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

UGT2B15 is an important androgen-metabolizing UDP-glucuronosyltransferase (UGT) and the mechanisms controlling its expression are of considerable interest. Recent studies showed that miR-376c regulates UGT2B15 in prostate cancer cells via a canonical target site in the 3' untranslated region ( 3'UTR ). The UGT2B15 3'UTR also contains a canonical miR-331-5p target site; previous work indicated that deleting this site reduced, but did not abolish, the ability of miR-331-5p to repress a luciferase reporter carrying the UGT2B15 3'UTR We report here the discovery and characterization of a second, noncanonical miR-331-5p target site in the UGT2B15 3'UTR miR-331-5p-mediated repression of a UGT2B15 3'UTR- reporter was partly inhibited by mutating either of the two miR-331-5p target sites separately, but completely abolished by mutating the two sites simultaneously, indicating that the two sites act cooperatively. miR-331-5p mimics significantly reduced both UGT2B15 mRNA levels and glucuronidation activity in prostate cancer cells, confirming that the native transcript is a miR-331-5p target. Transfection of either miR-331-5p or miR-376c mimics repressed the activity of the UGT2B15 3'UTR -reporter; however, cotransfection of both microRNAs (miRNAs) further reduced activity, indicating cooperative regulation by these two miRNAs. A significant negative correlation between miR-331 and UGT2B15 mRNA levels was observed in a tissue RNA panel, and analysis of The Cancer Genome Atlas (TCGA) hepatocellular carcinoma data set provided further evidence that miR-331 may play an important role in regulation of UGT2B15 in vivo. There was no significant correlation between miR-331 and UGT2B15 mRNA levels in the TCGA prostate adenocarcinoma cohort, which may reflect the complexity of androgen-mediated regulation in determining UGT2B15 levels in prostate cancer. Finally, we show that miR-331-5p does not regulate UGT2B17, providing the first evidence for a post-transcriptional mechanism that differentially regulates these two important androgen-metabolizing UGTs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The transcriptional regulation of UDP-glucuronosyltransferases UGT2B4 and UGT2B7 has been well studied using liver cancer cell lines, and post-transcriptional regulation of these two UGTs by microRNA (miRNA/miR) miR-216b-5p was recently reported. This study describes novel miRNA-mediated regulation of UGT2B4 and UGT2B7 in liver cancer cells. Bioinformatic analyses identified a putative miR-3664-3p binding site in the UGT2B7 3'-untranslated region (UTR) and binding sites for both miR-135a-5p and miR-410-3p in the UGT2B4 3'-UTR. These sites were functionally characterized using miRNA mimics and reporter constructs. A miR-3664-3p mimic induced repression of a luciferase reporter carrying the UGT2B7 3'-UTR in liver cancer cell lines; mutation of the miR-3664-3p site abrogated the response of the reporter to the mimic. Similarly, mutation of the miR-135a-5p site or miR-410-3p site in a luciferase reporter bearing UGT2B4 3'-UTR abrogated the ability of miR-135a-5p or miR-410-3p mimics to reduce reporter activity. Transfection of miR-3664-3p mimics in HepG2 liver cancer cells significantly reduced mRNA and protein levels of UGT2B7, and this led to reduced enzymatic activity. Transfection of miR-135a-5p or miR-410-3p mimics significantly decreased UGT2B4 mRNA levels in Huh7 liver cancer cells. The expression levels of miR-410-3p were inversely correlated with UGT2B4 mRNA levels in The Cancer Genome Atlas cohort of liver hepatocellular carcinoma (371 specimens) and a panel of ten normal human tissues. Similarly, there was an inverse correlation between miR-135a and UGT2B4 mRNA levels in a panel of 18 normal human liver tissues. Together, these data suggest that miR-135a and miR-410 control UGT2B4 and that miR-3664 controls UGT2B7 expression in liver cancer and/or normal liver cells., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Exemestane (EXE) is an aromatase inhibitor indicated for endocrine therapy of breast cancer in postmenopausal women. The primary active metabolite of EXE, 17-hydroexemestane (17-HE), is inactivated via glucuronidation, mainly by UDP-glucuronosyltransferase 2B17 (UGT2B17). UGT2B17 also has a primary role in inactivation of endogenous androgens testosterone and dihydrotestosterone and may play an important role in regulation of breast and prostate tumor intracrinology. We recently reported that UGT2B17 could be induced by both estrogenic and androgenic ligands in breast cancer cells via binding of the estrogen receptor α (ER α ) or the androgen receptor (AR) to a complex regulatory unit in the proximal UGT2B17 promoter. In this study we show that both EXE and 17-HE increase UGT2B17 mRNA levels in breast cancer MCF-7 and MDA-MB-453 cells, and increase glucuronidation of UGT2B17 substrates, including 17-HE and androsterone. Using antagonists of ER α and AR as well as inhibition mediated by small interfering RNA (siRNA) we demonstrate that EXE and 17-HE induce UGT2B17 expression primarily via the AR. This result is consistent with previous reports that 17-HE can act as an AR ligand. In vitro studies suggest that multiple steroid-responsive DNA elements within the proximal promoter are involved in the response to 17-HE-liganded AR. The up-regulation of UGT2B17 by EXE and 17-HE in breast cancer cells might enhance the local metabolism of 17-HE as well as that of endogenous androgens, hence impacting potentially on treatment outcomes., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Given the prime importance of UDP-glucuronosyltransferase (UGT) 2B15 and UGT2B17 in inactivating testosterone and dihydrotestosterone, control of their expression and activity in the prostate is essential for androgen signaling homeostasis in this organ. Although several studies provide evidence of transcriptional control of UGT2B15 and UGT2B17 by various endogenous and exogenous compounds, potential post-transcriptional regulation of UGT2B15 and UGT2B17 by microRNAs (miRs) in prostate cancer cells has not been examined. The present study identified a putative miR-376c target site in the 3'-untranslated regions (UTRs) of both UGT2B15 and UGT2B17 mRNAs. In accordance with the possibility that this miRNA negatively regulates UGT2B15 and UGT2B17 expression, there is an inverse correlation in the levels of miR-376c and UGT2B15/UGT2B17 mRNAs in prostate cancer cell lines versus normal prostate tissue. In LNCaP cells, transfection of miR-376c mimics inhibited the glucuronidations of testosterone, 4-methylumbelliferone (a substrate of UGT2B15), and androsterone (a substrate of UGT2B17). miR-376c reduced both UGT2B15 and UGT2B17 mRNA and protein levels and the activity of luciferase reporters containing UGT2B15 or UGT2B17 3'-UTRs. This microRNA-mediated repression was significantly abrogated by mutating the miR-376c binding site in the 3'-UTRs of both UGTs. Collectively, these data indicate that the expression of UGT2B15 and UGT2B17 is negatively regulated by the binding of miR-376c to the 3'-UTRs of UGT2B15 and UGT2B17 in prostate cancer cells. This represents the first evidence for post-transcriptional regulation of UGT2B15 and UGT2B17 by miRNAs in prostate cancer cells and may have importance in regulating androgen receptor signaling., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The human UDP glycosyltransferase (UGT) superfamily comprises four families of enzymes that catalyze the addition of sugar residues to small lipophilic chemicals. The UGT1 and UGT2 enzymes use UDP-glucuronic acid, and UGT3 enzymes use UDP-N-acetylglucosamine, UDP-glucose, and UDP-xylose to conjugate xenobiotics, including drugs and endobiotics such as metabolic byproducts, hormones, and signaling molecules. This metabolism renders the substrate more polar and more readily excreted from the body and/or functionally inactive. The fourth UGT family, called UGT8, contains only one member that, unlike other UGTs, is considered biosynthetic. UGT8 uses UDP galactose to galactosidate ceramide, a key step in the synthesis of brain sphingolipids. To date other substrates for this UGT have not been identified and there has been no suggestion that UGT8 is involved in metabolism of endo- or xenobiotics. We re-examined the functions of UGT8 and discovered that it efficiently galactosidates bile acids and drug-like bile acid analogs. UGT8 conjugates bile acids ∼60-fold more efficiently than ceramide based on in vitro assays with substrate preference deoxycholic acid > chenodeoxycholic acid > cholic acid > hyodeoxycholic acid > ursodeoxycholic acid. Activities of human and mouse UGT8 are qualitatively similar. UGT8 is expressed at significant levels in kidney and gastrointestinal tract (intestine, colon) where conjugation of bile acids is likely to be metabolically significant. We also investigate the structural determinants of UDP-galactose selectivity. Our novel findings suggest a new role for UGT8 as a modulator of bile acid homeostasis and signaling., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Anthracyclines are effective genotoxic anticancer drugs for treating human malignancies; however, their clinical use is limited by tumor resistance and severe cardiotoxicity (e.g., congestive heart failure). Epirubicin (EPI) is less cardiotoxic compared with other canonical anthracyclines (e.g., doxorubicin). This has been attributed to its unique glucuronidation detoxification pathway. EPI is primarily inactivated by UDP-glucuronosyltransferase 2B7 (UGT2B7) in the liver. Hence, the regulation of hepatic UGT2B7 expression is critical for EPI systemic clearance but remains poorly characterized. We show herein that EPI upregulates UGT2B7 expression in hepatocellular carcinoma (HCC) HepG2 and Huh7 cells. Our analyses of deleted and mutated UGT2B7 promoter constructs identified a p53 response element (p53RE) in the UGT2B7 promoter. EPI stimulated UGT2B7 promoter activity via this p53RE and enhanced in vivo p53 binding at this p53RE in HepG2 cells. Knockdown of p53 expression by small interfering RNA silencing technology significantly repressed the capacity of EPI to stimulate UGT2B7 transcription. Furthermore, the p53 activator nutlin-3α significantly enhanced UGT2B7 expression and recruited the p53 protein to the UGT2B7 p53RE in HepG2 cells. Collectively, our results demonstrated that EPI promotes its own detoxification via the p53-mediated pathway. This regulation may contribute to tumor resistance to EPI-containing HCC chemotherapy and may also provide a new explanation for the reduced cardiotoxicity of EPI compared with other anthracyclines. Our finding also suggests that upon exposure to genotoxic agents, detoxifying genes are activated by the p53-mediated pathway to clear genotoxic agents locally within the tumor site or even systemically through the liver.

UDP glucuronosyltransferase 2B17 is present in the prostate, where it catalyzes the addition of glucuronic acid to testosterone and dihydrotestosterone and their metabolites androsterone and androstane-3α,17β-diol. Hence, changes in UGT2B17 gene expression may affect the capacity of the prostate to inactivate and eliminate male sex hormones. In this work, we identify a prevalent polymorphism, -155G/A, in the proximal promoter of the UGT2B17 gene. This polymorphism modulates UGT2B17 promoter activity, because luciferase-gene reporter constructs containing the -155A allele were 13-fold more active than those containing the -155G allele in prostate cancer LNCaP cells. The -155G/A polymorphism is contained within a putative binding site for the transcription factor Forkhead Box A1 (FOXA1). Using gene reporter, electromobility shift, and chromatin immunoprecipitation analyses, we show that FOXA1 binds to this site and stimulates the UGT2B17 promoter. Furthermore, down-regulation of FOXA1 in LNCaP cells substantially reduces UGT2B17 mRNA levels. The binding of FOXA1 and subsequent stimulation of the UGT2B17 promoter is greatly reduced in the presence of the -155G allele compared with the -155A allele. Consonant with its capacity to be stimulated by FOXA1, the UGT2B17 -155A allele, compared with the -155G allele, is associated with higher levels of circulating androstane-3α,17β-diol glucuronide. Although the initial phases of prostate cancer are androgen-dependent and UGT2B17 inactivates androgens, there was no association of the UGT2B17 -155G/A polymorphism with prostate cancer risk. In summary, this work identifies FOXA1 as an important regulator of UGT2B17 expression in prostate cancer LNCaP cells and identifies a polymorphism that alters this regulation.

UDP-glucuronosyltransferase 2B15 and 2B17 expression is up-regulated by 17beta-estradiol in MCF-7 breast cancer cells, as assessed by quantitative real-time polymerase chain reaction. Using 5'-deletion mapping and site-directed mutagenesis, we demonstrate that 17beta-estradiol activation of UGT2B15 gene transcription is mediated by a 282-base pair fragment positioned -454 to -172 nucleotides from the translation start site. This region contains two putative activator protein-1 (AP-1) elements, one imperfect estrogen response element (ERE), and two consensus ERE half-sites. We propose that these five sites act as an estrogen response unit (ERU), because mutation in any site reduces activation of the UGT2B15 promoter by 17beta-estradiol. Despite the presence of two AP-1 elements, the UGT2B15 promoter is not responsive to the AP-1 activator phorbol 12-myristate 13-acetate. Although electrophoretic mobility shift assays (EMSA) indicate that the AP-1 proteins c-Jun and Fos-related antigen 2 (Fra-2) bound to the distal AP-1 site, binding of Jun or Fos family members to the proximal AP-1 site was not detected by EMSA. Chromatin immunoprecipitation assays showed a 17beta-estradiol-induced recruitment of estrogen receptor (ER) alpha, c-Jun, and Fra-2 to the 282-bp ERU. The involvement of these three transcription factors in the stimulation of UGT2B15 gene expression by 17beta-estradiol was confirmed by siRNA silencing experiments. Mutagenesis and siRNA experiments indicate that UGT2B17 expression is also regulated by 17beta-estradiol via the ERU, which is fully conserved in both promoters. Because UGT2B15 and UGT2B17 inactivate steroid hormones by glucuronidation, the regulation of their genes by 17beta-estradiol may maintain steroid hormone homeostasis and prevent excessive estrogen signaling activity.