Quantification of Hepatic UDP glucuronosyltransferase 1A splice variant expression and correlation of UDP glucuronosyltransferase 1A1 variant expression with glucuronidation activity.
The UDP glucuronosyltransferase (UGT) 1A gene cluster encodes nine UGT1A family members via splicing of individual first exons to common exons 2 through 5. Each of these nine UGT1As can also undergo alternative splicing at their 3' ends by using an alternate exon 5, resulting in 27 different UGT1A mRNA species with each UGT1A gene encoding three different combinations of 5A and 5B UGT1A exons. To examine the importance of UGT1A exon 5 splice variants on overall UGT1A activity, a nested quantitative polymerase chain reaction assay was developed to accurately assess the combined expression of exon 5 splice variants (termed v2/v3) versus the expression of wild-type (termed v1) for each specific UGT1A. v1 expression was 16-, 17-, 57- and 29-fold higher than that observed for the levels of v2/v3 for UGTs 1A1, 1A4, 1A6, and 1A9, respectively, in normal human liver specimens. In a series of 58 normal human liver specimens, the expression of both UGT1A1 v1 and v2/v3 mRNAs was positively correlated with raloxifene glucuronidation activity in corresponding microsomes prepared from the same specimens (p < 0.0001, r² = 0.720; p = 0.0002, r² = 0.241, respectively), with expression of both variants lower in individuals homozygous for the UGT1A1*28 allele (42% for v1, p = 0.041; 53% for v2/v3, p = 0.0075). The expression of UGT1A1 v2/v3 was 1.6-fold higher than v1 (p = 0.03) in HepG2 cells, and short interfering RNA knockdown of HepG2 v2/v3 increased raloxifene glucuronidation activity by 83%. Together, these data suggest that hepatic UGT1A v2/v3 mRNA species are minor form variants in human livers from most individuals.
Department of Pharmacology, Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA.
Nathan R
NR
Jones
Dongxiao
D
Sun
Willard M
WM
Freeman
Philip
P
Lazarus
D017398:Alternative Splicing; D002460:Cell Line; D045744:Cell Line, Tumor; D005091:Exons; D055785:Gene Knockdown Techniques; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D020849:Raloxifene Hydrochloride / Q000008:administration & dosage / Q000493:pharmacokinetics; D000099148:UGT1A1 Enzyme
D014453:Glucuronosyltransferase; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020849:Raloxifene Hydrochloride; D000099148:UGT1A1 Enzyme