Quantification of Hepatic UDP glucuronosyltransferase 1A splice variant expression and correlation of UDP glucuronosyltransferase 1A1 variant expression with glucuronidation activity

Nathan R Jones; Dongxiao Sun; Willard M Freeman; Philip Lazarus

doi:10.1124/jpet.112.192658

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Quantification of Hepatic UDP glucuronosyltransferase 1A splice variant expression and correlation of UDP glucuronosyltransferase 1A1 variant expression with glucuronidation activity

Journal article

Open access

Peer reviewed

Quantification of Hepatic UDP glucuronosyltransferase 1A splice variant expression and correlation of UDP glucuronosyltransferase 1A1 variant expression with glucuronidation activity

Nathan R Jones, Dongxiao Sun, Willard M Freeman and Philip Lazarus

The Journal of pharmacology and experimental therapeutics, Vol.342(3), pp.720-729

09/2012

DOI: https://doi.org/10.1124/jpet.112.192658

Handle:

https://hdl.handle.net/2376/116253

PMCID: PMC3422519

PMID: 22661630

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Abstract

Cell Line

RNA, Small Interfering - genetics

Liver - enzymology

Alternative Splicing

Exons

Humans

RNA, Messenger - genetics

Raloxifene Hydrochloride - pharmacokinetics

Gene Knockdown Techniques

Hep G2 Cells

Glucuronosyltransferase - genetics

Glucuronosyltransferase - metabolism

Raloxifene Hydrochloride - administration & dosage

Cell Line, Tumor

Microsomes, Liver - enzymology

RNA, Small Interfering - administration & dosage

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.

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Title: Quantification of Hepatic UDP glucuronosyltransferase 1A splice variant expression and correlation of UDP glucuronosyltransferase 1A1 variant expression with glucuronidation activity
Creators: Nathan R Jones - Department of Pharmacology, Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA
Dongxiao Sun
Willard M Freeman
Philip Lazarus
Publication Details: The Journal of pharmacology and experimental therapeutics, Vol.342(3), pp.720-729
Academic Unit: Pharmaceutical Sciences, Department of
Publisher: United States
Grant note: R01 DE013158 / NIDCR NIH HHS R01-DE13158 / NIDCR NIH HHS
Identifiers: 99900547646901842
Language: English
Resource Type: Journal article