Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines.
The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P<0.01) for all substrates tested. Functional data support a dominant-negative function for endogenous exon 5b-spliced forms of UGT1A, hence potentially affecting in vivo glucuronidation capacity. This new regulatory strategy may ensure an additional mean to modulate cellular response to endo/xeno stimulus.
Bellemare
J
J
Pharmacogenomics Laboratory, CHUQ Research Center and Faculty of Pharmacy, Laval University, Québec, Canada.
Rouleau
M
M
Harvey
M
M
Têtu
B
B
Guillemette
C
C
D017398:Alternative Splicing; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003106:Colon / Q000201:enzymology / Q000473:pathology; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D020719:Glucuronides / Q000096:biosynthesis*; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D050217:Metabolic Detoxication, Phase II; D008862:Microsomes, Liver / Q000201:enzymology / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D000099148:UGT1A1 Enzyme
D020719:Glucuronides; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme