Abstract
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.
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Abbreviations
- E2:
-
estradiol
- i1:
-
isoforms 1
- i2:
-
isoforms 2
- MPA:
-
mycophenolate acid
- PBS:
-
phosphate-buffered saline
- siRNA:
-
small-interfering RNA
- SN-38:
-
7-ethyl-10-hydroxycamptothecin
- UGT:
-
UDP-glucuronosyltransferase
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Acknowledgements
We thank Patrick Caron and Lyne Villeneuve for technical assistance. We are thankful to Michèle Orain for colon tissue preparation samples, to Dr Georges Pelletier, Johanne Ouellet and Marie-Odile Benoit-Biancamano for scientific advices for immunohistochemistry experiments. This work was supported by the Canadian Institutes of Health Research (CIHR MOP-88745) and the Canada Research Chair Program (CG). JB and MR are recipients of a CIHR Frederick Banting and Charles Best studentship award and a graduate studentship award from the Fonds de la recherche en santé du Québec (FRSQ), respectively. CG holds a Canada Research Chair in Pharmacogenomics.
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Bellemare, J., Rouleau, M., Harvey, M. et al. Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines. Pharmacogenomics J 10, 431–441 (2010). https://doi.org/10.1038/tpj.2009.64
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DOI: https://doi.org/10.1038/tpj.2009.64
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