Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines

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% (Po0.01) 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.

Dual Roles for Splice Variants of the Glucuronidation Pathway as Regulators of Cellular Metabolism

“…Further functional assays with human cells coexpressing both i1s and i2s clearly demonstrated a significant decrease in cellular glucuronidation activity for many different UGT1A substrates Bellemare et al, 2010b). It was previously shown that transient repression of i2 by small interfering RNAs leads to an increased glucuronidation activity in human colon and liver cells, revealing a novel regulatory mechanism by i2s (Bellemare et al, 2010c;Jones et al, 2012). UGT oligomerization is likely the underlying mechanism for the negative modulation of transferase activity by splice variant i2.…”

“…A multiplicity of infection of 1 was utilized to infect HT115 cells (European Collection of Cell Cultures, Salisbury, UK). Isoformspecific quantitative polymerase chain reaction strategies were used to confirm v2/v3 mRNA depletion (Bellemare et al, 2010c). Enzymatic metabolism assays for SN-38 glucuronidation were performed using 50 mg cell homogenates incubated for 60 minutes at 37°C with 200 mM SN-38 and rates of SN-38 glucuronide formation were measured by mass spectrometry (MS) (Gagné et al, 2002 Global Gene Expression Analysis.…”

“…To test this hypothesis, we developed a stable knockdown (KD) model of i2s using short hairpin RNA (shRNA) in HT115 colon cancer cells, which express both classes of i1 and i2 with an almost equivalent level of v2/v3 transcripts encoding i2 isoforms relative to v1 levels encoding UGT1A enzymes (0.8:1) (Bellemare et al, 2010c). We used, as a pharmacological model compound, the active metabolite of irinotecan (CPT-11), namely SN-38 (7-ethyl-10-hydroxycamptothecin), which is almost exclusively metabolized by the UGT1A pathway and is one of the most common cytotoxic agents used to treat metastatic colorectal cancer.…”

Quantification of Hepatic UDP Glucuronosyltransferase 1A Splice Variant Expression and Correlation of UDP Glucuronosyltransferase 1A1 Variant Expression with Glucuronidation Activity

“…The i2 protein has been previously shown to be expressed in human carcinoma tissue and cell lines, and siRNA knockdown of the v2/v3 variant resulted in increased activity against a range of substrates in colon cancer cell lines (Bellemare et al, 2010c(Bellemare et al, , 2011). In the current study, it was observed that the human hepatocellular carcinoma cell line HepG2 exhibited relatively high endogenous expression of exon 5b-containing variants for UGT1A1, with v2/v3 variant expression reaching levels that were slightly greater than that observed for UGT1A1 v1.…”

The Relative Protein Abundance of UGT1A Alternative Splice Variants as a Key Determinant of Glucuronidation Activity In Vitro

“…Active UGT1As_i1 and inactive UGT1As_i2 isoforms were shown to be coexpressed in human tissues including liver, kidney, stomach, intestine, and colon (Bellemare et al, 2011), further supporting a dominant-negative role of UGT1As_i2. In addition, their regulatory function is sustained by results of small interfering RNA-mediated depletion of endogenous i2 in two human colon cancer cellular models that lead to increased cellular UGT activity (Bellemare et al, 2010c). This observation was recently replicated using the hepatocellular carcinoma HepG2 cell line, which displays slightly higher mRNA transcript levels of UGT1A1_v2/v3 transcripts compared with UGT1A1_v1 (Jones et al, 2012), although protein levels were not measured.…”