DOWN-REGULATION OF ALPHA CLASS GLUTATHIONE S-TRANSFERASE BY INTERLEUKIN-1 IN HUMAN INTESTINAL EPITHELIAL CELLS (CACO-2) IN CULTURE LAURA ROMERO, MARNIE A. HIGGINS, JAMES GILMORE, KIM BOUDREAU, ANN MASLEN, HEATHER J. BARKER, AND GORDON M. KIRBY Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada (Received May 29, 2002; accepted August 2, 2002) This article is available online at http://dmd.aspetjournals.org ABSTRACT: The influence of pro-inflammatory cytokines on alpha class glutathi- one S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture. Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopo- lysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7). Neutralizing anti-interleukin-1 (IL-1) antibodies attenuated this repression of GSTA1/A2 expression by CM. Moreover, recombi- nant human IL-1 reduced GST expression at the mRNA, protein, and activity levels in a dose-related fashion. Reduction of GSTA1/A2 mRNA levels by IL-1 was attenuated by pretreatment with IL-1 receptor antagonist. GSTA1/A2 mRNA half-lives were similar in con- trol and IL-1-treated cells, indicating that IL-1 has no effect on mRNA stability. In reporter gene studies, IL-1 caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct. Using truncated constructs, IL-1 responsiveness was mapped to a region 286 base pairs upstream to the coding region. Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1-mediated repression of GSTA1 promoter activity. These results demonstrate that IL-1 down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site. Cellular resistance to cytotoxicity is conferred by various detoxifi- cation and antioxidant enzymes, including glutathione S-transferases (GSTs 1 ). GSTs plays an important role in protecting colonic epithelial cells against the effects of dietary mutagens and reactive oxygen species (Hayes and Pulford, 1995). Although the precise mechanisms are unclear, there is convincing evidence that the pathogenesis of inflammatory bowel disease is associated with increased oxidative damage due to a reduction in cytoprotection (Lih-Brody et al., 1996), including diminished GST activity (Bhaskar et al., 1995; Clapper and Szarka, 1998). Pro-inflammatory cytokines, notably tumor necrosis factor-, interleukin-1, and interleukin-6 are secreted by inflamma- tory cells and enterocytes during the intestinal inflammatory response (Stevens et al., 1992; Jung et al., 1995). There is substantial evidence that pro-inflammatory cytokines profoundly influence the constitutive expression of a variety of genes, including drug-metabolizing en- zymes such as the cytochromes P450 (Ghezzi et al., 1986; Bertini et al., 1988; Trautwein et al., 1992). Relatively few studies, however, have investigated the effects of cytokines on GST gene expression. In cultured primary hepatocytes, the effects of cytokines on GST gene expression are variable, resulting in either down-regulation (Adams and Czuprynski, 1994; Maheo et al., 1997; Navasa et al., 1998) or up-regulation (Voss et al., 1996), depending on the cytokine treat- ment. There are no reports of the influence of cytokines on GST expression in cultured human enterocytes. The human alpha class GST isoenzymes consist of homodimers or heterodimers of four major subunits (GSTA1, A2, A3, and A4) that are involved in detoxification of dietary carcinogens (Huber et al., 1997), and organic hydroperoxides (Hayes and Pulford, 1995). Induc- tion of GSTs, including those of the alpha class, by various structur- ally unrelated electrophilic compounds and pro-oxidants, represents a major mechanism of protection against oxidative and chemical stress. The regulation of alpha class GSTs has been extensively studied in rodents, and a complex set of regulatory elements has been charac- terized in the 5-flanking regions, which control basal and xenobiotic inducible expression (Daniel, 1993; Hayes and Pulford, 1995; Whalen and Boyer, 1998). In rats, for example, transcription of the GSTA2 gene is regulated through a “xenobiotic responsive element” that mediates induction by planar aromatic compounds and an “antioxi- dant responsive element”, which mediates induction by phenolic antioxidants and pro-oxidants (Rushmore and Pickett, 1990; Daniel, 1993; Whalen and Boyer, 1998). A similar “electrophile responsive element” has been identified in murine GSTA1 (Daniel, 1993; Hayes and Pulford, 1995; Whalen and Boyer, 1998). Although the 5- flanking regions of the human GSTA1 and GSTA2 genes are more than 95% homologous, they are very different from the promoter for rat GSTA and lack a functional antioxidant responsive element, suggest- ing that the mechanisms of transcriptional regulation are different in the human and rodent genes (Suzuki et al., 1994). Supported by funding from the Medical Research Council of Canada (Grant MT-13757). 1 Abbreviations used are: GST, glutathione S-transferase; IL-1, interleukin- 1; IL-6, interleukin-6; TNF, tumor necrosis factor-; GSTA1/A2, alpha class glutathione S-transferase A1 and A2; HNF-1, hepatic nuclear factor-1; CM, con- ditioned medium; LPS, lipopolysaccharide; FBS, fetal bovine serum; IL-1ra, in- terleukin-1 receptor antagonist; bp, base pair(s); LUC, luciferase; PCR, polymer- ase chain reaction; AP-1, activating protein-1; IBD, inflammatory bowel disease. Address correspondence to: Dr. Gordon M. Kirby, Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail: gkirby@uoguelph.ca 0090-9556/02/3011-1186–1193$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 11 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 811/1018833 DMD 30:1186–1193, 2002 Printed in U.S.A. 1186 at ASPET Journals on August 27, 2017 dmd.aspetjournals.org Downloaded from