The effect of GSTA1-1 (glutathione S-transferase Alpha 1-1) on JNK (c-Jun N-terminal kinase) activation was investigated in Caco-2 cells in which GSTA1 expression increases with degree of confluency, and in MEF3T3 cells with Tet-Off-inducible GSTA1 expression. Comparison of GSTA1 expression in pre-confluent, confluent and 8-day post-confluent Caco-2 cells revealed progressively increasing mRNA and protein levels at later stages of confluency. Exposure of pre-confluent cells to stress conditions including IL-1beta (interleukin-1beta), H2O2 or UV irradiation resulted in marked increases in JNK activity as indicated by c-Jun phosphorylation. However, JNK activation was significantly reduced in post-confluent cells exposed to the same stresses. Western-blot analysis of GSTA1-1 protein bound to JNK protein pulled down from cellular extracts showed approx. 4-fold higher GSTA1-1-JNK complex formation in post-confluent cells compared with pre-confluent cells. However, stress conditions did not alter the amount of GSTA1-1 bound to JNK. The role of GSTA1-1 in JNK suppression was more specifically revealed in Tet-Off-inducible MEF3T3-GSTA1-1 cells in which GSTA1 overexpression significantly reduced phosphorylation of c-Jun following exposure to IL-1beta, H2O2 and UV irradiation. Finally, the incidence of tumour necrosis factor alpha/butyrate-induced apoptosis was significantly higher in pre-confluent Caco-2 cells expressing low levels of GSTA1 compared with post-confluent cells. These results indicate that GSTA1 suppresses activation of JNK signalling by a pro-inflammatory cytokine and oxidative stress and suggests a protective role for GSTA1-1 in JNK-associated apoptosis.

Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine-and tobaccospecific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17␤-hydroxy-11␤-[4-dimethylamino phenyl]-17␣-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a Ϫ95 to ϩ12 region containing an hepatic nuclear factor 4 (HNF4) ␣ response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4␣-and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4␣ increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4␣ mRNA levels by 4-fold; however, the amount of HNF4␣ nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4␣ to the HNF4-RE and that an interaction of GR and HNF4␣ occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4␣ to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4␣ with an HNF4-RE rather than a glucocorticoid response element.

The regulation of human GSTA1 by chemical inducers of rodent glutathione S-transferases (GSTs) and the regulatory role of hepatic nuclear factor (HNF) 1 was investigated in Caco-2 cells. Treatment of preconfluent and confluent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), 3-methylcholanthrene (3-MC), 2-tert-butyl-4-hydroxy-anisol (BHA), and phenobarbital (PB) reduced GSTA1 mRNA levels in preconfluent and confluent cells. Constitutive levels of GSTA1 and HNF1␣ mRNA were elevated 6.25-and 50-fold, respectively, in postconfluent cells compared with preconfluent cells. Overexpression of HNF1␣ in cells transfected with a GSTA1 promoter-luciferase construct (pGSTA1-1591-luc) resulted in dose-related increases in reporter activity not observed when an HNF1 response element (HRE) in the proximal promoter was mutated (pGSTA1-⌬HNF1-luc). TPA, 3-MC, BHA, and PB reduced HNF1␣ mRNA levels in preconfluent and confluent cells and caused marked reductions in luciferase activity in pGSTA1-1591-luc transfectants. Transcriptional repression was abrogated with pGSTA1-⌬HNF1-luc and with truncated constructs that eliminated a functional HRE. Moreover, cotransfection of pHNF1␣ with pGSTA1-1591-luc partially prevented the reduction in luciferase activity by rodent GST inducers. Immunoblot analysis of DNA binding studies indicate that variant (v)HNF1-C binding to HRE is increased in preconfluent cells treated with 3-MC, BHA, and PB. In addition, overexpression of vHNF1-C repressed GSTA1 transcriptional activity in luciferase reporter assays. Finally, treatment with 3-MC, BHA, and PB increased vHNF1-C mRNA levels in preconfluent cells. These data demonstrate that repression of human GSTA1 transcription by chemical inducers of rodent GSTs occurs, in part, through a mechanism involving the repressive action of vHNF1-C.Glutathione S-transferases (GSTs) are a family of multifunctional proteins involved in the detoxification of a broad range of xenobiotics and therapeutic compounds, playing a critical role in protecting cells from reactive electrophiles. Rodent GSTs are inducible by drugs, carcinogens, antioxidants, and other dietary components. Inducers such as 2-tertbutyl-4-hydroxyanisole (BHA), phenobarbital (PB), 3-methylcholanthrene (3-MC), dithiolethiones, and ethoxyquin differentially control the regulation of mouse and rat hepatic GST isoenzymes and have led to the discovery of new inducible ␣-and -class subunits (Ding and Pickett, 1985;Hayes et al., 1991). Transcriptional activation of rodent ␣ class GSTs occurs via the antioxidant-responsive element (ARE), required for induction by dithiolethiones and antioxidants, as well as the xenobiotic-responsive element, which was first noted as a mediator in the induction of cytochrome P450 1A1 by polyaromatic hydrocarbons. (Rushmore et al., 1990;Hayes and Pulford, 1995;Eaton and Bammler, 1999).Only a few studies have addressed the regulation of human GST genes. Although the intron and exon structure of the two major human GSTA genes (A1 and A2) is highly homologous with tho...

Down-regulation of glutathione transferase A1 (GSTA1) expression has profound implications in cytoprotection against toxic by-products of lipid peroxidation produced during inflammation. We investigated the role of hepatic nuclear factor 1 (HNF-1) in repression of human GSTA1 expression by interleukin (IL)-1␤ in Caco-2 cells. In luciferase reporter assays, overexpression of HNF-1␣ increased GSTA1 transcriptional activity via an HNF-1 response element (HRE) in the proximal promoter. In addition, constitutive mRNA levels of GSTA1 and HNF-1␣ rose concurrently in Caco-2 cells with increasing stage of confluence. IL-1␤ reduced GSTA1 mRNA levels at all stages of confluence; however, HNF-1␣ mRNA levels were not altered. IL-1␤ repressed GSTA1 transcriptional activity, an effect that was abolished by mutating the HRE. Similar results were observed in HT-29 and HepG2 cells. Overexpression of HNF-1␣ did not counteract IL-1␤-mediated repression of GSTA1 transcription either in reporter assays or at the mRNA level. Involvement of the transdominant repressor C isoform of variant HNF-1 (vHNF-1C) in GSTA1 repression was demonstrated, because vHNF-1C overexpression significantly reduced GSTA1 transcriptional activity. Finally, IL-1␤ caused concentration-related up-regulation of vHNF-1C mRNA levels and increased binding of vHNF-1C protein to the HRE, whereas HNF-1␣-HRE complex formation was reduced. These findings indicate that IL-1␤ represses GSTA1 transcription via a mechanism involving overexpression of vHNF-1C.

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