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.

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.