Role of Human Glutathione S-Transferase Alpha in Modulating Cellular Stress and Cell Phase Transitions
As intestinal epithelial cells mature, they continuously transition from proliferation to differentiation to apoptosis under the influence of cell signalling pathways including c-Jun N-terminal kinase (JNK). Glutathione S-transferases (GSTs) are cytoprotective detoxification enzymes, some of which, including GSTA1, also sequester and inhibit JNK through complex formation. Thus, GSTA1 may be a key sensor of cellular state and regulator of responses to cell stress stimuli. The focus of this research study was to investigate the functional importance of GSTA1 in two contexts: 1) modulating complex integrity with JNK and activation of JNK by oxidative stress, 2) controlling cellular transitioning between proliferation, differentiation and apoptosis. In the first study, the impact of GSTA1 levels on dissociation of GSTA1-JNK complexes and JNK activation in response to cellular stress was investigated in the human colonic adenocarinoma Caco-2 cells. The pro-oxidant menadione caused GSTA1-JNK complex dissociation in preconfluent Caco-2 cells, whereas postconfluent cells were relatively resistant to this effect. Preconfluent cells were more sensitive than postconfluent cells to menadione-induced cytotoxicity. Additionally, menadione-induced JNK activation was transient since removal of the stimulus resulted in re-association of GSTA1 with JNK and significantly reduced cytotoxicity. Over-expression and knockdown of GSTA1 affected the degree of GSTA1-JNK complex association without altering the JNK activation. However, enhanced GSH levels by N-acetyl cysteine blocked menadione-induced complex dissociation and JNK activation in Caco-2 cells. The results suggest that the mechanism of menadione-mediated JNK activation involves the production of reactive oxygen species, likely superoxide anion, and that the level of intracellular GSH plays an important role in preventing menadione-induced GSTA1-JNK complex dissociation and subsequent JNK activation. The functional importance of GSTA1 in controlling cellular proliferation, differentiation and apoptosis was investigated. Sodium butyrate (NaB) is a short-chain fatty acid, physiologically present in the human large intestine and modulates transitioning of cell states in colon cancer cell lines. GSTA1 levels increased in association with differentiation markers in postconfluent Caco-2 cells. Forced expression of GSTA1 significantly reduced cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increased cells in S-phase and associated cell proliferation. NaB (1 mM) reduced Caco-2 cell proliferation, increased differentiation and up-regulated GSTA1 activity. In contrast, higher dose of NaB (10 mM) caused toxicity in preconfluent cells via apoptosis through caspase-3 activation in association with reduced GSTA1 activity. GSTA1 down-regulation by siRNA did not alter NaB-induced differentiation or the sensitivity of Caco-2 cells to NaB-induced apoptosis. Furthermore, NaB (10 mM) caused GSTA1-JNK complex dissociation but did not affect JNK activation. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.