Regulation of Na+ -H+ Exchanger 1 (NHE-1) gene expression by mild oxidative stress

Abstract

Owing to the historical focus on gene induction, most reports on the regulation of transcription factors by oxidative stress have described the activation of stress-inducible genes in response to various stimuli. In contrast to the wealth of literature on redox-dependent activation of transcription factors, very little is known in terms of the oxidative repression of transcription machinery. Recently, our laboratory showed that exposure of cells to non-toxic doses of H2O2 led to the inhibition of the ubiquituously expressed regulator of intracellular pH, NHE-1. Hence, the mechanism of NHE-1 gene repression upon exposure of cells to non-apoptotic concentrations of H2O2 was investigated. We show that the down-regulation of NHE-1 promoter activity and protein expression was abrogated by the presence of reducing agents such as beta mercaptoethanol. The pan-caspase inhibitor zVAD-fmk also blocked the effect of H2O2 on NHE-1 promoter activity and expression, but unlike beta mercaptoethanol, caspase inhibition was ineffective in rescuing the early phase of NHE1 repression. Interestingly, the effect of caspase inhibition was observed only after 9 hours of exposure to H2O2 and completely restored NHE-1 promoter activity by 18 to 24 hours. Using tetra-peptide inhibitors of a variety of caspases and siRNA-mediated gene silencing, caspases 3 and 6 were identified as mediators of H2O2-induced NHE-1 repression, independent of initiator caspase activation. Furthermore, incubation of cells with various iron chelators, not only blocked the activities of caspases 3 and 6, but also affected NHE-1 promoter and protein expression in a manner similar to zVAD-fmk. Activated caspases 3 was found in the nucleus of L6 cell upon stimulated by H2O2 in the absence of cell death. Interestingly, we also discovered that ONOO- plays an important role to regulate NHE-1 promoter activity as well. The production of ONOO- appears to be dependent on the presence of iron. We speculate that p38MAPK-HO-1 signaling pathway could be one pathway that releases the intracellular iron that is required for the down-regulation of NHE-1 gene expression. Taken together, our data show that a mild oxidative stress represses NHE-1 promoter activity and expression via an early oxidation phase blocked by reducing agents and a late phase requiring an iron-dependent increase in caspases 3 and 6 activities. Moreover, these data suggest that iron-mediated activation of caspase 3 and 6 may be a new pathway involved in the redox-mediated repression of gene transcription.