Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/17739
Title: Regulation of Na+ -H+ Exchanger 1 (NHE-1) gene expression by mild oxidative stress
Authors: CHANG KER XING
Keywords: NHE1,H202,caspase,iron,gene expression,ROS
Issue Date: 21-Aug-2009
Source: CHANG KER XING (2009-08-21). Regulation of Na+ -H+ Exchanger 1 (NHE-1) gene expression by mild oxidative stress. ScholarBank@NUS Repository.
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.
URI: http://scholarbank.nus.edu.sg/handle/10635/17739
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