Abstract
Purpose: :
Oxidative stress is an underlying determinant in the progression of age related cataract. The antioxidant protein peroxiredoxin 6 (PRDX6) protects cell by controlling H2O2 and mediating survival signaling. Given the role of H2O2 as a mediator of cell signaling, regulation of PRDX6 gene expression may require fine tuning to maintain cellular homeostasis. By using Prdx6–/– LECs (redox state) as a model system, we investigated the role(s) of redox–signaling in regulation of Prdx6 gene expression.
Methods: :
Matlnspector, a computer program was used to analyze putative redox–active transcription factor binding sites in Prdx6 gene promoter. 5’–flanking sequence spanning from –1139 to +109 bps was subcloned into pCAT–basic vector. Deletion mutants –839, –513, –339 and –131 constructs were prepared with common 3’ end (+109 bps).Gelshift and supershift assays, mutational analysis (site–directed mutagenesis) and CAT–ELISA were used to characterize transcriptional proteins that functionally binds to prdx6 promoter under redox–environment. Western blot and real time PCR was used to measure expression levels of NFkB family and IkB, and TGF ß and Smad3 in Prdx6–/–LECs and/or cells under oxidative stress.
Results: :
An analysis of Prdx6 gene promoter revealed the presence of the redox–active NFkB and six repressive Smad3–binding elements (RSBE, repressive Smad3 binding element). Western analysis showed expression of NFkB (p65/p50) and pIkB and bioactive TGFß in Prdx6–/– cells or cells under oxidative stress. Gel–shift assay showed that nuclear extract isolated from Prdx6–/– cells binds to RSBE and NFkB responsive elements. Transactivation experiment with Prdx6 gene promoter bearing NFkB and RSBE sites (–1139, –839 –513 and –339 to +109nts) showed reduced transcriptional activity, while disruption of RSBE sites revealed higher Prdx6 transcription in Prdx6–/– LECs, suggesting the involvement of NFkB and Smad3–mediated TGFß signaling. Interestingly, a supply of PRDX6 protein could restore NFkB activation of Prdx6 transcription by preventing Smad3–mediated deleterious TGF ß signaling in cells under redox environment.
Conclusions: :
Our findings provide insight into the regulation of Prdx6 gene expression and its regulatory role in normal cells as well as cells facing oxidative stress that in turn may provide a base to develop PRDX6 protein as therapeutic molecule to control oxidative stress induced deleterious cellular signaling.
Keywords: oxidation/oxidative or free radical damage • gene/expression • transcription