Purpose: To study the mechanism of activation of the human thioltransferase (hTTase) gene under oxidative stress.
Methods: Human lens epithelial cells (HLE-B3) were exposed to 0.1 mM H2O2 for 0, 5, 10, 15, 30, or 60 minutes; lysed; and used for gel mobility shift assay (GMSA) and supershift assay for activating protein (AP)-1 transcription factor. The search for transcriptional coactivators was performed with Western blot analysis. The ability of different parts and a mutated fragment of the hTTase gene promoter region to activate the gene expression under the oxidative stress conditions was examined by reporter gene assay.
Results: The AP-1-binding element was identified in the 5' region of the hTTase gene, and evidence was obtained that binding of AP-1 with this element in vivo was redox sensitive. In addition, the pattern of AP-1 binding under the oxidative stress was similar to the pattern of TTase activity and mRNA synthesis modulation. In contrast, direct exposure of the cell lysate to oxidants, reductants, or redox-regulating enzymes in vitro had no influence on AP-1 binding. AP-1 transcriptional coactivator redox factor (Ref)-1 was present in the lens epithelium and was association with the AP-1-binding complex during oxidative stress. In the reporter gene assay, only the fragments of the hTTase 5' region, which contained the AP-1-binding site, could activate the CAT reporter gene's expression in an oxidative stress-dependent manner. The mutant with a replaced AP-1-binding site failed to stimulate CAT expression in an oxidation-sensitive manner. The results showed that the c-Jun component in the AP-1-binding complex was transiently phosphorylated during H2O2 treatment. The c-Jun N-terminal kinase or SAPK/JNK, which responds to stress signaling and is the upstream protein kinase of c-Jun, was activated and translocated from cytosol to nucleus under the same conditions.
Conclusions: The data demonstrate that the activation of the hTTase gene under oxidative stress depends on the AP-1 transcription factor. The event was initiated only through an intact cell, possibly mediated through signal transduction by a phosphorylation-dephosphorylation mechanism. As far as the authors know, this is the first evidence of the association of AP-1 with the regulation of hTTase gene expression.