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1 From the Departments of Veterinary and Biomedical Sciences and 3 Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska; and the 4 Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska.
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.
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