Regulation of human thioltransferase (hTTase) gene by AP-1 transcription factor under oxidative stress.
OBJECTIVE
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.
Krysan
Kostyantyn
K
Departments of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
Lou
Marjorie F
MF
D015153:Blotting, Western; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D054477:Glutaredoxins; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D007908:Lens, Crystalline / Q000166:cytology*; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011490:Protein Disulfide Reductase (Glutathione); D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000502:physiology*
D054477:Glutaredoxins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases