Upstream regulatory elements in chick heme oxygenase-1 promoter: a study in primary cultures of chick embryo liver cells.
Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear protein binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5'UTR.
Lu
T H
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Department of Medicine, University of Massachusetts Medical School, Worcester 01655, USA.
Shan
Y
Y
Pepe
J
J
Lambrecht
R W
RW
Bonkovsky
H L
HL
D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003035:Cobalt / Q000494:pharmacology; D016384:Consensus Sequence; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics*; D051547:Heme Oxygenase-1; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017670:Sodium Compounds / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D018053:Arsenites; D011993:Recombinant Fusion Proteins; D017670:Sodium Compounds; D003035:Cobalt; C017947:sodium arsenite; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; C018021:cobaltous chloride