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 re gions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -15 73) 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 dep endent induction of cHO-1 were investigated further. DNA binding studies an d site-directed mutagenesis studies indicated that both the AP-1 and MRE/cM yc elements are important for the sodium arsenite induction, while cobalt c hloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear proteins binding to the AP-1 element was i ncreased 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, t o 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 suffi cient to reduce the cobalt chloride induction almost completely. The MRE/cM yc complex plays a major role in the basal level expression, and shares som e 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 becau se activation of AP-1 proteins has been shown to be a key step in the oxida tive stress response pathway, we also explored the possibility that the ind uction of the cHO-1 gene by sodium arsenite is mediated through oxidative s tress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction o f the endogenous cHO-1 message or cHO-1 reporter construct activities induc ed by sodium arsenite or cobalt chloride. These antioxidants also reduced t he protein binding activities to the AP-1 element in the electrophoretic mo bility shift assays. In summary, induction of the cHO-1 gene by sodium arse nite or cobalt chloride is mediated by activation of the AP-1 element locat ed at -1,573 to -1,580 of the 5' UTR.