Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent mechanism in Chinese hamster ovary cells - Regulation by Nrf2 and MafG transcription factors

We have shown previously that activation of the heme oxygenase-1 (ho-1) gen e by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking RIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia -mimetic CoCl2 was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIF-independent mechanis ms for ho-1 gene activation by hypoxia and CoCl2. In Ka13 cells, CoCl2 stim ulated expression of a luciferase reporter gene under the control of a 15-k ilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified t he cobalt-responsive sequences as the stress-response elements (StREs). In electrophoretic mobility shift, assays, two specific StRE-protein complexes were observed using extracts from Ka13 cells. In response to cobalt, the l evel of the slower migrating complex X increased, whereas that of complex Y decreased, in a time-dependent manner. Members of the AP-1 superfamily of basic-leucine zipper factors bind to the StRE. Antibody supershift electrop horetic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of comp lex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but no t of other bZIP factors, attenuated cobalt-mediated gene activation. Additi onal experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 bu t is dependent on cellular oxidative stress. Unlike cobalt, hypoxia did not stimulate pHO15luc expression and did not increase StRE binding activity, indicating distinct mechanisms for ho-1 gene activation by cobalt and hypox ia in Chinese hamster ovary cells.