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) gene by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking HIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia-mimetic CoCl(2) was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIF-independent mechanisms for ho-1 gene activation by hypoxia and CoCl(2). In Ka13 cells, CoCl(2) stimulated expression of a luciferase reporter gene under the control of a 15-kilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified the 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 level 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 electrophoretic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of complex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but not of other bZIP factors, attenuated cobalt-mediated gene activation. Additional experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 but 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 hypoxia in Chinese hamster ovary cells.