Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene.

Heme oxygenase is an essential enzyme in heme catabolism and is inducible by various environmental derangements such as cadmium. The activity and mRNA levels of heme oxygenase were remarkably increased in HeLa cells by the treatment with cadmium. As a first step in studying the molecular mechanisms of this induction, we performed transient expression assays in four human cell lines including HeLa to analyze the cadmium-mediated inducibility of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase gene promoter. By determining the luciferase activity expressed in the transfected cells, we found the region between about 4.5 and 4 kilobase pairs upstream from the transcriptional initiation site of the heme oxygenase gene which confers cadmium-mediated inducibility on the fusion gene. The region was then subjected to further functional analysis in HeLa cells, which allowed us to localize the cadmium-responsive element to 20 base pairs. Gel mobility shift assays demonstrated that this 20-base pair element is specifically bound by nuclear protein(s) of HeLa cells, the binding activities of which were however unchanged by the treatment with cadmium. Using the synthetic cadmium-responsive elements containing various base changes, we have identified a 10-base pair sequence, TGCTAGATTT, required for the cadmium-mediated inducibility and in vitro protein binding. We thus suggest that this binding protein(s) is involved in the cadmium-mediated activation of the heme oxygenase gene. Incidentally, the consensus sequence of AP-1 binding site, TGAGTCA, is present downstream of this cadmium-responsive element. However, we provide evidence that AP-1 is not directly involved in the cadmium-mediated induction of the human heme oxygenase gene.