Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation.

Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2.ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl(2), a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 min versus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl(2)-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl(2). These results indicate that ATF4 regulates basal and CdCl(2)-induced expression of the ho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.