Regulation of human apolipoprotein A-I gene expression by gramoxone

To induce oxidative stress, HepG2 cells were exposed to a compound known as gramoxone. This compound undergoes a one-electron reduction to form a stable free radical which is capable of generating reactive oxygen species. We demonstrated that exposure of HepG2 cells to gramoxone (0.1 microM) resulted in a 2-fold decrease in apoA-I mRNA with no significant change in apoB and apoE mRNA levels. To examine if increased rates of mRNA degradation were responsible for the reduction in apoA-I mRNA levels, mRNA half-lives were measured in the presence of actinomycin D with and without gramoxone treatment. These studies revealed a 4-fold increase in the rate of apoA-I mRNA degradation in cells exposed to gramoxone. In similarly treated cells, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was also increased 2-fold. Consistent with nuclear run-off assays, transient transfection experiments using a series of pGL2-derived luciferase reporter plasmids containing the human apoAI proximal promoter demonstrated that gramoxone treatment increased apoA-I promoter activity 2-fold. We have identified a potential "antioxidant response element" (ARE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gramoxone. Gel mobility shift assays with an ARE oligonucleotide revealed increased levels of a specific protein-DNA complex that formed with nuclear extracts from gramoxone-treated cells. UV cross-linking experiments with the ARE and nuclear extracts from either untreated or gramoxone-treated cells detected proteins of approximately 100 and 115 kDa. When a single copy of the ARE was inserted upstream of the SV40 promoter in a luciferase reporter plasmid, a significant 2-fold induction in luciferase activity was observed in HepG2 cells in the presence of gramoxone. In contrast, a plasmid containing a mutant apoAI-ARE did not confer responsiveness to gramoxone. Furthermore, pGL2 (apoAI-250 mutant ARE), in which point mutations eliminated the ARE in the apoAI promoter, showed no increase in luciferase activity in response to gramoxone. Taken together, the data suggest that gramoxone affects apoA-I mRNA levels by both transcriptional and post-transcriptional mechanisms.