Ascorbic acid and alpha-tocopherol down-regulate apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines

HepG2 cells and Caco-2 cells were treated with various concentrations of select antioxidants to study some of the molecular pathways underlying antioxidant-related changes in apolipoprotein A-I (apoA-I) expression. Both alpha-tocopherol and ascorbate treatment over a time course of 72 hours caused a significant reduction in apoA-I messenger RNA and protein levels in a dose-dependent fashion. Albumin levels did not change with any treatment, suggesting that the effect is protein-specific. Similar changes were seen in Caco-2 cells. In contrast, apoA-I messenger RNA and protein levels significantly increased after 28 and 280 micromol/L dimethyl sulfoxide (DMSO) treatment. Cells were transfected with chloramphenicol acetyltransferase (CAT) reporter gene plasmid containing the full-length apoA-I promoter to determine if these changes occur at a transcriptional level, and after 24 hours, the HepG2 or Caco-2 cells were treated with varying concentrations of ascorbate or alpha-tocopherol. At the highest concentration of ascorbate and alpha-tocopherol used (5 mmol/L), there was a significant reduction in apoA-I promoter activity. DMSO treatment up-regulated apoA-I promoter activity, whereas increasing oxidative load with 50, 100, and 200 micromol/L hydrogen peroxide treatment did not significantly alter apoA-I promoter activity. Studies with deletional constructs of the promoter containing or lacking the antioxidant response element (ARE) indicated that the effect of ascorbate and alpha-tocopherol, unlike that of DMSO, was independent of this ARE. Using a series of apoA-I deletion constructs, it was shown that site A-containing segment of the promoter has a critical regulatory element. However, electromobility shift assays indicated that there was no significant change in nuclear factor binding activity to site A as a result of treatment with ascorbate or alpha-tocopherol. As expected, treatment with DMSO increased factor binding to the previously described ARE. It is concluded that the apoA-I promoter-stimulating effect of DMSO may be independent of its antioxidant activity and that some antioxidants at very high concentrations may have suppressive effect on the apoA-I gene expression. It appears that the inhibitory effect of ascorbate or alpha-tocopherol on the apoA-I promoter is either indirect or is the result of posttranslational modifications of the nuclear binding factors. The previously described ARE is not a response element for the ascorbate or alpha-tocopherol.