The effect of 25-hydroxycholesterol on the regulation of apolipoprotein E mRNA levels and secretion in the human hepatoma HepG2

Abstract

The human hepatoma cell line, HepG2, was cultured with 25 OH cholesterol, a potent inhibitor of HMG-CoA reductase, in order to examine the effect of the oxysterol on apo E synthesis and secretion. Treatment of cells with oxysterol (2.5 μM) resulted in a greater than 90% inhibition of HMG-CoA reductase activity and a 3-fold reduction in its cognate mRNA level. However, apo E mRNA level and secretion were not affected after 24 h of drug treatment. This drug treatment was associated with a reduction in both cellular free and esterified cholesterol levels by 50% and 40%, respectively. Exposure of HepG2 cells to an ACAT inhibitor, the Sandoz compound (58-035) for 24 h, at a concentration of 5 μg/ml, resulted in a 30% increase and 70% decrease in the intracellular levels of free and esterified cholesterol, respectively. Under this regimen of drug treatment, the level of apo E mRNA was increased by approximately 70%, while HMG-CoA reductase mRNA level was decreased by 35%. When the cells were exposed to the combination of the ACAT inhibitor and 25 OH cholesterol, the cellular levels of free and esterified cholesterol were reduced by 30% and 80%, respectively. This combination of drugs had no effect on apo E mRNA; however, the level of HMG-CoA reductase mRNA was decreased by 3.5-fold. Taken together, the data suggested that reduction in the intracellular levels of either free or esterified cholesterol had no effect on apo E mRNA level. By contrast, a small increment in cellular free cholesterol content was associated with a significant induction in apo E mRNA level. Furthermore, 25 OH cholesterol caused a significant redistribution (50%) of apo E from the HDL fraction to the d > 1.21 g/ml infranatant. By using high performance liquid chromatography and molecular sieve columns, it was found that the appearance of a lipid-poor apo E particle was not an artifact of ultracentrifugation. This particle contained 85 wt% protein and 15 wt% of free cholesterol and phospholipid. The results suggested that a lipid-poor apo E particle was secreted by the HepG2 cells under certain circumstances.