Abstract
Apolipoprotein (apo) A-I is the principal protein component of high density lipoproteins and the major in vivo activator of lecithin-cholesterol acyltransferase. We have used the human hepatoma cell line, HepG2, as a model to examine the ability of estrogen to modulate hepatic synthesis of apo-A-I. Primer extension studies have demonstrated that the major transcriptional initiation site of the apo-A-I gene utilized in the hepatoma cells is the same as that used in human liver. The kinetics of induction of high- and low-affinity estrogen-binding sites, rates of secretion of apolipoproteins, and apo-A-I mRNA levels were examined following treatment of the cells with estrogen. Initial concentrations of 20 nM 17 beta-estradiol resulted in a 14-15-fold increase in the levels of high-affinity nuclear estrogen-binding sites within 8 h, while the level of low-affinity sites increased by only 10%. During the same period, the levels of apo-A-I mRNA and the rate of accumulation of the secreted protein increased by 55 and 50%, respectively. New steady state levels of apo-A-I mRNA and rates of accumulation of protein, approximately twice those in control cultures, were established within 24-48 h of exposure to hormone. Experiments with a 50-fold higher concentration of estrogen resulted in only an additional 10% increase in mRNA levels. The increase in mRNA levels following estrogen treatment was adequate to account for 85-90% of the elevation observed in the rate of accumulation of secreted apo-A-I. Comparison of the apo-A-I mRNA levels in HepG2 cells with those present in human liver revealed that the concentration of the mRNA was approximately 3-fold lower than that found in vivo.
Highlights
On the basis of in vivo turnover studies, it has been concluded in some cases that the hormone influences the rate of synthesis of apo-A-I [29], while in others, the conclusion has been drawn thatthe effect of estrogen is toalter metabolism of the apolipoprotein [30]
Detectionand Sizing of Apo-A-ImRNAinTotalRNA Isolated fromHumanLiver, Peripheral Lymphocytes, and HepG2 Cells-The triacontanucleotideprobe used in these studies was synthesized by the phosphoramidite procedure and was complementary to the region of human apo-A-I mRNA that specifies amino acids-10 to -1 of the preproprotein
End-labeled probe was hybridized initially to total RNA isolated from human fetal liver and HepG2 cells as detailed in the legend to Fig. 1.The probe detected a single size class of mRNA on blots prepared from agarose gels of samples of glyoxylated total RNA
Summary
We have analyze1d)the kinetics of induction of both high- and low-affinity nuclear estrogen-binding sites; 2) the rates of accumulation of the major apolipoproteins at various times after hormone addition; and 3) the rapidity with which apo-A-I mRNA levels increase upon exposure of the cells to estrogen.
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