Tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells

Tumor necrosis factor mediates most biological activities of endotoxin and also, in part, mediates endotoxin-induced disturbances in lipid metabolism. In this study, the effect of tumor necrosis factor on low-density lipoprotein receptor activity was investigated in cells of HepG2, a well-differentiated human hepatoma cell line. Pretreatment of the cells with tumor necrosis factor leads to enhanced binding, uptake and degradation of 125I-labeled low-density lipoprotein. This effect of tumor necrosis factor was dose and time dependent. Tumor necrosis factor-stimulated enhancement of low-density lipoprotein binding occurred at all stages of cell growth. However, addition of an excess of unlabeled low-density lipoprotein, to down-regulate low-density lipoprotein receptors before exposure to tumor necrosis factor of the cells, completely abolished the effects of tumor necrosis factor. Competition experiments using unlabeled low-density lipoprotein and blockage experiments with a monoclonal low-density lipoprotein receptor antibody showed that tumor necrosis factor-stimulated low-density lipoprotein binding takes place through stimulation of low-density lipoprotein receptors. Comparison of the kinetics of specific low-density lipoprotein binding in the unstimulated cells and in the tumor necrosis factor-stimulated cells indicated that tumor necrosis factor caused a 30% increase in maximum velocity with no significant change in Michaelis constant, suggesting that tumor necrosis factor increases the number of low-density lipoprotein receptors on the cells rather than changing binding affinity. Preincubation of the cells with cycloheximide or actinomycin D totally abolished the up-regulatory effect of tumor necrosis factor on low-density lipoprotein receptors. Tumor necrosis factor did not stimulate proliferation of HepG2 cells, as judged by cell protein determination or by [3H]thymidine incorporation. In conclusion, this study suggests that tumor necrosis factor up-regulates expression of low-density lipoprotein receptors on HepG2 cells by stimulation of de novo synthesis of receptors, independent of cell growth.