Characterization of HDL-cholesterol uptake in cells by a novel mechanism

The serum high density lipoprotein (HDL) cholesterol level is an excellent predictor of atherosclerosis and coronary artery disease, which is one of the major causes of death in our society. The protective effect of HDL is thought to be mainly due to its role in `reverse cholesterol transport` by which HDL delivers excess cholesterol from peripheral tissues back to the liver for disposal. In contrast to the low density lipoprotein (LDL) receptor pathway, in which the entire LDL particle is taken up by the cell, HDL delivers only its core lipids to the cells. This uptake process is referred to as selective cholesterol uptake. A few years ago the scavenger receptor class B type I (SR-BI) was described to mediate this well characterized selective uptake of cholesterol. In contrast to the selective uptake pathway in which the HDL particles remains intact, the fate of the holo - HDL particle and its degradation is still enigmatic. The fact that HDL binds to a variety of cells with different degrees of specificity has led to speculations about the existence of several `HDL receptors` and the description of a retrograde HDL transfer process. Up to date no receptors mediating holo-HDL uptake have been identified. Here we propose to characterize the mechanism that enables a line of Chinese hamster ovarian cells (CHO7) to survive at low levels of cholesterol by degrading HDL and delivering its released cholesteryl moiety to the endoplasmic reticulum. In this project we aim to clone the gene(s) involved and to describe in detail this novel uptake pathway for HDL- cholesterol.

The serum high density lipoprotein (HDL) cholesterol level is an excellent predictor of atherosclerosis and coronary artery disease, which is one of the major causes of death in our society. The hallmark of this chronic and progressive disease is the accumulation of cholesterol within the arterial wall. Macrophages are involved in the formation of plaques by acquiring lipids and thereby inducing atherosclerosis. This accumulation of cholesterol is the result of an imbalance between cholesterol delivery and removal. The pathway responsible for the removal of cholesterol from peripheral tissues followed by its transfer via plasma to the liver for either recycling or excretion from the body via bile is called reverse cholesterol transport (RCT). The first step of RCT is cholesterol efflux, the transfer of cholesterol from cells to an extracellular acceptor, mainly HDL. Targeting the expression of specific genes involved in cholesterol flux could prevent the accumulation of excessive cholesterol in the vessel wall, attenuating thus the development of atherosclerotic plaques. In this project we studied the fate of the holo - HDL particles in cells and an unusual HDL degradation in specific specialized cells. First we characterized the mechanism that enables a line of Chinese hamster ovarian cells (CHO7) to survive at low levels of cholesterol by degrading HDL. The delivery of cholesterol to the cell was shown by an increase in the total cholesterol content and the down-regulation of cholesterol responsive genes, like HMG-CoA reductase. Furthermore, HDL derived cholesterol was transported to the endoplasmic reticulum were excess cholesterol was deposited as cholesteryl ester. The degradation of HDL was mediated by a protein attached loosely to the cell surface, possibly a lipase. HDL degradation with similar properties was also seen in placental trophoblast cells. Second, we followed the holo-HDL particle uptake, its intracellular movement and resecretion in CHO cells as well as adrenal and liver cell lines. This HDL particle uptake was facilitated by the Scavenger Receptor Class B Type I. Part of the HDL taken up by the cells was resecreted. This HDL resecretion could mediate cholesterol efflux. Thus, we described a novel pathway of cholesterol efflux in macrophages and hepatocytes that could be possible important for the cholesterol homeostasis.