HDL RECEPTOR-MEDIATED CHOLESTEROL EFFLUX FROM CELLS AND ITS REGULATTION

HDL RECEPTOR-MEDIATED CHOLESTEROL EFFLUX FROM CELLS AND ITS REGULATTION

HD particles can nonspecifically pick up cholesterol from the plasma membrane by desorption, but specific high affinity interaction of particles containing intact apo AI with an HDL receptor protein (110 kDA) on the cell surface is required to signal cholesterol translocation from intracellular compartments to the cell surface, thereby enhancing cholesterol efflux (1). Once at the cell surface, cholesterol is removed from cells by appropriate acceptor particles including, but not limited to, HDL. Treatment of HDL particles with trypsin or tetranitromethane will destroy the ability of these particles to signal cholesterol translocation; however, the ability of trypsinized HDL to accept cholesterol from plasma membrane sterol is preserved (2). Thus, cells excrete intracellular cholesterol by a pathway that involves two major steps: the translocation of cholesterol from intracellular pools to the plasma membrane, and the removal of membrane­ associated cholesterol from cells by exogenous acceptors. It is only the translocation step of this pathway that requires receptor binding of HDL and this is the step that is regulated.

One of the major signals promoting intracellular translocation of sterol to the plasma membrane appears to involve protein kinase-C (3). Treatment of cells with protein kinase activators (phorbol ester, diacylglycerol, calcium ionophore) will mimic the action of HDL, while use of protein kinase-C inhibitors (e.g., sphingosine) will block this effect. Direct measurements of protein kinase-C have shown that HDL binding to cells will activate the enzyme. The nature of the proteins phosphorylated by protein kinase-C and the mechanisms of sterol translocation from intracellular sites remain to be elucidated.

The number of HDL receptors on the cell surface appears to be regulated by cell cholesterol content and their proliferative state. HDL binding and HDL receptor-mediated sterol efflux from cells (fibroblasts, smooth muscle cells, endothelial cells, monocyte-derived macrophages) are up-regulated by an increase in cellular unesterified cholesterol, whether added from exogenous sources (solubilized cholesterol, LDL cholesterol or acetyl LDL cholesterol to cells with scavenger receptors) or endogenous sources (inhibition of esterification with an ACAT inhibitor) (4) (5) (6) Modification of HDL apoproteins with tetranitromethane or treatment with proteases will block HDL binding and subsequent sterol translocation (1) (7).

Distinct cellular pools of cholesterol can be differentially radio­ labeled. The intracellular pool of cholesterol can be radiolabeled by incubating cholesterol-loaded cells with tritiated mevalonic acid at 1s•c. With this technique most of the label remains with newly synthesized intracellular sterol (1). The plasma membrane compartment can be labelled by brief incubation at 37•c with tritiated cholesterol. The specific translocation of intracellular cholesterol to the plasma membrane can be measured by washing and fixing cells and then incubating them with cholesterol oxidase, an enzyme that reacts only with sterol in accessible domains in the plasma membrane. Thus, cholesterol oxidase-sensitive sterol is presumed to be at the plasma membrane while cholesterol oxidase-resistant sterol is presumed to be in an intracellular compartment.

Since the growth state of cells influences cell cholesterol homeostasis, the question of whether alteration of cell proliferation would regulate HDL receptor activity was tested. Cholesterol-loaded fibroblasts were incubated 24 to 48 hours with growth factors including platelet-derived growth factor, insulin and IGF-1. All mitogens tested reduced HDL binding in a dose related manner in association with increased DNA synthesis (8) (9). HD ­ mediated efflux of intracellular sterols was diminished by growth factors in parallel with decreased HDL binding. Decreased HDL receptor activity was associated with reciprocally increased LDL receptor activity. Thus, both mechanisms responsive to growth factors appear to be designed to make more cell cholesterol available for new membrane synthesis.

Conversely, growth inhibition increased HDL receptor activity. Both HDL binding and HDL mediated efflux of intracellular sterols from cells were increased in the presence of the growth inhibitor gamma interferon (10). Receptor up-regulation was associated with increased HDL receptor number (by kinetic analysis) and increased binding (by ligand blot) to the 110 kilodalton HDL receptor protein. Thus, by altering intracellular cholesterol levels in regulatory pools, changes in the proliferative state of cells can modulate HDL receptor activity to finely regulate cell cholesterol homeostasis (Table 1). Mitogens, by down-regulating HDL receptor-mediated cholesterol efflux from cells, could contribute to arterial cell cholesterol accumulation.

The coupling of receptor binding of HDL to the cellular translocation pathway provides an efficient means for cells to protect themselves from accumulati n of unesterified cholesterol. In the absence of HDL particles, excess cholesterol that accumulates in cells by any of a variety of delivery pathways is esterified and stored as esterified cholesterol in lipid droplets. When HDL receptors become occupied with lipoprotein particles, cells receive a signal that sterol acceptors are present in the extracellular fluid, and excess intracellular cholesterol is diverted from the microsomal esterifying enzyme to the plasma membrane for removal. By this process, HDL depletes cells of cholesterol and prevents the formation of cholesterol ester-rich foam cells. This receptor-mediated sterol translocation pathway may be the biochemical mechanism that underlies the apparent anti-atherogenic effect of HDL.

References

1) Slotte JP, Oram JF, and Bierman EL, 1987, Binding of high density lipo­ proteins to cell receptors promotes translocation of cholesterol from intra-cellular membranes to the cell surface. J. Biol. Chern. 262:12904- 12907.

2) Oram JF, Johnson CJ, Brown TA, 1987, Interaction of high density lipoprotein with its receptor on cultured fibroblasts in macrophages: Evidence for reversible binding at the cell surface without internalization. J. Biol. Chern. 262:2405-2410.

3) Mendez, AJ, Oram JF, and Bierman EL, 1989, Sphingosine inhibits HDL­ mediated efflux of intracellular sterols. Arteriosclerosis 9:720a.

4) Oram JF, Brinton EA, Bierman EL, 1983, Regulation of high-density lipoprotein receptor activity in cultured human skin fibroblasts and human arterial smooth muscle cells. J. Clin. Invest. 72:1611-1621.

5) Brinton EA, Kenagy R, Oram JF, Bierman EL, 1985, Regulation of high density lipoprotein binding activity of aortic endothelial cells by treatment with acetylated low density lipoprotein. Arteriosclerosis 5:329-335.

6) Aviram M, Bierman EL, Oram JF, 1989, High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages. J. Lipid Res. 30:65-76.

7) Brinton EA, Oram JF, Chen C-H, Albers JJ, Bierman EL, 1986, Binding of high-density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues. J. Biol. Chern. 261:491- 503.

8) Oppenheimer MJ, Oram JF, Bierman EL, 1987, Downregulation of high density lipoprotein receptor activity of cultured fibroblasts by platelet-derived growth factor. Arteriosclerosis 7:325-332.

9) Oppenheimer MJ, Sundquist K, Bierman EL, 1989, Downregulation of high density lipoprotein receptor activity in human fibroblasts by insulin and IGF-I. Diabetes 38:117-122.

10) Oppenheimer MJ, Oram JF, Bierman EL, 1988, Up-regulation of high density lipoprotein receptor activity by 1-Interferon associated with inhibition of cell proliferation. J. Biol. Chern. 263:19318-19323.