Cellular mechanisms regulating CETP mediated selective uptake of HDL derived cholesteryl esters in mammalian cells

Abstract

Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein that mediates the transfer of neutral lipids between lipoproteins. Recently, we have demonstrated a novel role for CETP in directly mediating the selective acquisition of CE from HDL by hepatocytes, indicating a direct and potentially anti-atherogenic function in reverse cholesterol transport. Further studies have been carried out to address the cellular mechanisms of CETP-mediated selective uptake of HDL-CE. Using biochemical plasma membrane isolation followed by detergent extraction and sucrose gradient membrane fractionation, we demonstrate that CETP localizes in the low density, detergent-resistant membrane fractions in both COS-7 and primary murine hepatocytes over-expressing CETP. In an attempt to dissect the intracellular events following the selective uptake of HDL-derived CE mediated by CETP, immunofluorescence confocal microscopy was used. By incubating HeLa cells and primary hepatocytes overexpressing CETP with fluorescently labeled HDL and CE, we demonstrate that CETP colocalizes with both CE and HDL both on the cell surface and intracellularly consistent with internalization of a CETP/HDL complex. We also note that the CETP/HDL complex colocalizes with a subset of early endosomes. The mechanism of internalization is unknown but could involve raft-mediated endocytic route. At 1h post-treatment, HDL and CETP were seen to separate from the early endosomes and segregate in perinuclear structures that are identified as a Rab11 positive endocytic recycling compartment. Based on these findings, we speculate that HDL and CETP may be recycled through a retroendocytic pathway, during which CE is removed from the HDL particle and directed to the lipid droplets for storage. These studies provide new insight into CETP membrane localization and intracellular trafficking, relevant to its role in CE selective uptake.