Hepatocellular Membrane Trafficking of Cholesterol

Yeshiva University, New York NY

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Abstract

DESCRIPTION (provided by applicant): Reverse cholesterol transport is the metabolic pathway for movement of cholesterol from peripheral tissues to liver for secretion into bile. Consistent with their central role in reverse cholesterol transport, high density lipoproteins (HDL) are the principal source of biliary cholesterol. Nevertheless, hepatocellular mechanisms for transport and biliary secretion of HDL-derived cholesterol remain poorly understood. Scavenger receptor class B type I (SR-BI) is highly expressed in liver and mediates uptake of HDL lipids as well as cellular cholesterol efflux. Caveolae are cholesterol-and sphingolipid-rich membrane microdomains, which also participate in influx and efflux of cellular cholesterol. Both caveolin-1, the structural protein of caveolae, and SR-BI are expressed on the sinusoidal and canalicular domains of hepatocyte plasma membranes. Using primary cultures of hepatocytes derived from caveolin-1 transgenic mice and mice with targeted disruption of genes encoding caveolin-1 or SR-BI, we will explore contributions of caveolin-1 and SR-BI to hepatocellular uptake of HDL cholesterol, as well as to efflux of free cholesterol from the plasma membrane. Observations in tissue culture will be correlated with measurements of HDL clearance and biliary lipid rates in vivo. Based upon preliminary experiments, which demonstrate colocalization of fluorescence-labeled HDL with SR-BI and caveolin-1 in early endocytic vesicles, we will also examine a role for caveolae in trafficking of HDL through the hepatocyte. Real time fluorescence microscopy of HuH-7 hepatoma cells, which have been transfected with a green fluorescent protein-caveolin-1 fusion protein, will be employed to characterize uptake of HDL, processing within the endosomal compartment and trafficking to the canalicular membrane. Using an in vitro vesicle motility assay, we will explore a role for microtubules in transport and sorting of vesicles containing HDL, SR-BI and caveolin-1. Hepatocellular secretion of phosphatidylcholines into blood and bile is critical for HDL metabolism and bile formation, respectively. Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein of unknown function that is highly expressed in liver. Using PC-TP knockout mice, we will explore a role for this protein in reverse cholesterol transport. These studies should provide new insights into hepatic HDL metabolism and biliary lipid secretion, and may lead to early interventions in prevention and management of atherosclerosis, as well as cholesterol gallstones.