Abstract
SR-BI is a cell surface HDL receptor that mediates selective uptake of the lipid cargo of HDL, an important process in hepatocytes, driving reverse cholesterol transport from cells in the artery wall. To facilitate examination of factors that modulate SR-BI activity in hepatocytes, we have generated fluorescent protein-tagged versions of SR-BI that allow for facile monitoring of SR-BI protein levels and distribution in transfected cells. We show that deletion of the C-terminal cytosolic tail does not affect the distribution of SR-BI in HepG2 cells, nor is the C-terminal cytosolic tail required for SR-BI-mediated uptake of HDL lipids. We also demonstrate that the phorbol ester, PMA, increased, while protein kinase C inhibitors reduced SR-BI-mediated HDL lipid uptake in HepG2 cells. These data suggest that protein kinase C may modulate selective uptake of HDL lipids including cholesterol in hepatocytes, thereby influencing hepatic HDL cholesterol clearance and reverse cholesterol transport.
Highlights
High-density lipoproteins (HDL) mediate reverse cholesterol transport from cells in the artery wall to the liver where cholesterol and cholesteryl ester are taken up by the scavenger receptor class B type 1 via a process known as selective lipid uptake [1]
Lysates from HepG2[enhanced green fluorescent protein (eGFP)-SR-BI-ΔC] cells contained an eGFP immunoreactive band that migrated slightly faster than that in HepG2[eGFP-SR-BI] lysates, consistent with the expected size of eGFP fused to SR-BI lacking the C-terminal cytoplasmic tail. β-actin was used as a control to demonstrate equal loading across all lanes
We have utilized an approach in which SR-BI protein levels, distribution, and activity can be monitored in HepG2 cells in order to examine factors that may influence SR-BI function in hepatocytes and the activity of Reverse cholesterol transport (RCT)
Summary
High-density lipoproteins (HDL) mediate reverse cholesterol transport from cells in the artery wall to the liver where cholesterol and cholesteryl ester are taken up by the scavenger receptor class B type 1 via a process known as selective lipid uptake [1]. This is the uptake of the lipid components of the HDL particle without the net internalization and degradation of the particle itself [2]. Knocking out SR-BI expression in mice results in impaired RCT due to a lack of expression of SR-BI in livers [20] This leads to the appearance of unusually large, cholesterol laden HDL particles because cholesterol cannot be cleared from HDL by selective uptake. On the other hand, overexpressing SR-BI in livers of mice results in increased
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