Abstract
The scavenger receptor BI (SR-BI) is highly expressed in hepatocytes, where it mediates the uptake of lipoprotein cholesterol, promotes the secretion of cholesterol into bile, and protects against atherosclerosis. Despite a strong correlation between the hepatic expression of SR-BI and biliary cholesterol secretion, little is known about SR-BI trafficking in response to changes in sterol availability. Using a well characterized polarized hepatocyte cell model, WIF-B, we determine that in cholesterol-depleted cells, SR-BI is extensively located on the basolateral surface, where it can access circulating lipoproteins. However, in response to cholesterol loading, SR-BI undergoes a slow transcytosis to the apical bile canaliculus independently of lipoprotein binding and new protein synthesis. In cholesterol-replete WIF-B cells, SR-BI that resides on the canalicular membrane is dynamically associated with defined microdomains and does not rapidly recycle to and from the subapical or basolateral regions. Taken together, these data demonstrate that hepatic SR-BI transcytosis is regulated by cholesterol and suggest that SR-BI has a stationary function on the bile canaliculus.
Highlights
scavenger receptor BI (SR-BI) is a two-transmembrane domain cell surface glycoprotein with short intracellular N- and C-terminal domains [3]
Using both fluorescence and electron microscopy, we demonstrate that SR-BI localizes to the bile canaliculus and describe the canalicular microenvironment in which SR-BI resides
We find that cholesterol loading promotes SR-BI transcytosis from the basolateral membrane to the bile canaliculus
Summary
Cells and Reagents—WIF-B cells were a kind gift from Dr Ann Hubbard (The Johns Hopkins University, Baltimore, MD). Immunofluorescence—Differentiated WIF-B cells were washed two times quickly with PBS and incubated with 200 l of 3.3% paraformaldehyde for 10 min. The canalicular localization of SR-BI-YFP was determined by estimating the percentage of the BC surface area where SR-BI-YFP and MDR1 colocalized. This was achieved by individually analyzing each BC through multiple z planes. C and D, WIF-B cells infected with SR-BI-YFP (green) were labeled with an antibody against MDR1 (red). Processed using Advance Microscope Technology soft- BI-YFP and MDR1 compared with 7.1 Ϯ 2.6% colocalization ware and Adobe Photoshop
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