Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL

Margarita De La Llera-Moya,Margery A Connelly,Denise Drazul,Seth M Klein,Elda Favari,Patricia G Yancey,David L Williams,George H Rothblat

doi:10.1016/s0022-2275(20)31525-x

Margarita De La Llera-Moya, Margery A Connelly + Show 6 more

Open Access

https://doi.org/10.1016/s0022-2275(20)31525-x

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Abstract

Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.

Highlights

As expected, when COS-7 cells were incubated for 24 h with medium containing increasingly lower serum concentrations, a reciprocal increase in HMG-CoA reductase (HMGR) activity was obtained in both control and scavenger receptor class B type I (SR-BI)-expressing cells (Fig. 1), signifying a drop in cell cholesterol content
The increase in enzyme activity was most pronounced when COS-7 cells were exposed to lipoprotein-deficient serum (LPDS), and HMGR activity measured in these cells was significantly greater than the activity measured in cells exposed to 5% calf serum (CS) in both control and SR-BI cells
It is hypothesized that, to the LDL receptor, SR-BI must affect cell cholesterol balance, little is known about the effect that SR-BI has on cellular cholesterol homeostasis

Summary

MATERIALS AND METHODS

Human serum was obtained by the Lipoprotein Core Laboratory under an approved protocol and with approved consent from healthy, normolipemic volunteers. Transfected SR-BI-expressing clones were obtained fro m COS-7 cells transfected as described above with pcDNA 3(SR-BI). Transfected WI38-VA13 human fibroblastic cells were obtained after transfection with the same SR-BI-expressing plasmid, using geneticin selection (800 ␮g/ml). The reaction product obtained was corrected for recover y and enzyme activity was expressed as units (picomoles of mevalonic acid per minute per milligram cell protein). Cell cholesterol content was measured in aliquots of 2-propanol extracts from monolayers, using GLC as previously described [15]. Cell cholesterol efflux to HDL3 was measured as previously described [8]. Cells plated in multiwell plates were prelabeled by incubation for 24 – 48 h in growth medium containing serum labeled with [1,2- 3H]cholesterol. Transfected COS-7 clones were characterized for SR-BI expression by measuring the specific binding of HDL3 labeled with 125I-iodine. All statistical analyses were done using GraphPad (San Diego, CA) Prism

RESULTS

Methods

DISCUSSION