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. 1997 Dec 9;94(25):13600-5.
doi: 10.1073/pnas.94.25.13600.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

R E Temel  1 , B Trigatti, R B DeMattos, S Azhar, M Krieger, D L Williams
Affiliations

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells

R E Temel et al. Proc Natl Acad Sci U S A. .

Abstract

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

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Figures

Figure 1
Figure 1
Western blot analysis of anti-mSR-BI antibodies. Postnuclear supernatant (20 μg protein) from ldlA[mSR-BI] cells (lane 1), and Y1-BS1 cells treated without (lane 2) or with (lane 3) 1–24ACTH were separated by SDS/8% PAGE and transferred to nitrocellulose membranes. The membranes were incubated overnight at 4°C in the presence of either SWB1 nonimmune IgG (A), SWB2 nonimmune IgG (B), 355 anti-mSR-BI IgG (C), or 356 anti-mSR-BI IgG (D) at 4 μg/ml. IgG binding was visualized by enhanced chemiluminescence.
Figure 2
Figure 2
Effects of 356 anti-mSR-BI IgG on DiI uptake from DiI HDL by ldlA[mSR-BI] cells. (A) ldlA[mSR-BI] cells were incubated for 2 hr with DiI-HDL (10 μg protein/ml) in medium containing the indicated concentration of 356 anti-mSR-BI IgG and complementary amounts of nonimmune IgG to give a final IgG concentration of 6 mg/ml. Cells were then washed and processed for fluorescence determination by flow cytometry as described. Samples containing 6 mg/ml nonimmune IgG were taken as the 100% control value (arbitrary scale). (B) The uptake of DiI-HDL in the presence of no IgG (100% value) is shown in comparison with cells incubated with 6 mg/ml nonimmune IgG or with excess unlabeled HDL. Error bars represent the range of duplicate determinations.
Figure 3
Figure 3
Selective CE uptake and cell association of [125I,3H]hHDL3 by Y1-BS1 cells. Y1-BS1 cells were incubated with the indicated concentrations of [125I,3H]hHDL3 for 4 hr, after which the cells were processed to determine selective CE uptake (A) and cell association of HDL apolipoprotein (B). The high-affinity (▴) component for each of these parameters was resolved from the total measured value (•) as described. Error bars represent the range of duplicate determinations.
Figure 4
Figure 4
Effects of 356 anti-mSR-BI IgG on HDL-selective CE uptake and HDL cell association. Y1-BS1 cells were incubated for 2 hr with [125I,3H]hHDL3 (10 μg protein/ml) in medium containing the indicated concentration of 356 anti-mSR-BI IgG and complementary amounts of nonimmune IgG to give a final IgG concentration of 6 mg/ml. Cells were processed to determine HDL-selective CE uptake (A) and cell association of HDL apolipoprotein (B). The 100% of control value in each case refers to samples incubated with 6 mg/ml nonimmune IgG. (A and B) Results for 0.0 mg/ml and 6.0 mg/ml anti-mSR-BI IgG are the means of 22 samples (±SEM) from seven experiments. The results for the intermediate anti-mSR-BI IgG concentrations are the means of eight samples (±SEM) from four experiments. (C) HDL-selective CE uptake (open bars) and cell-associated HDL apolipoprotein (stippled bars) in the presence of no IgG (100% of control value) in comparison with cells incubated with 6 mg/ml nonimmune IgG or with excess unlabeled HDL (500 μg protein/ml). For the no IgG samples, the results are the means of 20 samples (±SEM) from seven experiments. The results for the 6 mg/ml NI (nonimmune) IgG are the means of 22 samples (±SEM) from seven experiments, and the 500 μg/ml cold HDL results are the means of 10 samples (±SEM) from 4 experiments.
Figure 5
Figure 5
Secretion of [3H] steroid by 1–24ACTH-stimulated Y1-BS1 cells incubated with [3H]hHDL3. Y1-BS1 cells were incubated for 24 hr with 25 μg protein/ml [3H]hHDL3 in the presence or absence of 1 mM aminogluthethimide. Steroids were extracted from the medium with CH2Cl2 and separated by HPLC as described. (A and B) Absorbance profile at 240 nm and the radioactivity profile, respectively. Arrows in A indicate the elution position of standards: corticosterone (I), 11β-hydroxyprogesterone (II), 20α-hydroxyprogesterone (III), and progesterone (IV).
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