Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol

Abstract

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high density lipoproteins (HDL) and mediates selective uptake of HDL cholesteryl esters (CE) in transfected cells. To address the physiological role of SR-BI in HDL cholesterol homeostasis, mice were generated bearing an SR-BI promoter mutation that resulted in decreased expression of the receptor in homozygous mutant (designated SR-BI att) mice. Hepatic expression of the receptor was reduced by 53% with a corresponding increase in total plasma cholesterol levels of 50-70% in SR-BI att mice, attributable almost exclusively to elevated plasma HDL. In addition to increased HDL-CE, HDL phospholipids and apo A-1 levels were elevated, and there was an increase in HDL particle size in mutant mice. Metabolic studies using HDL bearing nondegradable radiolabels in both the protein and lipid components demonstrated that reducing hepatic SR-BI expression by half was associated with a decrease of 47% in selective uptake of CE by the liver, and a corresponding reduction of 53% in selective removal of HDL-CE from plasma. Taken together, these findings strongly support a pivotal role for hepatic SR-BI expression in regulating plasma HDL levels and indicate that SR-BI is the major molecule mediating selective CE uptake by the liver. The inverse correlation between plasma HDL levels and atherosclerosis further suggests that SR-BI may influence the development of coronary artery disease.

Figure 1

Disruption of the mouse SR-BI locus. (A) Schematic representation and partial restriction maps of the SR-BI targeting vector, the wild-type locus, and the targeted allele. Open boxes represent SR-BI exons 1 and 2, the hatched boxes indicate the 5′ flanking probe and the 3′ probe, the shaded boxes are the lacZ gene and the PGK-neo expression cassette, and the dashed line is plasmid DNA. The arrows above lacZ and neo indicate the direction of transcription. Double-headed arrows indicated approximate predicted restriction fragment sizes. (B) Autoradiogram of Southern blot analysis of ES cell DNA. Genomic DNA from control (+/+) and targeted (+/−) ES cells was digested with the indicated enzymes and hybridized with radiolabeled 5′ probe, 3′ probe, or exon 1 probe, as indicated. When probed with the 5′ flanking probe, BamHI and PvuII digestion of genomic DNA from targeted ES cells produced the expected wild-type 5- and 7.5-kb bands, respectively, and the predicted 4- and 3.7-kb bands diagnostic of homologous recombination with the vector. Hybridization of these digests with an exon 1 probe revealed novel bands of 5.8 (BamHI) and 6.4 kb (PvuII) in the targeted clone, in addition to the wild-type 5- (BamHI) and 7.5-kb (PvuII) bands. Further analysis with the exon 1 probe demonstrated that the wild-type XbaI-BamHI and SacI bands of 2.8 and 1.6 kb were unaltered in the targeted allele. Lastly, hybridization with the 3′ probe detected a control BglI fragment of >12 kb and a novel 6-kb BglI band in the targeted clone. These data are consistent with homologous insertion of the entire targeting vector via single reciprocal recombination (reviewed in ref. 24) as diagrammed. In addition, typical of such sequence insertion recombination events, a genomic duplication was generated (reviewed in refs. and 26), such that in the targeted locus the targeting vector is bracketed by the 2-kb fragment originating from upstream of exon 1. (C) Autoradiogram of Southern blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) F2 littermates. Genomic DNA was digested with BamHI and hybridized with the 5′ flanking probe.

Figure 2

Immunoblot analysis of SR-BI protein expression in control and homozygous mutant mice. A total of 50 μg of protein from postnuclear supernatants of mSR-BI transfected CHO cells and from liver, spleen, brain, heart, testis, and kidney, and 20 μg from the adrenal gland, of representative control (+/+) and SR-BI att (−/−) mice was analyzed by gel electrophoresis and immunoblotting with a rabbit polyclonal anti-human SR-BI antibody. ∗, 2.5-fold less protein was loaded in this, relative to the other, lanes.

Figure 3

Plasma cholesterol and HDL analysis. (A) FPLC analysis of plasma lipoproteins. Plasma was pooled (n = 4) from homozygous mutant (▪) and control (□) female mice (ages 10, 11.5, and 15.5 weeks, and 11.5 and 15.5 weeks, respectively) and subjected to FPLC Superose-6 gel filtration, and the cholesterol content of the fractions was determined. (B) Nondenaturing PAGE of plasma from control and mutant mice. Ten microliters of plasma from control and SR-BI att male mice were analyzed.

Figure 4

SDS/PAGE of plasma lipoproteins. Lipoprotein fractions were separated by ultracentrifugation of pooled plasma (n = 4) from control and SR-BI att male mice and analyzed on a 4–20% gradient gel.

Figure 5

Plasma decay curves for ¹²⁵I-NMTC/[³H]CEt-labeled HDL. Mice were injected with labeled HDL, blood was collected periodically over 24 h, and plasma content of both tracers was determined as described in Materials and Methods. The values are means ± SEM of each of the n = 4 control and SR-BI att mice.

Figure 6

Uptake of labeled HDL by the liver. Liver FCRs, calculated as described in Materials and Methods, are shown for uptake of labeled protein (¹²⁵I, open bars) and cholesteryl ether (³H, solid bars). The shaded bars represent selective CE uptake (³H − ¹²⁵I). Significance was determined by Student’s one-tailed t test for unpaired data.