A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Abstract

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by approximately 31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Figure 1

Strategy for targeted disruption of the SR-BI locus in the mouse (A) and confirmation of the expected null mutation by PCR genotype analysis (B) and immunoblot analysis of liver membranes (C). (A) Restriction map of the genomic DNA surrounding the first coding exon of the murine gene encoding SR-BI. The targeting vector and the predicted structure of the targeted (mutant) allele are shown and described in the text. The locations of the sequences for the PCR primers used to specifically detect either the wild-type (primers 1 and 2) or targeted mutant (primers 1 and 3) alleles are indicated along with the predicted PCR product lengths. TK, herpes simplex thymidine kinase; neo, pol2sneobpA expression cassette, X, XbaI; B, BamHI; S, SacI; “ATG”, codon for the initiator methionine. (B) PCR genotype analysis. Two sets of primer pairs specific for the wild-type (primers 1 and 2) or targeted mutant (primers 1 and 3) alleles (see A) were used to screen genomic DNA by PCR as described. Representative results from individual F₁ wild-type (+/+, lanes 1 and 2), F₁ heterozygous (+/−, lanes 3 and 4), and F₂ homozygous mutant (−/−, lanes 5 and 6) animals are shown. (C) Immunoblot analysis of hepatic membranes (50 μg protein/lane) from unfasted wild-type (F₁ and F₂ generations, lanes 1 and 5), heterozygous (F₁ and F₂ generations, lanes 2 and 4), and homozygous mutant (F₂ generation, lane 3) male mice were performed using polyclonal antipeptide antibodies to SR-BI (≈82 kDa, Upper) or the internal control ɛ-COP (≈36 kDa, Lower), as described. Essentially identical results were obtained using specimens from female mice (data not shown).

Figure 2

FPLC profiles of plasma lipoprotein cholesterol (A) and apolipoproteins (B) for wild-type (srbI^+/+) and heterozygous (srbI^+/−) and homozygous (srbI^−/−) mutant F₂ male mice. (A) The chromatograms represent single analyses of pooled samples (150 μl of plasma from three animals per sample) from 4 to 8 h fasted wild-type (srbI^+/+, open squares), and heterozygous (srbI^+/−, partly filled squares) and homozygous (srbI^−/−, filled squares) mutant mice and are representative of multiple, independent determinations. Approximate positions of VLDL, IDL/LDL, and HDL elution are indicated by brackets and were determined both by analysis of human lipoprotein standards and by previous analysis of lipoproteins in murine plasma (28). (B) Combined immunoblot analysis of fractions 23–38 from the chromatograms shown in A were performed with polyclonal antibodies to apoE, apoA-I, and apoA-II as described. Additional analysis of these and independent chromatograms (data not shown) established that there were no additional peaks containing apoA-I in fractions containing larger lipoproteins (fractions 1–22) and that the only other peak containing a small amount of apoE was in fraction 6, which corresponds to VLDL.