Scavenger Receptor BI (SR-BI) Is Up-regulated in Adrenal Gland in Apolipoprotein A-I and Hepatic Lipase Knock-out Mice as a Response to Depletion of Cholesterol Stores

Abstract

Scavenger receptor BI (SR-BI), a putative high density lipoprotein (HDL) receptor, mediates the selective uptake of HDL cholesteryl ester into cells and is highly expressed in adrenal gland (Acton, S., Rigotti, A., Land-schulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Science 271, 518-520). Apolipoprotein A-I knockout (apoA-I0) mice have decreased HDL cholesterol, depleted adrenal cholesterol stores and impaired cortico-steroid synthesis (Plump, A. S., Erickson, S. K., Weng, W., Partin, J. S., Breslow, J. L., and Williams, D. L. (1996) J. Clin. Invest. 97, 2660-2671). We now show up-regulation of adrenal SR-BI mRNA and protein in apoA-I0 mice, but not in apoA-II0, LDL receptor 0, apoE0, or cholesteryl ester transfer protein transgenic mice. Ad-renal SR-BI mRNA and protein are also increased and cholesterol stores decreased in female mice with knockout of hepatic lipase, an enzyme previously shown to increase selective uptake in cell culture. SR-BI mRNA is increased in stressed wild type mice and in Y1 adrenal cells treated with adrenocorticotropic hormone; the latter effect is inhibited by HDL. These findings provide in vivo evidence showing SR-BI is a functional HDL receptor under feedback control. The action of hepatic lipase on apoA-I-containing lipoproteins may facilitate the SR-BI-mediated uptake of HDL lipid. High density protein (HDL) 1 metabolism plays a pivotal role in cholesterol homeostasis and development of atherosclerosis. Plasma HDL cholesterol levels show a general inverse relationship with coronary heart disease (1). The normal function of HDL and the mechanisms underlying the HDL-coronary heart disease relationship are poorly understood. There is evidence in rodents that HDL provides cholesterol for adrenal steroid hormone synthesis via selective cholesterol uptake, a putative receptor-mediated process for delivery of cholesteryl ester into the cells without degradation of HDL protein (2). Although long suspected, the molecular identification of a functional HDL receptor has proven to be elusive. In a major breakthrough, Acton et al. (3) recently demonstrated that murine scavenger receptor SR-BI, when expressed in transfected cells, binds HDL and mediates selective uptake of HDL cholesteryl ester. SR-BI protein is abundant in adrenal gland, ovary, testis, and, to a lesser extent, liver, precisely the tissues actively involved in selective uptake (2, 4). Therefore, SR-BI appears to be an authentic HDL receptor mediating selective uptake. We now provide in vivo evidence showing that adrenal SR-BI is a functional receptor for HDL under feedback regulation in response to changes of cellular cholesterol stores. EXPERIMENTAL PROCEDURES Animals-All animals used were between 3 and 4 months old. HL0 mice and wild type C57BL/6 mice were purchased from Jackson Laboratory (Maine). HL0 mice backcrossed with C57BL/6 mice were kindly provided by Dr. Nobuyo Maeda, University of North Carolina. ApoA-I0 and ApoA-II0 mice were created by gene targeting in embryonic stem cells and detailed characterization will be presented elsewhere. 2,3 Ribonuclease Protection Assay-Reverse transcription-polymerase chain reaction was used to obtain murine SR-BI cDNA from the adrenal gland. Murine SR-BI and-actin antisense riboprobes were prepared by in vitro transcription using murine SR-BI and-actin cDNA plasmid constructs. The protected hybrid fragments for SR-BI and-actin were 290 and 160 bp, respectively. The RNase protection assay was described in detail previously (5). In brief, 20 g of liver total RNA or 5 g of adrenal gland total RNA were hybridized with 510 5 cpm SR-BI and-actin riboprobes at 48 °C overnight in 30 l of a buffer consisting of 40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, and 80% formamide. The hybridization mixture was digested with 20 units of T 2 ribonuclease at 37 °C for 1 h, extracted with phenol/chloroform, precipitated with eth-anol, and dissolved in 5 l of RNA loading buffer. The protected RNA hybride fragments were resolved on a 5% polyacrylamide/urea gel and subjected to autoradiography. Other Assays and Reagents-Anti-SR-BI antisera were prepared by immunization of rabbits with a recombinant murine SR-BI fragment (amino acid 315-412) that was expressed in a bacterial expression system and purified. Western analysis was performed with the adrenal membrane preparation and equal quantity of membrane protein (50 g of protein/lane) was subjected to 7.5% reducing SDS-polyacrylamide gel. SR-BI protein immunoreactivity was identified at its anthentic molecular size (82 kDa) (3). Tissue cholesterol and cholesteryl ester content were determined as described (6) using chloroform/methanol extraction and cholesterol CII and free cholesterol C kit (Wako, Japan). Cholesteryl ester content was determined by subtracting free cholesterol from total cholesterol. Rat ACTH was purchased from Sigma. Human HDL was prepared by preparative ultracentrifugation between d 1.063 and 1.210 g/ml as described (7). For experiments with murine adrenal Y1 cells, the cells, obtained from ATCC, were maintained in Ham F-12 media plus 20% horse serum. On the day of experiments, the cells were treated for 8 h with or without horse serum. When indicated,