Liver X Receptor (LXR)- Regulation in LXR -Deficient Mice: Implications for Therapeutic Targeting

The nuclear receptors liver X receptor (LXR) LXR and LXR are differentially expressed ligand-activated transcription factors that induce genes controlling cholesterol homeostasis and lipogenesis. Synthetic ligands for both receptor subtypes activate ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol metabolism, increase reverse cholesterol transport, and provide atheroprotection in mice. However, these ligands may also increase hepatic triglyceride (TG) synthesis via a sterol response element binding protein 1c (SREBP-1c)-dependent mechanism through a process reportedly regulated by LXR . We studied pan-LXR / agonists in LXR knockout mice to assess the contribution of LXR to the regulation of selected target genes. In vitro dose-response studies with macrophages from LXR / and / mice confirm an equivalent role for LXR and LXR in the regulation of ABCA1 and SREBP-1c gene expression. Cholesterol-efflux studies verify that LXR can drive apoA1-dependent cholesterol mobilization from macrophages. The in vivo role of LXR in liver was further evaluated by treating LXR / mice with a pan-LXR / agonist. Highdensity lipoprotein (HDL) cholesterol increased without significant changes in plasma TG or very low density lipoprotein. Analysis of hepatic gene expression consistently revealed less activation of ABCA1 and SREBP-1c genes in the liver of LXR null animals than in treated wild-type controls. In addition, hepatic CYP7A1 and several genes involved in fatty acid/TG biosynthesis were not induced. In peripheral tissues from these LXR -null mice, LXR activation increases ABCA1 and SREBP-1c gene expression in a parallel manner. However, putative elevation of SREBP-1c activity in these tissues did not cause hypertriglyceridemia. In summary, selective LXR activation is expected to stimulate ABCA1 gene expression in macrophages, contribute to favorable HDL increases, but circumvent hepatic LXR -dominated lipogenesis. There is great interest in targeting LXR nuclear receptors and their modulation for the treatment of atherosclerosis. These transcription factors play a critical role in the control of cholesterol homeostasis and have been the topic of several recent reviews (Jaye, 2003; Joseph and Tontonoz, 2003; Tontonoz and Mangelsdorf, 2003; Cao et al., 2004). Their therapeutic potential resides in their ability to dramatically upregulate ABCA1 transcription and thereby stimulate cholesterol efflux from macrophages. It has been demonstrated that activation of LXR by cognate ligands promotes apoA1-mediated efflux, and this is believed to be a critical first step for the removal of cholesterol from the actual site of atherogenesis in the vasculature (Costet et al., 2000; Repa et al., 2000b; Schwartz et al., 2000). LXRs behave as cholesterol sensors to stimulate transcription from a number of genes, including ABCA1, ABCG1, apoE, CETP, and LPL, resulting in the coordinate up-regulation of the reverse cholesterol transport (RCT) process. RCT promotes the return of excess cholesterol from peripheral tissues, including arterial lesion sites, to the liver for conversion to bile acids and excretion from the body. As such, the process of RCT plays a central role in maintaining wholebody cholesterol homeostasis. The atheroprotective properThe generation of LXR isoform-deleted mice was supported by grants from the Swedish Science Council and from KaroBio AB. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.022608. ABBREVIATIONS: LXR, liver X receptor; ABCA1, ATP binding cassette transporter A1; VLDL, very low density lipoprotein; CYP7A1, cholesterol 7 hydroxylase; angptl3, angiopoietin-like protein 3; apoCI, apolipoprotein CI; DMEM, Dulbecco’s modified Eagle’s medium; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CETP, cholesteryl ester transfer protein; TG, triglyceride; RCT, reverse cholesterol transport; SCD1, stearoyl CoA desaturase-1; KO, knockout; FAS, fatty acid synthase; WT, wild-type; PCR, polymerase chain reaction; BSA, bovine serum albumin; FBS, fetal bovine serum; SREBP-1c, sterol-response element binding protein; TO901317, N-(2,2,2,-trifluoro-ethyl)-N-[4-(2,2,2,-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide; GW3965, 3-(3-(2-chloro-3-trifluoromethylbenzyl-2,2-diphenylethylamino) proproxy) phenylacetic acid. 0026-895X/06/7004-1340–1349$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 22608/3139560 Mol Pharmacol 70:1340–1349, 2006 Printed in U.S.A. 1340 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from ties of LXR nuclear receptors also include the regulation of key genes involved in inflammation (Joseph et al., 2003) and several intestinal cholesterol transporters (ABCA1, ABCG5, and ABCG8) limiting cholesterol absorption (Berge et al., 2000; Repa et al., 2000a,b). In mice but not humans, LXRs also induce the expression of cholesterol 7 hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis (Peet et al., 1998; Chiang et al., 2001). A potential obstacle in the pharmacologic targeting of nuclear receptors as a general class resides in their ability to regulate or integrate numerous gene responses, some of which may be deleterious. In this case, it is known that synthetic LXR agonists can exhibit the adverse property of increasing lipogenesis (Schultz et al., 2000; Grefhorst et al., 2002; Repa et al., 2002a) through transcriptional activation of sterol response element binding protein 1c (SREBP-1c) (Schultz et al., 2000; Yoshikawa et al., 2001; Grefhorst et al., 2002; Repa et al., 2002a), fatty acid synthase (FAS) (Joseph et al., 2002), angiopoietin-like protein 3 (angptl3) (Inaba et al., 2003) and/or inhibition of Apo AV (Jakel et al., 2004). Although purported to be transient, these effects are cause for concern because triglyceride (TG) elevations are an established independent risk factor for atherosclerotic heart disease (Assmann et al., 1998). For this reason, pharmacological modulators are being sought which separate the favorable LXR antiatherogenic properties from the less favorable lipogenic effects. Several possible approaches for achieving this have been put forward in recent reviews, and one strategy commonly cited is by the selective modulation of LXR isoforms (Jaye, 2003; Joseph and Tontonoz, 2003; Lund et al., 2003; Tontonoz and Mangelsdorf, 2003). The two known receptor subtypes, LXR and , exhibit differential expression patterns and may perform different functional roles. The apparent ubiquitous expression of LXR contrasts with preferential expression of LXR in liver, kidney, macrophages, and intestine. LXR / mice challenged with high-cholesterol diets accumulate hepatic lipid, thus pointing to a dominant role for LXR in liver (Peet et al., 1998; Alberti et al., 2001). Moreover, genetic ablation of LXR impairs CYP7A1 induction and hepatic conversion of cholesterol to bile acids. These studies also suggest that it is primarily the LXR subtype controlling liver lipogenesis though the activation of SREBP-1c transcription (Peet et al., 1998). LXR knockout mice handle excess cholesterol as effectively as wild-type mice (Alberti et al., 2001). However, the LXR subtype has been implicated in control of basal ABCA1 expression in LXR / macrophages and regulation of cholesterol efflux (Repa et al., 2000b). A recent report demonstrates that either receptor can play an atheroprotective role in macrophages and that the combined deficiency of both LXR and LXR is required for foam cell-lipid accumulation in aortic lesions (Schuster et al., 2002). These studies imply that LXR -selective targeting may avoid detrimental lipogenic effects dominated by LXR while achieving beneficial effects from ABCA1 gene activation and increased cholesterol efflux in macrophages. The current studies were undertaken to more definitively characterize the role of the LXR isoform in the regulation of selected LXR target genes and control of lipogenesis. For these studies, LXR / mice were treated with pan-LXR / agonists that have comparable binding activity for and isoforms. Materials and Methods Ligands. LXR agonists, Tularik TO901317 (Repa et al., 2000b; Schultz et al., 2000) and Glaxo GW3965 (Laffitte et al., 2001; Collins et al., 2002) were prepared by following standard chemical syntheses from the published literature. Human LDL was obtained from Wake Forest University, School of Medicine (Wake Forest, NC), and [1,2H(N)]cholesterol was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). In Vivo Studies: Animals and Diet. Mice of wild-type, LXR / , LXR / , and LXR / / genotype have been characterized in detail previously (Alberti et al., 2001; Juvet et al., 2003). Mice used for in vivo experiments and cultured macrophage preparations were Sv129/C57BL/6 hybrids backcrossed on C57BL/6 mice for three generations. Upon receipt, all mice were maintained on a 12-h light/dark cycle and fed a normal chow diet, Rodent Diet 5001 (PMI Nutritionals, St. Louis, MO) ad libitum. Peritoneal macrophages were prepared as described below and represent pools from four to six male mice (25–30 g) from each genotype. Age-matched adult mice (6–8 months) were used for in vivo studies for which ligands were administered once a day in the morning by oral gavage for 3 days. Control animals received vehicle, 1.3% Tween 80/0.25% sodium carboxymethylcellulose. At study termination, mice were fasted for 5 to 6 h, blood was recovered, and plasma was prepared using standard centrifugation techniques. Tissues were collected for RNA preparation and frozen in liquid N2 before storage at 70°C. Animal experiments were approved by the Institutional Animal Care and Use Committee of Wyeth (Collegeville, PA). Murine Peritoneal Macrophage Isolation and Culture. Thioglycollate-elicited peritoneal macrophages were isolated from nonfasted male mice 3 days after peritoneal injection of 4% Brewers thioglycollate media (25 ml/kg) (Joseph et al., 2000). The peritoneal cavity was flushed with 10 ml of ice-cold Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (10% FBS/DMEM), and cells were pelleted from the medium by centrifugation at 1500 rpm for 15 min (4°C). Cells were resuspended in DMEM containing 10% FBS and plated in 96-well plates (4 10 cells/well). Nonadherent cells were removed after 5 h. The media were replaced, and peritoneal macrophages were treated with ligands in DMEM containing 5% lipoprotein-deficient serum (Intracel, Frederick, MD). RNA was isolated after 18 to 20 h of ligand treat-