The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis
2014; Elsevier BV; Volume: 55; Issue: 6 Linguagem: Inglês
10.1194/jlr.m047548
ISSN1539-7262
AutoresTamer Sallam, Ayaka Ito, Xin Rong, Jason Kim, Caroline van Stijn, Brian T. Chamberlain, Michael E. Jung, Lily C. Chao, Marius Jones, Thomas Gilliland, Xiaohui Wu, Grace L. Su, Rajendra K. Tangirala, Peter Tontonoz, Cynthia Hong,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP−/− recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis. The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP−/− recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis. Despite recent advances in our treatment and understanding of its biology, CVD contributes to one in every three deaths (1Go A.S. Mozaffarian Roger D. Benjamin V.L. Berry E.J. Borden J.D. Bravata W.B. Dai D.M. Ford S. Fox E.S. et al.Heart disease and stroke statistics–2013 update: a report from the American Heart Association.Circulation. 2013; 127: e6-e245Crossref PubMed Scopus (4360) Google Scholar, 2Yang Q. Cogswell M.E. Flanders W.D. Hong Y. Zhang Z. Loustalot F. Gillespie C. Merritt R. Hu F.B. Trends in cardiovascular health metrics and associations with all-cause and CVD mortality among US adults.JAMA. 2012; 307: 1273-1283Crossref PubMed Scopus (549) Google Scholar). Additionally, CVD costs more than any other condition with an estimated annual burden of $312 billion, an astonishing figure in light of the growing interest in healthcare utilization and costs (1Go A.S. Mozaffarian Roger D. Benjamin V.L. Berry E.J. Borden J.D. Bravata W.B. Dai D.M. Ford S. 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All animals (C57Bl/6, greater than 10 generations backcrossed) were housed in a temperature-controlled room under a 12 h light/12 h dark cycle and under pathogen-free conditions. LXRα−/−, LXRβ−/−, and LXRαβ−/− mice were originally provided by David Mangelsdorf, University of Texas Southwestern Medical Center, Dallas, TX. LBP−/− mice (C57Bl/6 background) were obtained from Grace Su University of Michigan. Mice were fed either standard chow or Western diet as indicated (21% fat, 0.21% cholesterol; D12079B; Research Diets Inc.). For bone marrow transplantation studies, recipient LDLR−/− mice (11 weeks of age) were lethally irradiated with 900 rads and transplanted with 3 × 106 bone marrow cells from 8-week-old or older donors (WT or LBP−/−) via tail vein injection as previously described (22Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. 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Primary peritoneal macrophages were isolated 4 days after thioglycollate injection and prepared as described (19Bradley M.N. Hong C. Chen M. Joseph S.B. Wilpitz D.C. Wang X. Lusis A.J. Collins A. Hseuh W.A. Collins J.L. et al.Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE.J. Clin. Invest. 2007; 117: 2337-2346Crossref PubMed Scopus (221) Google Scholar). Bone marrow cells were harvested as described and cultured in L929 cell-conditioned media for 7 days to induce differentiation into macrophages (22Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. et al.Identification of macrophage liver X receptors as inhibitors of atherosclerosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 11896-11901Crossref PubMed Scopus (370) Google Scholar). Peritoneal macrophages were incubated in 0.5% FBS in DMEM, with 5 μM simvastatin and 100 μM mevalonic acid. Five to eight hours later, cells were pretreated with DMSO or an appropriate ligand overnight. For inflammation studies, cells were treated with 10 or 100 ng/ml LPS (Axxora, ALX-581-008-L002) for 4 h after ligand stimulation. Mouse primary hepatocytes were isolated as previously described and cultured in William's E medium with 5% FBS (54Rong X. Albert C.J. Hong C. Duerr M.A. Chamberlain B.T. Tarling E.J. Ito A. Gao J. Wang B. Edwards P.A. et al.LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition.Cell Metab. 2013; 18: 685-697Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Stable cell lines were made using pBABE retroviral vectors as described (56Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha.Proc. Natl. Acad. Sci. USA. 2000; 97: 12097-12102Crossref PubMed Scopus (838) Google Scholar). Electrophoretic mobility shift assay (EMSA) was performed as previously described (57Marathe C. Bradley M.N. Hong C. Lopez F. Ruiz de Galarreta C.M. Tontonoz P. Castrillo A. The arginase II gene is an anti-inflammatory target of liver X receptor in macrophages.J. Biol. Chem. 2006; 281: 32197-32206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, Klenow enzyme buffer system (Promega) was used for labeling and annealing oligonucleotides. TNT quick coupled transcription/translation system (Promega) was used for in vitro translation of RXRα and LXRα. The translated protein products were combined and incubated at room temperature for 10 min with labeled DNA and 100 M NaCl, 1 mM EDTA, 20 mM HEPES, 5% glycerol, 0.01% Nonidet P-40, and 2 μg/μl poly(dI:dC). Protein-DNA complexes were electrophoresed on a polyacrylamide gel and visualized by autoradiography. The following antibodies were used for immunohistochemistry: CD68 (MCA1957GA, AbD) 1:400 with secondary antibody biotin-SP-conjugated AffiniPure goat anti-rat IgG (H+L) (Jackson Laboratories) 1:1,000 and α-smooth actin rabbit monoclonal (E184) 1:100 with secondary antibody biotinylated goat anti-rabbit IgG (Vector Laboratories) 1:200. For immunoblot analysis the following antibodies were used: ABCA1 (Novus) 1:1,000, COX-2 (Santa Cruz) 1:200, LDLR (Cayman Chemicals) 1:1,000, and actin (Sigma) 1:10,000. Assays were performed as previously described (55Hong C. Bradley M.N. Rong X. Wang X. Wagner A. Grijalva V. Castellani L.W. Salazar J. Realegeno S. Boyadjian R. et al.LXRα is uniquely required for maximal reverse cholesterol transport and atheroprotection in ApoE-deficient mice.
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