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Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages

2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês

10.1194/jlr.m300100-jlr200

ISSN

1539-7262

Autores

Weerapan Khovidhunkit, Arthur H. Moser, Judy K. Shigenaga, Carl Grünfeld, Kenneth R. Feingold,

Tópico(s)

Nuclear Receptors and Signaling

Resumo

Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells.The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis. Several of the ATP binding cassette (ABC) transporters have recently been shown to play important roles in reverse cholesterol transport (RCT) and prevention of atherosclerosis. In the liver, ABCG5 and ABCG8 have been proposed to efflux sterols into the bile for excretion. ABCG5 and ABCG8 also limit absorption of dietary cholesterol and plant sterols in the intestine. In macrophages, ABCA1 and ABCG1 mediate cholesterol removal from these cells to HDL. Many of these ABC transporters are regulated by the liver X receptor (LXR). We have previously shown that endotoxin (lipopolysaccharide) down-regulates LXR in rodent liver. In the present study, we examined the in vivo and in vitro regulation of these ABC transporters by endotoxin. We found that endotoxin significantly decreased mRNA levels of ABCG5 and ABCG8 in the liver, but not in the small intestine. When endotoxin or cytokines (tumor necrosis factor and interleukin-1) were incubated with J774 murine macrophages, the mRNA levels of ABCA1 were decreased. This effect was rapid and sustained, and was associated with a reduction in ABCA1 protein levels. Endotoxin and cytokines also decreased ABCG1 mRNA levels in J774 cells. Although LXR is a positive regulator of ABCA1 and ABCG1, we did not observe a reduction in protein levels of LXR or in binding of nuclear proteins to an LXR response element in J774 cells. The decrease in ABCG5 and ABCG8 levels in the liver as well as a reduction in ABCA1 and ABCG1 in macrophages during the host response to infection and inflammation coupled with other previously described changes in the RCT pathway may aggravate atherosclerosis. In epidemiological studies, plasma levels of HDL cholesterol are inversely correlated with the risk of coronary artery disease; therefore, HDL is postulated to protect against atherosclerosis (1Gordon D.J. Rifkind B.M. High-density lipoprotein—the clinical implications of recent studies.N. Engl. J. Med. 1989; 321: 1311-1316Crossref PubMed Scopus (1413) Google Scholar). Reverse cholesterol transport (RCT) is an HDL-mediated pathway by which cholesterol is removed from peripheral cells and transported to the liver for excretion and/or catabolism (2Tall A.R. Plasma high density lipoproteins. Metabolism and relationship to atherogenesis.J. Clin. Invest. 1990; 86: 379-384Crossref PubMed Scopus (586) Google Scholar, 3Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). Cholesterol efflux, the first step of RCT, begins when HDL or its major apolipoprotein, apoA-I, accepts cholesterol from cells. Cholesterol on HDL is returned to the liver by several routes. Once delivered to the liver, most cholesterol is metabolized by a series of hepatic enzymes into bile acids. The remaining cholesterol is excreted directly into the bile, along with bile acids. Accumulating evidence has suggested a possible relationship between atherosclerosis and chronic infections and inflammatory diseases (4Libby P. Egan D. Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research.Circulation. 1997; 96: 4095-4103Crossref PubMed Scopus (494) Google Scholar). How infections and inflammatory states, especially those outside the arterial wall, can promote atherosclerosis is not clear. However, it is known that the acute-phase response (APR) is induced during infection and inflammation. During the APR, multiple alterations in lipid and lipoprotein metabolism occur (5Khovidhunkit W. Memon R.A. Feingold K.R. Grunfeld C. Infection and inflammation-induced proatherogenic changes of lipoproteins.J. Infect. Dis. 2000; 181: 462-472Crossref Scopus (333) Google Scholar). Plasma triglyceride levels increase, and there is an increase in small, dense LDL (6Feingold K.R. Krauss R.M. Pang M. 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Second, we and others have shown that levels of several proteins involved in RCT pathway decrease, including lecithin:cholesterol acyltransferase, cholesterol ester transfer protein, phospholipid transfer protein, hepatic lipase, and scavenger receptor class B type I (8Ettinger W.H. Miller L.D. Albers J.J. Smith T.K. Parks J.S. Lipopolysaccharide and tumor necrosis factor cause a fall in plasma concentration of lecithin:cholesterol acyltransferase in cynomolgus monkeys.J. Lipid Res. 1990; 31: 1099-1107Abstract Full Text PDF PubMed Google Scholar, 9Ly H. Francone O.L. Fielding C.J. Shigenaga J.K. Moser A.H. Grunfeld C. Feingold K.R. Endotoxin and TNF lead to reduced plasma LCAT activity and decreased hepatic LCAT mRNA levels in Syrian hamsters.J. Lipid Res. 1995; 36: 1254-1263Abstract Full Text PDF PubMed Google Scholar, 10Masucci-Magoulas L. Moulin P. Jiang X.C. Richardson H. Walsh A. Breslow J.L. Tall A. Decreased cholesteryl ester transfer protein (CETP) mRNA and protein and increased high density lipoprotein following lipopolysaccharide administration in human CETP transgenic mice.J. Clin. Invest. 1995; 95: 1587-1594Crossref PubMed Scopus (74) Google Scholar, 11Hardardóttir I. Moser A.H. Fuller J. Fielding C. Feingold K. Grünfeld C. Endotoxin and cytokines decrease serum levels and extra hepatic protein and mRNA levels of cholesteryl ester transfer protein in syrian hamsters.J. Clin. Invest. 1996; 97: 2585-2592Crossref PubMed Scopus (69) Google Scholar, 12Jiang X.C. Bruce C. Regulation of murine plasma phospholipid transfer protein activity and mRNA levels by lipopolysaccharide and high cholesterol diet.J. Biol. Chem. 1995; 270: 17133-17138Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 13Feingold K.R. Memon R.A. Moser A.H. Shigenaga J.K. Grunfeld C. Endotoxin and interleukin-1 decrease hepatic lipase mRNA levels.Atherosclerosis. 1999; 142: 379-387Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Khovidhunkit W. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines.J. Lipid Res. 2001; 42: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar). Third, there is an impairment of both cholesterol efflux from cells and cholesterol ester uptake into the liver (14Khovidhunkit W. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines.J. Lipid Res. 2001; 42: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar, 15Khovidhunkit W. Shigenaga J.K. Moser A.H. Feingold K.R. Grunfeld C. Cholesterol efflux by acute-phase high density lipoprotein. Role of lecithin:cholesterol acyltransferase.J. Lipid Res. 2001; 42: 967-975Abstract Full Text Full Text PDF PubMed Google Scholar, 16Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Role of serum amyloid A during metabolism of acute-phase HDL by macrophages.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 763-772Crossref PubMed Scopus (213) Google Scholar, 17Artl A. Marsche G. Pussinen P. Knipping G. Sattler W. Malle E. Impaired capacity of acute-phase high density lipoprotein particles to deliver cholesteryl ester to the human HUH-7 hepatoma cell line.Int. J. Biochem. Cell Biol. 2002; 34: 370-381Crossref PubMed Scopus (27) Google Scholar). Fourth, a series of enzymes that catabolize cholesterol into bile acids, and several proteins in the bile acid transport pathway, are down-regulated (18Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. Endotoxin, TNF, and IL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels and activity.J. Lipid Res. 1996; 37: 223-228Abstract Full Text PDF PubMed Google Scholar, 19Memon R.A. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. In vivo and in vitro regulation of sterol 27-hydroxylase in the liver during the acute phase response. potential role of hepatocyte nuclear factor-1.J. Biol. Chem. 2001; 276: 30118-30126Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20Trauner M. Arrese M. Lee H. Boyer J.L. Karpen S.J. Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors.J. Clin. Invest. 1998; 101: 2092-2100Crossref PubMed Scopus (228) Google Scholar, 21Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response.Biochem. Biophys. Res. Commun. 2002; 293: 145-149Crossref PubMed Scopus (171) Google Scholar). Collectively, several steps in RCT are impaired during the APR that may contribute to the increased risk of atherosclerosis. Recently, several key proteins in the RCT pathway have been discovered, a number of which are members of the ATP-binding cassette (ABC) transporter superfamily (22Schmitz G. Kaminski W.E. Orso E. ABC transporters in cellular lipid trafficking.Curr. Opin. Lipidol. 2000; 11: 493-501Crossref PubMed Scopus (116) Google Scholar). ABCA1 and ABCG1 are ABC transporters that are involved in the movement of cholesterol from cells to HDL and its apolipoproteins (23Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar, 24Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.J. Biol. Chem. 2000; 275: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 25Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 26Attie A.D. Kastelein J.P. Hayden M.R. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis.J. Lipid Res. 2001; 42: 1717-1726Abstract Full Text Full Text PDF PubMed Google Scholar, 27Oram J.F. Lawn R.M. ABCA1. The gatekeeper for eliminating excess tissue cholesterol.J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar, 28Santamarina-Fojo S. Remaley A.T. Neufeld E.B. Brewer Jr., H.B. Regulation and intracellular trafficking of the ABCA1 transporter.J. Lipid Res. 2001; 42: 1339-1345Abstract Full Text Full Text PDF PubMed Google Scholar). ABCG5 and ABCG8 regulate sterol absorption from the small intestine and its excretion from the liver into the bile (29Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1361) Google Scholar, 30Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption.Nat. Genet. 2001; 27: 79-83Crossref PubMed Scopus (0) Google Scholar, 31Yu L. Hammer R.E. Li-Hawkins J. Von Bergmann K. Lutjohann D. Cohen J.C. Hobbs H.H. Berge K.E. Horton J.D. Graf G.A. Li W.P. Gerard R.D. Gelissen I. White A. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16237-16242Crossref PubMed Scopus (610) Google Scholar, 32Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Graf G.A. Li W.P. Gerard R.D. Gelissen I. White A. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (612) Google Scholar). Identification of factors that regulate ABC transporters should provide insights into the mechanisms by which RCT is affected in physiological and pathological states. Previously, our laboratory has shown that in rodents, hepatic mRNA levels of liver X receptor (LXR) and retinoid X receptor (RXR) were rapidly decreased in response to endotoxin (33Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. The acute phase response is associated with retinoid X receptor repression in rodent liver.J. Biol. Chem. 2000; 275: 16390-16399Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). LXR is a nuclear hormone transcription factor that heterodimerizes with RXR and with activation increases the expression of ABCA1, ABCG1, ABCG5, and ABCG8 (25Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport.Proc. Natl. Acad. Sci. USA. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 29Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1361) Google Scholar, 34Venkateswaran 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 (848) Google Scholar, 35Schwartz K. Lawn R.M. Wade D.P. ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR.Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (376) Google Scholar, 36Costet P. Luo Y. Wang N. Tall A.R. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 37Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 38Repa J.J. Berge K.E. Pomajzl C. Richardson J.A. Hobbs H. Mangelsdorf D.J. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta.J. Biol. Chem. 2002; 277: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). We therefore hypothesized that endotoxin treatment would decrease these ABC transporters in the liver. In the present study, we report that endotoxin decreases mRNA levels of ABCG5 and ABCG8 in mouse liver. In J774 murine macrophages, endotoxin and cytokines decrease levels of ABCA1 and ABCG1. However, there was no reduction in levels of LXR or in binding of nuclear proteins to an LXR response element in macrophages. Thus, while LXR may mediate the decrease in these proteins in the liver, these data demonstrate that, in macrophages, the effects of endotoxin on the transporters may not be mediated through LXR. Endotoxin [lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5] was purchased from Difco Laboratories (Detroit, MI). Recombinant tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) were purchased from R and D Systems (Minneapolis, MN). Polyclonal antibody against ABCA1 was purchased from Novus Biologicals (Littleton, CO). Antibodies against LXRα and LXRβ were purchased from Affinity Bioreagents (Golden, CO). Antibodies against RXRα, RXRβ, and RXRγ were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). 8-(4-Chlorophenylthio) adenosine 3′, 5′-cyclic monophosphate (cpt-cAMP), 22(R)-hydroxycholesterol, and all other chemicals were obtained from Sigma (St. Louis, MO). Supplies for immunoblot analysis were purchased from Amersham Biosciences (Piscataway, NJ). C57BL/6 mice (∼6–8 weeks of age) were obtained from Jackson Laboratory (Bar Harbor, ME) and provided with rodent chow and water ad libitum. Animals were injected intraperitoneally with indicated doses of endotoxin (0.1–100 μg/mouse), whereas control animals were injected with normal saline. The highest dose of endotoxin used in our study (100 μg/animal) was able to induce the APR but is far below the lethal dose (LD50 ∼5 mg/100 g body weight) required to cause death in rodents in our laboratory. Because endotoxin can cause anorexia, food was withdrawn from endotoxin-injected mice and control mice after the injection. At indicated time points, the animals were euthanized using halothane, and the tissue was excised and stored at −80°C. The animal procedures were approved by the Animal Studies Subcommittee of the San Francisco Veterans Affairs Medical Center, and were performed in accordance with the guidelines. J774 murine macrophages were obtained from the American Type Culture Collection and maintained in minimum essential media supplemented with 10% fetal bovine serum under 5% CO2. In some experiments, cells were incubated with 0.3 mM cpt-cAMP or 10 μM 22(R)-hydroxycholesterol before being treated with endotoxin or cytokines. Endotoxin or cytokines was incubated with cells in media containing human serum albumin in the absence of serum, and at indicated time points, cells were washed and harvested. Isolation of RNA and RNA blot analyses were performed as previously described (14Khovidhunkit W. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines.J. Lipid Res. 2001; 42: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar). Total or poly(A)+ RNA was quantified by measuring absorption at 260 nm, and equal amounts of RNA were loaded on 1% agarose-formaldehyde gels and electrophoresed. The uniformity of sample applications was checked by UV visualization of the acridine orange-stained gels before transfer to Nytran membranes. Because endotoxin increased hepatic mRNA levels of actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin in rodent liver (39Feingold K.R. Hardardottir I. Memon R. Krul E.J. Moser A.H. Taylor J.M. Grunfeld C. Effect of endotoxin on cholesterol biosynthesis and distribution in serum lipoproteins in Syrian hamsters.J. Lipid Res. 1993; 34: 2147-2158Abstract Full Text PDF PubMed Google Scholar, 40Morrow J.F. Stearman R.S. Peltzman C.G. Potter D.A. Induction of hepatic synthesis of serum amyloid A protein and actin.Proc. Natl. Acad. Sci. USA. 1981; 78: 4718-4722Crossref PubMed Scopus (125) Google Scholar, 41Memon R.A. Feingold K.R. Moser A.H. Fuller J. Grunfeld C. Regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines.Am. J. Physiol. 1998; 274: E210-E217PubMed Google Scholar), the mRNA levels of actin, GAPDH, and cyclophilin, which are widely used for normalizing data, cannot be used to study endotoxin regulation of proteins in the mouse liver. However, the differing directions of changes in mRNA levels (increased for some proteins, and decreased for some proteins), the magnitude of alterations (8-fold increase or up to 75% decrease), and the relatively small standard error of the mean (SEM) make it unlikely that the changes observed were due to unequal loading of mRNA. RNA blots were hybridized with 32P-labeled cDNA probes generated by RT-PCR from the mouse liver (ABCG5, ABCG8, and ABCA1) or J774 murine macrophages (ABCG1) using the following primers: ABCG5 upper primer, 5′-TGC CCT TTC TGA GTC CAG AG-3′ and lower primer, 5′-GTG CTC TTT CAA TGT TCT CCA G-3′; ABCG8 upper primer, 5′-ATG AGC TGG AAG ACG GGC TG-3′ and lower primer, 5′-GCC AGT GAG AGC AAG GCT GA-3′; ABCA1 upper primer, 5′-TCT CTG CTA TCT CCA ACC TCA TC-3′ and lower primer, 5′-ACG TCT TCA CCA GGT AAT CTG AA-3′; ABCG1 upper primer, 5′-GAA GAC CTG CAC TGC GAC ATC-3′ and lower primer, 5′-GTT GCA TTG CGT TGC GTT AGT C-3′. After washing, the blots were exposed to X-ray films for various durations to ensure that measurements were done on the linear portion of the curve, and the bands were quantified using the Bio-Rad imaging densitometer. J774 cells were scraped into cold PBS and centrifuged to get cell pellets. Lysis buffer containing 10 mM HEPES (pH 7.9), 0.5% Nonidet P40, 1.5 mM MgCl2, 0.2 M sucrose, 10 mM KCl, 0.5 mM dithiothreitol, and 1% (v/v) protease inhibitor cocktail (Sigma) was incubated with cells for 5 min at 4°C. The supernatant was collected after centrifugation and the protein concentrations were measured. For immunoblot analysis of LXR and RXR, nuclear proteins were isolated as previously described (33Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. The acute phase response is associated with retinoid X receptor repression in rodent liver.J. Biol. Chem. 2000; 275: 16390-16399Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Proteins were resolved on polyacrylamide gels and transferred to Hybond-P PVDF membrane as previously described (14Khovidhunkit W. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines.J. Lipid Res. 2001; 42: 1636-1644Abstract Full Text Full Text PDF PubMed Google Scholar). For immunodetection, the blots were blocked in PBS/0.1% Tween containing 5% nonfat dry milk before incubation with a primary antibody and a secondary antibody conjugated with horseradish peroxidase. The blots were visualized using the ECL Plus chemiluminescence detection system and subjected to autoradiography. Quantification of the signals was performed by densitometry. Nuclear extracts were prepared as described above, and an electrophoretic mobility shift assay was performed as previously described (33Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. The acute phase response is associated with retinoid X receptor repression in rodent liver.J. Biol. Chem. 2000; 275: 16390-16399Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The oligonucleotides corresponding to the LXR response element of ABCA1 5′GCG CAG AGG TTA CTA TCG GTC AAA3′ (36Costet P. Luo Y. Wang N. Tall A.R. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar) and the mutated oligonucleotides 5′ GCG CAG TAG TTA CTA TCA CAC AAA3′ were used. Data are presented as mean ± SEM. Comparisons between groups were performed using a Student's t-test. P values less than 0.05 were considered significant. Our laboratory has previously shown that endotoxin decreased hepatic mRNA and protein levels of LXRα in hamsters (33Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. The acute phase response is associated with retinoid X receptor repression in rodent liver.J. Biol. Chem. 2000; 275: 16390-16399Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). We have obtained similar results in mouse liver (data not shown). Because ABCG5 and ABCG8 are regulated by LXRα, we examined whether these two transporters were affected by endotoxin. A single dose of 100 μg of endotoxin rapidly decreased hepatic mRNA levels of ABCG5 and ABCG8 in mice as shown in Fig. 1A and B. The mRNA levels started to decrease at 4–8 h and continued to be significantly suppressed for 24 h after endotoxin administration. Although the chronological pattern and the magnitude of the reduction of hepatic mRNA levels of both ABCG5 and ABCG8 were relatively similar, slight differences in the degree of the inhibition suggest that the mechanisms might not be completely identical. The dose response curve of the effects of endotoxin on hepatic mRNA levels of ABCG5 and ABCG8 was examined at the 16 h time point. Administration of endotoxin resulted in a dose-dependent decrease in hepatic mRNA levels of both ABCG5 and ABCG8 (Fig. 2A, B). The levels were significantly decreased by low doses of endotoxin. A half-maximal decrease in hepatic levels of ABCG5 and ABCG8 was produced by ∼0.1 μg and 2.0 μg of endotoxin, respectively. In addition to the liver, ABCG5 and ABCG8 are also expressed in the small intestine. Therefore, we examined whether endotoxin injection decreased intestinal mRNA levels of these two transporters. In contrast to those observed in the liver, we found that there were no changes in the mRNA levels of both ABCG5 and ABCG8 in the small intestine (ABCG5 expression in the control group was 100 ± 2% and in the endotoxin group was 79 ± 12%, P = nonsignificant; and ABCG8 expression in the control group was 100 ± 6% and in the endotoxin group was 115 ± 13%, P = nonsignificant). ABCA1 mRNA levels in the small intestine were also not decreased in response to endotoxin (100 ± 15% in the control group and 166 ± 25% in the endotoxin group, P = nonsignificant). To further determine whether the different results were due to differential regulation of LXR/RXR between the small intestine and the liver, we examined the mRNA levels of LXR and RXR in the small intestine in response to endotoxin. We found no significant changes in LXRβ or RXRα mRNA in the small intestine, and did not detect LXRα, RXRβ, or RXRγ (data not shown). Additionally, PPARα and PPARγ were not detected, and PPARβ/σ was abundantly expressed, but was not changed by endotoxin treatment. These results suggest that the difference in the responsiveness of ABCG5 and ABCG8 between the liver and the small intestine may be due to differential regulation of LXR and RXR by endotoxin in different tissues. In macrophages, ABCA1 plays a critical role in apolipoprotein-mediated cholesterol efflux (23Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar, 24Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.J. Biol. Chem. 2000; 275: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). We found that incubation of endotoxin with J774 murine macrophages resulted in a decrease in ABCA1 mRNA levels (Fig. 3). The maximal decrease in mRNA levels of ABCA1 was observed 8 h after endotoxin and was relatively sustained until 24 h (Fig. 3A). There was also a concentration-dependent decrease in mRNA levels of ABCA1 (Fig. 3B). It is of note that the half-maximal decrease in the mRNA levels of ABCA1 was produced by the concentrations of endotoxin between 1–10 ng/ml, which corresponds to the concentrations of endotoxin found in the circulation during sepsis (42Levin J. Poore T.E. Zauber N.P. Oser R.S. Detection of endotoxin in the blood of patients with sepsis due to gran-negative bacteria.N. Engl. J. Med. 1970; 283: 1313-1316Crossref PubMed Scopus (230) Google Scholar). Besides ABCA1, ABCG1 is another ABC

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