The Acute Phase Response Is Associated with Retinoid X Receptor Repression in Rodent Liver
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m000953200
ISSN1083-351X
AutoresAnne P. Beigneux, Arthur H. Moser, Judy K. Shigenaga, Carl Grünfeld, Kenneth R. Feingold,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor α (PPARα) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRα, RXRβ, and RXRγ proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRα (62%) and RXRβ (50%) and at 2 h for RXRγ (61%). These decreases were associated with a marked reduction in RXRα, RXRβ, and RXRγ mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decreaseRXRβ and RXRγ transcription and only marginally inhibited (38%) RXRα transcription. RXR repression was associated with decreased LXRα and PPARα mRNA levels and reduced RXR·RXR, RXR·PPAR and RXR·LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARα target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR. The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor α (PPARα) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRα, RXRβ, and RXRγ proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRα (62%) and RXRβ (50%) and at 2 h for RXRγ (61%). These decreases were associated with a marked reduction in RXRα, RXRβ, and RXRγ mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decreaseRXRβ and RXRγ transcription and only marginally inhibited (38%) RXRα transcription. RXR repression was associated with decreased LXRα and PPARα mRNA levels and reduced RXR·RXR, RXR·PPAR and RXR·LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARα target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR. retinoid X receptor peroxisome proliferator-activated receptor liver X receptor acute phase response fatty acid tumor necrosis factor interleukin lipopolysaccharide acyl-CoA synthetase body weight intraperitoneally acute phase protein mutant hepatocyte nuclear factor Small lipophilic compounds, such as steroids, thyroid hormones, vitamin D, and retinoids, regulate gene expression by binding to nuclear hormone receptors (1.Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (283) Google Scholar, 2.Kliewer S.A. Lehmann J.M. Willson T.M. Science. 1999; 284: 757-760Crossref PubMed Scopus (424) Google Scholar, 3.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar). Nuclear hormone receptors are the largest known family of transcription factors, with over 150 members currently. The nuclear hormone receptors share a common structural composition, including a central, highly conserved DNA-binding domain and a carboxyl-terminal domain that mediates ligand recognition, receptor dimerization, and ligand-dependent activation (1.Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (283) Google Scholar, 2.Kliewer S.A. Lehmann J.M. Willson T.M. Science. 1999; 284: 757-760Crossref PubMed Scopus (424) Google Scholar, 3.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar). The nuclear receptor superfamily has been divided into four major subgroups according to their dimerization and DNA binding properties (3.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar). Class II receptors consist of nuclear receptors that heterodimerize with the retinoid X receptor (RXR)1 and usually bind to direct repeats separated by a variable number of spacer nucleotides (3.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar,4.Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1510) Google Scholar). The class II subgroup includes the retinoic acid receptor (RAR), thyroid hormone receptor, vitamin D receptor, farnesoid X receptor, peroxisome proliferator-activated receptor (PPAR), and liver X receptor (LXR) (3.Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar, 5.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6002) Google Scholar). Three distinct RXR genes have been cloned:RXRα, RXRβ, and RXRγ. RXRα is strongly expressed in liver, kidney, muscle, lung, and spleen (6.Leid M. Kastner P. Lyons R. Nakshatri H. Saunders M. Zacharewski T. Chen J.Y. Staub A. Garnier J.M. Mader S. Chambon P. Cell. 1992; 68: 377-395Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 7.Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1056) Google Scholar). RXRα is also present in the brain and heart (6.Leid M. Kastner P. Lyons R. Nakshatri H. Saunders M. Zacharewski T. Chen J.Y. Staub A. Garnier J.M. Mader S. Chambon P. Cell. 1992; 68: 377-395Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 7.Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1056) Google Scholar). RXRβ is expressed ubiquitously, and it is present at low level in liver, intestine, and testis (6.Leid M. Kastner P. Lyons R. Nakshatri H. Saunders M. Zacharewski T. Chen J.Y. Staub A. Garnier J.M. Mader S. Chambon P. Cell. 1992; 68: 377-395Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 7.Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1056) Google Scholar). RXRγ is expressed only in liver, kidney, muscle, brain, heart, and adrenal (6.Leid M. Kastner P. Lyons R. Nakshatri H. Saunders M. Zacharewski T. Chen J.Y. Staub A. Garnier J.M. Mader S. Chambon P. Cell. 1992; 68: 377-395Abstract Full Text PDF PubMed Scopus (1011) Google Scholar, 7.Mangelsdorf D.J. Borgmeyer U. Heyman R.A. Zhou J.Y. Ong E.S. Oro A.E. Kakizuka A. Evans R.M. Genes Dev. 1992; 6: 329-344Crossref PubMed Scopus (1056) Google Scholar). To date, the 9-cis retinoic acid isomer has been identified as the most potent endogenous ligand for RXR (8.Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1549) Google Scholar). The acute phase response (APR), which is induced during infection, inflammation, and injury, is associated with numerous changes in lipid metabolism (9.Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Crossref PubMed Scopus (56) Google Scholar). Hypertriglyceridemia, decreased high density lipoprotein cholesterol levels, accelerated lipolysis, decreased hepatic fatty acid (FA) oxidation, and inhibition of the synthesis of bile acids are some of the alterations in lipid metabolism that occur during the APR (9.Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Crossref PubMed Scopus (56) Google Scholar). In most instances, these changes are mediated by pro-inflammatory cytokines such as tumor necrosis factor (TNF) or interleukin (IL)-1 and are due to alterations in gene transcription (9.Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Crossref PubMed Scopus (56) Google Scholar). However, the molecular mechanisms underlying these alterations in gene transcription that account for the changes in lipid metabolism during the APR remain to be identified. Both PPAR and LXR have been implicated in the regulation of genes important in lipid metabolism. PPARα activation in the liver stimulates FA metabolism and transport, and LXR activation leads to an increase in bile acid synthesis. Specifically, PPARα increases the expression of carnitine palmitoyltransferase I, 3-hydroxy-3-methylglutaryl-CoA synthase, acyl-CoA oxidase, acyl-CoA synthetase (ACS), cytochrome P450 4A enzymes, FA transport protein, and FA-binding protein (10.Dreyer C. Krey G. Keller H. Givel F. 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Ther. 1999; 290: 1250-1257PubMed Google Scholar) have shown that the expression or activity of each of these proteins involved in lipid metabolism in the liver is rapidly and markedly decreased following induction of the APR by LPS or cytokine administration. One potential mechanism by which the expression of many genes could be coordinately decreased during the APR is by the reduction of the levels of specific transcription factors. Because heterodimerization with RXR is crucial for the action of several nuclear hormone receptors including PPAR and LXR, we hypothesized that a decrease in RXR levels in the liver may occur during APR. Previous studies by Sugawaraet al. (27.Sugawara A. Uruno A. Nagata T. Taketo M.M. Takeuchi K. Ito S. Endocrinology. 1998; 139: 3030-3033Crossref PubMed Scopus (18) Google Scholar) showed that TNF decreases the expression of aRXRβ promoter construct in rat GH3 cells. Here we report that RXRα, RXRβ, and RXRγ proteins and mRNA levels decline during the APR in hamster. RXR repression is associated with LXRα and PPARα repression, resulting in an overall decreased ability of RXR·RXR homodimers, RXR·PPAR, and RXR·LXR heterodimers to bind to their respective response elements. LPS (Escherichia coli 55:B5) was obtained from Difco Laboratories and freshly diluted to desired concentration in pyrogen-free 0.9% saline. Human TNF-α (specific activity, 5 × 107 units/mg) was provided by Genentech, Inc. Recombinant human IL-1β (specific activity, 4 × 108 units/mg) was a gift from Dr. Charles A. Dinarello (University of Colorado, Denver, CO). The cytokines were freshly diluted to desired concentrations in pyrogen-free 0.9% saline containing 0.1% human serum albumin. Oligo(dT)-cellulose type 77F was from Amersham Pharmacia Biotech. Wy-14,643 was purchased from Sigma and freshly resuspended in corn oil at the appropriate concentration. [α-32P]dCTP (3,000 Ci/mmol), [γ-32P]dATP (3,000 Ci/mmol), and [α-32P]dUTP (800 Ci/mmol) were purchased from NEN Life Science Products. Male Syrian hamsters (140–160 g) were purchased from Simonsen Laboratories (Gilroy, CA). The animals were maintained in a normal-light-cycle room and were provided with rodent chow and waterad libitum. Anesthesia was induced with halothane. To assess the effect of the acute phase response on RXR, hamsters were injected intraperitoneally with 0.1–100 μg/100 g of body weight (BW) LPS, 25 μg/100 g of BW TNF-α, or 1 μg/100 g of BW IL-1β in 0.5 ml of saline or with saline alone. To assess the effect of LPS treatment on PPARα activation, hamsters were injected IP daily with Wy-14,643 at a dosage of 5 mg/100 g of BW or with corn oil alone for 5 days. On the fifth day, 100 μg/100 g of BW LPS or saline alone was administered IP. Food was withdrawn at the time of injection because LPS and cytokines induce marked anorexia in rodents (28.Grunfeld C. Zhao C. Fuller J. Pollack A. Moser A. Friedman J. Feingold K.R. J. Clin. Invest. 1996; 97: 2152-2157Crossref PubMed Scopus (820) Google Scholar). Livers were removed after treatment at the times indicated below. The doses of LPS used in this study have significant effects on triglyceride and cholesterol metabolism without causing death (23.Feingold K.R. Pollock A.S. Moser A.H. Shigenaga J.K. Grunfeld C. J. Lipid. Res. 1995; 36: 1474-1482Abstract Full Text PDF PubMed Google Scholar, 29.Feingold K.R. Hardardottir I. Memon R. Krul E.J. Moser A.H. Taylor J.M. Grunfeld C. J. Lipid. Res. 1993; 34: 2147-2158Abstract Full Text PDF PubMed Google Scholar). Similarly, the nonlethal doses of TNF-α and IL-1β used in this study reproduce many of the effects of LPS on lipid metabolism, causing marked changes in serum lipid and lipoprotein levels (9.Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Crossref PubMed Scopus (56) Google Scholar, 30.Hardardottir I. Moser A.H. Memon R. Grunfeld C. Feingold K.R. Lymphokine Cytokine Res. 1994; 13: 161-166PubMed Google Scholar). Fresh liver (1.5–2 g) was homogenized in 10 mm HEPES (pH 7.9), 25 mm KCl, 0.15 mm spermine, 1 mm EDTA, 2 msucrose, 10% glycerol, 50 mm NaF, 2 mm sodium metavanadate, 0.5 mm dithiothreitol, and 1% protease inhibitor mixture (Sigma) at the times indicated below after LPS or saline treatment. Immediately following homogenization, nuclear proteins were extracted as described by Neish et al. (31.Neish A.S. Khachigian L.M. Park A. Baichwal V.R. Collins T. J. Biol. Chem. 1995; 270: 28903-28909Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), except that 1 mm NaF, 0.1 mm metavanadate, and 1% protease inhibitor mixture (Sigma) were added to all buffers. Nuclear protein content was determined by the Bradford assay (Bio-Rad), and yields were similar in control and LPS-treated groups. Denatured nuclear protein (25 μg) was loaded onto 10% polyacrylamide precast gels (Bio-Rad) and subjected to electrophoresis. After electrotransfer onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blots were blocked with phosphate-buffered saline containing 0.10% Tween and 5% dry milk for 1 h at room temperature and incubated for 1 h at room temperature with the following polyclonal rabbit antibodies (Santa Cruz Biotechnology) at a dilution of 1:5000: anti-RXRα, anti-RXRβ, and anti-RXRγ. Immune complexes were detected using horseradish peroxidase-linked donkey anti-rabbit IgG (dilution 1:20,000) according to the ECL Plus Western blotting kit (Amersham Pharmacia Biotech). Immunoreactive bands obtained by autoradiography were quantified by densitometry. Total RNA was isolated from 300–400 mg of snap-frozen whole liver tissue by a modified acid guanidinium thiocyanate-phenol-chloroform method (32.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar) as described earlier (21.Memon R.A. Fuller J. Moser A.H. Smith P.J. Feingold K.R. Grunfeld C. Am. J. Physiol. 1998; 275: E64-E72PubMed Google Scholar). Poly(A)+ RNA was purified using oligo(dT) cellulose and quantified by measuring absorption at 260 nm. Ten micrograms of poly(A)+ or 20 μg of total RNA were denatured and electrophoresed on a 1% agarose/formaldehyde gel. The uniformity of sample applications was checked by UV visualization of the acridine orange-stained gel before electrotransfer to Nytran membrane (Schleicher & Schuell), or when indicated, p18S was used as a control probe. We and others have found that LPS increases actin mRNA levels in liver by 2–5-fold in rodents (29.Feingold K.R. Hardardottir I. Memon R. Krul E.J. Moser A.H. Taylor J.M. Grunfeld C. J. Lipid. Res. 1993; 34: 2147-2158Abstract Full Text PDF PubMed Google Scholar, 33.Morrow J.F. Stearman R.S. Peltzman C.G. Potter D.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4718-4722Crossref PubMed Scopus (123) Google Scholar). TNF and IL-1 produced a 2-fold increase in actin mRNA levels. LPS also produces a 2-fold increase in glyceraldehyde-3-phosphate dehydrogenase and a 2.6-fold increase in cyclophilin mRNA (20.Memon R.A. Feingold K.R. Moser A.H. Fuller J. Grunfeld C. Am. J. Physiol. 1998; 274: E210-E217PubMed Google Scholar). Thus, the mRNA levels of actin, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin, which are widely used to normalize data, cannot be used to study LPS or cytokine-induced regulation of proteins in liver. However, the differing direction of the changes in mRNA levels for specific proteins after LPS or cytokine administration, the magnitude of the alterations, and the relatively small standard error of the mean make it unlikely that the changes observed were due to unequal loading of mRNA (20.Memon R.A. Feingold K.R. Moser A.H. Fuller J. Grunfeld C. Am. J. Physiol. 1998; 274: E210-E217PubMed Google Scholar, 23.Feingold K.R. Pollock A.S. Moser A.H. Shigenaga J.K. Grunfeld C. J. Lipid. Res. 1995; 36: 1474-1482Abstract Full Text PDF PubMed Google Scholar, 24.Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. J. Lipid. Res. 1996; 37: 223-228Abstract Full Text PDF PubMed Google Scholar, 29.Feingold K.R. Hardardottir I. Memon R. Krul E.J. Moser A.H. Taylor J.M. Grunfeld C. J. Lipid. Res. 1993; 34: 2147-2158Abstract Full Text PDF PubMed Google Scholar, 34.Memon R.A. Shechter I. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Lipid. Res. 1997; 38: 1620-1629Abstract Full Text PDF PubMed Google Scholar). Prehybridization, hybridization, and washing procedures were performed as described previously (21.Memon R.A. Fuller J. Moser A.H. Smith P.J. Feingold K.R. Grunfeld C. Am. J. Physiol. 1998; 275: E64-E72PubMed Google Scholar). Membranes were probed with [α-32P]dCTP labeled cDNAs using the random priming technique. mRNA levels were detected by exposure of the membrane to x-ray film and quantified by densitometry. hRXRα cDNA was a gift from Dr. Daniel D. Bikle (University of California, San Francisco, CA). mouse RXRβ, mouse RXRγ, human LXRα, and human LXRβ cDNAs were kindly provided by Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). RACS cDNA was kindly provided by Dr. Pamela J. Smith (Ross Products Division, Abbott Laboratories, Columbus, OH). rPPARα, mPPARδ, and mPPARγ cDNAs were a gift from Dr. Anthony Bass (University of California San Francisco, CA). Isolation of liver nuclei from fresh tissue, the procedure for in vitro nuclear transcription and hybridization was essentially as described by Clarkeet al. (35.Clarke C.F. Fogelman A.M. Edwards P.A. J. Biol. Chem. 1985; 260: 14363-14367Abstract Full Text PDF PubMed Google Scholar). Briefly, nuclei were incubated with 200 μCi of [α-32P]UTP, and after labeling nascent transcripts for 30 min at 30 °C, total RNA was recovered according to Chomczynski and Sacchi (32.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). After prehybridization, all of thein vitro labeled RNA isolated (2–9 × 106cpm total) from nuclei of control and LPS-treated hamsters were hybridized to prepared nylon membrane (Schleicher & Schuell). After being washed and autoradiographed, the filters were air-dried, and the amount of in vitro labeled RNA that hybridized to each dot containing 10 μg of cDNA for RXRα, RXRβ, RXRβ, actin, and vector pUC19 was measured by liquid scintillation counting. 10 μg of crude nuclear extract were incubated on ice for 30 min with 6 × 104 cpm of 32P-labeled oligonucleotides in 15 μl of binding buffer (20% glycerol, 25 mm Tris-HCl (pH 7.5), 40 mm KCl, 0.5 mm MgCl2, 0.1 mm EDTA, 1 mm dithiothreitol), 2 μg of poly(dI-dC), and 1 μg of salmon sperm DNA. Double-stranded oligonucleotide probes were end-labeled with T4-polynucleotide kinase in presence of 50 μCi of [γ-32P]dATP and purified on a Sephadex G-25 column (Amersham Pharmacia Biotech), and the LXR oligonucleotide was subsequently gel-purified. DNA-protein complexes were separated by electrophoresis (constant voltage of 300 V) on a 5% nondenaturing polyacrylamide gel in 1× TBE at 4 °C. The gel was dried, exposed to x-ray film, and quantified by densitometry. In the competition assay, a 100-fold molar excess of the specific or mutated unlabeled oligonucleotide was preincubated on ice for 1 h with 10 μg of nuclear extract from control hamster in the binding buffer before adding the oligonucleotide probe. The following oligonucleotides were used: PPAR response element, 5′-GATCCTCCCGAACGTGACCTTTGTCCTGGTCCA-3′ (36.Ren B. Thelen A. Jump D.B. J. Biol. Chem. 1996; 271: 17167-17173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar); mut-PPAR response element, 5′-GATCCTCCCGAACGCAGCTGTCAGCTGGGTCCA-3′; CRBPII, 5′-GATACTGCTGTCACAGGTCACAGGTCACAGTTCAA-3′ (36.Ren B. Thelen A. Jump D.B. J. Biol. Chem. 1996; 271: 17167-17173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar); (37.Mangelsdorf D.J. Umesono K. Kliewer S.A. Borgmeyer U. Ong E.S. Evans R.M. Cell. 1991; 66: 555-561Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 38.Nagao Y. French B.A. Cai Y. French S.W. Wan Y.J. J. Cell. Biochem. 1998; 69: 189-200Crossref PubMed Scopus (14) Google Scholar); mut-CRBPII, 5′-GATACTGCTGTCACAGCACACAGCACACAGTTCAA-3′; CYP7-LXR response element, 5′-GATCCCTTTGGTCACTCAAGTTCAAGTGGATC-3′ (18.Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar); and mut-CYP7-LXR response element, 5′-GATCCCTTTGGTCACTCAAGAACAAGTGGATC-3′ (18.Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar). In supershift studies, control nuclear extract was preincubated with 2 μl of one of the following antibodies (Santa Cruz Biotechnology) for 1 h at room temperature prior to the addition of the labeled probe: anti-RXRα, anti-RXRβ, anti-RXRγ, and anti-rabbit IgG. Data are expressed as mean ± S.E. of experiments from 3–5 animals per group for each time point. The difference between two experimental groups was analyzed using the unpaired t test. Differences among multiple groups were analyzed using one-way analysis of variance with the Dunnett's post-test correction. A p value < 0.05 was considered significant. We initially determined the effect of LPS administration on RXRα protein levels in the nuclei from liver of Syrian hamsters. RXRα is the most abundant RXR isoform in liver. As shown in Fig. 1 A,LPS (100 μg/100 g of BW) produced a maximum decrease (62%) in RXRα protein levels at 4 h. A similar decrease was also present at 8 h following LPS treatment, but by 16 h, RXRα protein was returning toward normal levels (23% decrease at 16 h). As shown in Fig. 2 A, the LPS-induced decrease in RXRα protein levels was dose-dependent, with the half-maximal effect occurring at approximately 2 μg/100 g of BW. Thus, LPS at relatively low doses (LD50 for LPS in rodents is approximately 5 mg/100 g of BW) rapidly decreases RXRα protein levels in the liver of Syrian hamsters.Figure 2Dose response of the regulation by LPS of RXR α (A), RXR β (B), and RXR γ (C) proteins in hamster liver. Syrian hamsters were injected IP with either saline or LPS at the doses indicated, and the animals were sacrificed 4 h after LPS administration. Hepatic nuclear extracts were prepared, and Western blot analysis was carried out as described under "Experimental Procedures." Data (means ± S.E., n = 5) are expressed as a percentage of controls. **, p < 0.01; ***, p < 0.005 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next determined whether this decrease in RXRα protein levels was associated with alterations in RXRα mRNA levels in the liver. As shown in Fig. 3 A, LPS administration resulted in a marked reduction (97%) in RXRα mRNA levels in the liver of Syrian hamsters at 4 h. To determine whether this decrease in mRNA levels was due to an inhibition of transcription, nuclear run-on assays were performed on nuclei prepared from hamster liver 4 h after LPS or saline injection. As shown in Fig. 4 A, LPS treatment resulted in a 38% decrease in RXRα transcription compared with control. Therefore, the decrease in RXRα protein levels following LPS administration is associated with a decrease in mRNA levels that is partially accounted for by LPS inhibition ofRXRα gene transcription. However, the modest reduction in transcription compared with the marked decrease in mRNA levels suggests that post-transcriptional factors in addition to inhibition of transcription contribute to the LPS-induced decrease in RXRα mRNA levels.Figure 4Effect of LPS treatment on RXRα(A), RXRβ(B), and RXRγ(C) gene transcription rates in hamster liver. Syrian hamsters were injected IP with either saline or LPS (100 μg of LPS/100 g of BW). Four hours later, livers were removed, and nuclei for in vitro transcription were prepared as described under "Experimental Procedures." Data (means ± S.E., n = 5) are expressed as a percentage of controls. ***, p < 0.005 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because cytokines, such as TNF and IL-1, mediate many of the changes induced by LPS administration, we next examined the effect of TNF and/or IL-1 on RXRα mRNA levels. As shown in Fig. 3 B,2 h after the administration of TNF, IL-1, or TNF plus IL-1, there was a 63, 60, and 80% reduction in RXRα mRNA levels, respectively. Thus, the combination of TNF and IL-1 can reproduce the effects of LPS. Both RXRβ and RXRγ are also present in the liver but are expressed at lower levels than RXRα. As shown in Fig. 1, B andC, LPS treatment (100 μg/100 g of BW) resulted in a decrease in RXRβ and RXRγ protein levels in the liver of Syrian hamsters. RXRγ was decreased by 61% as early as 2 h after LPS administration but returned to normal by 8 h (Fig. 1 C). RXRβ protein levels also rapidly decreased following LPS treatment, but in contrast to RXRγ, this decrease was sustained for at least 16 h (Fig. 1 B). The decrease in protein levels of RXRγ and RXRβ induced by LPS was a sensitive response, with the half-maximal effect seen at less than 1 μg of LPS/100 g of BW for RXRγ (Fig. 2 C) and approximately 1 μg of LPS/100 g of BW for RXRβ (Fig. 2 B). Thus, LPS treatment not only decreases RXRα protein levels but also decrease
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