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• W2088201364 abstract "The levels of plasma HDL cholesterol and apoA-I in NFκB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFκBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFκB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IκBα super-repressor ameliorated the reduction. This IκBα-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor α (PPARα), suggesting that NFκB inactivation induces apoA-I through activation of PPARα. To further support this idea, the expression of IκBα increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARα-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFκB increases apoA-I and HDL cholesterol through activation of PPARα in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease. The levels of plasma HDL cholesterol and apoA-I in NFκB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFκBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFκB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IκBα super-repressor ameliorated the reduction. This IκBα-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor α (PPARα), suggesting that NFκB inactivation induces apoA-I through activation of PPARα. To further support this idea, the expression of IκBα increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARα-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFκB increases apoA-I and HDL cholesterol through activation of PPARα in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease. Nuclear Factor κB (NFκB) 1The abbreviations used are: NFκB, nuclear factor κB; HDL, high density lipoprotein; LDL, low density lipoprotein; LPS, lipopolysaccharide; PPRE, peroxisome proliferator response element; PPAR, peroxisome proliferator-activated receptor.1The abbreviations used are: NFκB, nuclear factor κB; HDL, high density lipoprotein; LDL, low density lipoprotein; LPS, lipopolysaccharide; PPRE, peroxisome proliferator response element; PPAR, peroxisome proliferator-activated receptor. transcription factors are homodimeric and heterodimeric complexes of five family members: p50, p52, c-Rel, RelB, and p65 (RelA). Most of the cells in the body contain only a heterodimeric complex of p50 and p65 (1Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1924) Google Scholar, 2Sen R. Baltimore D. Cell. 1986; 47: 921-928Abstract Full Text PDF PubMed Scopus (1459) Google Scholar). Activation of NFκB is involved in the pathogenesis of many chronic inflammatory diseases, such as asthma and rheumatoid arthritis (3Tak P.P. Firestein G.S. J. Clin. Invest. 2001; 107: 7-11Crossref PubMed Scopus (3230) Google Scholar). It may also be involved in some neurodegenerative disorders such as Alzheimer's disease (4Mattson M.P. Camandola S. J. Clin. Invest. 2001; 107: 247-254Crossref PubMed Scopus (751) Google Scholar), ischemic brain injury (5Schneider A. Martin-Villalba A. Weih F. Vogel J. Wirth T. Schwaninger M. Nat. Med. 1999; 5: 554-559Crossref PubMed Scopus (42) Google Scholar), a variety of human cancers (6Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2242) Google Scholar), and atherosclerosis (7Collins T. Cybulsky M.I. J. Clin. Invest. 2001; 107: 255-264Crossref PubMed Scopus (642) Google Scholar). Mice lacking the p65 subunit (RelA) display embryonic death at 15–16 days of gestation, concomitant with massive degeneration of the liver by apoptosis (8Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1629) Google Scholar). To the contrary, mice lacking the p50 subunit of NFκB show no developmental abnormalities and live for at least 1 year after birth but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection (9Sha W.C. Liou H.C. Tuomanen E.I. Baltimore D. Cell. 1995; 80: 321-330Abstract Full Text PDF PubMed Scopus (1054) Google Scholar). Although many researchers have demonstrated abnormalities in the immune responses of NFκB p50 subunit-deficient mice, to our knowledge no one has reported any abnormality in lipid metabolism of them. In this study, we found that the plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) in NFκB p50 subunit-deficient mice were significantly higher than those in wild-type littermates under a regular or high fat diet. In addition, the level of apoA-I mRNA in the liver was significantly higher in NFκB-deficient mice than in wild-type littermates (data not shown). The plasma levels of apoE and apoB did not appear to differ between these NFκB-deficient and wild-type mice. ApoA-I is mainly synthesized in the liver and small intestine (10Zannis V.I. Cole F.S. Jackson C.L. Kurnit D.M. Karathanasis S.K. Biochemistry. 1985; 24: 4450-4455Crossref PubMed Scopus (147) Google Scholar). Because the levels of plasma apoA-I are positively correlated with hepatic apoA-I mRNA (11Srivastava R.A. Tang J. Krul E.S. Pfleger B. Kitchens R.T. Schonfeld G. Biochim. Biophys. Acta. 1992; 1125: 251-261Crossref PubMed Scopus (45) Google Scholar, 12Srivastava R.A. Methods Mol. Biol. 1998; 86: 103-112PubMed Google Scholar, 13Sorci-Thomas M. Prack M.M. Dashti N. Johnson F. Rudel L.L. Williams D.L. J. Biol. Chem. 1988; 263: 5183-5189Abstract Full Text PDF PubMed Google Scholar), factors influencing the level of apoA-I may be mediated through the level of apoA-I gene expression. The distinct enhancer region in the human apoA-I gene contains the regulatory element necessary for maximal expression in a human hepatoma cell line, called peroxisome proliferator response element (PPRE) (14Sastry K.N. Seedorf V. Karathanasis S.K. Mol. Cell. Biol. 1988; 8: 605-614Crossref PubMed Scopus (80) Google Scholar). In this cell line activated PPARα binds to this element, thereby enhancing the expression of apoA-I (15Martin G. Duez H. Blanquart C. Berezowski V. Poulain P. Fruchart J.C. Najib-Fruchart J. Glineur C. Staels B. J. Clin. Invest. 2001; 107: 1423-1432Crossref PubMed Scopus (398) Google Scholar, 16Peters J.M. Hennuyer N. Staels B. Fruchart J.C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 17Bocher V. Pineda-Torra I. Fruchart J.C. Staels B. Ann. N. Y. Acad. Sci. 2002; 967: 7-18Crossref PubMed Scopus (149) Google Scholar). In this report we demonstrated that inactivation of NFκB enhances the expression and secretion of apoA-I from HepG2 cells through activation of the transcription factor PPARα. Reagents—Lipopolysaccharide (LPS) and WY-14643 (synthetic PPARα agonist) were purchased from Sigma, and MK886 (selective inhibitor of PPARα) was from Wako Pure Chemicals (Osaka, Japan). LPS was dissolved in pure water, whereas WY-14643 and MK886 were dissolved in Me2SO. Goat polyclonal anti-apoA-I antibody was purchased from Rockland (Gilbertsville, PA), rabbit anti-apoB antibody was from BioDesign (Saco, ME), and goat anti-apoE antibody and mouse anti-β-actin monoclonal antibody were from Chemicon International (Temecula, CA). Animals—Animal experiments were performed in compliance with the Guide for Animal Experimentation and with the approval of the Committee of Animal Experimentation, Ehime University School of Medicine. Animals were maintained in a specific pathogen-free facility under a 12-h dark/light cycle. Homozygous NFκB p50 subunit-deficient mice Nfkb1tm1Bal, NFκB – / –) were originally purchased from The Jackson Laboratory (Bar Harbor, ME). The gene-targeting strategy used to generate this mouse line was described previously (9Sha W.C. Liou H.C. Tuomanen E.I. Baltimore D. Cell. 1995; 80: 321-330Abstract Full Text PDF PubMed Scopus (1054) Google Scholar). These mice were back-crossed at least six times to C57BL/6 mice (Clea Japan Inc., Osaka, Japan). Genomic DNA was extracted from the mouse tail and genotyped by PCR following the protocol of The Jackson Laboratory. Eight-week-old female mice were divided into two groups. Each group, composed of five NFκB – / – mice and five control mice, was kept on a regular diet containing 5% (wt/wt) fat or a high fat diet containing 1.25% (wt/wt) cholesterol, 17.8% (wt/wt) butter, 1.0% (wt/wt) corn oil, and 0.5% (wt/wt) sodium cholate (Oriental Yeast Co. Ltd., Tokyo, Japan) for 2 weeks. After an overnight fast, mice were anesthetized with ketamine hydrochloride (Sankyo Co. Ltd., Tokyo, Japan) for phlebotomy via the femoral vein. Blood samples were centrifuged at 2500 rpm for 10 min, and about 200 μl of plasma was obtained per mouse. Plasma Lipid Analysis—The concentrations of total cholesterol, triglyceride, and HDL cholesterol in plasma were measured using enzymatic kits (Wako Pure Chemical Industries Ltd.) following the manufacturer's protocols. Plasma lipoprotein profile was determined by polyacrylamide disc-gel electrophoresis (18Narayan K.A. Narayan S. Kummerous F.A. Nature. 1965; 205: 246-248Crossref PubMed Scopus (82) Google Scholar). Cell Culture—HepG2 cells were purchased from RIKEN Cell Bank (Ibaraki, Japan) and routinely propagated at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Invitrogen), 100 μg/ml penicillin, and 100 μg/ml streptomycin. Recombinant Adenovirus—Ad5IκB (donated by Dr. Iimuro, Kyoto University, Kyoto, Japan) expresses IκBα super-repressor under the control of the cytomegalovirus promoter, where serine to alanine mutations at amino acids 32 and 36 prevent the phosphorylation that is necessary for it to dissociate from the NFκB complex (19DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). Ad5LacZ, which expresses Escherichia coli β-galactosidase under the control of the cytomegalovirus promoter, was used as a control for adenovirus-mediated transfection (20Tamatani M. Che Y.H. Matsuzaki H. Ogawa S. Okado H. Miyake S. Mizuno T. Tohyama M. J. Biol. Chem. 1999; 274: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). Amplification, purification, and titration of the adenoviruses and adenovirus-mediated gene transfer were carried out as described previously (21Mitsuda N. Ohkubo N. Tamatani M. Lee Y.D. Taniguchi M. Namikawa K. Kiyama H. Yamaguchi A. Sato N. Sakata K. Ogihara T. Vitek M.P. Tohyama M. J. Biol. Chem. 2001; 276: 9688-9698Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Cells were pretransfected with these adenoviruses 16 h before adding reagents. Reverse Transcriptase-PCR Analysis—HepG2 cells were seeded on 6-well tissue culture plates (3 × 105 cells/well) and incubated for 24 h. The cells were transfected with multiplicity of infection 10 Ad5IkB or Ad5LacZ as described previously (21Mitsuda N. Ohkubo N. Tamatani M. Lee Y.D. Taniguchi M. Namikawa K. Kiyama H. Yamaguchi A. Sato N. Sakata K. Ogihara T. Vitek M.P. Tohyama M. J. Biol. Chem. 2001; 276: 9688-9698Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and incubated for an additional 16 h, exposed to 10 μg/ml LPS or vehicle (5% v/v pure water), and cultured for 12 h. Total RNA was extracted from the cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. Oligo-dT primers (Takara Bio Inc., Shiga, Japan) together with 3 μg of DNase-treated total RNA and Moloney murine leukemia virus reverse transcriptase (Invitrogen) were used to obtain first strand cDNA. PCR was performed using ExTaq polymerase (Takara Bio Inc.) and the oligonucleotide primers listed in Table I.Table IPrimers used for mRNA detection by reverse transcriptase-PCRGeneSense primerAntisense primerReferenceApoA-I5′-ATGAAAGCTGCGGTGCTGACC-3′5′-CACCTTCTGGCGGTAGAGCTCC-3′22Hsu M.H. Savas U. Griffin K.J. Johnson E.F. J. Biol. Chem. 2001; 276: 27950-27958Abstract Full Text Full Text PDF PubMed Scopus (155) Google ScholarApoE5′-ACTGGCACTGGGTCGCTTT-3′5′-GTTGTTCCTCCAGTTCCGATT-3′23Malek G. Li C.M. Guidry C. Medeiros N.E. Curcio C.A. Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google ScholarApoB5′-TAGACACCAACTTCTTCCACG-3′5′-GGCGACCTCAGTAATTTTCTTG-3′23Malek G. Li C.M. Guidry C. Medeiros N.E. Curcio C.A. Am. J. Pathol. 2003; 162: 413-425Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholarβ-Actin5′-CAAGAGATGGCCACGGCTGCT-3′5′-TCCTTCTGCATCCTGTCGGCA-3′24Sumida A. Fukuen S. Yamamoto I. Matsuda H. Naohara M. Azuma J. Biochem. Biophys. Res. Commun. 2000; 267: 756-760Crossref PubMed Scopus (56) Google Scholar Open table in a new tab Immunoblot Analysis—HepG2 cells were seeded on 6-well tissue culture plates. If adenovirus-mediated transfection had been necessary, the cells were transfected with adenovirus as described above. The cells were exposed to 10 μg/ml LPS or vehicle (5% v/v pure water) or 10 μm MK886 or vehicle (0.1% v/v Me2SO) and cultured for 24 h. Culture media were subjected to SDS-polyacrylamide gel electrophoresis using 4–20% gradient polyacrylamide gel (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) and Western blot analysis with the specific anti-apoA-I antibody, anti-apoB antibody, or anti-apoE antibody, as previously described (25Ohkubo N. Mitsuda N. Tamatani M. Yamaguchi A. Lee Y.D. Ogihara T. Vitek M.P. Tohyama M. J. Biol. Chem. 2001; 276: 3046-3053Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Mouse plasma was also subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis by the same method. Construction of ApoA-I Promoter Plasmids—The human apoA-I promoter fragment between positions –330 to +69 relative to the transcription start site was prepared by PCR with primers 5′-TATAGCTAGCAACACAATGGACAATGGCAACTG-3′ and 5′-TATAAAGCTTGAACCTTGAGCTGGGGAGC-3′. The amplified fragment, which contains the PPRE element (26Ladias J.A. Karathanasis S.K. Science. 1991; 251: 561-565Crossref PubMed Scopus (306) Google Scholar, 27Vu-Dac N. Schoonjans K. Laine B. Fruchart J.C. Auwerx J. Staels B. J. Biol. Chem. 1994; 269: 31012-31018Abstract Full Text PDF PubMed Google Scholar), was doubly digested with NheI and HindIII and inserted between the NheI and HindIII sites of pGL2-basic vector (Promega, Madison, WI) to make an “ApoAI-wt” plasmid. Mutation in the PPRE element was generated by PCR using a sense primer (5′-ACTGATCCCTTGTCCCCTGCCCTGCAGCCCCCGCA-3′) and an antisense primer (5′-AGGGGACAAGGGATCAGTGGGGGCGGGAGGGGAGT-3′) carrying mutations (underlined) to make an “ApoAI-mut” plasmid. The shorter promoter fragment between positions –142 to +69, which does not contain the PPRE element, was also prepared by PCR with primers 5′-TATAGCTAGCAGGGACAGAGCTGATCCTTGAAC-3′ and 5′-TATAAAGCTTGAACCTTGAGCTGGGGAGC-3′ and inserted between the NheI and HindIII sites of pGL2-basic vector to make an “ApoAI-short” plasmid. Transient Transfection and Reporter Gene Assays—HepG2 cells were seeded on 12-well tissue culture plates (1 × 105 cells/well). The cells were co-transfected with 0.4 μg of the reporter plasmid and pRL-TK (Promega) internal control plasmid using LipofectAMINE plus reagent (Invitrogen), according to the manufacturer's protocol. The cells were subsequently transfected with multiplicity of infection 10 recombinant adenovirus as described above. After the cells were incubated at 37 °C for 16 h, they were treated with 10 μg/ml LPS, 10 μm MK886 or vehicle (0.1% v/v Me2SO) and incubated for an additional 24 h. The activities of firefly luciferase from apoA-I promoter-luciferase plasmid and Renilla luciferase from pRL-TK plasmid in the cell extracts were evaluated with a dual luciferase assay kit (Promega) using a luminometer (TD-20/20, Promega) according to the manufacturer's protocol. Statistical Analysis—Experimental data were evaluated by twotailed Student's t test. All unspecified data were presented as the mean ± S.D. Plasma Lipids—There was no significant difference in the concentration of total cholesterol in the plasma of female NFκB-deficient (NFκB – / –) mice and wild-type (NFκB + / +) controls after6hof food removal. After the mice were fed a high fat diet for 2 weeks, both types of mice had an about 2.5-fold increase in the fasting cholesterol level, and there was no significant difference between NFκB-deficient mice and wild-type littermates. NFκB-deficient mice showed a significantly higher HDL cholesterol level than wild-type littermates under both a regular and high fat diet. NFκB-deficient mice also showed a significantly lower triglyceride level than wild-type littermates only under a regular diet (Table II). Polyacrylamide disc-gel electrophoresis of whole plasma confirmed that the ratio of HDL to total lipoprotein was significantly higher in NFκB-deficient mice than in wild-type control, whereas there was no significant difference in the ratios of low density lipoprotein (LDL) plus intermediate density lipoprotein and very low density lipoprotein (Table III).Table IIFasting plasma lipids in NFκB-deficient (NFκB-/-) and wild-type (NFκB+/+) miceDietNFκBnTotal cholesterolHDL cholesterolTriglyceridemg/dl, mean ± S.E.Normal+/+562.0 ± 2.831.9 ± 1.7ap < 0.01, compared to wild-type controls.174.0 ± 23.5bp < 0.05, compared to wild-type controls.-/-564.5 ± 2.445.5 ± 1.7ap < 0.01, compared to wild-type controls.112.7 ± 5.3bp < 0.05, compared to wild-type controls.High fat+/+5152.4 ± 17.537.9 ± 4.1ap < 0.01, compared to wild-type controls.170.3 ± 17.8-/-5161.7 ± 16.251.7 ± 7.3ap < 0.01, compared to wild-type controls.164.8 ± 12.9a p < 0.01, compared to wild-type controls.b p < 0.05, compared to wild-type controls. Open table in a new tab Table IIIFasting plasma lipoprotein fractionation in NFκB-deficient (NFκB-/-) and wild-type (NFκB+/+) mice, measured by polyacrylamide disc-gel electrophoresisDietNFκBnHDLLDL + IDLVLDL% of total lipoprotein, mean ± S.E.Normal+/+540.4 ± 5.7ap < 0.05, compared to wild-type controls.21.1 ± 4.038.5 ± 4.7-/-551.8 ± 3.3ap < 0.05, compared to wild-type controls.16.1 ± 0.632.5 ± 1.2High Fat+/+519.3 ± 0.5bp < 0.01, compared to wild-type controls.33.7 ± 6.447.0 ± 3.5-/-525.0 ± 2.0bp < 0.01, compared to wild-type controls.26.4 ± 4.348.6 ± 5.6a p < 0.05, compared to wild-type controls.b p < 0.01, compared to wild-type controls. Open table in a new tab Plasma Apolipoproteins—The levels of plasma apoA-I, apoB-100, and apoE were determined by immunoblot with specific antibodies (Fig. 1A). Plasma apoA-I levels showed a 10-fold increase in NFκB-deficient mice compared with control under a regular diet and a 2-fold increase under a high fat diet (Fig. 1B). There were no significant differences in the levels of apoE and apoB between these mouse types irrespective of diet. mRNA of Apolipoproteins in HepG2 Cells—To activate NFκB we treated HepG2 cells with LPS, and to inactivate NFκB directly we used adenovirus-mediated overexpression of IκBα super-repressor. When intracellular NFκB was activated by LPS treatment, the level of apoA-I mRNA in HepG2 cells was significantly decreased. Adenovirus-mediated overexpression of IκBα super-repressor, which leads to inactivation of NFκB, ameliorated this LPS-induced decrease. There was no significant difference in the levels of apoB and apoE mRNA, although NFκB was activated by LPS and/or inhibited by IκBα super-repressor (Fig. 2, A and B).Fig. 2Expression of apolipoproteins in HepG2 cells. HepG2 cells were transfected with multiplicity of infection 10 Ad5LacZ or Ad5IκB. 16 h after the transfection, the cells were treated with 10 μg/ml LPS or vehicle and incubated for additional 12 h. A, the levels of mRNA encoding apolipoprotein A-I, B, and E in HepG2 cells were semiquantified by reverse transcriptase-PCR analysis. B, mean relative level of apoA-I mRNA was compared with the level in Ad5LacZ-transfected and LPS-untreated cells. Values are the mean ± S.D. of five independent experiments. *, p < 0.05; **, p < 0.01. Note that Ad5IκB-mediated overexpression of IκBα super-repressor ameliorated the LPS-induced decrease in the level of apoA-I mRNA. C, apolipoprotein A-I, B, and E secreted from HepG2 cells were quantified by immunoblot analysis. D, mean relative level of apoA-I in culture medium of cells were compared with the level in Ad5Laz-transfected and LPS-untreated cells. Values are the mean ± S.D. of five independent experiments. *, p < 0.05; **, p < 0.01. Note that Ad5IκB-mediated overexpression of IκBα super-repressor ameliorated the LPS-induced decrease in the level of apoA-I in culture medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Apolipoproteins Secreted from HepG2 Cells—The level of apoA-I protein secreted from HepG2 cells was significantly decreased by LPS treatment. Adenovirus-mediated overexpression of IκBα super-repressor ameliorated this decrease. There was no significant difference in the levels of apoB and apoE, although NFκB is activated by LPS and/or inhibited by IκBα super-repressor (Fig. 2, C and D). PPARα Inhibitor Blocked the Effect of NFκB on ApoA-I Secretion—A control study in which HepG2 cells were treated with 10 μm WY14643, a PPARα agonist, for 24 h showed that apoA-I protein secretion from the cells was increased. This increase was blocked by treatment with 10 μm MK886, a selective PPARα inhibitor (28Kehrer J.P. Biswal S.S. La E. Thuillier P. Datta K. Fischer S.M. Vanden Heuvel J.P. Biochem. J. 2001; 356: 899-906Crossref PubMed Scopus (138) Google Scholar). These results indicate that apoA-I secretion from HepG2 cells is regulated by activation/inactivation of PPARα (Fig. 3, A and B). Likewise, when IκBα super-repressor was overexpressed in the cells, which leads to inactivation of NFκB, the secreted apoA-I protein was increased. This increase was blocked by treatment with 10 μm MK886 (Fig. 3, C and D). These results suggest that NFκB inactivation induces apoA-I secretion from HepG2 cells through activation of PPARα. Unlike apoA-I, there was no difference in the levels of apoB and apoE in the culture medium, although NFκB was inhibited by IκBα super-repressor or PPARα was inhibited by MK886 (Fig. 3C). Transactivation of ApoA-I Promoter by Inhibition of NFκB Was Mediated by PPARα—To confirm whether this induction of apoA-I mRNA by inhibition of NFκB really derives from activation of the apoA-I promoter and to determine whether this induction is mediated by the transcription factor PPARα, we constructed human apoA-I promoter-luciferase plasmids carrying the wild-type PPRE element (ApoAI-wt) or a mutant PPRE element (ApoAI-mut) and a shorter plasmid that does not carry the PPRE element (ApoAI-short) (Fig. 4A). HepG2 cells were transiently transfected with one of these promoter-reporter plasmids, transfected with adenovirus expressing IκBα super-repressor or LacZ, and then treated with LPS, MK886, or vehicle (5% v/v pure water and/or 0.1% v/v Me2SO). The relative promoter activity in the cells where IκBα super-repressor was overexpressed was significantly higher than the activity in LacZ-overexpressing cells (2.7-fold, p < 0.01, Fig. 4B). When the cells were transfected with ApoAI-wt plasmid and IκBα super-repressor, treatment with 10 μm MK886 significantly decreased relative promoter activity (2.7–1.4-fold, p < 0.01). When the cells were transfected with ApoAI-mut plasmid or apoAI-short plasmid, overexpression of IκBα super-repressor appeared to increase relative promoter activity a little, but these increases were much less than the increase when ApoAI-wt plasmid was used and were not significant. Cholesterol circulates in plasma as two main lipoprotein particles, LDL and HDL. LDL transports cholesterol synthesized in the liver to the peripheral tissues, whereas HDL returns cholesterol from the peripheral tissues to the liver for bile acid excretion. The major structural component of the HDL particle is apoA-I, whereas that of the LDL particle is apoB (29Malmendier C.L. Delcroix C. Atherosclerosis. 1985; 43: 557-564Google Scholar, 30Breslow J.L. Circulation. 1993; 87: 16-21Google Scholar). 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Nature. 1991; 353: 265-267Crossref PubMed Scopus (859) Google Scholar). Inhibitors of the NFκB pathway include a variety of agents, including antioxidants, proteasome inhibitors, decoy oligonucleotides, IκB phosphorylation and/or degradation inhibitors, and IκB super-repressor (43Epinat J-C. Gilmore T.D. Oncogene. 1999; 18: 6896-6909Crossref PubMed Scopus (205) Google Scholar). Some of them act as general inhibitors, whereas others act as specific inhibitors. Among them, adenovirus-mediated overexpression of IκBα super-repressor is a good tool for direct and selective inhibition of NFκB activity in vitro (44Jobin C. Panja A. Hellerbrand C. Iimuro Y. Didonato J. Brenner D.A. Sartor R.B. J. Immunol. 1998; 160: 410-418PubMed Google Scholar). In most cell types, the NFκB p50-p65 dimer is bound to one of the closely related endogenous inhibitory proteins, collectively referred to as IκB, and is held inactive in the cytoplasm. A variety of extracellular stimuli, including viral infection, LPS, cytokines, and stress factors have been reported to activate NFκB through phosphorylation of IκB at serine-32 and serine-36, dissociation of IκB from the complex, and translocation of the p50-p65 dimer from the cytoplasm to the nucleus. Phosphorylation of IκB also leads to its ubiquitination and proteasomal degradation. Adenovirus-mediated overexpression of IκBα super-repressor, where serine-32 and serine-36 of the IκBα are substituted by alanines, suppresses phosphorylation and degradation of IκBα (19DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar), which leads to selective and constitutive inactivation of NFκB. Transcriptional regulation of apoA-I by activated PPARα may be a species-specific phenomenon. In rats, Staels et al. (45Staels B. van Tol A. Andreu T. Auwerx J. Arterioscler. Thromb. 1992; 12: 286-294Crossref PubMed Google Scholar) report that treatment with a PPARα agonist, fenofibrate, markedly reduced hepatic apoA-I mRNA. In contrast, Vu-Dac et al. (46Vu-Dac N. Chopin-Delannoy S. Gervois P. Bonnelye E. Martin G. Fruchart J.C. Laudet V. Staels B. J. Biol. Chem. 1998; 273: 25713-25720Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) report that fibrates increase human apoA-I production due to stimulation of apoA-I gene expression in the liver. In this report they clearly demonstrated that this species-specific regulation of apoA-I expression was because of sequence differences in two distinct enhancer regions in the rodent and human apoA-I promoter. Similarly, using transgenic mice containing 21 copies of an 11-kilobase human genomic DNA fragment, Berthou et al. (47Berthou L. Duverger N. Emmanuel F. Langouet S. Auwerx J. Guillouzo A. Fruchart J.C. Rubin E. Denefle P. Staels B. Branellec D. J. Clin. Invest. 1996; 97: 2408-2416Crossref PubMed Scopus (243) Google Scholar) also clearly demonstrate that 7 days of treatment with fenofibrate (5% wt/wt) increased plasma human apoA-I up to 750% and HDL cholesterol up to 200% in the transgenic mice, whereas it decreased plasma mouse apoA-I to 59% in non-transgenic mice. Fibrates are widely used hypolipidemic drugs, and they activate PPARα that binds to the PPRE element on the human apoA-I promoter and positively regulate the expression of apoA-I. Fibrates also induce the expression of Rev-erbα, which binds to a RebRE site in the rat apoA-I promoter and then repress the expression of apoA-I. They showed that the transcription from human apoA-I gene was promoted via PPARα binding to a positive PPRE. Because of three single nucleotide differences, this site is not conserved in rats and mice. In contrast, rodent apoA-I transcription is repressed by Rev-erbα, whose binding site RebRE is adjacent to the TATA box in the rodent apoA-I promoter but not in the human apoA-I promoter (48Staels B. Auwerx J. Atherosclerosis. 1998; 137: 19-23Abstract Full Text Full Text PDF Scopus (106) Google Scholar). However, the question arises as to whether the three single nucleotide differences completely inactivated rodent PPRE or not. In our study, the levels of plasma apoA-I and HDL cholesterol were significantly higher in NFκB p50 subunit-deficient mice than wild-type littermates. To support our data, Bisgaier et al. (49Bisgaier C.L. Essenburg A.D. Barnett B.C. Auerbach B.J. Haubenwallner S. Leff T. White A.D. Creger P. Pape M.E. Rea T.J. Newton R.S. J. Lipid Res. 1998; 39: 17-30Abstract Full Text Full Text PDF PubMed Google Scholar) report that 7 days of treatment of rats with a novel PPARα activator PD72935 at a daily dose of 100 mg/kg significantly increased serum apoA-I and HDL cholesterol to 148 and 185% compared with control, respectively. Formation of the p65-p50 dimer and its translocation to the nucleus are completely lost in NFκB p50 subunit-deficient mice. Therefore, unlike the fenofibrate-treatment, nuclear PPARα could not interact with the p65 subunit, and the transcriptional activity of PPARα might be selectively on a high level (50Delerive P. De Bosscher K. Besnard S. Vanden Berghe W. Peters J.M. Gonzalez F.J. Fruchart J.C. Tedgui A. Haegeman G. Staels B. J. Biol. Chem. 1999; 274: 32048-32054Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar). Similarly, unlike with fenofibrate treatment, lack of the NFκB-p50 gene did not seem to induce the expression of Rev-erbα. To further support our concept, specific inhibition of NFκB by IκBα super-repressor significantly induced apoA-I expression in murine hepatoma Hepa1–6 cells, and this induction was blocked by treatment with a PPARα inhibitor, MK886 (data not shown). In such a way our result that the lack of NFκB p50 subunit increased plasma apoA-I and HDL cholesterol in mice is mostly compatible with the results of Berthou et al. (47Berthou L. Duverger N. Emmanuel F. Langouet S. Auwerx J. Guillouzo A. Fruchart J.C. Rubin E. Denefle P. Staels B. Branellec D. J. Clin. Invest. 1996; 97: 2408-2416Crossref PubMed Scopus (243) Google Scholar) and Bisgaier et al. (49Bisgaier C.L. Essenburg A.D. Barnett B.C. Auerbach B.J. Haubenwallner S. Leff T. White A.D. Creger P. Pape M.E. Rea T.J. Newton R.S. J. Lipid Res. 1998; 39: 17-30Abstract Full Text Full Text PDF PubMed Google Scholar). To assess our hypothesis that inactivation of NFκB facilitates expression of apoA-I through activation of the transcription factor PPARα, we performed reverse transcriptase-PCR analysis to semi-quantify apoA-I mRNA in HepG2 cells and immunoblot analysis to quantify its secretion into the medium from HepG2 cells. Activation of NFκB by LPS treatment significantly decreased apoA-I at both the mRNA and protein levels. Specific inactivation of NFκB by overexpression of IκBα super-repressor ameliorated the decreases in both levels (Fig. 2). This IκBα super-repressor-induced apoA-I elevation was completely blocked by pretreatment of the cells with MK886, a selective PPARα inhibitor (Fig. 3, C and D). When the NFκB p65-p50 dimer translocates into the nucleus, the p65 subunit interacts with PPARα and represses PPARα transactivation of a PPRE-driven promoter (50Delerive P. De Bosscher K. Besnard S. Vanden Berghe W. Peters J.M. Gonzalez F.J. Fruchart J.C. Tedgui A. Haegeman G. Staels B. J. Biol. Chem. 1999; 274: 32048-32054Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar). In the cells where IκBα super-repressor is overexpressed, the dimer tightly binds to the super-repressor, and translocation of the dimer into the nucleus and interaction of the p65 subunit with PPARα are suppressed (50Delerive P. De Bosscher K. Besnard S. Vanden Berghe W. Peters J.M. Gonzalez F.J. Fruchart J.C. Tedgui A. Haegeman G. Staels B. J. Biol. Chem. 1999; 274: 32048-32054Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar, 51Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1682) Google Scholar, 52Collart M.A. Baeuerle P. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar). To further support our hypothesis, activity of the human apoA-I promoter was also increased 2.7-fold by overexpression of IκBα super-repressor. The 2.7-fold increase was reduced to 1.5-fold by pretreatment with MK886, a selective PPARα inhibitor. When the apoA-I promoter, carrying mutations in the PPRE element, or the shorter promoter, which did not contain the PPRE element, was used instead of the wild-type promoter, the increase in promoter activity induced by IκBα super-repressor was blocked. These results indicate that NFκB activation/inactivation controls expression of apoA-I at the transcriptional level through PPARα. We have demonstrated here that lack of the NFκB p50 subunit in mice increased plasma apoA-I and HDL cholesterol without any difference in total cholesterol in vivo. We have also demonstrated that adenovirus-mediated inactivation of NFκB increased the expression of apoA-I at the mRNA and protein level in vitro. The reciprocal function between PPARα and NFκB to the PPRE in the apoA-I promoter was responsible for this transactivation. In summary, this report demonstrated that NFκB activity indirectly controls apoA-I expression both in vivo and in vitro. We thank Dr. Iimuro, Kyoto University, for Ad5IκB and Ad5LacZ." @default.
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