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Originally published In Press as doi:10.1074/jbc.M306336200 on July 25, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38188-38193, October 3, 2003
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NF{kappa}B Regulates Plasma Apolipoprotein A-I and High Density Lipoprotein Cholesterol through Inhibition of Peroxisome Proliferator-activated Receptor {alpha}*

Atsuyuki Morishima {ddagger} §, Nobutaka Ohkubo {ddagger}, Nobuji Maeda {ddagger}, Tetsuro Miki § and Noriaki Mitsuda {ddagger} 

From the {ddagger}Departments of Physiology and the §Department of Geriatric Medicine, School of Medicine, Ehime University, Shigenobu, Ehime 791-0295, Japan

Received for publication, June 16, 2003 , and in revised form, July 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The levels of plasma HDL cholesterol and apoA-I in NF{kappa}B 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 NF{kappa}Bin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NF{kappa}B reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of I{kappa}B{alpha} super-repressor ameliorated the reduction. This I{kappa}B{alpha}-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), suggesting that NF{kappa}B inactivation induces apoA-I through activation of PPAR{alpha}. To further support this idea, the expression of I{kappa}B{alpha} increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPAR{alpha}-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NF{kappa}B increases apoA-I and HDL cholesterol through activation of PPAR{alpha} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear Factor {kappa}B (NF{kappa}B)1 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 (1, 2). Activation of NF{kappa}B is involved in the pathogenesis of many chronic inflammatory diseases, such as asthma and rheumatoid arthritis (3). It may also be involved in some neurodegenerative disorders such as Alzheimer's disease (4), ischemic brain injury (5), a variety of human cancers (6), and atherosclerosis (7).

Mice lacking the p65 subunit (RelA) display embryonic death at 15–16 days of gestation, concomitant with massive degeneration of the liver by apoptosis (8). To the contrary, mice lacking the p50 subunit of NF{kappa}B 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 (9). Although many researchers have demonstrated abnormalities in the immune responses of NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B-deficient mice than in wild-type littermates (data not shown). The plasma levels of apoE and apoB did not appear to differ between these NF{kappa}B-deficient and wild-type mice.

ApoA-I is mainly synthesized in the liver and small intestine (10). Because the levels of plasma apoA-I are positively correlated with hepatic apoA-I mRNA (11–13), 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) (14). In this cell line activated PPAR{alpha} binds to this element, thereby enhancing the expression of apoA-I (15–17). In this report we demonstrated that inactivation of NF{kappa}B enhances the expression and secretion of apoA-I from HepG2 cells through activation of the transcription factor PPAR{alpha}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Lipopolysaccharide (LPS) and WY-14643 (synthetic PPAR{alpha} agonist) were purchased from Sigma, and MK886 (selective inhibitor of PPAR{alpha}) 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-{beta}-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 NF{kappa}B p50 subunit-deficient mice Nfkb1tm1Bal, NF{kappa}B–/–) were originally purchased from The Jackson Laboratory (Bar Harbor, ME). The gene-targeting strategy used to generate this mouse line was described previously (9). 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 NF{kappa}B–/– 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 (18).

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—Ad5I{kappa}B (donated by Dr. Iimuro, Kyoto University, Kyoto, Japan) expresses I{kappa}B{alpha} 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 NF{kappa}B complex (19). Ad5LacZ, which expresses Escherichia coli {beta}-galactosidase under the control of the cytomegalovirus promoter, was used as a control for adenovirus-mediated transfection (20). Amplification, purification, and titration of the adenoviruses and adenovirus-mediated gene transfer were carried out as described previously (21). 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 x 105 cells/well) and incubated for 24 h. The cells were transfected with multiplicity of infection 10 Ad5IkB or Ad5LacZ as described previously (21) 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.


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  		TABLE I
Primers used for mRNA detection by reverse transcriptase-PCR

 

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 (25). 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 (26, 27), 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 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma Lipids—There was no significant difference in the concentration of total cholesterol in the plasma of female NF{kappa}B-deficient (NF{kappa}B–/–) mice and wild-type (NF{kappa}B+/+) 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 NF{kappa}B-deficient mice and wild-type littermates. NF{kappa}B-deficient mice showed a significantly higher HDL cholesterol level than wild-type littermates under both a regular and high fat diet. NF{kappa}B-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 NF{kappa}B-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).


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  		TABLE II
Fasting plasma lipids in NF{kappa}B-deficient (NF{kappa}B-/-) and wild-type (NF{kappa}B+/+) mice

Eight-week-old female mice were kept on a regular diet or high fat diet for 2 weeks. After an overnight fast, blood samples were obtained.

 

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  		TABLE III
Fasting plasma lipoprotein fractionation in NF{kappa}B-deficient (NF{kappa}B-/-) and wild-type (NF{kappa}B+/+) mice, measured by polyacrylamide disc-gel electrophoresis

All the mice used were female. IDL, intermediate density lipoprotein; VLDL, very low density lipoprotein.

 

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 NF{kappa}B-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 NF{kappa}B we treated HepG2 cells with LPS, and to inactivate NF{kappa}B directly we used adenovirus-mediated overexpression of I{kappa}B{alpha} super-repressor. When intracellular NF{kappa}B was activated by LPS treatment, the level of apoA-I mRNA in HepG2 cells was significantly decreased. Adenovirus-mediated overexpression of I{kappa}B{alpha} super-repressor, which leads to inactivation of NF{kappa}B, ameliorated this LPS-induced decrease. There was no significant difference in the levels of apoB and apoE mRNA, although NF{kappa}B was activated by LPS and/or inhibited by I{kappa}B{alpha} super-repressor (Fig. 2, A and B).



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  		FIG. 1.
Fasting plasma apolipoproteins in NF{kappa}B-deficient (NF{kappa}B–/–) and wild-type (NF{kappa}B+/+) mice. Mouse plasma was subjected to Western blot analysis with specific anti-apoA-I antibody, anti-apoB antibody, or anti-apoE antibody. A, immunoblot of apolipoproteins. Each lane is from the plasma of each mouse. B, relative plasma apolipoprotein levels. Values are the mean ± S.D. of 5 mice. **, p < 0.01, compared with wild-type control.

 


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  		FIG. 2.
Expression of apolipoproteins in HepG2 cells. HepG2 cells were transfected with multiplicity of infection 10 Ad5LacZ or Ad5I{kappa}B. 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 Ad5I{kappa}B-mediated overexpression of I{kappa}B{alpha} 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 Ad5I{kappa}B-mediated overexpression of I{kappa}B{alpha} super-repressor ameliorated the LPS-induced decrease in the level of apoA-I in culture medium.

 

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 I{kappa}B{alpha} super-repressor ameliorated this decrease. There was no significant difference in the levels of apoB and apoE, although NF{kappa}B is activated by LPS and/or inhibited by I{kappa}B{alpha} super-repressor (Fig. 2, C and D).

PPAR{alpha} Inhibitor Blocked the Effect of NF{kappa}B on ApoA-I Secretion—A control study in which HepG2 cells were treated with 10 µM WY14643, a PPAR{alpha} 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 PPAR{alpha} inhibitor (28). These results indicate that apoA-I secretion from HepG2 cells is regulated by activation/inactivation of PPAR{alpha} (Fig. 3, A and B). Likewise, when I{kappa}B{alpha} super-repressor was overexpressed in the cells, which leads to inactivation of NF{kappa}B, 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 NF{kappa}B inactivation induces apoA-I secretion from HepG2 cells through activation of PPAR{alpha}. Unlike apoA-I, there was no difference in the levels of apoB and apoE in the culture medium, although NF{kappa}B was inhibited by I{kappa}B{alpha} super-repressor or PPAR{alpha} was inhibited by MK886 (Fig. 3C).



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  		FIG. 3.
Effect of PPAR{alpha} inhibitor MK886 on the secretion of apolipoprotein A-I, B, and E. A and B, 10 µM WY14643, a PPAR{alpha} agonist, increased apoA-I in the culture medium. This increase was blocked by treatment with 10 µM MK886, a selective inhibitor of PPAR{alpha}. C and D, Ad5I{kappa}B-mediated overexpression of I{kappa}B{alpha} super-repressor in HepG2 cells increased the apoA-I in the culture medium compared with Ad5LacZ-mediated overexpression of {beta}-galactosidase. This increase was blocked by 10 µM MK866. A and C, the levels of apolipoprotein A-I, B, and E secreted from HepG2 cells and {beta}-actin in the cell lysate were quantified by immunoblot analysis. B and D, mean relative level of apoA-I in culture medium of HepG2 cells compared with the level in untreated cells (C) or Ad5LacZ-transfected cells (D). Values are mean ± S.D. of five independent experiments. *, p < 0.05; **, p < 0.01.

 

Transactivation of ApoA-I Promoter by Inhibition of NF{kappa}B Was Mediated by PPAR{alpha}—To confirm whether this induction of apoA-I mRNA by inhibition of NF{kappa}B really derives from activation of the apoA-I promoter and to determine whether this induction is mediated by the transcription factor PPAR{alpha}, 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 I{kappa}B{alpha} 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 I{kappa}B{alpha} 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 I{kappa}B{alpha} 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 I{kappa}B{alpha} 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.



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  		FIG. 4.
Transactivation of apoA-I promoter by inhibition of NF{kappa}B was mediated by PPAR{alpha}. A, human apoA-I promoter-luciferase constructs used in this study. The top line represents the region of the apoA-I promoter, where a hatched box for the putative PPAR{alpha}-binding site and open boxes for exon 1 and exon 2 are labeled PPRE, Exon 1, and Exon 2. A vertical arrow represents the transcription start site at position +1. A bent arrow represents the translation start site at position +236. Open boxes represent genomic DNA fragments corresponding to the human apoA-I gene (top line), which are cloned upstream of the firefly luciferase reporter gene in the plasmid pGL2-basic. Open boxes are labeled on the left with the name of the promoter-reporter plasmid. Numbers to the immediate left of the open box denote the 5'-end of the promoter fragment, whereas numbers to the immediate right denote the 3'-end. ApoAI-wt plasmid contains the wild-type PPRE sequence, whereas ApoAI-mut plasmid contains the mutant PPRE sequence. The ApoAI-short plasmid does not contain PPRE. B, mean relative promoter activity (RPA). HepG2 cells were transiently transfected with one of these promoter-reporter plasmids, transfected with adenovirus expressing I{kappa}B{alpha} super-repressor or LacZ, and then treated with LPS, MK886 or vehicle (0.1% v/v Me2SO (DMSO)). Values are the mean ± S.D. of five independent experiments. *, p < 0.05; **, p < 0.01.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 (29, 30). Plasma HDL cholesterol and apoA-I levels are correlated with each other (31–34), and they are negatively correlated with the prevalence of coronary heart disease (35–37) and atherosclerosis (31, 38–42).

Inhibitors of the NF{kappa}B pathway include a variety of agents, including antioxidants, proteasome inhibitors, decoy oligonucleotides, I{kappa}B phosphorylation and/or degradation inhibitors, and I{kappa}B super-repressor (43). Some of them act as general inhibitors, whereas others act as specific inhibitors. Among them, adenovirus-mediated overexpression of I{kappa}B{alpha} super-repressor is a good tool for direct and selective inhibition of NF{kappa}B activity in vitro (44). In most cell types, the NF{kappa}B p50-p65 dimer is bound to one of the closely related endogenous inhibitory proteins, collectively referred to as I{kappa}B, 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 NF{kappa}B through phosphorylation of I{kappa}B at serine-32 and serine-36, dissociation of I{kappa}B from the complex, and translocation of the p50-p65 dimer from the cytoplasm to the nucleus. Phosphorylation of I{kappa}B also leads to its ubiquitination and proteasomal degradation. Adenovirus-mediated overexpression of I{kappa}B{alpha} super-repressor, where serine-32 and serine-36 of the I{kappa}B{alpha} are substituted by alanines, suppresses phosphorylation and degradation of I{kappa}B{alpha} (19), which leads to selective and constitutive inactivation of NF{kappa}B.

Transcriptional regulation of apoA-I by activated PPAR{alpha} may be a species-specific phenomenon. In rats, Staels et al. (45) report that treatment with a PPAR{alpha} agonist, fenofibrate, markedly reduced hepatic apoA-I mRNA. In contrast, Vu-Dac et al. (46) 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. (47) 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 PPAR{alpha} 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-erb{alpha}, 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 PPAR{alpha} 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-erb{alpha}, whose binding site RebRE is adjacent to the TATA box in the rodent apoA-I promoter but not in the human apoA-I promoter (48).

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 NF{kappa}B p50 subunit-deficient mice than wild-type littermates. To support our data, Bisgaier et al. (49) report that 7 days of treatment of rats with a novel PPAR{alpha} 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 NF{kappa}B p50 subunit-deficient mice. Therefore, unlike the fenofibrate-treatment, nuclear PPAR{alpha} could not interact with the p65 subunit, and the transcriptional activity of PPAR{alpha} might be selectively on a high level (50). Similarly, unlike with fenofibrate treatment, lack of the NF{kappa}B-p50 gene did not seem to induce the expression of Rev-erb{alpha}. To further support our concept, specific inhibition of NF{kappa}B by I{kappa}B{alpha} super-repressor significantly induced apoA-I expression in murine hepatoma Hepa1–6 cells, and this induction was blocked by treatment with a PPAR{alpha} inhibitor, MK886 (data not shown). In such a way our result that the lack of NF{kappa}B p50 subunit increased plasma apoA-I and HDL cholesterol in mice is mostly compatible with the results of Berthou et al. (47) and Bisgaier et al. (49).

To assess our hypothesis that inactivation of NF{kappa}B facilitates expression of apoA-I through activation of the transcription factor PPAR{alpha}, 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 NF{kappa}B by LPS treatment significantly decreased apoA-I at both the mRNA and protein levels. Specific inactivation of NF{kappa}B by overexpression of I{kappa}B{alpha} super-repressor ameliorated the decreases in both levels (Fig. 2). This I{kappa}B{alpha} super-repressor-induced apoA-I elevation was completely blocked by pretreatment of the cells with MK886, a selective PPAR{alpha} inhibitor (Fig. 3, C and D). When the NF{kappa}B p65-p50 dimer translocates into the nucleus, the p65 subunit interacts with PPAR{alpha} and represses PPAR{alpha} transactivation of a PPRE-driven promoter (50). In the cells where I{kappa}B{alpha} 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 PPAR{alpha} are suppressed (50–52).

To further support our hypothesis, activity of the human apoA-I promoter was also increased 2.7-fold by overexpression of I{kappa}B{alpha} super-repressor. The 2.7-fold increase was reduced to 1.5-fold by pretreatment with MK886, a selective PPAR{alpha} 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 I{kappa}B{alpha} super-repressor was blocked. These results indicate that NF{kappa}B activation/inactivation controls expression of apoA-I at the transcriptional level through PPAR{alpha}.

We have demonstrated here that lack of the NF{kappa}B 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 NF{kappa}B increased the expression of apoA-I at the mRNA and protein level in vitro. The reciprocal function between PPAR{alpha} and NF{kappa}B to the PPRE in the apoA-I promoter was responsible for this transactivation. In summary, this report demonstrated that NF{kappa}B activity indirectly controls apoA-I expression both in vivo and in vitro.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 81-89-960-5245; Fax: 81-89-960-5246; E-mail: mitsuda{at}m.ehime-u.ac.jp.

1 The abbreviations used are: NF{kappa}B, nuclear factor {kappa}B; HDL, high density lipoprotein; LDL, low density lipoprotein; LPS, lipopolysaccharide; PPRE, peroxisome proliferator response element; PPAR, peroxisome proliferator-activated receptor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Iimuro, Kyoto University, for Ad5I{kappa}B and Ad5LacZ.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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