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Originally published In Press as doi:10.1074/jbc.M605132200 on September 5, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31245-31253, October 20, 2006
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Transcription Factor Activating Enhancer-binding Protein-2beta

A NEGATIVE REGULATOR OF ADIPONECTIN GENE EXPRESSION*

Kazuhiro Ikeda{ddagger}, Hiroshi Maegawa{ddagger}1, Satoshi Ugi{ddagger}, Yukari Tao{ddagger}, Yoshihiko Nishio{ddagger}, Shuichi Tsukada§, Shiro Maeda§, and Atsunori Kashiwagi{ddagger}

From the {ddagger}Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, 520-2192 Japan and §Laboratory for Diabetic Nephropathy, SNP Research Center, The Institute of Physical and Chemical Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan

Received for publication, May 30, 2006 , and in revised form, August 4, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported the association between the activating enhancer-binding protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2beta showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study we demonstrated that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2beta on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2beta modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2beta overexpression and enhanced by knockdown of endogenous AP-2beta, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2beta and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2beta binding site abolished the inhibitory effect of AP-2beta. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2beta and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2beta directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2beta might modulate the expression of adiponectin by directly inhibiting its transcriptional activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently identified the human AP-2beta transcription factor gene (TFAP2B) located on chromosome 6p12 as a susceptibility gene for type 2 diabetes in a genome-wide association study (1). Several variations in the TFAP2B gene were significantly associated with obese type 2 diabetes in Japanese and British individuals (1). We also demonstrated that AP-2beta is preferentially expressed in human adipose tissue and that its expression is increased during adipocyte differentiation in mouse 3T3-L1 adipocytes (1). Moreover, polymorphism in the first intron of TFAP2B directly affects the transcriptional activity of the gene (2), and subjects with the disease-susceptible allele have stronger expression of AP-2beta in their adipose tissue than those without the susceptible allele. Recently we also found that overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport, thereby inducing insulin resistance in 3T3-L1 adipocytes (3). These results suggest that TFAP2B is important in the pathogenesis of type 2 diabetes through the dysregulation of adipocyte function and that polymorphisms in TFAP2B affect expression of the gene, which thus, confers disease susceptibility.

Adipose tissue is an important source of metabolically active secretory factors, including free fatty acids, leptin, tumor necrosis factor-{alpha}, interleukin-6 (IL-6),2 monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, resistin, visfatin, and adiponectin (4). Although small adipocytes secrete insulin-sensitizing hormones such as adiponectin, hypertrophied adipocytes exhibit lower expression levels of these factors and high levels of insulin-resistant hormones such as tumor necrosis factor-{alpha} and IL-6, resulting in the insulin resistance observed in obesity (5). These phenomena are considered to have crucial roles in the pathogenesis of obesity-related diseases such as metabolic syndrome and type 2 diabetes.

Adiponectin has anti-diabetic (6–11) and anti-atherogenic (12–17) properties. Plasma concentrations of adiponectin are low in obesity, diabetes (12), and ischemic heart disease (12, 13). Several factors regulate adiponectin gene expression including other adipocytokines such as tumor necrosis factor-{alpha} (10) and IL-6 (18), transcription factors such as peroxisome proliferator-activated receptor-{gamma}, liver receptor homolog-1 (19, 20), CCAAT/enhancer binding protein (C/EBP), nuclear transcription factor-Y (NF-Y) (21), and sterol regulatory element-binding protein-1c (22). Although fat accumulation in the body seems to be the most powerful modulator of adiponectin expression, the molecular mechanism underlying this is largely unknown.

In this study, we demonstrated that overexpression of AP-2beta in 3T3-L1 adipocytes decreased both the expression and secretion of adiponectin and increased those of IL-6. We found that the effects of AP-2beta on the expression of adiponectin and IL-6 and the mechanisms by which AP-2beta modulate their expressions were different. We herein demonstrated that AP-2beta directly inhibits adiponectin gene expression by binding to its gene promoter and displacing NF-Y, subunit A (NF-YA). We concluded that AP-2beta might modulate the expression of adiponectin by directly inhibiting its transcriptional activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human insulin was kindly provided by Eli Lilly (Indianapolis, IN). Anti-AP-2beta antibody (H-87), anti-Acrp30 antibody (A-13), anti-IL-6 antibody (M-19), anti-CCAAT-binding transcription factor (CBF)-B antibody (G-2), C/EBP consensus oligonucleotide (sc-2525), CBF gel shift oligonucleotide (sc-2591), AP-2 consensus oligonucleotide (sc-2513), horseradish peroxidase-linked anti-rabbit, and anti-goat antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The pGL3-Basic luciferase vector and phRL-null vector were purchased from Promega (Madison, WI), and the piGENE mU6 vector was purchased from iGENE (Ibaragi, Japan) and TaKaRa BIO (Shiga, Japan). Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Invitrogen. All radioisotopes were obtained from ICN (Costa Mesa, CA). BioMax MR film was obtained from Eastman Kodak Co. (Rochester, NY). All other reagents and chemicals were from standard suppliers.

Cell Culture—3T3-L1 cells, which were provided by Dr. J. M. Olefsky (University of California, San Diego, CA), were cultured and differentiated into adipocytes as described previously (23). Before each experiment, the adipocytes were trypsinized and reseeded in appropriate culture dishes. The Ad-E1A-transformed human embryonic kidney cell line, 293 cells, was cultured as described previously (24).

Preparation of Recombinant Adenovirus—Adenovirus vector encoding the human AP-2beta gene (Ad5-AP-2beta) was generated as described previously (3). Adenovirus encoding the LacZ gene (Ad5-LacZ), as described previously (25), was used for the control.

Preparation of Expression Plasmid Vectors—Plasmid vector encoding the mouse AP-2beta gene (pcDNA3.1/AP-2beta) and mutant AP-2beta that lacks DNA binding ability (pcDNA3.1/AP-2beta R225C) were generated as described previously (3). Mouse NF-YA was cloned and inserted into pcDNA3.1 to generate the expression vector for mouse pcDNA3.1 (pcDNA3.1/NF-YA).

Infection—Ten days after induction of differentiation, 3T3-L1 adipocytes were infected with adenoviruses at the indicated multiplicity of infection for 24 h. Transfected cells were incubated for 48 h at 37 °C in an atmosphere of 10% CO2 in Dulbecco's modified Eagle's medium with 22.5 mM glucose and 2% heat-inactivated serum followed by serum starvation as required for the assay.

Nuclear Extraction—Nuclear extracts were prepared as described previously (26). Briefly, cells were rinsed twice with phosphate-buffered saline and then lightly trypsinized and pelleted by centrifugation at 650 x g for 5 min. The pellet was washed twice with phosphate-buffered saline, then suspended in lysis buffer A (10 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 1 µM phenylmethylsulfonyl fluoride, 2 µM sodium vanadate (Na3VO4), 2 µM leupeptin, 1 µM aprotinin, and 1 µM pepstatin). The cell suspension was homogenized, and nuclei were pelleted by centrifugation at 8000 x g for 5 min. The pellet was resuspended in buffer C (20 mM Tris-HCl, pH 7.5, 0.42 mM KCl, 20% glycerol, 1.5 mM MgCl2, Na3VO4, dithiothreitol, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and pepstatin at concentrations used for buffer A). The lysate was rotated for 30 min at 4 °C and centrifuged at 15,000 x g for 30 min.

Western Blotting—Cells were lysed in a solubilizing buffer containing 20 mM Tris-HCl, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 50 units/ml of aprotinin, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 50 mM NaF, pH 7.5, for 30 min at 4 °C. Whole cell lysates were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE, then electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). The specific proteins were detected by enhanced chemiluminescence.

Enzyme-linked Immunosorbent Assay—Conditioned media were collected from 3T3-L1 adipocyte cultures. The levels of adiponectin were determined using a mouse/rat adiponectin enzyme-linked immunosorbent assay kit (Otsuka, Tokyo, Japan).

Transfection Study—Cell transfection was performed using the Amaxa Nucleofector technology (Amaxa, Cologne, Germany) as described previously (3). Briefly, on day 5 after induction of differentiation, the cell suspension was mixed with 5 µg of luciferase reporter vector and phRL-null with various expression vectors or small interfering RNA (siRNAs) and electroporated using the program U-28. After transfection, cells were immediately transferred to 1 ml of growth medium and cultured for reporter assays, quantitative reverse transcription (RT)-PCR, and Western blotting.

RNA Preparation from Adipocytes and Quantitative RT-PCR—Total RNA was isolated with TRIzol reagent (Invitrogen). RT-PCR reactions were performed using the reverse transcription reagent (TaKaRa BIO). Real-time PCR was performed on a LightCycler machine (Roche Applied Science) using Light-Cycler-FastStart DNA Master SYBR Green I. Primer sets were as follows: mouse AP-2beta, 5'-GCGTCCTCAGAAGAGCCAAATC-3' and 5'-GTGCGTGATGAGACTGAAGTGC-3'; mouse adiponectin, 5'-GAAGATGACGTTACTACAAC-3' and 5'-TCAGTTGGTATCATGGAAGA-3'; mouse IL-6, 5'-ACAACCACGGCCTTCCCTACTT-3' and 5'-CACGATTTCCCAGAGAACATGTG-3'; mouse beta-actin, 5'-CGTGCGTGACATCAAAGAGAA-3' and 5'-TGGATGCCACAGGATTCCAT-3'.

Measurement of Luciferase Reporter Gene Activity—The luciferase reporter plasmid for human adiponectin promoter expression (pGL3/adiponectin promoter luc) was kindly provided by Dr. Iichiro Shimomura (Osaka University, Osaka, Japan) (20). The luciferase reporter plasmid for mouse IL-6 promoter expression (pGL3/IL-6 promoter luc) was generated by excising the promoter fragment (–1819/+70) from the genomic clone of IL-6 and inserting it into the MluI and BglII sites of the pGL3-Basic luciferase vector. Luciferase activities were measured using the dual-luciferase reporter assay system (Promega) using the protocol provided by the manufacturer. Luciferase values of phRL-null were measured for normalization.


Figure 1
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  		FIGURE 1.
Effect of overexpressing AP-2beta on expression of various adipocytokine genes. 3T3-L1 adipocytes were infected with either Ad5-LacZ or Ad5-AP-2beta at 50 multiplicity of infection. A, total RNA was isolated after 4 days, and quantitative RT-PCR was performed. Data are presented as the increase (n-fold) compared with LacZ control cells and are the mean ± S.E. of three independent experiments. *, p < 0.05 compared with the LacZ value. **, p < 0.01 compared with the LacZ value. B and C, culture media were collected 11 days after infection, and the protein levels of IL-6 (B) and adiponectin (C) were determined by enzyme-linked immunosorbent assay. The graph represents the mean ± S.E. of three independent experiments. D, LacZ- or AP-2beta-infected cells were lysed and analyzed by Western blotting with anti-AP-2beta (top), anti-IL-6 (second), anti-Acrp30 (third), and anti-beta-actin antibody (bottom). IB, immunoblot.

 
Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed using radiolabeled double-strand oligonucleotides as described previously (26). The oligonucleotide sequences of candidate 1, candidate 2, and mutant candidate 2 are shown in Fig. 6A. The protein-DNA binding reaction was performed at room temperature for 30 min, and the resultant complexes were resolved on 4% polyacrylamide gels. Competition and supershift assays were performed by adding a 50-fold molar excess of unlabeled oligonucleotide or 2 µg of anti-AP-2beta antibody (H-87X), respectively.

Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay protocol described by Latasa et al. (27) was used with some modification. Briefly, ~ 1 x 106 differentiated 3T3-L1 adipocytes were cross-linked for 10 min by adding formaldehyde directly to the tissue culture medium to a final concentration of 1%. Cross-linking was stopped by the addition of glycine to a final concentration of 0.125 M. Cross-linked cells were washed twice with phosphate-buffered saline and scraped. Nuclei were pelleted by centrifugation and resuspended in SDS lysis buffer. The chromatin solution was sonicated for 10-s pulses at maximum power. After centrifugation, the supernatant was divided into aliquots for 10-fold dilution in ChIP dilution buffer and precleared with protein G-agarose containing salmon sperm DNA for 1 h. The antibodies were added and incubated for 18 h at 4 °C followed by incubation with protein G-agarose for 3 h. The precipitates were washed, and chromatin complexes were eluted. After reversal of the cross-linking, the DNA was purified, and 5 µg of input control or ChIP samples were used as a template for PCR using the primer sets for regions containing the candidate AP-2 responsive elements. The sequences of primers used for ChIP assay were as follows: 5'-AGAAGCTCTACTTGGCTTCCC-3' and 5'-GCAGACCCCAGCTTACCA-3'.

Generation of Mutant Adiponectin Promoter—A QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used for mutagenesis. The putative AP-2 binding site in the proximal promoter region of the human adiponectin gene was mutated using the oligonucleotides 5'-CCCTCACTGAGTTGGAAAATAAGAAATGACAATTGTGAGG-3' and 5'-CCTCACAATTGTCATTTCTTATTTTCCAACTCAGTGAGGG-3' as primers in the in vitro mutagenesis reaction.

Transfection Using siRNAs—The target sequences for designing the siRNA against AP-2beta were obtained from Hokkaido System Science Co. (Hokkaido, Japan), and the sequence for the scrambled control was designed with four base mutations. Sense and antisense DNA oligonucleotides were inserted into piGENE mU6 vector. The target sequences for designing the siRNAs against AP-2beta and scrambled control were as follows: AP-2beta, 5'-CTACTCAGTTCAACTTCAAAGTACA-3'; scrambled control, 5'-CTACTCAGCCCAACGGCAAAGTACA-3' (underlining indicates the mutated bases).

Statistical Analysis—All values are expressed as the mean ± S.E. unless otherwise stated. Scheffe's multiple comparison test was used to determine the significance of any differences among more than three groups. A p value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of AP-2beta Modulates the Gene Expression of Adiponectin and IL-6—Adipocyte hypertrophy has been proposed as the primary cause of dysregulation of expression and secretion of adipocytokine in obesity, leading to metabolic syndrome and type 2 diabetes (5). We reported previously that overexpression of AP-2beta leads to lipid accumulation through enhanced glucose transport in 3T3-L1 adipocytes (3). We, therefore, sought to evaluate the expression of adipocytokine genes under the same conditions. AP-2beta was transfected into 3T3-L1 adipocytes with adenoviral vector 10 days after differentiation, and the mRNA expression of adiponectin and IL-6 was measured by real-time quantitative RT-PCR at 14 days. As shown in Fig. 1A, the mRNA expression of the IL-6 gene was augmented by 12.5-fold. In contrast, the mRNA expression of the adiponectin gene was inhibited by 62%. Consistent with these findings, IL-6 and adiponectin secretion into the media was increased by 2.2-fold and decreased by 48%, respectively, in cells overexpressing AP-2beta (Fig. 1, B and C). The cellular level of IL-6 and adiponectin was also increased and decreased, respectively, in the cells expressing AP-2beta (Fig. 1D).

Effects of AP-2beta on Adiponectin and IL-6 Expression Is Independent of Adipocyte Hypertrophy—It was difficult to distinguish the effect of adipocyte hypertrophy on the expression of adipocytokines in AP-2beta-overexpressing cells. Because 3T3-L1 adipocytes at 5–7 days after induction of differentiation contain only a small number of lipid droplets, the effect of adipocyte hypertrophy could be minimized. Thus, we next transfected the vector expressing AP-2beta (pcDNA3.1/AP-2beta) into 3T3-L1 adipocytes at 5 days after induction of differentiation and measured the mRNA expression of adiponectin and IL-6 after 48 h. As expected, the mRNA expression of AP-2beta was increased in a vector dose-dependent manner (Fig. 2, top panel). In this condition, the mRNA expression of IL-6 was increased, but that of adiponectin was decreased in a vector dose-dependent manner (Fig. 2, middle and bottom panels). These results suggest that the effect of AP-2beta on the expression of both adiponectin and IL-6 is independent of adipocyte hypertrophy.

Overexpression of AP-2beta Inhibits Adiponectin but Not IL-6 Promoter Activity—To explore the possibility that AP-2beta directly modulates the gene expression levels of adiponectin or IL-6, we next tested the effect of AP-2beta on the transcriptional activities of adiponectin and IL-6 by luciferase reporter assays. As shown in Fig. 3A, overexpression of AP-2beta did not affect the IL-6 promoter activity but inhibited the activity of adiponectin promoter in a vector dose-dependent manner (Fig. 3B).


Figure 2
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  		FIGURE 2.
Effects of AP-2beta on adiponectin mRNA level is independent of adipocyte hypertrophy. 3T3-L1 adipocytes were nucleofected with various amount of either pcDNA3.1 or pcDNA3.1/AP-2beta plasmid at 5 days after induction of differentiation, and total RNA was isolated after 48 h. mRNA levels of AP-2beta, IL-6, and adiponectin were measured. Data are presented as the increase (n-fold) compared with the pcDNA3.1-transfected control cells and are the mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 
Endogenous AP-2beta Knockdown by siRNA Enhances Adiponectin Promoter Activity—To evaluate the physiological role of AP-2beta, we electroporated siRNA against AP-2beta into 3T3-L1 adipocytes to deplete endogenous AP-2beta protein. Forty-eight hours after electroporation, mRNA expression and the amount of AP-2beta protein in the nuclear fraction were decreased by 70% in AP-2beta siRNA-transfected cells compared with scrambled control siRNA-transfected cells (Fig. 4, A and B). In this condition, knockdown of AP-2beta did not affect the mRNA expression of IL-6 but augmented that of adiponectin (Fig. 4C). Consistent with this, the promoter activity of adiponectin was also augmented by knockdown of AP-2beta (Fig. 4D). These results indicate that endogenous AP-2beta modulates adiponectin promoter activity and that the regulatory effect of AP-2beta on IL-6 expression occurs via a different mechanism to its effect on adiponectin.


Figure 3
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  		FIGURE 3.
Effect of overexpressing AP-2beta on adiponectin and IL-6 promoter activity. A and B, 3T3-L1 adipocytes were nucleofected with various concentrations of AP-2beta expression vector (pcDNA3.1/AP-2beta) and luciferase reporter constructs containing the 5'-flanking region of the mouse IL-6 gene (pGL3/IL-6 promoter luc) (A) or human adiponectin gene (pGL3/adiponectin promoter luc) (B), and the promoter activity of adiponectin was measured after 48 h. Data are presented as the increase (n-fold) compared with cells transfected with pGL3-Basic and pcDNA3.1 and are the mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 


Figure 4
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  		FIGURE 4.
Effects of depletion of AP-2beta by siRNA on promoter activity and mRNA expression of adiponectin. 3T3-L1 adipocytes were nucleofected with 5 µg of pcDNA3.1 vector (mock), AP-2beta siRNA (siRNA), or scrambled control siRNA (scramble) with (D) or without (A–C) pGL3/adiponectin promoter luc and analyzed after 48 h. A, total RNA was isolated, and mRNA expression of AP-2beta was measured. Data are presented as the increase (n-fold) compared with cells transfected with pcDNA3.1 and are the mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value. B, whole-cell lysates were prepared and analyzed by Western blotting with anti-AP-2beta antibody. IB, immunoblot. C, total RNA was isolated, and mRNA expressions of IL-6 and adiponectin were measured. Data are presented as the increase (n-fold) compared with cells transfected with pcDNA3.1 and are the mean ± S.E. of three independent experiments. D, data are presented as the increase (n-fold) in adiponectin promoter activity compared with cells transfected with pcDNA3.1 and are the mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 
DNA Binding Activity of AP-2beta Is Required for Its Inhibitory Effect on Adiponectin Promoter—To determine whether the AP-2beta effect on adiponectin promoter activity is mediated via its role as a transcription factor, we prepared a mutant AP-2beta lacking the ability to bind DNA. Arginine 225 of AP-2beta is located in the DNA binding domain, and replacement of this arginine with cysteine (R225C) extinguishes DNA binding ability (28, 29). We transiently expressed the wild-type (WT) and mutant (R225C) AP-2beta in 3T3-L1 adipocytes and then measured adiponectin promoter activity. Adiponectin promoter activity was inhibited in WT-transfected cells; however, the R225C mutant had no effect despite identical expression levels of the recombinant WT and mutated AP-2beta (Fig. 5, A and B). These results suggest that the DNA binding activity of AP-2beta is required for its inhibitory effect on the adiponectin promoter.

Identification of AP-2beta-responsive Element in Adiponectin Promoter—The AP-2 transcription factor family consists of five members, AP-2{alpha}, AP-2beta, AP-2{gamma}, AP-2{delta}, and AP-2{epsilon}, with each encoded by a separate gene, and all members recognize the same consensus sequence (5'-GCCN3GGG/C-3') through the basic domain that lies immediately N-terminal of the dimerization motif (30–34). There is 55.9% sequence homology between the proximal promoter regions of human and mouse adiponectin (22). As shown in Fig. 6A, we found that the human adiponectin promoter has two putative AP-2 binding sites (–110 to –102, candidate 1, and –88 to –80, candidate 2). The mouse adiponectin promoter has only one putative binding site that corresponds to the candidate 2 human promoter. Interestingly, these regions are close to NF-Y and C/EBP binding sites (21).

To identify the exact AP-2 binding sites, we performed EMSA using nuclear extracts from 3T3-L1 adipocytes. The oligonucleotide probe corresponding to candidate 1 did not form a complex (Fig. 6B, first lane), whereas candidate 2 oligonucleotide successfully formed the complex (Fig. 6B, second lane). This binding was not affected by unlabeled competitor oligonucleotide for C/EBP (Fig. 6B, third lane) but was ablated by unlabeled competitor oligonucleotide for NF-Y (Fig. 6B, fourth lane). The intensity of this binding was also attenuated by unlabeled competitor oligonucleotide for AP-2 (Fig. 6B, fifth lane). As shown in Fig. 6A, the binding sites for AP-2 and NF-Y seem to overlap, and it is possible that the expression levels of endogenous AP-2beta are lower than that of NF-Y. To address this, we repeated the assay using nuclear extract from cells overexpressing AP-2beta. As shown in Fig. 6C, the signal derived from AP-2beta-overexpressing cells was strong and broad; it also was partially ablated by either the competitive AP-2 consensus oligonucleotide or a blocking anti-AP-2beta antibody (Fig. 6D, third through fifth lanes). The signal derived from AP-2beta-overexpressing cells did not appear in 32P-radiolabeled candidate 1 oligonucleotide (Fig. 6D, first and second lanes). To further confirm the precise binding site, we generated a mutant oligonucleotide probe of candidate 2. We point mutated CC to AA at the –87/–86 position and GG to AA at the –82/–81 position (Fig. 6, box). These replacements totally abolished the complex formation (Fig. 6D, sixth and seventh lanes), suggesting that the candidate 2 sequence (–88 to –80) is the AP-2 binding site.


Figure 5
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  		FIGURE 5.
DNA binding activity of AP-2beta is required for its inhibitory effect on the adiponectin promoter. A, 3T3-L1 adipocytes nucleofected either pcDNA3.1 (mock), pcDNA3.1/AP-2beta (AP-2beta), or pcDNA3.1/AP-2beta R225C (R225C) were lysed and analyzed by Western blotting with anti-AP-2beta antibody. IB, immunoblot. B, 3T3-L1 adipocytes were nucleofected with either mock, AP-2beta, or R225C together with the pGL3/adiponectin promoter luc. Data are presented as the increase (n-fold) compared with cells transfected with pcDNA3.1 and are mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 


Figure 6
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  		FIGURE 6.
Identification of AP-2beta binding site in adiponectin promoter. A, comparison of human and mouse adiponectin promoter sequences. Binding sites of AP-2 (candidates 1 and 2), NF-Y, and C/EBP are shown. DNA oligonucleotide sequence of wild-types (candidate 1 and candidate 2) are boxed. In mutated probes (candidate 2MT) point mutations of CC to AA and GG to AA were introduced at –87/–86 and –82/–81, respectively. B, 32P-labeled double-stranded candidate 1 (lane 1) and 2WT (second through fifth lanes) oligonucleotides were used as probes. Unlabeled oligonucleotides with the consensus sequences of C/EBP, NF-Y, and AP-2 were added (third through fifth lanes). C, nuclear extracts from 3T3-L1 adipocytes overexpressing either LacZ or AP-2beta were incubated with 32P-labeled double-stranded 2WT oligonucleotides.D, nuclear extracts from 3T3-L1 adipocytes overexpressing either LacZ or AP-2beta were incubated with 32P-labeled probe of either candidate 1 (first and second lanes), 2WT (third through fifth lanes), or 2MT (sixth and seventh lanes). Unlabeled oligonucleotides of consensus sequences of AP-2 (fourth lane) or blocking anti-AP-2beta antibody (fifth lane) were added.

 
AP-2beta and NF-Y Bind to the Same Region of Adiponectin Promoter in Vivo—To further evaluate the interaction between transcriptional factors, AP-2beta, NF-YA, and C/EBP beta, we performed a ChIP assay using the corresponding antibodies. The DNA encompassing the region containing the AP-2, NF-Y, and C/EBP binding sites was amplified by PCR from chromatin immunoprecipitated with antibodies against C/EBP beta, NF-YA, and AP-2beta (Fig. 7A). The DNA binding of NF-YA was greater than that of AP-2beta but decreased when AP-2beta was overexpressed (Fig. 7A, lower panel). These results suggest that AP-2beta and NF-YA may competitively bind to an identical region of the adiponectin gene promoter.


Figure 7
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  		FIGURE 7.
AP-2beta competitively antagonizes NF-Y in the promoter region of the adiponectin gene. A, cross-linked chromatin from intact 3T3-L1 adipocytes infected with either LacZ or AP-2beta adenovirus was incubated with or without antibodies (no Ab) against C/EBP beta, NF-YA, or AP-2beta (second through fourth lanes). DNA isolated from input chromatin was amplified as a control. The amplified region matched the adiponectin promoter from –202 to –62. B, 3T3-L1 adipocytes were nucleofected with vector expressing either mock or mouse NF-YA. Whole-cell lysates were analyzed by Western blotting using anti-NF-YA antibody. IB, immunoblot. C, 3T3-L1 adipocytes were nucleofected with either mock, NF-YA, AP-2beta, or both NF-YA and AP-2beta with pGL3/adiponectin promoter luc. Data are presented as the increase (n-fold) compared with cells transfected with pcDNA3.1 and are mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 
To further analyze this putative competitive binding, mouse NF-YA, AP-2beta, or both were transiently overexpressed in 3T3-L1 adipocytes, and the adiponectin promoter activity was measured. Overexpression of NF-YA alone did not affect the adiponectin promoter activity compared with pcDNA3.1 alone (Fig. 7C), consistent with the report that NF-YA may have a crucial role in maintaining basal adiponectin gene expression (21). This was based on the findings that overexpression of dominant-negative mutant NF-YA but not wild-type NF-YA, affected adiponectin promoter activity. Based on these findings, we speculated that NF-Y is constitutively expressed and composed of three different subunits, which are collectively required to bind to the sequence (21). Co-expression of NF-YA and AP-2beta partially rescued the inhibition by AP-2beta alone. These results further support the competitive binding of NF-YA and AP-2beta on identical regions of the adiponectin gene promoter.

Introduction of a Mutation in the Adiponectin Promoter Region Abolishes the Inhibitory Effect of AP-2beta—Finally, to further validate the importance of AP-2beta binding to the adiponectin promoter, we introduced point mutations in the adiponectin promoter region in the same positions as the mutant candidate 2 oligonucleotide (Fig. 6A). Replacement of four nucleotides led to decreased luciferase activity due to the missing NF-Y binding site. Furthermore, these mutations completely abolished the inhibitory effect of AP-2beta on adiponectin promoter activity (Fig. 8). These results suggest that AP-2beta directly binds to the adiponectin promoter (position –88 to –80) and inhibits its activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We proposed previously that AP-2beta transcription factor is a candidate gene for causing metabolic syndrome and type 2 diabetes through the induction of adipocyte dysfunction, based on the following findings. We first identified the AP-2beta gene as a susceptibility gene for type 2 diabetes by conducting a genome-wide association study (1). Several variations in the AP-2beta were significantly associated with type 2 diabetes. Second, AP-2beta is preferentially expressed in adipose tissue, and its expression is increased upon induction of differentiation in 3T3-L1 adipocytes (1). Third, subjects with the disease susceptible allele have stronger expression of AP-2beta in adipose tissue than subjects without the susceptible allele (2). Finally, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and induces insulin resistance in 3T3-L1 adipocytes (3).

In the present study, overexpression of AP-2beta augmented the expression and secretion of IL-6, whereas it inhibited the expression and secretion of adiponectin. These results are consistent with the changes observed in obesity (35, 36). Because AP-2beta is a transcription factor, it may directly bind to the promoter regions of adipocytokines and thereby modulate their transcriptional activities. We identified putative AP-2 binding sites in the promoter region of adiponectin; the promoter activity of this cytokine was inhibited by overexpression of AP-2beta and enhanced by its knockdown, whereas that of IL-6 was unaffected. These results are consistent with the fact that putative AP-2 binding site exists in the adiponectin but not in the IL-6 promoter region. We also demonstrated a direct interaction between the adiponectin promoter and AP-2beta by both EMSA and ChIP assay. These results suggest that different molecular mechanisms operate to regulate the expressions of adiponectin and IL-6. AP-2beta may not only modulate the expressions of certain adipocytokines through direct binding of its promoter region but may also indirectly modulate adipocytokine expression in some cases. Further studies are needed to explore the mechanism by which AP-2beta modulates IL-6 expression as well as other adipocytokines.


Figure 8
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  		FIGURE 8.
Introduction of mutation in adiponectin promoter region abolished the inhibitory effect of AP-2beta. Mutant adiponectin promoter was generated. 3T3-L1 adipocytes were nucleofected with AP-2beta expression vector together with either pGL3/adiponectin promoter luc (WT) or pGL3/adiponectin (mutant) promoter luc (MT). Data are presented as the increase (n-fold) compared with cells transfected with pGL3-Basic and are mean ± S.E. of three independent experiments. *, p < 0.05 compared with the control value.

 
The newly identified AP-2beta binding site is close to those of C/EBP beta and NF-YA transcriptional factors, which bind to the adiponectin gene promoter region, and NF-YA was reported to play a crucial role in maintaining basal adiponectin gene expression (21). In particular, AP-2beta and NF-YA seemed to share the identical binding site. In fact, DNA binding of AP-2beta was ablated by unlabeled competitor oligonucleotide for NF-Y and decreased by the AP-2 oligonucleotide by EMSA. These results raised the possibility that AP-2beta and NF-YA form a molecular complex, which might bind to the adiponectin promoter. However, we did not observe complex formation of these two molecules by coimmunoprecipitation assay (data not shown). Furthermore, our ChIP assay suggested that AP-2beta and NF-YA may competitively bind to an identical region of the adiponectin promoter. Overexpression of AP-2beta decreased the DNA binding of NF-YA but did not affect the DNA binding of C/EBP beta, whereas overexpression of NF-Y decreased the DNA binding of AP-2beta, and NF-YA overexpression partially rescued the inhibitory effect of AP-2beta on adiponectin promoter activity. The AP-2 transcriptional factor modulated several genes, some of which were up-regulated (34, 37–44), whereas others are down-regulated (45, 46). Competitive binding of AP-2 with other transcription factors such as NF-Y is a possible molecular mechanism underlying the diverse effects of AP-2.

The finding that AP-2beta leads to lipid accumulation by enhancing glucose transport in 3T3-L1 adipocytes led us to propose AP-2beta as the candidate gene behind adipocyte hypertrophy. However, expression of AP-2beta was increased upon induction of differentiation, reaching a peak at 14 days (when the cells were most mature as assessed by insulin-stimulated glucose transport activity) and declined thereafter, even when the adipocytes continued to accumulate lipids in 3T3-L1 adipocytes (data not shown). These results suggest that AP-2beta plays a role in the differentiation and maturation of adipocytes rather than in their hypertrophy. Furthermore, our preliminary experiments revealed no increased, but rather reduced expression of AP-2beta in obese mice such as the ob/ob, db/db, and KKAy animals (data not shown). We observed that subjects with disease-susceptible alleles have stronger expression of AP-2beta in adipose tissue (2). Thus, AP-2beta expression may be predominantly regulated by genetic factors such as polymorphisms that affect disease susceptibility, whereas environmental factors may not regulate AP-2beta expression and acquired obesity may not affect its expression.

In conclusion, we identified the AP-2beta binding site in the promoter region of the human and mouse adiponectin genes and found that AP-2beta inhibits the transcriptional activity of the adiponectin gene through its binding in 3T3-L1 adipocytes. Based on these findings, we hypothesize that the AP-2beta transcriptional factor is a candidate gene causing dysfunction in adipocytes, leading to metabolic syndrome and type 2 diabetes.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to H. M.). 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

1 To whom correspondence should be addressed. Tel.: 81-77-548-2222; Fax: 81-77-543-3858; E-mail: maegawa{at}belle.shiga-med.ac.jp.

2 The abbreviations used are: IL-6, interleukin-6; Acrp30, adipocyte complement-related protein of 30 kDa; AP-2beta, activating enhancer-binding protein-2beta; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; luc, luciferase; NF-Y, nuclear transcription factor-Y; NF-YA, NF-Y, subunit A; RT, reverse transcription; siRNA, small interfering RNA; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank J. M. Olefsky (University of California, San Diego, CA) and Iichiro Shimomura (Osaka University, Osaka, Japan) for providing 3T3-L1 adipocytes and the luciferase reporter plasmid of human adiponectin promoter, respectively. We also thank Yukie Nakai for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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