Transcription Factor Activating Enhancer-binding Protein-2β
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84037-1
ISSN1083-351X
AutoresKazuhiro Ikeda, Hiroshi Maegawa, Satoshi Ugi, Yukari Tao, Yoshihiko Nishio, Shuichi Tsukada, Shiro Maeda, Atsunori Kashiwagi,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoWe previously reported the association between the activating enhancer-binding protein-2β (AP-2β) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2β showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2β 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-2β in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2β on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2β modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2β overexpression and enhanced by knockdown of endogenous AP-2β, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2β and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2β binding site abolished the inhibitory effect of AP-2β. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2β and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2β directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2β might modulate the expression of adiponectin by directly inhibiting its transcriptional activity. We previously reported the association between the activating enhancer-binding protein-2β (AP-2β) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2β showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2β 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-2β in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2β on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2β modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2β overexpression and enhanced by knockdown of endogenous AP-2β, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2β and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2β binding site abolished the inhibitory effect of AP-2β. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2β and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2β directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2β might modulate the expression of adiponectin by directly inhibiting its transcriptional activity. We recently identified the human AP-2β transcription factor gene (TFAP2B) located on chromosome 6p12 as a susceptibility gene for type 2 diabetes in a genome-wide association study (1Maeda S. Tsukada S. Kanazawa A. Sekine A. Tsunoda T. Koya D. Maegawa H. Kashiwagi A. Babazono T. Matsuda M. Tanaka Y. Fujioka T. Hirose H. Eguchi T. Ohno Y. Groves C.J. Hattersley A.T. Hitman G.A. Walker M. Kaku K. Iwamoto Y. Kawamori R. Kikkawa R. Kamatani N. McCarthy M.I. Nakamura Y. J. Hum. Genet. 2005; 50: 283-292Crossref PubMed Scopus (63) Google Scholar). Several variations in the TFAP2B gene were significantly associated with obese type 2 diabetes in Japanese and British individuals (1Maeda S. Tsukada S. Kanazawa A. Sekine A. Tsunoda T. Koya D. Maegawa H. Kashiwagi A. Babazono T. Matsuda M. Tanaka Y. Fujioka T. Hirose H. Eguchi T. Ohno Y. Groves C.J. Hattersley A.T. Hitman G.A. Walker M. Kaku K. Iwamoto Y. Kawamori R. Kikkawa R. Kamatani N. McCarthy M.I. Nakamura Y. J. Hum. Genet. 2005; 50: 283-292Crossref PubMed Scopus (63) Google Scholar). We also demonstrated that AP-2β is preferentially expressed in human adipose tissue and that its expression is increased during adipocyte differentiation in mouse 3T3-L1 adipocytes (1Maeda S. Tsukada S. Kanazawa A. Sekine A. Tsunoda T. Koya D. Maegawa H. Kashiwagi A. Babazono T. Matsuda M. Tanaka Y. Fujioka T. Hirose H. Eguchi T. Ohno Y. Groves C.J. Hattersley A.T. Hitman G.A. Walker M. Kaku K. Iwamoto Y. Kawamori R. Kikkawa R. Kamatani N. McCarthy M.I. Nakamura Y. J. Hum. Genet. 2005; 50: 283-292Crossref PubMed Scopus (63) Google Scholar). Moreover, polymorphism in the first intron of TFAP2B directly affects the transcriptional activity of the gene (2Tsukada S. Tanaka Y. Maegawa H. Kashiwagi A. Kawamori R. Maeda S. Mol. Endocrinol. 2006; 20: 1104-1111Crossref PubMed Scopus (45) Google Scholar), and subjects with the disease-susceptible allele have stronger expression of AP-2β in their adipose tissue than those without the susceptible allele. Recently we also found that overexpression of AP-2β leads to lipid accumulation by enhancing glucose transport, thereby inducing insulin resistance in 3T3-L1 adipocytes (3Tao Y. Maegawa H. Ugi S. Ikeda K. Nagai Y. Egawa K. Nakamura T. Tsukada S. Nishio Y. Maeda S. Kashiwagi A. Endocrinology. 2006; 147: 1685-1696Crossref PubMed Scopus (35) Google Scholar). 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. 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Diabetes. 2001; 50: 2094-2099Crossref PubMed Scopus (1503) Google Scholar), CCAAT/enhancer binding protein (C/EBP), nuclear transcription factor-Y (NF-Y) (21Park S.K. Oh S.Y. Lee M.Y. Yoon S. Kim K.S. Kim J.W. Diabetes. 2004; 53: 2757-2766Crossref PubMed Scopus (86) Google Scholar), and sterol regulatory element-binding protein-1c (22Seo J.B. Moon H.M. Noh M.J. Lee Y.S. Jeong H.W. Yoo E.J. Kim W.S. Park J. Youn B.S. Kim J.W. Park S.D. Kim J.B. J. Biol. Chem. 2004; 279: 22108-22117Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). 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-2β 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-2β on the expression of adiponectin and IL-6 and the mechanisms by which AP-2β modulate their expressions were different. We herein demonstrated that AP-2β directly inhibits adiponectin gene expression by binding to its gene promoter and displacing NF-Y, subunit A (NF-YA). We concluded that AP-2β might modulate the expression of adiponectin by directly inhibiting its transcriptional activity. Materials—Human insulin was kindly provided by Eli Lilly (Indianapolis, IN). Anti-AP-2β 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 (23Ugi S. Imamura T. Maegawa H. Egawa K. Yoshizaki T. Shi K. Obata T. Ebina Y. Kashiwagi A. Olefsky J.M. Mol. Cell. Biol. 2004; 24: 8778-8789Crossref PubMed Scopus (192) Google Scholar). 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 (24Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Preparation of Recombinant Adenovirus—Adenovirus vector encoding the human AP-2β gene (Ad5-AP-2β) was generated as described previously (3Tao Y. Maegawa H. Ugi S. Ikeda K. Nagai Y. Egawa K. Nakamura T. Tsukada S. Nishio Y. Maeda S. Kashiwagi A. Endocrinology. 2006; 147: 1685-1696Crossref PubMed Scopus (35) Google Scholar). Adenovirus encoding the LacZ gene (Ad5-LacZ), as described previously (25Egawa K. Sharma P.M. Nakashima N. Huang Y. Huver E. Boss G.R. Olefsky J.M. J. Biol. Chem. 1999; 274: 14306-14314Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), was used for the control. Preparation of Expression Plasmid Vectors—Plasmid vector encoding the mouse AP-2β gene (pcDNA3.1/AP-2β) and mutant AP-2β that lacks DNA binding ability (pcDNA3.1/AP-2β R225C) were generated as described previously (3Tao Y. Maegawa H. Ugi S. Ikeda K. Nagai Y. Egawa K. Nakamura T. Tsukada S. Nishio Y. Maeda S. Kashiwagi A. Endocrinology. 2006; 147: 1685-1696Crossref PubMed Scopus (35) Google Scholar). 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 (26Hashimoto T. Nakamura T. Maegawa H. Nishio Y. Egawa K. Kashiwagi A. J. Biol. Chem. 2005; 280: 1893-1900Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Briefly, cells were rinsed twice with phosphate-buffered saline and then lightly trypsinized and pelleted by centrifugation at 650 × 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 × 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 × 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 (3Tao Y. Maegawa H. Ugi S. Ikeda K. Nagai Y. Egawa K. Nakamura T. Tsukada S. Nishio Y. Maeda S. Kashiwagi A. Endocrinology. 2006; 147: 1685-1696Crossref PubMed Scopus (35) Google Scholar). 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-2β, 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 β-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) (20Maeda N. Takahashi M. Funahashi T. Kihara S. Nishizawa H. Kishida K. Nagaretani H. Matsuda M. Komuro R. Ouchi N. Kuriyama H. Hotta K. Nakamura T. Shimomura I. Matsuzawa Y. Diabetes. 2001; 50: 2094-2099Crossref PubMed Scopus (1503) Google Scholar). 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. Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed using radiolabeled double-strand oligonucleotides as described previously (26Hashimoto T. Nakamura T. Maegawa H. Nishio Y. Egawa K. Kashiwagi A. J. Biol. Chem. 2005; 280: 1893-1900Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). 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-2β antibody (H-87X), respectively. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay protocol described by Latasa et al. (27Latasa M.J. Griffin M.J. Moon Y.S. Kang C. Sul H.S. Mol. Cell. Biol. 2003; 23: 5896-5907Crossref PubMed Scopus (85) Google Scholar) was used with some modification. Briefly, ∼ 1 × 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-2β 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-2β and scrambled control were as follows: AP-2β, 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. Overexpression of AP-2β 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 (5Kadowaki T. Hara K. Yamauchi T. Terauchi Y. Tobe K. Nagai R. Exp. Biol. Med. 2003; 228: 1111-1117Crossref PubMed Scopus (163) Google Scholar). We reported previously that overexpression of AP-2β leads to lipid accumulation through enhanced glucose transport in 3T3-L1 adipocytes (3Tao Y. Maegawa H. Ugi S. Ikeda K. Nagai Y. Egawa K. Nakamura T. Tsukada S. Nishio Y. Maeda S. Kashiwagi A. Endocrinology. 2006; 147: 1685-1696Crossref PubMed Scopus (35) Google Scholar). We, therefore, sought to evaluate the expression of adipocytokine genes under the same conditions. AP-2β 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-2β (Fig. 1, B and C). The cellular level of IL-6 and adiponectin was also increased and decreased, respectively, in the cells expressing AP-2β (Fig. 1D). Effects of AP-2β 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-2β-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-2β (pcDNA3.1/AP-2β) 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-2β 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-2β on the expression of both adiponectin and IL-6 is independent of adipocyte hypertrophy. Overexpression of AP-2β Inhibits Adiponectin but Not IL-6 Promoter Activity—To explore the possibility that AP-2β directly modulates the gene expression levels of adiponectin or IL-6, we next tested the effect of AP-2β on the transcriptional activities of adiponectin and IL-6 by luciferase reporter assays. As shown in Fig. 3A, overexpression of AP-2β did not affect the IL-6 promoter activity but inhibited the activity of adiponectin promoter in a vector dose-dependent manner (Fig. 3B). Endogenous AP-2β Knockdown by siRNA Enhances Adiponectin Promoter Activity—To evaluate the physiological role of AP-2β, we electroporated siRNA against AP-2β into 3T3-L1 adipocytes to deplete endogenous AP-2β protein. Forty-eight hours after electroporation, mRNA expression and the amount of AP-2β protein in the nuclear fraction were decreased by 70% in AP-2β siRNA-transfected cells compared with scrambled control siRNA-transfected cells (Fig. 4, A and B). In this condition, knockdown of AP-2β 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-2β (Fig. 4D). These results indicate that endogenous AP-2β modulates adiponectin promoter activity and that the regulatory effect of AP-2β on IL-6 expression occurs via a different mechanism to its effect on adiponectin. DNA Binding Activity of AP-2β Is Required for Its Inhibitory Effect on Adiponectin Promoter—To determine whether the AP-2β effect on adiponectin promoter activity is mediated via its role as a transcription factor, we prepared a mutant AP-2β lacking the ability to bind DNA. Arginine 225 of AP-2β is located in the DNA binding domain, and replacement of this arginine with cysteine (R225C) extinguishes DNA binding ability (28Satoda M. Zhao F. Diaz G.A. Burn J. Goodship J. Davidson H.R. Pierpont M.E. Gelb B.D. Nat. Genet. 2000; 25: 42-46Crossref PubMed Scopus (220) Google Scholar, 29Zhao F. Weismann C.G. Satoda M. Pierpont M.E. Sweeney E. Thompson E.M. Gelb B.D. Am. J. Hum. Genet. 2001; 69: 695-703Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We transiently expressed the wild-type (WT) and mutant (R225C) AP-2β in 3T3-L1 adipocytes and then measured adiponectin promoter activity. Adiponectin promoter activity was inhibited in WT-transfected cells; however, the R225C mutant had n
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