Báo cáo khoa học: AdipoR2 is transcriptionally regulated by ER stress-inducible ATF3 in HepG2 human hepatocyte cells pdf

AdipoR2 is transcriptionally regulated by ER stress-inducible ATF3 in HepG2 human hepatocyte cells In-uk Koh1,2, Joo H Lim1, Myung K Joe1, Won H Kim1, Myeong H Jung3, Jong B Yoon2 and Jihyun Song1 Division of Metabolic Disease, Department of Biomedical Science, National Institutes of Health, Seoul, South Korea Department of Biochemistry, College of Science, Yonsei University, Seodaemoon-Gu, Seoul, South Korea School of Korean Medicine, Pusan National University, Yangsan-si, Gyeongnam, South Korea Keywords AdipoR2; ATF3; ER stress; insulin resistance; obesity Correspondence M H Jung, School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735, South Korea Fax: +82 51 510 8437 Tel: +82 51 510 8468 E-mail: jung0603@pusan.ac.kr J B Yoon, Department of Biochemistry, College of Science, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, South Korea Fax: +82 392 3488 Tel: +82 2123 2704 E-mail: yoonj@yonsei.ac.kr J Song, Division of Metabolic Disease, Department of Biomedical Science, National Institutes of Health, Nokbun-dong, Eunpyung-gu, Seoul 122-701, South Korea Fax: +82 354 1057 Tel: +82 380 1530 E-mail: jhsong10@korea.kr (Received 22 September 2009, revised 22 February 2010, accepted March 2010) Adiponectin acts as an insulin-sensitizing adipokine that protects against obesity-linked metabolic disease, which is generally associated with endoplasmic reticulum (ER) stress The physiological effects of adiponectin on energy metabolism in the liver are mediated by its receptors We found that the hepatic expression of adiponectin receptor (AdipoR2) was lower, but the expression of markers of the ER stress pathway, 78 kDa glucose-regulated protein (GRP78) and activating transcription factor (ATF3), was higher in the liver of ob/ob mice compared with control mice To investigate the regulation of AdipoR2 by ER stress, we added thapsigargin, an ER stress inducer, to a human hepatocyte cell line, HepG2 Addition of the ER stress inducer increased the levels of GRP78 and ATF3, and decreased that of AdipoR2, whereas addition of a chemical chaperone, 4phenyl butyric acid (PBA), could reverse them Up- or down-regulation of ATF3 modulated the AdipoR2 protein levels and AdipoR2 promoter activities Reporter gene assays using a series of 5¢-deleted AdipoR2 promoter constructs revealed the location of the repressor element responding to ER stress and ATF3 In addition, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we identified a region between nucleotides )94 and )86 of the AdipoR2 promoter that functions as a putative ATF3-binding site in vitro and in vivo Thus, our findings suggest that the ER stress-induced decrease in both protein and RNA of AdipoR2 results from a concomitant increase in expression of ATF3, which may play a role in the development of obesity-induced insulin resistance and related ER stress in hepatocytes doi:10.1111/j.1742-4658.2010.07646.x Introduction Obesity and/or obesity-linked insulin resistance, one of the key features of type diabetes, are regarded as risk factors for metabolic syndrome and atherosclerosis [1] Adiponectin, which is abundantly expressed in adipose tissue, is a circulating peptide hormone with direct insulin-sensitizing activity This adipokine has ameliorative effects on insulin resistance in peripheral tissues, and plays a central role in the regulation of energy Abbreviations AdipoR2, adiponection receptor 2; ATF3, activating transcription factor 3; GRP78, 78 kDa glucose-regulated protein; PBA, 4-phenyl butyric acid 2304 FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al homeostasis [2–4] In obesity, adiponectin activity declines as a result of decreased adiponectin expression and/or a defect in downstream adiponectin signalling The combined actions of genetic factors such as singlenucleotide polymorphisms in the adiponectin gene and environmental factors such as a high-fat diet and sedentary lifestyle promoting obesity are thought to be one of the mechanisms leading to insulin resistance [5] Several drugs are known to affect adiponectin levels in the plasma and expression in tissues Plasma adiponectin levels increase in response to the PPAR (peroxisome proliferator-activated receptor) agonists, thiazolidinediones, but decrease in response to antiHIV drugs and the well-known endoplasmic reticulum (ER) stressor, thapsigargin ER stress is a malfunction of the organelle caused by the influx of immature proteins and/or depletion of calcium ions [6–8], and this perturbation of ER homeostasis occurs in diet-induced and genetic models of obesity [9] Studies of the regulation of obesity-linked insulin resistance have led to the suggestion that ER stress plays a role in diabetic insulin resistance [10] ER dysfunction and the integrated stress response could lead to abnormal activation of c-Jun N-terminal kinase (JNK) and/or activating transcription factor (ATF3), which in turn modify the lipogenic pathway, insulin signaling and the expression levels of genes related to insulin action, such as insulin receptor substrates and adiponectin [8,9], resulting in obesity-related insulin resistance [11–13] In an in vitro model of ER stress induced by proteasome inhibition, the stress induced transcription of the transcription factors GADD153, ATF4 and ATF3, and regulation of lipogenic pathways by the ER stress response was also shown in hepatocytes [14] ER stressors such as thapsigargin or tunicamycin reduced insulin signaling by serine phosphorylation of insulin receptor substrate [9,15] We recently demonstrated that an agent causing ER stress activated JNK and consequently induced ATF3, with a reduction in adiponectin transcription [8] Nakatani et al [16] identified a molecular chaperone that protects cells from ER stress and its effect on insulin sensitivity in the liver The chemical chaperones 4-phenyl butyric acid (PBA) and tauroursodeoxycholic acid, which have the ability to decrease ER stress, act as potent anti-diabetic agents [10] Because ER is abundant in hepatocytes, and the liver is a primary target organ of insulin and adiponectin [3,14,17], we focused on regulation of the adiponectin receptor under ER stress-induced conditions in liver cells In humans, adiponectin receptors and (AdipoR1 and AdipoR2, respectively) serve as receptors for globular and/or full-length adiponectin AdipoR1 is ubiqui- Transcriptional regulation of AdipoR2 by ATF3 tously expressed and is particularly abundant in skeletal muscle, whereas AdipoR2 is expressed primarily in liver [17] These receptors have seven transmembrane domains, and share 67% amino acid homology In contrast to G protein-coupled receptors, their N-terminus is intracellular [5] Although the intracellular adaptor protein APPL1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) has recently been proposed as a modulator of insulin action by binding to adiponectin receptors [18], the overall mechanism of adiponectin signaling is largely unknown As expression of adiponectin receptors, as well as adiponectin itself, is known to be decreased in obesity-related insulin resistance but increased by PPAR agonists [1,19– 21], both adiponectin and its receptors are regarded as potential therapeutic targets for control of obesitylinked insulin resistance [22,23] However, there are very few studies that have examined a specific agent or the mechanisms responsible for regulating adiponectin receptor expression [17,24–26] In this study, we demonstrate that AdipoR2 is negatively regulated in liver cells in response to ER stress or induced expression of ATF3 In addition, by analyzing the promoter region of the AdipoR2 gene, we have identified a putative ATF3-binding site in the 5’ flanking region, suggesting that direct binding of this transcription factor might negatively regulate AdipoR2 expression We hypothesize that ER stress-inducible ATF3 plays an important role in regulating AdipoR2 in the liver Results Down-regulation of AdipoR2 with up-regulation of ATF3 expression under increased ER stress conditions As shown in previous studies of obesity and ER stress [9,10], the expression of the ER stress marker proteins GRP78 and ATF3 was increased 1.4- and 2.0-fold, respectively, in ob/ob mice compared with lean controls, whereas that of AdipoR2 was decreased 0.8-fold (Fig 1A) AdipoR2 is the major hepatic receptor for adiponectin [5] ER stress-induced disruption of adiponectin action and increased insulin resistance in hepatocytes could be attributed to down-regulation of the AdipoR2 level, and thus we studied the effect of ER stress on the AdipoR2 level in human hepatocytes When HepG2 cells were exposed to 1.0 lm thapsigargin, a well-known inducer of ER stress, for 24 h, both the ER molecular chaperone GRP78 and also ATF3, which has been shown to repress transcription of the adiponectin gene in adipocytes [8], were induced FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2305 Transcriptional regulation of AdipoR2 by ATF3 A In-uk Koh et al C57BL6/J ob/ob 2.5 C57BL6/J ob/ob * Arbitrary units GRP78 ATF3 AdipoR2 1.5 * 0.5 Actin GRP78 B ATF3 C + – + + GRP78 ATF3 C * Thap Thap/PBA atf3 adipor2 * * gapdh * * AdipoR2 * * 1.5 (AU) – – Arbitrary units Thap (1.0 µM) PBA (20 mM) AdipoR2 GRP78 Actin Control ATF3 Thap 0.5 AdipoR2 Thap/PBA atf3 D adipor2 E Adv-ATF3 (MOI) – 10 Adv-YFP Ad YFP – – – (MOI) ATF3 Thap siATF3 Neg.RNAi ATF3 – – – + – – + + – + – + Control Thap Thap + siATF3 Thap + Neg.RNAi 2.5 * * * * 1.5 AdipoR2 AdipoR2 Actin Actin 0.5 ATF3 AdipoR2 Fig Changes in expression of AdipoR2 under conditions of ER stress and ATF3 over-expression (A) Relative levels of GRP78, ATF3 and AdipoR2 protein in C57BL6/J and ob/ob mice (n = for each group; 30 lg protein per lane) (B) Relative levels of GRP78, ATF3 and AdipoR2 protein in HepG2 cells treated with or without pre-incubation in 20 mM PBA for 24 h prior to treatment with 1.0 lM thapsigargin (30 lg protein per lane; b-actin as control) (C) Relative levels of ATF3 and AdipoR2 mRNA in treated cells Semi-quantitative RT-PCR analysis was performed using GAPDH as the internal control and the values were normalized to control (untreated) (D) Changes in expression of AdipoR2 after infection with ATF3-expressing adenovirus HepG2 cells were infected with an adenoviral vector expressing human ATF3 (Adv-ATF3) at a multiplicity of infection of 2–10 and incubated for 48 h HepG2 cells infected with Adv-YFP at a multiplicity of infection of were used as control (E) Changes in expression of endogenous AdipoR2 upon thapsigargin-induced ER stress with or without silencing of ATF3 siATF3 or Neg.RNAi was introduced to the cells 24 h prior to treatment with 1.0 lM thapsigargin For western blot analysis, b-actin was used as a protein loading control The asterisks indicate a P value < 0.05 for the bracketed comparisons Interestingly, the level of AdipoR2 protein was decreased coincidentally with the increase of ATF3 (Fig 1B and Table S1) To determine whether the observed changes in protein expression were caused by thapsigargin-induced 2306 ER stress, cells were pre-incubated with 20 mm PBA for 24 h prior to thapsigargin exposure In cells pre-incubated with PBA, thapsigargin-induced increases in GRP78 and ATF3 protein levels did not occur, and the ER stress-induced decrease in AdipoR2 level was FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al specifically rescued (Fig 1B, lane 3) In addition, we measured the mRNA levels for ATF3 and AdipoR2 in HepG2 cells with or without thapsigargin or PBA treatment, and the results showed a trend similar to the protein level changes (Fig 1C) We next examined the effect of ATF3 over-expression in hepatocytes on changes in the AdipoR2 protein level We introduced an adenoviral vector carrying recombinant ATF3 (Adv-ATF3) into HepG2 cells, and analyzed the resulting protein expression using western blotting As expected, transduction of Adv-ATF3 resulted in a dose-dependent increase in the ATF3 protein level The increase in ATF3 protein level resulted in a decrease in the AdipoR2 protein level, but in a non-dose-dependent way (Fig 1D) The absence of dose dependence for the reduction of AdipoR2 may be due to cellular systemic utilization of the proteins In order to further investigate whether ATF3 plays an important role in ER stress-induced down-regulation of AdipoR2, we assessed the effect of knocking down ATF3 on the AdipoR2 level Within 48 h after introducing siRNA against ATF3 (siATF3) to HepG2 cells, ATF3 was mostly repressed, but the endogenous AdipoR2 level was relatively increased The expected changes in ATF3 and AdipoR2 levels as a result of thapsigargin treatment were significantly ameliorated by siATF3 (Fig 1E and Table S2) These changes were not observed in cells treated with control siRNA (Neg.RNAi) These data confirm the negative regulatory effect of transcription factor ATF3 on AdipoR2 levels Localization of a repressor element in the AdipoR2 promoter To further investigate the changes in AdipoR2 expression as a result of increased ER stress in hepatocytes, we examined the AdipoR2 promoter activities in HepG2 cells using the reporter gene construct AR2P()1974), comprising nucleotides )1974 to +0 Exposure of cells transfected with AR2P()1974) to 1.0 lm thapsigargin caused an approximately 80% repression of transcription from the promoter region of AdipoR2 in 24 h (Fig 2A) In addition, to assess the effect of ATF3, a known ER stress-induced transcriptional repressor in adipocytes [8], on AdipoR2 regulation in hepatocytes, we measured the transcriptional activity of the AdipoR2 promoter when AR2P()1974) was co-transfected with an ATF3expressing vector (ATF3/pcDNA3.1) As for thapsigargin exposure (Fig 2A), ATF3 expression in HepG2 cells down-regulated the promoter activity in a dose-depen- Transcriptional regulation of AdipoR2 by ATF3 dent manner (Fig 2B) Compared with Neg.RNAi treatment, silencing of ATF3 reduced the repressive effect of thapsigargin on the promoter activity of AdipoR2 (Fig 2C) To investigate whether ATF3 affects AdipoR2 expression directly, in other words to locate the repressor element in the AR2P()1974) promoter region, as suggested by the above results, we analyzed the promoter activity of 5¢ serially deleted human AdipoR2 promoter constructs in pGL3-Basic vector (Fig 2D,E) Four plasmid constructs containing portions of the promoter region of various lengths were transfected into HepG2 cells with or without co-transfection of the ATF3-expressing vector (ATF3/ pcDNA3.1) As shown in Fig 2D, ATF3 co-transfection repressed the promoter activities of the transfected AdipoR2 reporter constructs AR2P()1974), AR2P ()870) and AR2P()343) However, the activity of the shortest construct AR2P()72) was as low as that in the control (pGL3) group In another experiment, various amounts of ATF3/pcDNA3.1 (0, 0.2 and 0.4 lg) were co-transfected with AR2P()343) or AR2P()72), and significant dose-dependent repression by ATF3 was observed in cells transfected with AR2P()343) but not in those transfected with AR2P()72) (Fig 2E) ATF3 co-transfection with this shortest construct AR2P()72) showed a tendency to decrease the reporter activity (approximately 50%) but without statistical significance (P = 0.15) (Table S3 and Fig S1) Given that AR2P()72) was not responsive to ATF3, we presume that more than 72 nucleotides of promoter region are required for the expression of AdipoR2, and that at least one of the repressive elements of AdipoR2 is located between nucleotides )343 and )72 ATF3 binds to the AdipoR2 promoter in vitro and in vivo To confirm that the above results are an effect of ATF3 on AdipoR2 expression, we searched for a putative repressor binding site by observing sequences without the aid of computer software between nucleotides )343 and )72 of the human AdipoR2 gene and using TESS analysis (http://www.cbil.upenn.edu/cgi-bin/tess/ tess) with TRANSFAC database version 6.0 (available online; http://www.gene-regulation.com) We isolated the sequence 5¢-TGCGCGTCA-3¢ located at nucleotides )94 to )86 (Fig 3A), which is similar to the consensus palindromic ATF/CRE site (TGACGTCA) to which members of the ATF3 family are known to homo- or heterodimerize for DNA binding and transcriptional regulation [27] We performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts from HepG2 cells and FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2307 Transcriptional regulation of AdipoR2 by ATF3 A B * 120 80 60 40 20 C * * 100 80 60 40 20 D 150 + – + – + – * – + + AR2P(–1974) – ATF3 – * 75 50 NS AR2P(–343) – AR2P(–72) – ATF3 – + – + + Luciferase activity (%) 100 AR2P(–870) – 80 60 40 20 + AR2P(–1974) + ++ Thap (1 µM) – siATF3 – Neg.RNAi – + + – – * 120 125 AR2P( AR2P(–1974) + 100 E * 25 * * 0 pGL3 + AR2P(–1974) – Thap – * 120 Luciferase activity (%) 100 Luciferase activity (%) * 120 Luciferase activity (%) Luciferase activity (%) In-uk Koh et al + + + – * + + – + * 100 80 60 40 NS NS 20 NS + – – – + – + – – – – + – – + – – + – – – – + – + – – – + – – – – + + AR2P(–72) + ( + + – – – AR2P(–343) – – – + + + ATF3 0.4 0.4 Fig Changes in the promoter activity of AdipoR2 under conditions of ER stress and ATF3 over-expression (A) Activity of the AdipoR2 promoter in HepG2 human liver cells with ER stress induction by 1.0 lM thapsigargin for 24 h The pGL3-Basic-derived reporter construct comprising nucleotides )1974 to +0 of the AdipoR2 promoter [AR2P(–1974)] was transfected into HepG2 cells, followed by treatment with thapsigargin (B) Activity of the AdipoR2 promoter in HepG2 cells with ATF3 over-expression by co-transfection of 0.2 or 0.4 lg of ATF3 expression vector (C) Activity of the AdipoR2 promoter in HepG2 cells upon thapsigargin-induced ER stress with or without silencing of ATF3 siATF3 or Neg.RNAi was introduced to the cells 24 h prior to treatment with 1.0 lM thapsigargin Luciferase activity values were measured in triplicate and expressed as arbitrary units (D) Promoter activities of reporter gene constructs containing 0.6 lg of various lengths of 5¢ deleted fragments of the promoter region subcloned into the pGL3-Basic plasmid vector and transfected with or without 0.4 lg of ATF3-expressing vector (E) ATF3-dose-dependent repression of the promoter activity upon co-transfection of 0, 0.2 or 0.4 lg of ATF3-expressing plasmids with 0.6 lg of reporter plasmid into HepG2 cells The asterisks indicate a P value < 0.05 for the bracketed comparisons NS, not significant 22 bp radiolabeled DNA probes (nucleotides )79 to )100) containing the putative ATF3-binding site 5¢-TGCGCGTCA-3¢ to determine whether ATF3 directly interacts with the AdipoR2 promoter The EMSA results revealed that this oligonucleotide formed a DNA–protein complex with the hepatocyte nuclear extracts (Fig 3B, C) A specific interaction between the putative ATF3-binding site and the repressor ATF3 was confirmed by competition with unlabeled oligonu2308 cleotides (Fig 3C) and by dose-dependent inhibition by antibody against ATF3 (Fig 3D) As a negative control for binding of the bZIP (basic leucine zipper) transcription factor ATF3 to the putative binding site, we used probe ‘X’, containing a sequence that recruits one of the zinc-finger DNA-binding transcription factor, also known to interact with the CREB-binding protein In the competition EMSA shown in Fig 3C, a 100 x excess of non-specific probe ‘X’ did not showed FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al Transcriptional regulation of AdipoR2 by ATF3 A : –94/–86 of promoter Consensus ATF/CRE NE: HepG2 cells Competitor Non-specific Ab N 4μg 2μg 1μg μ w/o Ab w NE: HepG2 cells o No Ext x100 ATF/CRE x x100 Cold x Competitor o No Comp o No Ext x100 x10 No Comp No Ext NE: HepG2 cells D ATF3 A C x100 Non-specific c x B ATF3 Ab + Ab * Labeled probes Labeled probes * Labeled probes * Fig ATF3 binds to the promoter of AdipoR2 in vitro (A) Comparison of the sequence of the EMSA probe containing the putative ATF3/ CRE-binding site (TGCGCGTCA) from the promoter of AdipoR2 with that of the palindromic consensus ATF3-binding sequence (TGACGTCA) (B–D) The putative ATF3-binding site exhibited specific binding with the HepG2 nuclear extract Nuclear extracts were prepared from HepG2 cells, and 5.0 mg of extract was used in EMSA reactions with 100 ng of radiolabeled double-stranded probe containing the putative ATF3binding site between nucleotides )94 and )86 (B) Competition EMSAs were performed with a 10- or 100-fold excess of the unlabeled wildtype nucleotide )94/)86 probe, a 100-fold excess of the consensus ATF/CRE-binding site sequence, or a 100-fold excess of the non-specific probe (B,C) Competition assays with ATF-specific antibody (1–4 lg) were also performed (D) competition in binding to ATF3, but assays using a 100 x excess of cold/unlabeled )94/)86 or the ATF/ CRE positive control probe did show competition with tested probes containing the putative )94/)86 site, indicating the specificity of this binding assay To determine the physiological relevance of ER stress and/or stress-related expression of ATF3 on formation of the protein–DNA complex in vitro, we increased the expression of ATF3 in HepG2 cells by treatment with 1.0 lm thapsigargin or transduction with ATF3-expressing adenovirus, Adv-ATF3 (Fig 4A,B, upper panel) More protein–DNA complex was formed between radiolabeled oligonucleotides containing the putative ATF3-binding site, or the ATF/CRE consensus sequence, and nuclear extracts of cells when the cells were thapsigargin-treated Nuclear extracts of the cells adenovirally over-expressing ATF3 also formed more DNA–protein complex with both the ATF/CRE consensus sequence and the )94/)86 oligonucleotide probe (Fig 4A,B) To further investigate this interaction in vivo, we validated the predicted ATF3-binding site in the regulatory region of the human AdipoR2 gene using chromatin immunoprecipitation (ChIP) This showed specific in vivo binding of ATF3 to the putative ATF3-binding element at nucleotides )94/)86 of the promoter region of AdipoR2 (Fig 4C) In addition to the EMSA results (Figs and 4A,B), showing that recruitment of ATF3 was increased by treatment with thapsigargin in a time-dependent manner, these results confirm that the transcription factor ATF3 binds to the promoter of AdipoR2 both in vitro and in vivo Decreased responsiveness as a result of deletion of the putative ATF3-responsive repressor element in the nucleotide )343/)72 region of the promoter As the putative ATF3-binding site 5¢-TGCGCGTCA-3¢ from the promoter region of human AdipoR2 showed FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2309 Transcriptional regulation of AdipoR2 by ATF3 Thap (1.0 µM) + Adv-ATF3 Mock B – ATF3 ATF3 β-Actin β-Actin ATF/CRE –94 / –86 Mock k Adv-ATF3 No Ext E No Ext E ap – Tha ap + Tha No Ext E – Tha ap ap + Tha No Ext E ATF/CRE Mock k NE: HepG2 cells NE: HepG2 cells Adv-ATF3 A In-uk Koh et al –94 / –86 * Labeled probes Labeled probes * (–94/–86) C Exon1 Thapsigargin (1.0 μM) – h h h 12 h – h h h 12 h IgG Input ATF3 Promoter, 323 bp Exon1, 328 bp binding ability in EMSA and ChIP experiments, we generated a mutant promoter construct lacking a 22 bp fragment of the promoter between nucleotides )94 and )86 (Fig 5A), to investigate whether this region responds to ATF3 and ER stress and plays a role in the transcriptional regulation of AdipoR2 2310 Fig Binding of ATF3 to the AdipoR2 promoter region increases under conditions of ER stress and/or ATF3 over-expression in vitro and in vivo (A) ATF3 expression was increased in HepG2 cells exposed to 1.0 lM thapsigargin for 24 h compared to control The amount of protein–DNA complex formed with the ATF/CRE consensus sequence and nucleotide )94/)86 doublestranded oligonucleotide probes was higher for thapsigargin-exposed samples (B) Transfection of an ATF3-expressing adenoviral vector (Adv-ATF3) resulted in over-expression of recombinant human ATF3 in HepG2 cells compared to control (Adv-YFP) Adenoviral vectors were infected at a multiplicity of infection of for each sample Nuclear extracts from cells overexpressing ATF3 from Adv-ATF3 showed increased binding affinity for both the putative ATF3-binding site and the consensus ATF/CRE-binding site (C) ChIP assays were performed with anti-ATF3 antibody (ATF3) or without it (IgG) PCR was used to amplify immunoprecipitated DNA fragments from HepG2 cells exposed to 1.0 lM thapsigargin for 0–12 h, showing a timedependent increase in recruitment of ATF3 to the putative binding element as a result of ER stress The activity of this construct, AR2P()343D), was then analyzed with or without co-transfection of ATF3/pcDNA3.1 (Fig 5B) Co-transfection with ATF3 dramatically decreased the promoter activity of the wild-type promoter construct [AR2P()343)] to one-tenth that of untreated cells, but attenuated the FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al Transcriptional regulation of AdipoR2 by ATF3 (–343 bp) A Luc AR2P(–343Δ) Luc AR2P(–343) Luc AR2P(–72) (–343 bp) (–72 bp) (–94/–86) Luciferase activity (%) 120 * C * 100 80 60 40 20 AR2P(–343Δ) AR2P(–343) ATF3 * 100 80 60 40 20 pGL3 * 120 Luciferase activity (%) B + – – – + – – + – + – – – + – + – – + – – – + + AR2P(–343Δ) + + AR2P(–343) – – + + Thapsigargin – + – + – – Fig Decreased responsiveness by deletion of the putative ATF3-responsive repressor element in the nucleotide )343/)72 region of the promoter (A) Sequences of the deletion mutant construct used in the luciferase reporter assay In the AR2P()343D) reporter construct, the putative ATF3/CRE-binding sequence (nucleotides )94/)86: TGCGCGTCA) and six 5¢ and seven 3¢ flanking nucleotides are deleted (B) Reduced ATF3-induced repression of the promoter activity was observed for the deletion mutant promoter construct AR2P()343D) lacking the putative ATF3/CRE-binding site at nucleotides )94/)86 Reporter construct derivatives (0.6 lg) were transfected into HepG2 cells with or without 0.4 lg of ATF3-expressing vector (C) Rescued ER stress-induced repression of promoter activity for AR2P()343D) Cells were treated with 1.0 lM thapsigargin for 24 h to induce ER stress The asterisks indicate a P value < 0.05 for the bracketed comparisons All luciferase assays were performed in triplicate, and error bars indicate the SEM of or experiments activity of the mutant construct without the )94/)86 putative binding element [AR2P()343D)] to only half that of untreated cells (Fig 5B) As shown in Fig 5C, the ER stress inducer thapsigargin had less of a repressor effect on the mutant reporter construct ()56%) than on the wild-type construct ()85%) Discussion Many groups have confirmed the anti-diabetic/insulinsensitizing effect of adiponectin, and thus plasma adiponectin and its receptors in peripheral organs have been proposed as therapeutic targets for the treatment of diabetes and obesity-linked insulin resistance [2,28,29] The action of adiponectin is known to be transduced via regulation of AMP-activated protein kinase (AMPK) function, and, given the report of a putative adaptor protein that interacts with adiponectin receptors, insulin and adiponectin signaling are now considered to be linked in the peripheral organs of insulin action, such as the liver and skeletal muscle [16,30,31] Despite the fact that the action and plasma level of adiponectin have been reported to be reduced in diseases associated with obesity, including peripheral insulin resistance and related ER stress cascades [2,3,8,9], the relationship between obesity-related ER stress and the consequent reduction in adiponectin action is obscure We found that hepatic expression of AdipoR2 was lower but expression of the markers of the ER stress pathway, GRP78 and ATF3, was higher in the liver of ob/ob mice compared with control mice To determine the molecular mechanisms of this relationship, we studied the regulation of human AdipoR2 in ER stress-induced hepatocytes ATF3, a member of the ATF/CREB family of transcription factors, is known to be a transcriptional repressor that is induced by many stress signals, including ER stress [31,32], and has also been proposed to play a role in liver dysfunction involving defects in glucose homeostasis [33] We and other investigators have also reported that adiponectin is negatively regulated by ATF3 and by the ER stress-mediated protein CHOP (C/EBP homologous protein) under obesity-related hypoxic conditions in adipocytes [8,34] In particular, in a transcriptional context, ATF3 functioned in response to thapsigargininduced ER stress as a negative regulator of adiponectin expression by direct binding to the promoter [8] These reports imply that a relationship exists between the decreased transcriptional activity of AdipoR2 and subsequently-induced ATF3 in ER stress (Fig 1B) In thapsigargin-treated hepatocytes, AdipoR2 expression was inversely correlated with the induction of GRP78 and/or ATF3 by ER stress (Fig 1) Meanwhile, in cells pre-treated with PBA, the rescued FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2311 Transcriptional regulation of AdipoR2 by ATF3 In-uk Koh et al AdipoR2 level showed strong support for an ER stress-based mechanism of AdipoR2 decrease in human hepatocytes (Fig 1B) To determine the mechanism of changes in AdipoR2 expression resulting from ER stress and ATF3 over-expression or silencing in the liver (Fig 1B–E), we measured the promoter activity of the AdipoR2 gene Up- or down-regulation of ATF3 modulated the AdipoR2 promoter activity (Fig 2B,C) By analysing the promoter region of the AdipoR2 gene, we identified a putative ATF3-binding sequence (Fig 3) As shown in Figs and 4, the decrease in AdipoR2 expression by ATF3 in ER stress was mediated by this putative sequence which recruited ATF3 in vitro and in vivo This result provides an explanation for the role of ER stress and induced ATF3 in obesity-linked insulin resistance through regulation of adiponectin action These transcription-repressing mechanisms of ER stress-induced ATF3 have been shown to contribute to the development of insulin resistance and type diabetes Insulin receptor substrates and were found to be repressed by ATF3 in myocytes [35] and pancreatic bcells [11], respectively The level of the insulin-sensitizing hormone adiponectin was decreased by ATF3 in adipocytes [8], and the major receptor in hepatocytes, AdipoR2, was also negatively regulated The above effects of ATF3 on insulin signaling and glucose homeostasis involve the action of adiponectin in peripheral tissues In particular, given that the cause and effect relationship between adiponectin and insulin action is not fully understood, the inappropriate actions of adiponectin in obesity-linked insulin resistance are described as a ‘vicious cycle’ of adiponectin and insulin resistance [36] For example, insulin receptor transgenic/knockout mice exhibit decreased AdipoR2 levels in liver and muscle, as well as decreased expression of the peroxisome proliferator-activated receptor gamma (PPARc) target genes of fatty acid oxidation, showing that AdipoR2 defects are relevant to diabetes susceptibility [37] In addition, a decrease in levels of expression of adiponectin receptors was reported to be associated with type diabetes [21], as well as reductions in plasma adiponectin levels in various cases associated with insulin resistance [21, 38, 39] and alterations in the adiponectin gene [40–42] On the other hand, despite decreased responsiveness to thapsigargin and induced ATF3, a mutant AdipoR2 reporter construct lacking the putative ATF3-binding site still showed repression of transcriptional activity to some extent In addition, absence of the putative ATF3-binding site reduced expression of the reporter gene itself (Fig 5B,C) Co-transfection of ATF3 reduced the promoter activity of wild-type AdipoR2 dose-dependently, and mutant AdipoR2 to a lesser 2312 degree (Fig S2 and Table S4), but co-transfection of ATF3 had a non-specific effect on the activity of the pGL3-basic control vector (Fig S3 and Table S5) Thus the decrease in promoter activity itself (Table S4) and the smaller but remaining responsiveness to ATF3 for the mutant reporter gene suggests that, in addition to the ATF3-binding site ()94/)86), an indirect effect of ATF3 on the promoter region of AdipoR2 may exist through an unidentified binding site In addition, this putative ATF3-binding site could recruit the transcription factor complex for dichotomous actions, possibly through the action of uncharacterized dimerization partner(s) of ATF3, such as ATF2, c-Jun, JunB, JunD, etc [43] Given the nature of the deleted ‘semi-palindromic’ sequence )94/)86 (TGCGCGTCA) and bZip transcription factors including ATF3 [27], these dimerization partner(s) of ATF3 may have very complicated transcription factor/co-factor relationships These possibilities must be studied further to clarify the ATF3-mediated negative effect on transcription of AdipoR2 under ER stress in the liver In this study, exposure to the ER stress inducer thapsigargin and the accompanying induction of ATF3 were inversely correlated with changes in the expression level of AdipoR2 in human HepG2 cells, and this correlation was the result of direct transcriptional regulation of AdipoR2 by the repressor ATF3 via the putative binding site between nucleotides )94 and )86 of the promoter region This finding of decreased AdipoR2 levels as a result of the regulation by ATF3 is noteworthy, and suggests that obesity-related ER stress may affect the development of hepatic insulin resistance, at least in part by transcriptional repressing activity of ATF3 Experimental procedures Animals and materials To compare the expression levels of ER stress markers and the adiponectin receptor in animals of various genetic backgrounds, ob/ob mice and age-matched lean control C57BL6/J mice (10 weeks, three mice per group) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) After overnight fasting, the mice were killed and liver was collected for further analysis The Animal Care and Use Committee of the National Institutes of Health and the Korean Food and Drug Administration approved all animal protocols The expression plasmid encoding ATF3 was kindly provided by Dr T Hai (Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH, USA) Rabbit polyclonal antibodies against GRP78, ATF3 and AdipoR2 (sc-13968, sc-188 and sc-46754, respectively) and siRNA for ATF3 (sc-29758) FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) Cell culture and treatments Human hepatocyte HepG2 cells and human embryonic kidney HEK 293 cells (both American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium containing 4.5 gỈL)1 glucose (Invitrogen, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (GibcoBRL, Gaithersburg, MD, USA) To investigate the effect of ER stress, cells were treated with 1.0 lm thapsigargin (Sigma, St Louis, MO, USA) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 24 h To reduce the effect of ER stress, cells were pre-incubated for 24 h in culture medium containing 20 mm 4-phenyl butyric acid (PBA) (Calbiochem, San Diego, CA, USA) prior to treatment with 1.0 lm thapsigargin Over-expression of adenoviral ATF3 After PCR amplification, the ATF3 gene was ligated into the adenovirus shuttle vector pShuttle-CMV (Stratagene, La Jolla, CA, USA), which includes GFP (green fluorescent protein) tagged to the C-terminus of the ATF3 protein Recombinant adenoviral genomes were produced by recombination between the shuttle vector constructed above and the pAdEasy vector (Stratagene), according to the manufacturer’s protocol [44] The genomes were subsequently transfected into HEK 293 cells using Lipofectamine reagent (Invitrogen) ATF3-expressing adenovirus particles (AdvATF3) were obtained as a viral mixture in culture medium 7–9 days after transfection, with the viral particle number of the adenoviral mixture ranging between 1.0 and 2.0 · 1010 IFU (inclusion-forming units)ỈmL)1 depending on the sample The recombinant virus was propagated in HEK 293 cells before transduction into HepG2 cells Control adenovirus (mock, Adv-YFP) was generated by the same method using an empty adenoviral shuttle plasmid HepG2 cells were infected with the adenoviral mixture at a multiplicity of infection between and 10 for over-expression of recombinant ATF3, while Adv-YFP was infected into HepG2 at a multiplicity of infection of as a control (Figs 1C and 5B) To maximize ATF3 expression, cells were lysed 48 h after infection Knock-down of ATF3 Commercially available siRNA against ATF3 (siATF3, Santa Cruz Biotechnology) was used HepG2 cells grown in six-well plates were transfected with siATF3 using LipofectAMINE reagent according to the manufacturer’s protocol Briefly, the transfection reaction included Transcriptional regulation of AdipoR2 by ATF3 optimized amount of siATF3 (100 pm), · 106 cells and lL of Lipofectamine reagent A possible non-specific gene silencing effect was assessed using a non-targeting negative control siRNA (46-2001; Invitrogen) Promoter region constructs Portions of the AdipoR2 promoter region (approximately kb) were amplified using PCR with human genomic DNA as the template The AR2P()1974) primer pair sequences were 5¢-AGCACACGGTGAACTGTTCCA GAGG-3¢ and 5¢-ACTTCTTGGGAGCCACCGCTGAG3¢ A series of deletion constructs of the AdipoR2 promoter were PCR-generated using pairwise combinations of the antisense primer 5¢-ACTGGCGGCCGCTCGAG-3¢ with one of the sense primers AR2P()870), AR2P()343) or AR2P()72) (5¢-GGTACCTTCCCCCTCCTACTGAATGT-3¢, 5¢-GGTACCCCTCCTCCTCAGCTCCAAAT-3¢ and 5¢-GGTACCTCGTGGGGGCGGGGAGA-3¢, respectively) Plasmids were constructed as derivatives of pGL3-Basic luciferase reporter vectors (Promega, Madison, WI, USA) using the KpnI and XhoI restriction sites AR2P()343D), a deletion mutant lacking the putative ATF3-binding site, was PCR-generated from the AR2P()343) plasmid using the additional internal primers 5¢-GAGGCGGTTCGAG CCAATA-3¢ and 5¢-CGTGCGGTCGTGGGGG-3¢, which hybridized upstream and downstream, respectively, of the 22 bp promoter region containing the putative ATF3-binding site at nucleotide positions )94 to )86 Luciferase activity assay HepG2 cells were grown in six-well plates to 70% confluence and then transfected with pGL3-Basic-derived reporter constructs containing the AdipoR2 promoter region and a pcDNA3.1-derived ATF3 expression plasmid using LipofectAMINE reagent (Invitrogen) according to the manufacturer’s instructions [45] b-galactosidase (CMV-b-gal) expression vectors were used to correct differences in transfection efficiency The cells were lysed 24 h after transfection, and their luciferase activity was measured using a luciferase assay system (Promega) Semi-quantitative RT-PCR We used the following primers for RT-PCR: atf3-sense, 5¢-GGTTTGCCATCCAGAACAAG-3¢; atf3-antisense, 5¢-CC TCCCAGGAGAAGGTAAGC-3¢; adipor2-sense, 5¢-TAGC CTTTGGTTTGCTTTGG-3¢; adipor2-antisense, 5¢-CATAT CTCCAGGCGTCAACC-3¢; gapdh-sense, 5¢-ATGACATC AAGAAGGTGGTG-3¢; gapdh-antisense, 5¢-CCAAATTC GTTGTCATACCA-3¢ Total RNA was obtained from HepG2 cells using an RNeasy kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2313 Transcriptional regulation of AdipoR2 by ATF3 In-uk Koh et al We obtained first-strand cDNA using the SuperScriptÔ first-strand synthesis system for RT-PCR according to the manufacturer’s protocol (Invitrogen), and then performed PCR using the cDNA as a template and Taq polymerase The intensity of ethidium bromide-stained bands was analyzed using an i-MAX gel image analysis system (CoreBioSystem, Seoul, Korea) and Alpha EasyÔ FC software (Alpha Innotech, San Leandro, CA, USA) The relationship between the inverse of band intensity and the number of PCR cycles was linear The number of PCR cycles was 45 for ATF3, AdipoR2 and GAPDH Western blot analysis Mouse liver extract was obtained by homogenizing the same amount of liver tissue from each of two groups of mice, and HepG2 cells were lysed in PRO-PREP lysis reagent according to the manufacturer’s protocol (Intron Biotechnology, Sungnam, Korea) Lysed samples were centrifuged at 12 000 g for 10 min, and equal amounts of protein were separated by 12% SDS/PAGE, transferred to polyvinylidene difluoride membranes, and incubated with primary antibodies in blocking solution (5% skim milk in phosphate buffer, pH 7.2) The immune complexes were identified using enhanced chemiluminescence detection reagents (Amersham Biosciences, Uppsala, Sweden) with appropriate secondary antibodies Each blot was probed with an anti-actin antibody to verify equal loading of extracted protein The band intensity, i.e the expression of each protein (GRP78, ATF3 or AdipoR2), was measured densitometrically, and was normalized to the level of b-actin Then the protein level for ob/ob mice was compared with that of C57BL6/J mice to obtain the relative ratio value versus the mean of the control group Electrophoretic mobility shift assay (EMSA) Nuclear extracts of HepG2 cell were prepared as described previously [46] Probes corresponding to the putative ATF3/ CRE-binding site on the AdipoR2 promoter region were synthesized and radiolabeled with [c-32P]dATP (sense 5¢GTGCGATGCGCGTCACGGCGA-3¢; antisense 5¢-TC GCCGTGACGCGCATCGCAC-3¢) Labeled probes were then incubated with mg of nuclear extract protein in the presence or absence of competitor DNA or antibodies The resulting complexes were electrophoresed on a 5% non-denaturing polyacrylamide gel in 0.5· Tris borate/EDTA electrophoresis buffer (45 mm Tris borate, mm EDTA, pH 8.0) After drying, gels were visualized using autoradiography Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed with a ChIP assay kit (Upstate Biotechnology, Lake Placid, 2314 NY, USA) according to the manufacturer’s protocol, modified as previously described [47] After 0–12 h of thapsigargin-induced ER stress, · 106 HepG2 cells in a 100 mm plate were cross-linked with 1% formaldehyde in Dulbecco’s modified Eagle’s medium for 10 at room temperature The cells were collected, and the chromatin was sheared into fragments averaging 300–500 bp 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Mol Cell Biol 23, 5896–5907 Supporting information The following supplementary material is available: Fig S1 Arbitrary values corresponding to the luciferase activities of AR2P(–72) with or without ATF3 induction Fig S2 Determination of ATF3-dose-dependent repression of promoter activity by co-transfection of various amounts of ATF3-expressing plasmids in HepG2 cells Fig S3 Comparison of reporter activity of the pGL3basic vector with or without ATF3 co-transfection Table S1 Densitometric values of proteins analyzed in ER stress-induced HepG2 cells determined by western blot Table S2 Densitometric values of proteins analyzed in HepG2 cells by western blot Table S3 Statistical analysis of arbitrary values corresponding to the luciferase activities of AR2P(–72) with or without ATF3 induction Table S4 Arbitrary values of the reporter activities of WT AR2P(-343) and its mutant and the changes by ATF3 co-transfection Table S5 Arbitrary values for the reporter activity of pGL3-basic vector with or without ATF3 co-transfection FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS In-uk Koh et al This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer- Transcriptional regulation of AdipoR2 by ATF3 reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2304–2317 ª 2010 The Authors Journal compilation ª 2010 FEBS 2317 ... transcription of AdipoR2 under ER stress in the liver In this study, exposure to the ER stress inducer thapsigargin and the accompanying induction of ATF3 were inversely correlated with changes in the... proteins analyzed in ER stress-induced HepG2 cells determined by western blot Table S2 Densitometric values of proteins analyzed in HepG2 cells by western blot Table S3 Statistical analysis of arbitrary... predicted ATF3- binding site in the regulatory region of the human AdipoR2 gene using chromatin immunoprecipitation (ChIP) This showed specific in vivo binding of ATF3 to the putative ATF3- binding element