Novel repressor regulates insulin sensitivity through interaction with Foxo1
2012; Springer Nature; Volume: 31; Issue: 10 Linguagem: Inglês
10.1038/emboj.2012.97
ISSN1460-2075
AutoresJun Nakae, Yongheng Cao, Fumihiko Hakuno, Hiroshi Takemori, Yoshinaga Kawano, Risa Sekioka, Takaya Abe, Hiroshi Kiyonari, Toshiya Tanaka, Juro Sakai, Shin‐Ichiro Takahashi, Hiroshi Itoh,
Tópico(s)Plant Molecular Biology Research
ResumoArticle17 April 2012Open Access Novel repressor regulates insulin sensitivity through interaction with Foxo1 Jun Nakae Corresponding Author Jun Nakae Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan 21st Century COE Program for Signal Transduction Disease: Diabetes Mellitus as Model, Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Search for more papers by this author Yongheng Cao Yongheng Cao 21st Century COE Program for Signal Transduction Disease: Diabetes Mellitus as Model, Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Department of Molecular Metabolic Regulation, Research Institute, International Medical Center of Japan, Tokyo, Japan Search for more papers by this author Fumihiko Hakuno Fumihiko Hakuno Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Takemori Hiroshi Takemori Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan Search for more papers by this author Yoshinaga Kawano Yoshinaga Kawano Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Risa Sekioka Risa Sekioka Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Takaya Abe Takaya Abe Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan Search for more papers by this author Hiroshi Kiyonari Hiroshi Kiyonari Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan Search for more papers by this author Toshiya Tanaka Toshiya Tanaka Division of metabolic medicine; Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan Search for more papers by this author Juro Sakai Juro Sakai Division of metabolic medicine; Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan Search for more papers by this author Shin-Ichiro Takahashi Shin-Ichiro Takahashi Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Itoh Hiroshi Itoh Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Jun Nakae Corresponding Author Jun Nakae Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan 21st Century COE Program for Signal Transduction Disease: Diabetes Mellitus as Model, Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Search for more papers by this author Yongheng Cao Yongheng Cao 21st Century COE Program for Signal Transduction Disease: Diabetes Mellitus as Model, Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Department of Molecular Metabolic Regulation, Research Institute, International Medical Center of Japan, Tokyo, Japan Search for more papers by this author Fumihiko Hakuno Fumihiko Hakuno Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Takemori Hiroshi Takemori Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan Search for more papers by this author Yoshinaga Kawano Yoshinaga Kawano Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Risa Sekioka Risa Sekioka Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Takaya Abe Takaya Abe Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan Search for more papers by this author Hiroshi Kiyonari Hiroshi Kiyonari Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan Search for more papers by this author Toshiya Tanaka Toshiya Tanaka Division of metabolic medicine; Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan Search for more papers by this author Juro Sakai Juro Sakai Division of metabolic medicine; Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan Search for more papers by this author Shin-Ichiro Takahashi Shin-Ichiro Takahashi Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Itoh Hiroshi Itoh Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Author Information Jun Nakae 1,2, Yongheng Cao2,3, Fumihiko Hakuno4, Hiroshi Takemori5, Yoshinaga Kawano1, Risa Sekioka1, Takaya Abe6, Hiroshi Kiyonari6, Toshiya Tanaka7, Juro Sakai7, Shin-Ichiro Takahashi4 and Hiroshi Itoh1 1Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan 221st Century COE Program for Signal Transduction Disease: Diabetes Mellitus as Model, Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan 3Department of Molecular Metabolic Regulation, Research Institute, International Medical Center of Japan, Tokyo, Japan 4Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan 5Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan 6Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan 7Division of metabolic medicine; Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan *Corresponding author. Frontier Medicine on Metabolic Syndrome, Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan. Tel.:+81 3 3352 1211; Fax:+81 3 3359 2745; E-mail: [email protected] The EMBO Journal (2012)31:2275-2295https://doi.org/10.1038/emboj.2012.97 There is a Have you seen? (May 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Forkhead box-containing protein o (Foxo) 1 is a key transcription factor in insulin and glucose metabolism. We identified a Foxo1-CoRepressor (FCoR) protein in mouse adipose tissue that inhibits Foxo1's activity by enhancing acetylation via impairment of the interaction between Foxo1 and the deacetylase Sirt1 and via direct acetylation. FCoR is phosphorylated at Threonine 93 by catalytic subunit of protein kinase A and is translocated into nucleus, making it possible to bind to Foxo1 in both cytosol and nucleus. Knockdown of FCoR in 3T3-F442A cells enhanced expression of Foxo target and inhibited adipocyte differentiation. Overexpression of FCoR in white adipose tissue decreased expression of Foxo-target genes and adipocyte size and increased insulin sensitivity in Leprdb/db mice and in mice fed a high-fat diet. In contrast, Fcor knockout mice were lean, glucose intolerant, and had decreased insulin sensitivity that was accompanied by increased expression levels of Foxo-target genes and enlarged adipocytes. Taken together, these data suggest that FCoR is a novel repressor that regulates insulin sensitivity and energy metabolism in adipose tissue by acting to fine-tune Foxo1 activity. Introduction Forkhead transcription factor (Foxo1) is a key transcription factor in insulin and glucose metabolism that is phosphorylated, subsequently exported to the cytoplasm, and inhibited by insulin/IGF1 in a PI3 kinase-dependent manner (Accili and Arden, 2004). Foxo1 plays an important role in mediating insulin action in several insulin-responsive tissues. Specifically, Foxo1 promotes glucose production in the liver, inhibits compensatory β-cell proliferation in insulin-resistant states, activates feeding by promoting orexigenic peptide expression in the hypothalamic arcuate nucleus, inhibits differentiation of preadipocytes and myoblasts into mature adipocytes or myotubes, and regulates energy storage and expenditure in adipose tissue (Nakae et al, 2008a, 2008b). Because of its involvement in so many physiological processes, it is important to elucidate the mechanism that regulates Foxo1 transcriptional activity. Adipogenesis, during which preadipocytes differentiate into adipocytes, experiences several stages, including mesenchymal precursor, committed preadipocytes, growth-arrested preadipocyte, mitotic clonal expansion, terminal differentiation, and mature adipocyte (Lefterova and Lazar, 2009). Peroxisome proliferator-activated receptor-γ (PPARγ) regulates both the terminal differentiation and metabolism in mature adipocytes. Foxo1 is a PPARγ-interacting protein that antagonizes PPARγ activity (Dowell et al, 2003; Armoni et al, 2006; Fan et al, 2009). SirT2-mediated deacetylation of Foxo1 increases the association of Foxo1 with PPARγ, leading to inhibition of adipocyte differentiation (Jing et al, 2007). Furthermore, constitutively nuclear (CN) Foxo1 inhibits differentiation of the preadipocyte cell line 3T3-F442A cells by arresting the cell cycle that is required in the early stages of adipose conversion, whereas haploinsufficiency of Foxo1 restores the size of white adipocytes under high-fat diet (HFD) (Nakae et al, 2003; Kim et al, 2009). Overexpression of transactivation-defective Foxo1 in white adipose tissue (WAT) increases fat mass and number of small adipocytes. Overexpression of the same mutant Foxo1 in brown adipose tissue (BAT) increases oxygen consumption (Nakae et al, 2008a). Therefore, Foxo1 can be an attractive target to improve the energy homeostasis in adipose tissue. Targeting Foxo1 can increase energy store in WAT and increase energy expenditure in BAT. Foxo1 is primarily regulated by post-translational modifications. In addition to Akt-induced phosphorylation, Foxo1 is acetylated by histone acetyltransferases (HATs) such as CREB-binding protein (CBP)/P300; Foxo1 deacetylation is mediated by class I–III deacetylases, including the nicotin amide adenine dinucleotide (NAD)-dependent histone deacetylase (HDAC), Sirt1 (Accili and Arden, 2004). Interaction with other proteins also regulates the transcriptional activity of Foxo family proteins (Foxos). For example, PPAR-γ co-activator 1α (PGC-1α) interacts with Foxo1 and stimulates gluconeogenesis in the liver (Puigserver et al, 2003). SMK-1 modulates the transcriptional response of DAF-16, the Foxo1 orthologue in C. elegans (Wolff et al, 2006), and the Drosophila Melted gene product interacts with both Tsc1 and FOXO to inhibit FOXO activity (Teleman et al, 2005). In the present study, we identified a novel Foxo1-binding protein termed as Foxo1 CoRepressor (FCoR) in adipose tissue using a yeast two-hybrid screen of a mouse 3T3-L1 cDNA library. We demonstrated that FCoR inhibits Foxo1 transcriptional activity through increased Foxo1 acetylation, which is accompanied by preventing Foxo1 interaction with the deacetylase Sirt1 and by direct acetylation. Knockdown of FCoR in 3T3-F442A cells inhibited adipocyte differentiation, while knockout of Fcor led to a lean phenotype, glucose intolerance, and insulin resistance. In contrast, overexpression of FCoR in adipose tissue decreased adipocyte size, increased insulin sensitivity, and decreased PGC-1α expression in brown adipocytes, indicating that FCoR plays important roles in glucose and energy homeostasis. Results Identification of FCoR, a novel Foxo1-binding protein To identify Foxo1-interacting proteins, we performed a yeast two-hybrid screen, using a GAL4-Foxo1 fragment (amino acids 1–154) as bait and a mouse 3T3-L1 cDNA library as prey. Screening of about 1.5 × 106 primary transformants yielded 224 clones. We selected 17 clones that met the following criteria: (1) they possessed a nuclear localization signal, (2) they encoded transcription factors, or (3) unknown proteins, and (4) they were restricted to or enriched in adipose tissue and/or differentiated 3T3-F442A cells. In all, 5 of 17 clones thus identified encoded partial transcripts of the RIKEN cDNA 2400009B08. The gene is predicted to encode a peptide with an Mr of 13.71 kDa LOC68234 (gb∣EDL21945.1∣mCG1048501). Because further characterization showed that it acted as a Foxo1 CoRepressor, it was termed as 'FCoR'. We performed 5′- or 3′-rapid amplification of cDNA ends (RACE) to determine the transcription start site (Supplementary Figure S1A). Sequencing of the 5′- and 3′-RACE products determined that FCoR is a 106-amino acid protein (Supplementary Figure S1B). cDNA cloning and in-vitro translation studies confirmed that the peptide has a molecular weight of 13 kDa (Figure 1A). Functional domain analysis using the Eukaryotic Linear Motif server ( http://elm.eu.org/; Teleman et al, 2005) revealed the presence of a forkhead-associated ligand domain (LIG_FHA_1) from amino acids 78 to 84 (Supplementary Figure S1B). Figure 1.Interaction between FCoR and Foxos and expression profiling of FCoR. (A) In-vitro translation of FCoR. Lysates from liver (lane 1), WAT (lane 2), and BAT (lane 3) from wild-type mice, along with in vitro-translated FCoR were analysed by western blotting using anti-mouse FCoR antiserum. (B) Interaction between exogenous FCoR and Foxo1. HEK293 cells were co-transfected with pFLAG-CMV2-WT Foxo1 or pFLAG-CMV2-Δ256 Foxo1 plus pCMV5-cMyc-WT FCoR and cultured in the presence of serum. At 48 h after transfection, cells were harvested and lysates were immunoprecipitated with anti-cMyc (lanes 1 and 5), anti-FLAG antibody (lanes 3 and 7), or normal mouse IgG (lanes 2, 4, 6, and 8) and blotted with anti-FLAG (lanes 1, 2, 5, and 6) or anti-cMyc antibody (lanes 3, 4, 7, and 8). (C) Interaction between endogenous Foxo1 and FCoR in WAT and BAT. Lysates from WAT (lanes 1, 2, 5, and 6) and BAT (lanes 3, 4, 7, and 8) were immunoprecipitated with anti-FOXO1 (lanes 1 and 3), anti-FCoR (lanes 5 and 6), or normal rabbit IgG (lanes 2, 4, 6, and 8) and blotted with anti-FCoR (lanes 1–4) or anti-FOXO1 (lanes 5–8), respectively. (D) Direct interaction of FCoR with Foxo1. GST-FCoR was subjected to a pull-down assay. Aliquots of in vitro-translated WT Foxo1 were incubated with glutathione-Sepharose beads coated with bacterially expressed GST-P13 (lane 2) or GST alone (lane 3) for 6 h at 4°C. The in vitro-translated Foxo1 proteins retained on the column were eluted and separated by SDS–PAGE followed by western blotting with anti-FOXO1 antibody. The bottom panel shows GST or GST-GST-P13 (10% of input, lane 3) blotted with anti-GST antibody. (E) Expression profiling of Fcor in various tissues. Total RNA isolated from WAT (lane 1), BAT (lane 2), liver (lane 3), skeletal muscle (lane 4), and whole brain (lane 5) of wild-type mice was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (F) Real-time PCR of Fcor using the adipocyte or stromal vascular fractions of fractionated WAT. (G) Northern blotting of 3T3-F442A cells during differentiation. Total RNA isolated from 3T3-F442A cells on the indicated day after induction of differentiation was subjected to Northern blotting with Fcor (top panel) or β-actin (bottom panel). (H) Western blotting of FCoR protein from 3T3-F442A cells during differentiation. Lysates from 3T3-F442A cells on the indicated day after induction of differentiation were subjected to western blotting using anti-FCoR polyclonal antiserum (top panel) or anti-tubulin monoclonal antibody (bottom panel). (I) Effects of the feeding state on Fcor gene expression. Total RNA from liver (lanes 1–3), WAT (lanes 4–6), and BAT (lanes 7–9) from C57Bl6J mice in the fed, fasting, or refed states was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (J) Western blotting of the FCoR protein from WAT (lanes 1 and 2) and BAT (lanes 3 and 4) from C57Bl6J mice in fed (lanes 1 and 3) or fasting state (lanes 2 and 4). Tissue lysates were subjected to western blotting using anti-FCoR polyclonal antiserum (top panel) or anti-tubulin monoclonal antibody (bottom panel). (K) Fcor gene expression in WAT and BAT from Leprdb/db mice. Total RNA isolated from WAT (lanes1 and 2) and BAT (lanes 3 and 4) of C57Bl6J (lanes 1 and 3) or Leprdb/db mice (lanes 2 and 4) was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (L) Effect of cold exposure on Fcor gene expression in BAT. Total RNA isolated from the BAT of C57Bl6J mice exposed to the cold (4°C for 6 h) was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (M) Immunohistochemistry of WAT (left panel) and BAT (right panel) from C57Bl6J mice using anti-FCoR anti-sera. Scale bars indicate 20 μm. Download figure Download PowerPoint To confirm the interaction between Foxo1 and FCoR, we co-transfected HEK293 cells with FLAG-tagged Foxo1 and cMyc-tagged FCoR and performed reciprocal immunoprecipitation/immunoblotting experiments in the presence of serum using anti-FLAG and anti-cMyc antibodies. These experiments showed that FCoR interacted with Foxo1 (Figure 1B, lanes 1–4). To identify the FCoR-binding site of Foxo1, we co-transfected pFLAG-CMV2-Δ256 Foxo1, which encoded a Foxo1 mutant lacking the carboxyl terminal transactivation domain (Nakae et al, 2000), and pCMV5-cMyc-FCoR and performed immunoprecipitation studies. These experiments demonstrated that FLAG-Δ256 Foxo1 bound to cMyc-FCoR (Figure 1B, lanes 5–8), suggesting that FCoR binds the Foxo1 N-terminus. Foxo family members, including Foxo1, Foxo3a, and Foxo4, are expressed in WAT and/or BAT. Foxo1 and Foxo3a can be expressed in both WAT and BAT but Foxo4 is mainly expressed in BAT (Supplementary Figure S2). To investigate whether endogenous Foxo1 associates with FCoR, mouse WAT and BAT extracts were immunoprecipitated with an anti-FOXO1 antibody or anti-FCoR antiserum, followed by immunoblotting with antibodies against FCoR or Foxo1. The results showed that endogenous Foxo1 associated with endogenous FCoR (Figure 1C). Taken together, these results suggest that FCoR interacts with Foxo1 in vivo. To determine whether the interaction between FCoR and Foxo1 was a direct interaction, we performed a GST-fusion protein pull-down assay. We detected an interaction between FCoR and Foxo1 (Figure 1D), indicating that the two proteins bind directly to one another. FCoR is expressed in adipose tissue and differentiated 3T3-F442A cells Fcor mRNA is expressed in mouse WAT and BAT, but not in liver, skeletal muscle, or brain (Figure 1E). Fractionation of WAT revealed that Fcor is expressed mainly in the adipocyte fraction (Figure 1F). Analysis of Fcor mRNA and protein levels indicated that FCoR is expressed in 3T3-F442A cells in a differentiation-dependent manner (Figure 1G and H). To investigate whether FCoR expression was modulated in physiological conditions or insulin-resistant states, we examined Fcor mRNA regulation during fasting and feeding in C57bl6J mice. Fcor mRNA and FCoR protein levels in WAT and BAT decreased during fasting (Figure 1I and J), and Fcor mRNA expression levels were lower in the WAT and BAT of insulin-resistant Leprdb/db mice compared with control mice (Figure 1K). BAT is the main organ responsible for adaptive thermogenesis in rodents (Cannon et al, 1998). Interestingly, mouse BAT Fcor mRNA expression increased after 6 h of cold exposure (Figure 1L). Immunohistochemistry experiments demonstrated that the endogenous FCoR protein was expressed mainly in the nucleus in both WAT and BAT (Figure 1M). These data suggest that FCoR may have a functional role in adipose tissues in vivo. FCoR inhibits the transcriptional activity of Foxo1 and Foxo3a, but not of FOXO4 We next addressed the question of whether FCoR affects Foxo1 transcriptional activity using reporter assays with the Foxo1-target genes Igfbp1 and G6pc (Nakae et al, 2006). A constitutively nuclear Foxo1 mutant (CN Foxo1) increased IGFBP1 or G6PC promoter activity by 3.5- and 22.5-fold, respectively, in the presence of 8-Br-cAMP/IBMX/dexamethasone (Figure 2A and B). FCoR had no effect on the activity of either promoter in basal conditions, but it inhibited the Foxo1-dependent promoter activity of both genes in a dose-dependent manner (Figure 2A and B). In the transactivation assay, FCoR repressed the forskolin-induced luciferase activity of a GAL4-Foxo1 fusion protein (Figure 2C). Furthermore, co-expression of FCoR inhibited the effect of CN Foxo1 on the expression of the endogenous Igfbp1 gene in SV40-transformed hepatocytes (Figure 2D). In these cells, FCoR localized to both the cytosol and nucleus (Supplementary Figure S3). These data indicate that FCoR and CN Foxo1 colocalize in the nucleus and that FCoR inhibits Foxo-dependent transcription. Figure 2.FCoR inhibits Foxo1 transcriptional activity. FCoR inhibits CN Foxo1-induced IGFBP1 (A) and G6PC (B) promoter activity. After transient transfection with IGFBP1/luciferase (p925GL3) (A) or a G6Pase/luciferase reporter vector (PicaGene/human G6Pase promoter-luciferase) (B), SV40-transformed hepatocytes were infected with the indicated adenovirus. phRL-SV40 was used as an internal control for transfection efficiency. After overnight serum deprivation and induction with dexamethasone/8-Br-cAMP/IBMX, cells were harvested and luciferase activity was measured. Single and double asterisks indicate statistically significant difference between luciferase activity in the absence and the presence of FCoR (*P<0.005 and **P<0.05, respectively, by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. (C) FCoR inhibits Foxo1-induced 5XGAL4-luciferase activity. After transient transfection, HEK293 cells were stimulated with forskolin (20 μM) for 6 h, harvested, and luciferase activity was measured. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of FCoR (*P<0.02 by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. (D) Effect of overexpression of FCoR on endogenous Igfbp1 gene expression. SV40-transformed hepatocytes were transduced with adenovirus encoding FLAG-CN Foxo1. After 2 h, cells were transduced again with adenovirus encoding cMyc-FCoR. After 36 h, cells were incubated with dexamethasone/cAMP/IBMX for 8 h and harvested. Total RNA was isolated from cells and subjected to real-time PCR to analyse the Igfbp1 and β-actin levels. Data were corrected using the β-actin expression level and then represented as relative mRNA abundance. Data represent the mean values±s.e.m. from three independent experiments. An asterisk indicates a statistically significant difference between endogenous Igfbp1 expression induced by FLAG-CN Foxo1 in the absence and presence of cMyc-FCoR (*P<0.001 by one-way ANOVA). (E) Interaction between Foxo1, Foxo3a or FOXO4 and FCoR. HEK293 cells were co-transfected with pCMV5-cMyc-Foxo1, pCMV5-cMyc-Foxo3a or pCMV5/cMyc-FOXO4 and pFLAG-CMV2-WT FCoR and immunoprecipitated with anti-FLAG, anti-cMyc or normal mouse IgG and blotted with anti-cMyc or anti-FLAG antibody. (F) FCoR inhibits Foxo1-induced and Foxo3a-induced, but not FOXO4-induced 5XGAL4-luciferase activity. After transient transfection with FCoR, HEK293 cells were stimulated with forskolin (20 μM) for 6 h, harvested, and luciferase activity was measured. The grey bar indicates mock-transfected cells and the blue bar indicates pCMV5-cMyc-WT FCoR-transfected cells. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of FCoR (*P<0.01 by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint We also investigated whether other Foxo proteins (Foxo3a and Foxo4) co-immunoprecipitated with FCoR in the absence of serum and in the presence of forskolin. Transfection studies indicated that epitope-tagged Foxo3a also interacted with FCoR, but Foxo4 did not (Figure 2E). Furthermore, the transactivation assay demonstrated that FCoR repressed the forskolin-induced luciferase activity of a GAL4-Foxo1 fusion protein and a GAL4-Foxo3a fusion protein but did not repress the luciferase activity of a GAL4-FOXO4 fusion protein (Figure 2F). These data suggest that of the Foxo family members, FCoR interacts with Foxo1 and Foxo3a. FCoR enhances the acetylation of Foxo1 through disruption of the interaction between Foxo1 and Sirt1 and through direct acetylation Foxo is acetylated by CBP/P300 and deacetylated by Sirt1 or Sirt2 (Accili and Arden, 2004; Jing et al, 2007). We examined Foxo1 acetylation in HEK293 cells in the presence or absence of FCoR. Foxo1 was acetylated in the presence of H2O2 (Figure 3A, lane 1) and overexpression of FCoR enhanced Foxo1 acetylation (Figure 3A, lane 2). Sirt1 binds to acetylated Foxo1 (Brunet et al, 2004; Kitamura et al, 2005). Accordingly, Sirt1 bound to Foxo1 in the presence of H2O2 (Figure 3B, lane 1). However, co-transfection with FCoR decreased the amount of Sirt1 recovered in Foxo1 immunoprecipitates (Figure 3B, lane 2). These data suggest that FCoR disrupts the interaction between Foxo1 and Sirt1. Furthermore, we performed 5XGAL4-luciferase assays using 'constitutively acetylated' (6KQ) and 'acetylation-defective' (6KR) mutant Foxo1 (Kitamura et al, 2005). WT Foxo1 increased luciferase activity by ∼18-fold, while FCoR decreased WT Foxo1-induced luciferase activity by 40% (Figure 3C). However, FCoR failed to affect reporter activity induced by 6KQ or 6KR mutant Foxo1 (Figure 3C). These data suggest that acetylation is required for FCoR inhibition of Foxo1 transcriptional activity. Figure 3.FCoR enhances Foxo1 acetylation. (A) Effect of FCoR on Foxo1 acetylation. After transfection with pFLAG-CMV2-WT Foxo1 with or without pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with H2O2 (500 μM), nicotinamide (NAM) (50 mM), and trichostatin A (TSA) (2 μM) for 3 h and harvested. Lysates were immunoprecipitated with anti-FLAG mouse monoclonal antibody (M2) and subjected to western blotting with the indicated antibodies. The bottom panel shows western blotting of the lysates with and anti-cMyc mouse monoclonal antibody. (B) FCoR disrupts the interaction between Foxo1 and Sirt1. After transfection with pCMV5-cMyc-WT Foxo1 and pTOPO-Sirt1 with or without pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with H2O2 for 3 h and harvested. Cell lysates were immunoprecipitated with an anti-cMyc mouse monoclonal antibody and subjected to western blotting with the indicated antibodies. (C) 5XGAL4-luciferase assay of PM-WT, -6KQ, and -6KR Foxo1. At 36 h after transfection with pTAL-5XGAL4, phRL-SV40, and the indicated PM-Foxo1 with or without the FCoR expression vector, HEK293 cells were incubated with forskolin (20 μM) for 6 h and harvested. Luciferase activity was measured in the cell lysates. The grey bar indicates mock-transfected cells and the blue bar indicates pCMV5-cMyc-WT FCoR-transfected cells. Data represent the mean values±s.e.m. from three independent experiments. Asterisk indicates statistically significant difference (*P<0.005 by one-way ANOVA). (D) Sequence alignment of FCoR with various acetyltransferases. Residues that are identical or chemically similar to those in mouse FCoR are shown with a red background. FCoR has sequence similarity to the acetyl-CoA binding motifs of the MYST family of HATs (yeast Esa1, yeast Sas3, Drosophila MOF, human TIP60, and human P/CAF). (E) In-vitro acetylation assay of Foxo1. The GST-Foxo1-C1 (aa251–409) protein was subjected to in-vitro acetylation assays with GST (lanes 1, 4, and 5) or GST-FCoR (lanes 2 and 3) as described in 'Materials and methods'. Reaction products were analysed by Coomassie brilliant blue staining and autoradiography (14C). (F) In-vitro acetylation assay of Foxo1. The GST-Foxo1-C1 (aa251–409) protein was subjected to in-vitro acetylation assays with GST-FCoR (lane 1), GST (lane 2), or recombinant p300 (lane
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