Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic β-cells

FEBS Letters 587 (2013) 3883–3890

journal homepage: www.FEBSLetters.org

Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic b-cells Claire Mazuy, Maheul Ploton, Jérôme Eeckhoute, Wahiba Berrabah, Bart Staels, Philippe Lefebvre, Audrey Helleboid-Chapman ⇑ European Genomic Institute for Diabetes (EGID), FR 3508, F-59000 Lille, France UNIV LILLE 2, F-59000 Lille, France Inserm UMR 1011, F-59000 Lille, France IPL, F-59000 Lille, France

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Article history: Received 23 April 2013 Revised 18 September 2013 Accepted 15 October 2013 Available online 26 October 2013 Edited by Laszlo Nagy Keywords: Palmitate Nur77 Sp1 ZBP89 Pancreatic b-cell

a b s t r a c t Nur77 is a stress sensor in pancreatic b-cells, which negatively regulates glucose-stimulated insulin secretion. We recently showed that a lipotoxic shock caused by exposure of b-cells to the saturated fatty acid palmitate strongly increases Nur77 expression. Here, using dual luciferase reporter assays and Nur77 promoter deletion constructs, we identified a regulatory cassette between 1534 and 1512 bp upstream from the translational start site mediating Nur77 promoter activation in response to palmitate exposure. Chromatin immunoprecipitation, transient transfection and siRNA-mediated knockdown assays revealed that palmitate induced Nur77 promoter activation involves Sp1 recruitment and ZBP89 release from the gene promoter. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Peripheral insulin resistance combined with pancreatic b-cell dysfunction leads to type 2 diabetes, which is favored by genetic backgrounds and environmental factors. Hyperglycemia and hyperlipidemia induce additional damages or toxic effects in bcells. Notably, the increased release of free fatty acids (FFAs) contributes to the deterioration of b-cell functions [1,2] and plays a pathogenic role in the early stages of this disease. Chronically elevated FFA levels impair glucose-stimulated insulin secretion (GSIS) and induce apoptosis of insulin-secreting b-cells [3,4]. Several in vitro studies using isolated islets showed lowered insulin gene expression and impaired GSIS after exposure to FFAs [5,6]. However, the mechanism(s) responsible for palmitate-induced b-cell defect are incompletely understood. Triglyceride storage, reactive oxygen species production, dysregulation of the glucose metabolic pathway [7] and GPR40 FFA membrane receptor activation [8] are involved in FFA-induced GSIS inhibition. Notably, palmitatederived ceramide alters pancreatic and duodenal homeobox-1 ⇑ Corresponding author. Address: Faculté de Médecine de Lille-Pôle Recherche, Institut National de la Santé et de la Recherche Médicale (INSERM) U1011-Bâtiment J et K, Boulevard du Pr. Leclerc, 59045 Lille Cedex, France. E-mail address: [email protected] (A. Helleboid-Chapman).

(PDX-1) subcellular localization and MafA expression, which play a key role in activation of the insulin gene [9]. More recently, [10] identified by a proteomic approach several pathways responsible for palmitate-induced b-cell dysfunction and death. Palmitate induces b-cell dysfunction by triggering endoplasmic reticulum stress and generation of harmful metabolites during triglyceride synthesis, hampering insulin maturation and altering intracellular trafficking. The nuclear orphan receptor (NR4A) protein subclass contains three members (Nur77/NR4A1; Nurr1/NR4A2 and Nor1/NR4A3) implicated in multiple cellular and metabolic functions in the liver, skeletal muscle, brain, heart and adipose tissues [11]. These transcription factors are known to be rapidly and transiently induced by a diverse range of signals including extracellular stresses such as lipotoxic fatty acids, glucose or proinflammatory cytokines. The transcriptional activity of these receptors is primarily regulated through altered expression and/or posttranslational modification. Our recent study revealed a previously unrecognized function for Nur77 as a lipotoxicity sensor regulating glucoseinduced insulin secretion in b-cells [12]. As the most common saturated nonesterified fatty acid in human plasma, palmitate has been used to evaluate the contribution of NR4As to b-cell biology. Functional interaction of Nur77 with FOXO1 revealed a mechanistic basis for the Nur77-mediated inhibition of insulin biosynthesis

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.10.024

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though impaired mafA expression. However, how Nur77 expression is induced by a lipotoxic stress has not been characterized. Thus, we aimed to identify how fatty acids induce Nur77 expression in b-cells. In this study, we show that palmitate-mediated induction of Nur77 gene expression involves Sp1 recruitment together with ZBP89 release from the gene promoter potentially through direct competitive binding to overlapping sites.

The Nur77 promoter (2000/+1) was amplified from murin genomic DNA (http://www.informatics.jax.org; ID: 1352454) and cloned into the pGL3TK-luc vector. The following primers were used: 50 -AGTCGCTAGCGGTGGAGAGATAAAATGGGG-30 and 50 AGTCCTCGAGTCCTGCACTGGGGCTCCCCT-30 .

2. Materials and methods

2.4. Site directed mutagenesis

2.1. Reagents

The Nur77 promoter construct was modified using a site-directed mutagenesis kit (QuickChange, Stratagene). Primers were designed to delete 1525–1523 bp from the sequence of Nur77 (2000/+1) promoter according to the manufacturer’s recommendations (http:/labtools.stratagene.com). Mutagenesis was performed using primers 50 -GAAATAGCAGGCTGGTTGGTAAGGGG GGTCTTTTGTGGTGA-30 and 50 -TCACAACAAAAGACCCCCCTTACCA ACCAGCCTGCTATTTC-30 .

Sodium palmitate (P9767) and oleate (O7501) were from Sigma (St. Louis, MO). Stock solutions (100 mM) of sodium palmitate and oleate were prepared in ultrapure water and then were added to 10% FA-free BSA to a 5 mM final concentration and filtered. 2.2. Cell preparation MIN6 cells were grown in 75-cm2 flasks with 20 ml of Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-Invitrogen) containing 25 mM glucose, 50 lM b-mercaptoethanol, 110 U/ml penicillin, 110 U/ml streptomycin supplemented with 10% heat inactivated fetal bovine serum (growth medium) at 37 °C in a humidified atmosphere of 95% air/5% CO2.

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2.3. Cloning of the Nur77 (2000/+1) promoter luciferase construct

2.5. Dual luciferase reporter assay MIN6 cells were seeded in 48-well plates and transfected 48 h later with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) using 0.4 lg promoter reporter constructs and/or empty plasmid vector (pGL3TK-luc) and/or varying quantities of Sp1 or ZBP89 expression

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Fig. 1. Isolation of the activating region of Nur77 using promoter deletion constructs. Dual luciferase assays were performed in MIN6 cells using Nur77 promoter deletion constructs shortened stepwise from upstream. (A) Palmitate activation (24 h) on Nur77 promoter fragments (2000 to +1) were tested for luciferase activity. (B) Nur77 promoter fragments from 2000 to 1445 bp were used for sub-localization. Quantification represents five independent experiments. Data are means ± S.D. (Student’s ttest).

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Fig. 2. Verification of ZBP89 and Sp1 activation of mutated Nur77 promoter regulatory cassette in MIN6 cells. (A) ZBP89 binding site was mutated using site-directed mutagenesis as described in experimental procedures. (B) For dual luciferase reporter assay, MIN6 cells were transfected with the ZBP89 mutated binding site within the Nur77 promoter regulatory cassette located on the 1525 bp-Nur77 pGL3TK construct. Quantification represents five independent experiments. Data are means ± S.D.

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Fig. 3. Verification of ZBP89 and Sp1 bindings at their response elements of the Nur77 promoter by chromatin immunoprecipitation assays. MIN6 cells were chemically crosslinked with formaldehyde. Chromatin was extracted, fragmented and immunoprecipitated with ZBP89, Sp1 and RNApol II antibodies. The postulated binding regions of the Nur77 promoter were amplified by QPCR and results were normalized to myoglobin. Results are plotted as the fold enrichment over background.

vector according to the manufacturer’s instructions. The medium was replaced 5 h later by fresh growth medium containing palmitate or oleate (0.4 mM). Luciferase activity was measured (Luciferase assay system; Promega Corp., Madison, WI) with a Victor Light (Perkin Helmer) plate reader and normalized to cellular DNA. Transfection experiments were done at least three times in triplicate.

expression was calculated by the DDCt method. Final results were expressed as the fold difference in gene expression normalized to 18S rRNA and relative to control conditions. For ZBP89 and Sp1 gene expression, QPCR were carried out using SyberGreen-based chemistry. Primers used were: 50 -TTCTGCAGCAGGCTTTGGAC-30 and 50 -TGGGCATGGCTGAATAGACT-30 for ZBP89 and 50 -TCAGAACC CACAAGCCCAGA-30 and 50 -AGGAATGGAGGCAGCTGAGG-30 for Sp1.

2.6. RT-QPCR analysis

2.7. Cellular extract preparation and western blotting

Total RNA was extracted using RNeasy (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions. DNase I-treated total RNA was reverse transcribed using High-Capacity cDNA archive kit (Applied Biosystems, Foster City, CA). For Nur77 gene expression, cDNA were analysed using TaqMan Gene Expression Assays as recommended by the manufacturer. Relative gene

The following antibodies were used: anti-ZBP89 (Sc-48811, Santa-Cruz Biotech.), anti-Sp1 (Sc-14027, Santa-Cruz Biotech.) and anti-HSP a/b (Sc-7947). Approximatively 2  107 cells were lysed in cell lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 lg/ml leupeptin

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and 1 lM PMSF. Proteins were boiled 5 min in 2 Laemmli buffer, separated by 10% SDS–PAGE and revealed by Western blotting using SuperSignal West Dura Extended Duration Substrate (ThermoScientific, Illkirch, France). Images were acquired on a G-BOX (Syngene, Cambridge, UK) and quantification were performed using ImageQuant software.

2.9. Gene knockdown MIN6 cells were transfected with siRNA directed against ZBP89 (sc-38640) or Sp1 (sc-29488) or with a negative control siRNA using Lipofectamine according to the manufacturer’s instructions (Santa Cruz, CA). After 5 h, the transfection medium was replaced with growth medium, and the cells were further cultured with 0.4 mM palmitate for 24 h. The knockdown efficiency was verified by QPCR. Oligonucleotides used for ZBP89 and Sp1 were respectively 50 -TTCTGCAGCAGGCTTTGGAC-30 , 50 -TGGGCATGGCTGCATAG ACT-50 and 50 -TCAGAACCCACAAGCCCAGA-30 , 50 -AGGAATGGAGGC AGCTGAGG-30 .

2.8. Chromatin immunoprecipitation assays MIN6 cells were seeded in 10-cm dishes and incubated with 0.4 mM palmitate for 24 h. Chromatin immunoprecipitation (ChIP) assays were then performed as described by Sérandour [13]. Antibodies used in chromatin immunoprecipitation were raised against ZBP89 (sc-48811), Sp1 (sc-14027) and RNA Pol II (sc-899) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primers for ChiP PCR were 50 -TTCTGCAGCAGGCTTTGGAC-30 , 50 -TGGGCATGGCTG CATAGACT-50 for ZBP89 and 50 -TCAGAACCCACAAGCCCAGA-30 , 50 AGGAATGGAGGCAGCTGAGG-30 for Sp1. For Nur77-TSS, primers for ChiP PCR were 50 -CCCTTGTATGGCCAAAGCTC-30 , 50 -CATCTTA AGCGCTCCGTGAC-30 .

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2.10. Statistical analysis Statistical significance was evaluated using an unpaired twotailed t test or Anova test for siRNA experiments. Data are expressed as means ± standard error and differences were considered as significant (P < 0.05; ⁄); very significant (P < 0.005; ⁄⁄) and highly significant (P < 0.001; ⁄⁄⁄).

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Fig. 4. (A and B) Confirmation of ZBP89 knockdown using MIN6 cells treated with 0.4 mM palmitate for 24 h. Cells were transfected with nontargeted or ZBP89 siRNA prior to mRNA and qPCR analysis (A) or Western blot analysis (B). (C) Same cells were used to quantify Nur77 gene expression using Taqman probes. Quantification represents three independent experiments. Data are means ± S.D.

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involved in the response to free fatty acids, we performed reporter assay experiments using progressively shorter promoter constructs (Fig. 1B). These experiments identified the region comprised between 1668 and 1445 bp upstream from the transcriptional start site as essential for palmitate-mediated Nur77 promoter activation. A search for transcription factor binding motifs using MatInspector (www.genomatix.de) within the identified palmitate-responsive region of the Nur77 promoter identified a ZBP89 binding site overlapping a Sp1 binding site at 1534/1512 bp. To investigate the relative function of these two factors, we first introduced a mutation in the Sp1/ZBP89 binding motif between 1525 and 1523 bp from transcription start (Fig. 2A). Potential Sp1 and ZBP89 binding sites could not be individually mutated as they strongly overlap. The introduced mutation had a drastic effect, as the mutated promoter exhibited a 95% decreased activity in response to palmitate compared to control (Fig. 2B).

3. Results 3.1. Nur77 promoter activity is regulated by palmitate in MIN6 cells To define whether free fatty acids regulation of Nur77 expression can be ascribed to its promoter, we first investigated the effect of palmitate on a promoter fragment of 2000 bp cloned in pGL3TK-luc vector. As shown in Fig. 1A, increasing concentration of palmitate enhanced Nur77 promoter activity in a dose-dependant manner in MIN6 pancreatic beta-cells. Higher concentrations of palmitate up to 0.4 mM provide a maximum stimulation (2.3-fold compared with control) of Nur77 promoter in a physiological range of plasma free fatty acid concentrations. The up-regulation (2.8-fold induction) was also observed with the monounsaturated fatty acid oleate. We conclude that saturated or unsaturated free fatty acids induce Nur77 promoter activity. To delineate further the promoter region

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Fig. 5. (A and B) Confirmation of Sp1 knockdown using MIN6 cells treated with 0.4 mM palmitate for 24 h. Cells were transfected with nontargeted or Sp1 siRNA prior to mRNA extraction and qRT-PCR (A) or Western blot analysis (B). (C) Same cells were used to quantify Nur77 gene expression using Taqman probes. Quantification represents three independent experiments. Data are means ± S.D. (Student’s t-test).

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3.2. Palmitate treatment modulates the balance between ZBP89 and Sp1 binding to the Nur77 promoter ChIP assays using MIN6 cells treated or not with palmitate were performed to monitor ZBP89, Sp1 and RNA pol II binding to the identified palmitate-responsive region within the Nur77 promoter (Fig. 3). PCR amplification of immunoprecipitated DNA revealed an enhanced recruitment of RNA pol II in the presence of palmitate in line with Nur77 transcriptional induction by palmitate [12] (Fig. 3B). This was correlated with an increase in Sp1 binding (6.4-fold increase) and a concomitant loss of ZBP89 recruitment (Fig. 3A).

control) in the presence of an empty expression vector. ZBP89 overexpression abolished the Nur77 promoter response to palmitate, whereas overexpression of Sp1 increased palmitate-induced Nur77 promoter activity (3.7-fold compared with control) and gene expression level (data not shown). In contrast, coexpression of ZBP89 or Sp1 had no effect on Nur77 induction of the mutated, non palmitate-responsive promoter fragment. In conclusion, these results show that ZBP89 and Sp1 modulate Nur77 gene expression upon palmitate treatment. Thus, we propose that the palmitate-induced activation of the Nur77 promoter activity is linked to Sp1 recruitment and concommitant ZBP89 release potentially through direct competitive binding to the promoter (Fig. 7).

3.3. Activation of Nur77 promoter by palmitate is modulated by ZBP89 and Sp1

4. Discussion

To investigate the role of ZBP89 and Sp1 in regulating Nur77 expression, we used a siRNA-based approach. ZBP89 expression was down-regulated in MIN6 cells, by 50% (Fig. 4A), whereas the control siRNA showed an unaltered ZBP89 expression. Western blot analysis confirmed the decreased expression level of ZBP89 protein (Fig. 4B). Nur77 mRNA expression was upregulated in ZBP89-depleted MIN6 cells treated with 0.4 mM palmitate for 24 h to a higher extent than in control cells as shown in Fig. 4C. The loss of ZBP89 expression potentiated activation of Nur77 transcription by about 50% in response to palmitate. Thus, ZBP89 acts as a repressor of the Nur77 promoter by binding to the identified activation cassette. A similar approach was applied to Sp1. Using a siRNA targeting Sp1 (Fig. 5A and B), we observed a decreased response of Nur77 gene expression (Fig. 5C) after palmitate stimulation. The functional significance of these interactions was tested in a transactivation assay, in which Nur77 promoter fragments were cotransfected with ZBP89 or Sp1 expression vectors (Fig. 6). Palmitate enhanced Nur77 promoter activity (2.5-fold compared with

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Fig. 6. ZBP89 and Sp1 overexpression modulates Nur77 promoter activity induced by palmitate. (A) Min6 cells were transfected either with a control reporter gene (Pgl3TK-luc) or using Nur77 promoter deletion constructs (2000; 1525; 1525 mut), with a fixed amount of an expression vector coding either for ZBP89 or Sp1. Luciferase activity was assayed after 24 h palmitate treatment. Quantification represents three independent experiments. Data are means ± S.D.

As extracellular signals induce NR4A receptors in a context- and cell type-dependent manner, the target genes and functions of NR4A receptors are also likely to be tissue-and context-dependent. Several transcription factors have been shown to regulate Nur77 promoter activity like b-catenin, which binds to AP-1 binding sites in the Nur77 promoter [14] in colon cancer cells or C/EBPb, which cooperates with the p50 subunit of NF-jB to further enhance Nur77 promoter activity in Leydig cells [15]. The NF-jB signaling pathway was identified as regulating Nur77 expression, whose promoter harbors two highly conserved NF-jB response elements in macrophages [16]. It was also known that Nur77 promoter is controlled by CREB protein (c-AMP response element binding protein) in PC12 cells [17] and MEF2 protein in T-cells [18]. Although Nur77 up-regulation is a marker of cellular stress in cultured insulinoma b-cells, how fatty acids induce Nur77 expression remains unclear. We recently reported an increase in Nur77 expression in b-pancreatic cell lines and in isolated human pancreatic islets upon exposure to the saturated FA palmitate. This increased expression of Nur77 induced an inhibition of insulin biosynthesis through mafA expression [12]. In this report, we provide mechanistic bases for the fatty acid-induced activation of Nur77 in pancreatic b-cells at physiological concentration of 0.4 mM. Using transactivation assays with Nur77 promoter deletion constructs, we identified a regulatory region within the Nur77 promoter between 1668 and 1445 bp from the transcriptional start responsible for pancreatic b-cells Nur77 promoter activation by palmitate. An activation cassette with an overlapping ZBP89/ SP1 binding site was identified as a potential candidate by sequence comparison with a transcription factor binding site database (Matinspector). Indeed, a mutation in this activation cassette had a strong effect on palmitate-induced Nur77 promoter activity. Although ubiquitous, ZBP89 is a Krüppel-type zinc finger protein not only expressed in T-cells [19,20], but also in normal human pancreatic islets and insulinomas cells [21]. ZBP89 regulates diverse biological functions through direct promoter binding as well as through protein–protein interactions. In addition, ZBP89 regulates cell growth by inhibiting cell proliferation [22,23] and induces apoptosis in human cells [24]. This protein binds a GC-rich responsive element from several promoters like collagen [25]; benolase [26] or vimentin [27]. ZBP89 has bifunctional regulatory domains suggesting that it may function as both a transcriptional activator and repressor. ZBP89 inhibits cell growth via the repression of gastrin and ornithine decarboxylase [25,26]. More recently, [28] revealed that neuronal insulin receptor substrate 2 (IRS2) expression is regulated by ZBP89 and Sp1 binding to the IRS2 promoter. A regulatory cassette composed of four ZBP89/SP1 binding sites controlled the IRS2 promoter activity in response to the PI3K pathway activation. Similarly, our chromatin immunoprecipitation assay confirmed the specific DNA binding of ZBP89 and Sp1 on the described region in the Nur77 promoter. Treatment with

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ZBP89

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palmitate resulted in enhanced DNA binding activity of Sp1 to the identified sequence, while a complete loss of ZBP89 DNA binding activity was observed. Palmitate is known to induced Sp1 DNA binding to a Sp1 consensus sequence indicative of an increased activity of this transcription factor [29]. This binding activity may be mediated by enhanced O-GlcNac modification through the hexosamine pathway. To understand the influence of ZBP89 and Sp1 binding to the Nur77 promoter, we used knockdown experiments using siRNA. Without changing Sp1 expression, we performed palmitate stimulation using MIN6 cells transfected with siRNA against ZBP89. In this condition, ZBP89 repressed palmitate-induced Nur77 gene expression in MIN6 cells. In contrast, Sp1 knockdown decreased Nur77 gene expression indicating that Sp1 could promote Nur77 gene expression induction by palmitate. All these results showed that a differential binding of ZBP89 and Sp1 could regulate Nur77 promoter activity. Consequently, we assumed that the regulation of promoter activity might be controlled via a competitive binding of the two proteins to overlapping binding sites. Interestingly, direct competitive binding of ZBP89 and Sp1 to a shared DNA binding sequence has also been implicated into regulation of the ODC gene promoter activity [30]. As Nur77 stimulates glucose production [31] and decreased pancreatic b-cell insulin secretion [12], a functional interaction could be involved in metabolic regulation. Acknowledgements We thank Pr. Juanita Merchant (University of Michigan) for providing rat ZBP89 cDNA (Public Health Service Grant R01 DK055 732) and Pr. Guntram Suske (University of Marburg) for providing human Sp1 cDNA . CM was supported by a fellowship from INSERM and Region Nord-Pas-de-Calais. This work was supported by grants from ‘‘European Genomic Institute for Diabetes’’ (EGID, ANR-10-LABX46). B. Staels is a member of the Institut Universitaire de France. References [1] Boden, G., Chen, X. and Iqbal, N. (1998) Acute lowering of plasma fatty acids lowers basal insulin secretion in diabetic and nondiabetic subjects. Diabetes 47, 1609–1612.

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