Characterization of the Human CIDEA Promoter in Fat Cells

International Journal of Obesity (2008) 32, 1380–1387 & 2008 Macmillan Publishers Limited All rights reserved 0307-0565/08 $32.00 www.nature.com/ijo ORIGINAL ARTICLE Characterization of the human CIDEA promoter in fat cells

AT Pettersson1, J Laurencikiene1, EA Nordstro¨m1, BM Stenson1, V van Harmelen1, C Murphy2, I Dahlman1 and M Ryde´n1

1Department of Medicine, Huddinge, Lipid Laboratory, Novum, Karolinska Institutet, Stockholm, Sweden and 2Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

Background: Cell death-inducing DFFA (DNA fragmentation factor-a)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-a (TNF-a). TNF-a downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA. Methods: A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitro- differentiated human adipocytes. The effect of TNF-a was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein–DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA). Results: Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-a reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kB (NF-kB) site at position À163/À151. Conclusion: We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-a negatively regulates transcription of this gene, which at least in part involves NF-kB activation. International Journal of Obesity (2008) 32, 1380–1387; doi:10.1038/ijo.2008.101; published online 8 July 2008

Keywords: adipocyte; TNF-a; adipose tissue; NF-kB

Introduction We recently showed that cell death-inducing DFFA (DNA fragmentation factor-a)-like effector A (CIDEA) is an Obesity is associated with dyslipidemia, insulin resistance important regulator of fat cell metabolism.4 CIDEA is and type 2 diabetes mellitus.1 These are all well-documented downregulated in obese patients and CIDEA mRNA levels risk factors for cardiovascular disease.2 This association is correlate inversely with markers for the metabolic syndrome mediated through several mechanisms including altered in a manner that appears to be human-specific.4 In a gene transcription in human fat cells, which leads to increased profiling study of subcutaneous white adipose tissue (WAT) release of inflammatory mediators (for example, tumor in obese women following diet-induced weight reduction, necrosis factor-a (TNF-a)) and free fatty acids into the CIDEA was the most upregulated gene,5 indicating a role for circulation (for reviews see1,3). CIDEA in body weight regulation. Moreover, a single nucleotide polymorphism (SNP) in exon 4 of CIDEA, Correspondence: AT Pettersson, Department of Medicine Huddinge, C19787G-T, resulting in a V115F amino acid substitution, Karolinska Institutet, Lipid Laboratory Novum, Ha¨lsova¨gen 7, 14186 is associated with body mass index (BMI) in a Swedish cohort Stockholm, Sweden. of both genders.6 CIDEA mRNA is downregulated by TNF-a,a E-mail: [email protected] Received 19 March 2008; revised 30 May 2008; accepted 4 June 2008; cytokine overexpressed in obesity. It is not known whether published online 8 July 2008 this is mediated through direct effects on transcription or Transcriptional control of human CIDEA AT Pettersson et al 1381 post-transcriptional mechanisms, for example decreased which colonies were inoculated. DNA was isolated with a kit mRNA stability. Although the molecular action of CIDEA from Qiagen GmbH (Hilden, Germany) standard protocol. in human adipocytes is not clear, in vitro studies in human DNA sequencing confirmed the identities of all clones. adipocytes show that CIDEA inhibits basal lipolysis and CIDEA promoter reporter constructs were generated by TNF-a production in these cells.4 Thus, CIDEA may regulate amplifying a 1.1-kb fragment of the 50 region of human insulin sensitivity indirectly through effects on TNF-a- CIDEA (from À1124 to þ 2 relative to the TSS) by PCR using mediated adipocyte lipolysis. Pfu DNA polymerase (Promega). The amplified fragments Thus far, little is known regarding the transcriptional were cloned into the pGL3-basic vector (Promega). Constructs regulation of human CIDEA. To date only the transcriptional were verified by DNA sequencing (MWG Biotech, Germany). regulation of the murine CIDEA promoter in liver has been Site directed mutagenesis of the SNP rs7230534, located characterized.7 In this study, we characterized the human at À635 relative to the TSS, and of a putative nuclear factor- CIDEA promoter in human fat cells and 3T3-L1 cells and kB (NF-kB) binding site located at À163/À151 was performed studied the effect of TNF-a on promoter activity. Further- using QuikChange II Site-Directed Mutagenesis Kit more, several SNPs in the 50 flanking region of human CIDEA (Stratagene, La Jolla, California, USA). The sequences for have recently been described6 and we assessed the effect of the forward primers used in the mutagenesis were as follows the three most common haplotypes in this region on the (bold letters indicate mutated bases and the putative binding transcriptional regulation of CIDEA. site for NF-kB is underlined): 50-CATGCAGTCCAAAGGTTC CTTTCCCCTCCCG-30 and 50-CCAGGACGACCTTGGGCTC AAAGAGCGCGAAGTCGCGATC-30 respectively. The reverse Materials and methods primer was complementary to these sequences.

Cell cultures and patient samples Subcutaneous adipose tissue was obtained from healthy Transient transfection patients undergoing elective cosmetic liposuction. The study In vitro-differentiated human adipocytes were transfected as 11 was approved by the ethical committee at Karolinska described in detail previously. 3T3-L1 cells were transfected University Hospital and informed consent was obtained at 80% confluence with 0.5 mg of reporter DNA and 0.1 mgof from each patient. We certify that all applicable institutional CMV-b-galactosidase-containing vector using 2 ml lipofecta- and governmental regulations concerning the ethical use of mine and 8 ml plus reagent (Invitrogen, Carlsbad, CA, USA). human volunteers were followed during this research. At 24 h post-transfection, cells were incubated in differentia- Experiments were performed in in vitro-differentiated human tion medium with or without 10 mM of the peroxisome adipocytes derived either from human mesenchymal stem proliferator-activated receptor gamma (PPARg) agonist cells (hMSCs) or from human preadipocytes isolated from BRL49653 or TNF-a (50 ng/ml) (Sigma, St Louis, MO, USA) adipose tissue. Isolation, culture and differentiation of for an additional 24 h, respectively. 3T3-L1 cells and in vitro- hMSCs and of human preadipocytes were performed as differentiated human adipocytes were lysed and luciferase described previously.8,9 In vitro-differentiated hMSCs are and b-galactosidase activity were measured according to somewhat sensitive to transfections and concomitant the manufacturer’s protocol (Luciferase Assay System and TNF-a treatments. For transfection studies with simultaneous b-Galactosidase Enzyme Assay System, both from Promega) TNF-a incubations, our analyses were therefore complemented using an infinite 200 luminometer (Tecan, Salzburg, Austria). with experiments in the well-established murine adipocyte cell Luciferase activity was normalized for b-galactosidase activity line 3T3-L1. These cells were cultured as described.10 and experiments with high variability in b-galactosidase activity were excluded. All experiments were run in triplicate and repeated at least four times with similar results. 50 rapid amplification of cDNA ends (50 RACE), cloning and mutagenesis A50 RACE study was performed to identify the CIDEA Electrophoretic mobility shift assay transcriptional start site (TSS). CIDEA mRNA 50 ends were For electrophoretic mobility shift assays (EMSA) in vitro- amplified from Human Adipocyte and Adipose Tissue differentiated human adipocytes were incubated in the Marathon-Ready cDNA (Clontech, Mountain View, California, presence or absence of a peptide termed wild type NF-kB USA) with the CIDEA-specific primer 50-ATGCGTGTTGTCT essential modulator (NEMO)-binding domain (NBD) peptide CCCAAGGTCTGA-30 and nested primer 50-CAGAGTGACC (WP) that inhibits NF-kB signaling in several cell-based AGTCCGGTAGCGATG-30 using the Advantage 2 Polymerase systems including human fat cells11,12 or a biologically Mix (Clontech) according to the manufacturer’s instructions. inactive (mutated) peptide (MP) at a concentration of 33 mM. The PCR products were gel purified and ligated into a pGEM WP is a cell-permeable peptide consisting of the region from T Easy-vector (Promega, Madison, Wisconsin, USA). T735 to E745 of IKKb fused with a sequence derived from the Ligations were transformed into competent bacteria and Drosophila antennapedia homeodomain protein that mediates plated onto appropriate antibiotic-containing plates from membrane translocation. The amino acid sequence for WP

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1382 and MP were DRQIKIWFQNRRMKWKKTALDWSWLQTE and webgene/) from WebGene, we identified putative TATA- DRQIKIWFQNRRMKWKKTALDASALQTE respectively. After boxes in a 2.1-kb-long sequence upstream of the human 2 h, TNF-a was added at a concentration of 100 ng mlÀ1 and CIDEA translation initiation codon ATG (NT_010859). The cells were incubated for 24 h. Cells were lysed and nuclear TATA-box closest to the TSS was predicted at À40/À31 extracts were prepared using a Nuclear Extract Kit (Active (TTTAAGAGGC) (Figure 1a). A corresponding TATA-box Motif, Carlsbad, CA, USA). Nuclear extracts containing 5 mg sequence was also predicted in the murine 50 UTR. protein were incubated in 18 ml binding reaction (10 mM We compared the human CIDEA sequence, 2.0-kb

Tris/HCl pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, upstream of ATG (NT_010859), with that of a region À1 4% glycerol, 50 mgml poly (dIdC), 0.5 mM DTT) with or comprising 3.0-kb upstream of the translation initiation without unlabeled probe (200-fold excess of either probe codon of the corresponding mouse sequence (EF451058) and À242/À202 or À202/À151). In addition, another unlabeled the corresponding predicted rat sequence using zPicture probe was used comprising À202/À151 with a mutated (http://zpicture.dcode.org/). The analyzed sequences include NF-kB site (À163/À151). Two microliters of the 32P-labeled the 14-bp 50 UTR and the putative promoter region for probe (10 000 cpm mlÀ1) was added and the sample was human CIDEA and the 745-bp-long 50 UTR and promoter incubated 30 min at room temperature. Reaction mixtures region of the murine CIDEA. Using a threshold of at least were analyzed at 41C on 4% polyacrylamide gels in 50 mM 70% identity over 100-bp, no evolutionarily conserved Tris base/45 mM boric acid/0.5 mM EDTA. The gel was dried regions (ECRs) shared between the rat, mouse and human and developed in a Hypercassette (Amersham, UK) with an promoter regions were identified. However, ECRs between intensifying screen (Kodak, Rochester, New York, USA). The mouse, rat and human were found when threshold was set to sequences for the oligonucleotides/probes used were as 80% over 20-bp (Figure 1a). These ECRs are located in the follows (a putative binding site for NF-kB is underlined and 50UTR of the murine CIDEA transcript7 or in the human mutated bases are shown in bold letters): À242/À202: promoter between À811 and À61. We also analyzed the two 50-CATCTGGGCTTCCAAGGTCCTCGGTACGGCCCAAGGCA promoters for CpG islands with CpG Island Searcher (http:// GCG-30 and À202/À151: 50-GAAGGACGCGCGGCTCC www.uscnorris.com/cpgislands2/). Using a 55% limit for GC- AGGCTGCGGGAGCCAGGACGACCGGGGGCTCCCAG-30 as content and a length of at least 500-bp, one CpG island in well as mutated À202/À151 GAAGGACGCGCGGCTC the human promoter (À607/ þ 21) as well as one island in CAGGCTGCGGGAGCCAGGACGACCTTGGGCTCAAAG-30. the murine 50 UTR ( þ 183/ þ 784) was predicted. These Complementary oligonucleotides to À242/À202, À202/ regions overlap with the ECRs predicted by the zPicture. À151 and mutated À202/À151 were used to create double The human CIDEA sequence was analyzed for putative stranded probes. transcription factor binding sites using MatInspector software from Genomatix BiblioSphere (Figure 1b). Core simi- Statistical analyses larity was set to 0.75. Consensus sequences for, among Statistical differences were determined by nonparametric others, the vitamin D receptor (VDR), Sp1 and NF-kB were methods. Different groups were compared using predicted in the promoter region. Mann–Whitney U test. P-values are indicated when appropriate and P-values less than 0.05 were considered statistically significant. The minimal promoter for transcription of CIDEA is present within 97-bp from the transcriptional start site To identify the minimal promoter region needed for Results transcription, we cloned a 1.1-kb-long region from À1124 to þ 2 of TSS of CIDEA. We generated eight fragments (A-H) 0 Identification of the transcriptional start site of the human with deleted 5 ends and tested their transcriptional activity CIDEA gene in a luciferase reporter system in in vitro-differentiated Using 50 RACE we identified a single transcript of CIDEA in human adipocytes. The activity for all deletion fragments human adipocytes with the TSS at nucleotide A, 14-bp was significantly higher (A: Po0.001; B: Po0.01; C: upstream of the translational start codon ATG (Figure 1a). Po0.001; D: Po0.001; E: Po0.001; F: Po0.001; This identified transcript corresponds to the previously G: Po0.001 and H: Po0.05) than that of cells transfected published CIDEA transcript NM_001279. The alternative with empty vector alone (Figure 2a). The highest activity was CIDEA transcript NM_198289, which contains another observed with fragments C (À325/ þ 2), D (À279/ þ 2), E exon 1, was not detected in our cDNA libraries. (À244/ þ 2) and F (À123/ þ 2), whereas fragment A (À1124/ þ 2) and B (À409/ þ 2) displayed a significantly lower activity compared with fragment C, D, E and F. Furthermore, Bioinformatic comparison of the putative human adipose the transcriptional activity of the shortest fragment H (À46/ promoter sequence and the murine liver CIDEA promoter þ 2) was significantly different from fragment G (À97/ þ 2) Using the Hamming–Clustering Method for TATA Signal (Po0.05) as well as the other mutation fragments, indicating Prediction in Eukaryotic Genes (http://www.itb.cnr.it/sun/ that most of the transcriptionally important elements

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1383

Figure 1 CIDEA 50 flanking region and transcriptional start site (TSS). (a) The CIDEA 50 flanking region from À893 to þ 21 was aligned with the murine CIDEA 50 UTR from þ 190 to þ 784. Evolutionarily conserved regions between rat, mouse, and human as predicted by zPicture are shown as underlined sequences. The predicted TATA-box is shown as a boxed sequence. Translational start sites (ATG) are underlined and þ 1 denotes TSS in human. (b) The CIDEA 50 flanking region (Genbank accession number NT_010859) was searched for putative transcription factor binding sites using the MatInspector software from Genomatix BiblioSphere. Consensus sequences for transcription factors are shown in bold and SNPs in the human CIDEA promoter are indicated by numbers. The SNPs are all in strong linkage disequilibrium with each other (same haploblock) and allele frequency is 45% (6). ERE ¼ estrogen response element, RXR ¼ retinoid X receptor, Sp1 ¼ stimulating protein 1, HNF4 ¼ hepatocyte nuclear factor 4.

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1384 pGL3-basic Luc three different haplotypes. However, we observed no differ- -46+2 Luc H ence in luciferase activity among the constructs (Figure 2b). -97 +2 Luc G -123 +2 Luc F -244 +2 Luc E TNF-a, but not PPARg agonists, regulates human CIDEA -279 +2 Luc D promoter activity -325 +2 Luc C CIDEA mRNA expression is downregulated in vitro by TNF-a. -409 +2 Luc B To define putative TNF-a-regulated regions, we studied the -1124 +2 Luc A effect of TNF-a on human fat cells transfected with promoter construct A (À1124/ þ 2). Luciferase assays in in vitro- 010203040506070 differentiated human adipocytes demonstrated that TNF-a Promoter construct Relative luciferase activity (fold over pGL3-basic) significantly reduced the transcriptional activity of this construct (Figure 2c). We and others have previously shown

Haplotype that TNF-a promotes a slight increase in human adipocyte III 13,14 I II III apoptosis in culture. This effect is somewhat more II NS SNP 1. G G T pronounced in cells subjected to concomitant transfections I which preclude firm conclusions when differences among SNP 2. G G A promoter constructs are studied. We therefore used the well- SNP 3. T C T pGL3- ** basic established murine cell line 3T3-L1 to investigate the TNF-a- SNP 4. T T C 024681012 mediated regulation of the human CIDEA promoter in more SNP 5. - - G ins Relative luciferase activity detail. Similar to the findings in human adipocytes, TNF-a (fold over pGL3-basic) 18 attenuated the transcriptional activity of the CIDEA promo- 16 ter in murine 3T3-L1 cells (Figure 3a, left panel). Moreover, 14 12 this effect was specific, as incubation with the PPARg agonist 10 BRL49653 did not result in any significant difference in 8 * 6 transcriptional activity (Figure 3a, right panel). 4 To further investigate the TNF-a-induced regulation of the 2 (fold over pGL3-basic)

Relative luciferase activity 0 CIDEA promoter, 3T3-L1 cells were transfected with the ctrl TNFα -1124 +2 previously generated deletion fragments followed by incuba- Luc tions with TNF-a for 24 h. Luciferase assays demonstrated Figure 2 Transcriptional activity of the human CIDEA promoter in in vitro- that the TNF-a effect was confined to the region À244/À123, differentiated human adipocytes. (a) In vitro-differentiated human adipocytes, k derived from hMSCs, were transfected with various constructs of the human which includes a binding site for NF- B(À163/À151) CIDEA promoter. Luciferase activity was normalized for b-galactosidase (Figure 3b). We mutated this binding site in fragment E activity. The activity of each promoter fragment was tested in triplicate and (À244/ þ 2) and transfected 3T3-L1 cells with either wild the results are mean±s.e.m. of four independent experiments. (b) In vitro- type (wt) or mutated (mut) fragment. The attenuating effect differentiated human adipocytes, were transfected with three different a haplotypes (1, 2 and 3) of the human CIDEA promoter. The activity of each of TNF- treatment was significantly reduced in cells promoter fragment was tested in triplicate and the results are mean±s.e.m. of transfected with the mutated promoter fragment compared three independent experiments. (c) In vitro-differentiated human adipocytes, with cells treated with wt fragments (Figure 3c), suggesting were transfected with the human CIDEA promoter, construct A (À1124/ þ 2), that this site is important for TNF-a regulation in vivo. and incubated in differentiation media with or without TNF-a for 24 h. Finally, to investigate the transcription factor binding Samples were run in triplicate and the results are mean±s.d. of one representative experiment. This experiment was repeated at least three times ability of this region, we studied DNA–protein interactions with similar results. Relative luciferase activity was expressed as fold over by EMSAs using two double-stranded oligonucleotide probes empty vector (pGL3-basic). Different groups were compared using Mann– comprising the region between À242/À202 and À202/À151, Whitney U test. *P 0.05 and **P 0.01. o o respectively. TNF-a treatment of in vitro-differentiated human adipocytes resulted in an increased binding of proteins are found in the region upstream of À46 and that the to probe À202/À151 which was blocked by pre-incubation minimal transcriptional activity is present within 97-bp from with an excess of unlabeled probe À202/À151 (Figure 4). In the TSS. contrast, no binding was observed using probe À242/À202 (data not shown). Oligonucleotide À202/À151 comprises one NF-kB binding site (À163/À151) predicted by MatInspector. Effect of SNPs in the human CIDEA 50 flanking region To assess whether NF-kB could bind to this region we We investigated the effect on transcription of five common pretreated human adipocytes with a peptide (WP) that SNPs, highlighted in Figure 1b, in the 50 flanking region of inhibits NF-kB signaling. This resulted in a loss of the lower CIDEA, which form three common haplotypes that were protein complex, whereas pretreatment with a described previously.6 In vitro-differentiated human adipo- mutated, biologically inactive form of the peptide (MP) did cytes were transfected with reporter constructs containing not affect this interaction. Incubation with WP or MP alone

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1385 NF-κB site BRL49653 -163/-151 14 -244 +2 Luc 14 TNFα 12 E 12 NS Probe -202/-151 10 10 8 8 * ctrl TNFα 6 6 4 4 - - - - + EMSA competitor 2 2 No extract - - WP MP - NF-κB-inhibitor

0 pGL3-basic) (fold over (fold over pGL3-basic) pGL3-basic) (fold over α 0 ctrl activity luciferase Relative Relative luciferase activity luciferase Relative TNF ctrl BRL -1124 +2 Luc -1124 +2 A Luc A

-123 +2 Luc F NS -244 +2 Luc E * -325 +2 Luc C * TNFα pGL3-basic Luc control

0 20406080100 Promoter construct Relative luciferase activity (fold over pGL3-basic)

wt -163/-151 TNFα treatment -244 +2 123456 Luc E mut -163/-151 Figure 4 TNF-a-induced protein binding to the human CIDEA promoter. Control treated in vitro-differentiated human adipocytes (lane 2) or TNF-a- -244 +2 treated adipocytes (lane 3) were incubated with a 32P-labeled oligonucleotide Luc E * probe spanning the region À202/À151. Adipocytes were incubated with an inhibitory peptide (WP) (lane 4) or a biologically inactive peptide (MP) (lane 5) 0 20406080100 for 2 h before TNF-a treatment. DNA–protein interactions (indicated by an Promoter construct Relative luciferase activity arrow) were detected in samples treated with TNF-a alone or TNF-a and MP but not in samples treated with TNF-a and WP. Specificity of the protein–DNA (% of ctrl treated cells) interaction was tested with unlabeled oligonucleotide À202/À151 (lane 6). Figure 3 Effect of TNF-a and PPARg on the human CIDEA promoter in 3T3-L1 The picture depicts one representative example and was repeated three times cells. (a) Murine 3T3-L1 adipocytes were transfected with the human CIDEA with similar results. promoter, construct A (À1124/ þ 2), and incubated in differentiation media with or without TNF-a (left panel) or BRL49653 (right panel) for 24 h. Samples were run in triplicate and the results are mean±s.d. of one representative experiment. Relative luciferase activity was expressed as fold over empty vector (pGL3-basic). (b)The for CIDEA and the presence of one single transcript in effect of TNF-a was also tested on shorter fragments of the promoter as indicated. 0 Sampleswererunintriplicateandtheresultsaremean±s.d. of one representative human adipocytes as demonstrated by 5 RACE. This identi- experiment. (c) The effect of the indicated NF-kB site on promoter activity was fied transcript corresponds to the previously published tested in 3T3-L1 cells transfected with wt or mutated fragment E (À244/ þ 2) CIDEA transcript NM_001279. Furthermore, we analyzed ± treated with TNF-a. Samples were run in triplicate and the results are mean s.d. of transcriptional activity in a 1.1-kb genomic region upstream one representative experiment. Relative luciferase activity was expressed as percent of control treated cells (cells transfected as above but without addition of TNF-a). All of the TSS of CIDEA. The shortest deletion fragment (À46/ experiments described in this figure were repeated at least three times with similar þ 2) displayed a significantly lower transcriptional activity results. Different groups were compared using Mann–Whitney U test. *Po0.05. compared with the longer fragments, indicating that basal activity of CIDEA is confined to a 97-bp-long sequence had no effect on nuclear translocation of NF-kB, adipocyte upstream of the TSS. We therefore suggest that the core survival or differentiation.11 promoter for human CIDEA is found À97 to þ 2 relative to the TSS and that the region À97/À46 is critical for transcriptional activity of the gene. In addition, we found Discussion that the region between À325 and À97 was more transcriptionally active than the region À1125 to À325 indicating In this study we have characterized the 50 flanking region of that transcription activating elements are present in the human CIDEA in adipocytes. We were able to define the TSS more proximal portion, whereas repressing elements may be

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1386 located in the more distal part of the CIDEA promoter. During liver.7 We therefore analyzed the putative human promoter the preparation of this manuscript Li et al.15 published a study CIDEA sequence for possible PPRE elements and tested the on the tissue-specific expression of human CIDEA. Although effect of the PPARg agonist BRL49653 on murine adipocytes the focus in that work is different than that in our present transfected with the CIDEA promoter. By bioinformatic study, Li et al., describe a core promoter of human CIDEA that analysis, we could not detect any PPREs in the human corresponds to the one we described herein. sequence. Moreover, the PPARg agonist BRL49653 did not The murine CIDEA promoter was recently described in affect the transcriptional activity in 3T3-L1 cells transfected liver cells7 and we therefore compared the 50 flanking region with the human promoter. Therefore, at least the part of the of the human CIDEA with the murine promoter. Interest- human promoter studied here, does not seem to be directly ingly, the murine sequence contains a long (approx 745-bp) regulated by PPARs. These differences between the murine 50UTR from the TSS to the ATG, whereas the corresponding and human CIDEA promoter could account for the species- human sequence is only 14-bp. Shift of the TSS to a more 30 specific expression pattern of CIDEA, where the gene in end of the gene could indicate a species-specific change in mice, in contrast to humans, is predominantly expressed in gene regulation. It could be speculated that transcription brown but not in white adipose tissue. factor binding sites close to the translational start site are Finally, we studied the effects of TNF-a on CIDEA important for CIDEA regulation in human white adipocytes. expression. TNF-a suppressed the activity of the luciferase These could shift the TSS closer to translation, leaving a short reporter driven by the CIDEA promoter in both murine and 50UTR. Alternatively, the length of the 50UTR could influence human adipocytes and we therefore conclude that TNF-a mRNA stability, which could be another important directly controls CIDEA transcription. In our system, TNF-a mechanism of CIDEA regulation. regulation of transcription was confined to the region À244/ CpG island are regions in the genome with a high À123 upstream of the TSS This region contains a consensus frequency of cytosine-guanine (CG) dinucleotides. These sequence for NF-kB(À163/À151) a transcription factor regions typically occur at or near TSS of genes. Methylation regulated by TNF-a. Our EMSA experiments demonstrate of the cytosine in CG dinucleotides represses transcription that TNF-a induces protein binding to the probe À202/À151 and CpG islands are therefore implicated in the regulation of encompassing the NF-kB site. We tested two commercially gene expression. We found ECRs in the human as well as in available antibodies for NF-kB proteins (p50 and p65), but the murine promoter that overlapped with CpG islands were unable to detect any super shifts on the EMSAs (data suggesting that these regions are of importance in the not shown). It is however still possible that the DNA–protein regulation of CIDEA. In the recent study by Li et al.15 they complex is composed of other members of the NF-kB family. demonstrate that the ubiquitously expressed transcription This would be in line with the finding that protein binding factors Sp1 and Sp3 as well as DNA methylation in a CpG to probe À202/À151 is abolished with a specific NF-kB island is important for differential CIDEA expression. This inhibitor, WP. WP is a well characterized NBD peptide that CpG island spans the 50 upstream region as well as parts of has been shown to selectively inhibit NF-kB activation exon 1 of the human CIDEA and corresponds to the CpG induced by proinflammatory cytokines in several in vitro island we found in our human sequence. and in vivo systems.12,16,17 Furthermore, when the NF-kB site CIDEA expression in human adipose tissue correlates is mutated, the inhibitory effect of TNF-a on CIDEA inversely with different clinical parameters of insulin promoter activity is attenuated. Taken together these data resistance.4 It is therefore possible that genetic differences suggest that TNF-a, at least in part, mediates it’s action on in the transcriptional regulation of this gene may be of CIDEA transcriptional regulation through this (and possibly importance for weight regulation and/or insulin sensitivity. other) NF-kB binding site(s). Nevertheless, our results do not A set of SNPs in the 50 flanking region of the CIDEA sequence preclude that yet other transcription factors may be involved were recently described and could putatively influence in the negative regulation of CIDEA by TNF-a.Itis transcriptional regulation of CIDEA. However, when analy- interesting to note that TNF-a has previously been shown zing the reporter gene activity of the three most common to regulate CIDEA expression through the c-Jun N-terminal haplotypes we could not detect any significant difference, kinase (JNK).4 JNK phosphorylates and activates homodi- suggesting that the SNPs analyzed in this context do not play mers or heterodimers of c-jun and/or c-fos proteins termed any major part in transcriptional regulation. Admittedly, we AP-1 which act as transcription factors binding to AP-1 cannot exclude the possibility that the promoter SNPs might regulatory elements in the promoter regions of several be of importance in vivo when linked to other coding as well mammalian genes.18 Although we could not detect any AP- as noncoding polymorphisms. It is also possible that the 1 consensus sequences in the human CIDEA promoter using effect of the different haplotypes on the CIDEA promoter is MatInspector, another software (AliBaba 2.1, TRANSFAC) too small to be detected in our assays. predicted one putative AP-1 site at position À270/À258. The murine liver CIDEA promoter contains a set of PPAR However, mutation or deletion of this site did not affect response elements (PPREs) critical for transcriptional transcriptional regulation or modulation by TNF-a (data not regulation.7 Moreover, PPARg is essential for adipocyte shown), suggesting that JNK may either affect CIDEA differentiation and regulates CIDEA expression in murine transcription through indirect mechanisms probably

International Journal of Obesity Transcriptional control of human CIDEA AT Pettersson et al 1387 involving other transcription factors or influence CIDEA energy-restricted diets in obese women. Am J Clin Nutr 2005; 81: gene regulation by chromatin remodeling. 1275–1285. In conclusion, we have identified the TSS for human 6 Dahlman I, Kaaman M, Jiao H, Kere J, Laakso M, Arner P. The CIDEA gene V115F polymorphism is associated with obesity in CIDEA and the minimal promoter needed for transcription. Swedish subjects. Diabetes 2005; 54: 3032–3034. Furthermore, we demonstrate that three common haplo- 7 Viswakarma N, Yu S, Naik S, Kashireddy P, Matsumoto K, types in the CIDEA promoter appear to be of little Sarkar J et al. Transcriptional regulation of Cidea, mito- importance for transcriptional activity of the gene, at least chondrial cell death-inducing DNA fragmentation factor alpha-like effector A, in mouse liver by peroxisome in vitro. Finally, we have also defined a region in the CIDEA proliferator-activated receptor alpha and gamma. J Biol Chem promoter regulated by TNF-a, which most probably involves 2007; 282: 18613–18624. NF-kB activation. These results are an important basis for 8 Dicker A, Le Blanc K, Astrom G, van Harmelen V, Gotherstrom C, Blomqvist L et al. Functional studies of mesenchymal stem cells further studies of CIDEA in human adipocytes and may also derived from adult human adipose tissue. Exp Cell Res 2005; 308: enable the development of substances that would increase 283–290. CIDEA expression, which could be of clinical importance in 9 van Harmelen V, Skurk T, Hauner H. Primary culture and the treatment of a number of metabolic disorders. differentiation of human adipocyte precursor cells. Methods Mol Med 2005; 107: 125–135. 10 Shimizu M, Blaak EE, Lonnqvist F, Gafvels ME, Arner P. Agonist and antagonist properties of beta 3-adrenoceptors in human Acknowledgements omental and mouse 3T3-L1 adipocytes. Pharmacol Toxicol 1996; 78: 254–263. 11 Laurencikiene J, van Harmelen V, Arvidsson Nordstrom E, Dicker We thank Gaby A˚ stro¨m for excellent technical assistance. A, Blomqvist L, Naslund E et al. NF-{kappa}B is important for TNF- This work was supported by grants from the Swedish {alpha}-induced lipolysis in human adipocytes. J Lipid Res 2007; Research Council, Swedish Heart and Lung Foundation, 48: 1069–1077. 12 May MJ, D’Acquisto F, Madge LA, Glockner J, Pober JS, Ghosh S. Swedish Diabetes Association, The memorial foundation of Selective inhibition of NF-kappaB activation by a peptide that Sigurd and Elsa Golje, Storstockholms Diabetesfo¨rening, blocks the interaction of NEMO with the IkappaB kinase Torsten och Ragnar So¨derbergs stiftelse Novo Nordisk complex. Science 2000; 289: 1550–1554. Foundation, the European Union (HEPADIP, LSHM-CT- 13 Ryden M, Dicker A, van Harmelen V, Hauner H, Brunnberg M, Perbeck L et al. Mapping of early signaling events in tumor 2005-018734), the Swedish Medical Association and the necrosis factor-alpha-mediated lipolysis in human fat cells. J Biol Swedish Diabetes Foundation. Chem 2002; 277: 1085–1091. 14 Prins JB, Niesler CU, Winterford CM, Bright NA, Siddle K, O’Rahilly S et al. Tumor necrosis factor-alpha induces apoptosis of human adipose cells. Diabetes 1997; 46: 1939–1944. References 15 Li D, Da L, Tang H, Li T, Zhao M. CpG methylation plays a vital role in determining tissue- and cell-specific expression of the 1 Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity human cell-death-inducing DFF45-like effector A gene through to insulin resistance and type 2 diabetes. Nature 2006; 444: the regulation of Sp1/Sp3 binding. Nucleic Acids Res 2008; 36: 840–846. 330–341. 2 Van Gaal LF, Mertens IL, De Block CE. Mechanisms linking 16 Chapoval SP, Al-Garawi A, Lora JM, Strickland I, Ma B, obesity with cardiovascular disease. Nature 2006; 444: 875–880. Lee PJ et al. Inhibition of NF-kappaB activation reduces 3 Langin D, Arner P. Importance of TNFalpha and neutral lipases in the tissue effects of transgenic IL-13. J Immunol 2007; 179: human adipose tissue lipolysis. Trends Endocrinol Metab 2006; 17: 7030–7041. 314–320. 17 Tas SW, Vervoordeldonk MJ, Hajji N, May MJ, Ghosh S, Tak PP. 4 Nordstrom EA, Ryden M, Backlund EC, Dahlman I, Kaaman M, Local treatment with the selective IkappaB kinase beta inhibitor Blomqvist L et al. A human-specific role of cell death-inducing NEMO-binding domain peptide ameliorates synovial inflamma- DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) tion. Arthritis Res Ther 2006; 8: R86. in adipocyte lipolysis and obesity. Diabetes 2005; 54: 1726–1734. 18 Chinenov Y, Kerppola TK. Close encounters of many kinds: 5 Dahlman I, Linder K, Arvidsson Nordstrom E, Andersson I, Liden Fos-Jun interactions that mediate transcription regulatory speci- J, Verdich C et al. Changes in adipose tissue gene expression with ficity. Oncogene 2001; 20: 2438–2452.

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