eicosapentaenoic acid demethylates a single cpg … · eicosapentaenoic acid demethylates a single...
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EICOSAPENTAENOIC ACID DEMETHYLATES A SINGLE CpG THAT MEDIATES THE EXPRESSION OF TUMOR SUPPRESSOR CCAAT/ENHANCER-BINDING PROTEIN DELTA
IN U937 LEUKEMIA CELLS Veronica Ceccarelli1, Serena Racanicchi2, Maria Paola Martelli2, Giuseppe Nocentini2, Katia Fettucciari2, Carlo Riccardi2, Pierfrancesco Marconi2, Paolo Di Nardo3, Francesco Grignani2,
Luciano Binaglia1 and Alba Vecchini1 Department of Experimental Medicine and Biochemical Sciences1,
Department of Clinical and Experimental Medicine2, University of Perugia, Perugia 06126, Italy
Department of Internal Medicine3, University “Tor Vergata” Rome, Italy Running head: EPA induces tumor suppressor C/EBP� expression
Address correspondence to: Alba Vecchini, Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry, Faculty of Medicine, University of Perugia, Via del Giochetto 3, 06122 Perugia, Italy +39-075-585-7425; Fax : +39-075-585-7428; E-mail: [email protected]
Polyunsaturated fatty acids (PUFA) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, alpha-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFA affected proliferation, eicosapentaenoic acid (EPA) being the most potent on cell cycle progression. EPA enhanced the expression of the myeloid-specific transcription factors, CCAAT/enhancer-binding proteins (C/EBPββββ, C/EBPδδδδ), PU.1, and c-jun, resulting in increased expression of the monocyte-lineage specific target gene, the macrophage colony-stimulating factor (M-CSF) receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of M-CSF receptor promoter, determinant for the expression. We demonstrated that C/EBPββββ and C/EBPδδδδ bind the same response element as heterodimer. We focused on the enhanced expression of C/EBPδδδδ that has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the -370/-20 CpG island on C/EBPδδδδ promoter region, we found a site-specific CpG demethylation that was determinant for the binding activity of Sp1, an essential factor for C/EBPδδδδ gene basal expression. Our results evidence a new role of PUFA in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor
C/EBPδδδδ is controlled by the methylation state of a site-specific CpG dinucleotide.
Increasing evidence from animal and in
vitro studies indicates that fatty acids, especially the long-chain polyunsaturated fatty acids (PUFA) affect carcinogenesis (1). n-3 PUFA inhibit the growth of tumor cells both in vivo and in vitro (2, 3), decrease metastasis and cachexia (4, 5) increase cytotoxic effects of some chemotherapeutic agents (6), athough the results are not always consistent (7, 8). In addition, n-3 PUFA reduce cell proliferation and induce differentiation and apoptosis in hepatocarcinoma and leukaemia cells (9-12). Although not completely known, several molecular mechanisms whereby n-3 PUFA may modify the carcinogenic process have been proposed. These include alteration in cell membrane composition and function, modulation of mitochondrial calcium homeostasis, suppression of biosynthesis of proinflammatory molecules, influence on signal transduction, alteration of hormone-stimulated cell growth, inhibition of angiogenic mediators, production of free radicals and reactive oxygen species, and influence on transcription factors activity and gene expression (2, 13-17).
In hepatocarcinoma cells, n-3 PUFA modulate the expression of CCAAT enhancer binding proteins (C/EBPs) transcription factors (18, 19). C/EBPs (�, �, �, �, � and �) represent a family of master regulators that play an essential role in controlling cell proliferation and differentiation processes of several cell types, including myeloid cells (20). Members of C/EBP family are structurally related, each consisting of an N-terminal transactivating region, a central basic DNA-binding domain, and a C-terminal leucine zipper motif for dimerization (20).
http://www.jbc.org/cgi/doi/10.1074/jbc.M111.253609The latest version is at JBC Papers in Press. Published on June 9, 2011 as Manuscript M111.253609
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
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Within hematopoiesis, C/EBPα, C/EBPβ, and C/EBPδ are predominantely expressed in the granulocyte and monocyte lineages; whereas C/EBP� is found in the middle-to-later stages of differentiation of granulocytes and T cells. (21-25). The most compelling evidence for a crucial role of the C/EBPs in myeloid cell differentiation and maturation has come from studies on knockout mice. C/EBPα-deficient mice fail to undergo myeloid differentiation beyond myeloblast stage and, therefore, lack mature neutrophils (26), whereas the phenotype of C/EBPβ-deficient mice indicates a potential role in the activation and/or differentiation of macrophages (27). Moreover, overexpression of C/EBPα, C/EBPβ, C/EBPδ, and C/EBP� induce granulocyte differentiation in myeloblastic cell lines (28), suggesting that in myeloid cells C/EBP family members can compensate in vivo for the lack of one of the other C/EBP proteins.
Besides C/EBPs, other transcription factors and co-activators contribute to myeloid cell fate (29). First of all, PU.1 drives the transcription of monocyte-specific genes, including the macrophage colony-stimulating factor (M-CSF) receptor (30, 31). PU.1 and C/EBPs can bind to and activate the M-CSF receptor promoter and their combinatorial activities are essential to mediate the M-CSF receptor expression level (32). In addition, the co-activator partner protein c-jun cooperates with PU.1 (33) and C/EBPs (34) during monocyte differentiation, although able itself to induce partial monocyte differentiation in a variety of myeloid cell lines (35, 36). C-jun does not directly bind to the M-CSF receptor promoter, but enhances the ability of PU.1 to transactivate it (37). Synergism among PU.1, C/EBPs, and c-jun activation is essential to activate monocyte target genes (34). Among these, M-CSF receptor is critical for monocyte cell survival and proliferation, and is activated early during the monocyte differentiation process (38-40).
In the present study we evaluated the effects of fatty acids conditioning of U937 promonocytic cell line on proliferation, cell cycle progression, and differentiation program, related to chain length and number of double bonds. We found that eicosapentaenoic acid (EPA) treatment reduced cell cycle progression, induced the monocyte-specific M-CSF receptor expression by enhancing C/EBPβ, C/EBPδ, PU.1, and c-jun expression. Considering that
C/EBPδ was reported to be a tumor suppressor gene (41, 42) that is silenced by promoter hypermethylation in U937 cells and re-expressed by proximal promoter demethylation (43), we analyzed the same promoter region (-370 to -20) after EPA conditioning of U937 cells. We found a site-specific CpG demethylation that was determinant for the binding activity of Sp1 transcription factor to induce C/EBPδ gene expression.
Experimental Procedures Materials- Stearic acid (18:0; SA), oleic acid (18:1, n-9; OA), linoleic acid (18:2, n-6; LA), α-linolenic acid (18:3, n-3; LNA), arachidonic acid (20:4, n-6; AA), eicosapentenoic acid (20:5, n-3; EPA), dochosaexaenoic acid (22:6, n-3; DHA), bovine serum albumin fraction V (BSA, fatty acid free), and 5-aza-2’-deoxy-cytidine were from Sigma Chemical Co, (St. Luis, MO, U.S.A.). Preparation of albumin-bound fatty acids- A stock solution of each fatty acid (5 or 10 mM) was prepared by diluting the free fatty acid in ethanol and precipitating it with the addition of NaOH (final concentration of 0.25 M). The precipitate was dried under nitrogen, reconstituted with 0.9% (w/v) NaCl, and stirred at room temperature for 10 min with defatted BSA (final concentration at 10% (w/v) in 0.15 M NaCl). Each solution was adjusted to pH 7.4 with NaOH and stored in aliquots at -20°C protected from light under nitrogen. The fatty acid/BSA molar ratio was 3:1 or 6:1. Cell culture- Human promonocytic cell line U937 (CRL-1593.2) obtained from the American Type Culture Collection (Rockville, MP, U.S.A.) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin-streptomycin at 37°C in an humidified incubator aerated with 5% CO2. U937 cells were seeded at a density of 0.3x106 cells per ml for all experiments. Cells were incubated with fatty acid/BSA solutions at the indicated concentrations and times. Flow cytometry analysis- i) Cell cycle and apoptosis. U937 cells were treated with fatty acids for 24 hours (50-200 µM final concentration) and analyzed by flow cytometry as indicated below. After washing, the 200 x g cell pellets were resuspended in 1 ml of hypotonic PI solution (50 µg ml-1 in 0.1% sodium citrate plus 0.1 % Triton X-100, Sigma).
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The samples were placed overnight in the dark at 4°C and PI fluorescence of individual nuclei measured using an EPICS XL-MCLTM flow cytometer (Instrumentation Laboratory, Beckman Coulter, Miami, FL). Analysis of apoptosis was performed as described by Nicoletti et al. (44) and data were processed by an Intercomp computer and analyzed with SYSTEM IITM software (Instrumentation Laboratory, Beckman Coulter). Cell-cycle was analyzed measuring DNA-bound PI fuorescence in the orange-red fuorescence channel (FL2) through a 585/42-nm bandpass filter with linear amplification. Analysis of distribution profiles was performed with ModFit LT software (Verity Software House, Topsham, ME) to determine fractions of the population in each phase of the cell cycle (G0/G1, S, G2/M). At least 15,000 events were collected for each sample. Cells were gated on FL2-Area versus FL2-Width plots to exclude aggregates and debris from analysis (45). ii) Forward and side scatter. Intact U937 cells were recovered after treatment with 100 µM fatty acids, resuspended in 1 ml PBS, and identified by forward and right angle (side) light-scatter using the same flow cytometer equipped with a 15-mW argon-ion laser (488 nm). Cell viability was determined by counting triplicate samples for trypan bleu dye-excluding cells. [3H]Thymidine incorporation assay- Triplicate samples of 1 x 105 U937 cells suspended in 200 µl RPMI 1640 were cultured in the presence or in the absence of 100 µM fatty acids for 24 hours. [3H]thymidine (specific activity 6.7 Ci/mmol) (Amersham International) was added to the cultures at 2.5 µCi per well. After 4 hours incubation, cells were harvested with a multiple suction-filtration apparatus (Mash II) on a fiberglas filter (Bio Whittaker) and counted in a beta counter apparatus (Packard). Real-time PCR- Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer’s guidelines. cDNA was preparated by using Quanti Tect Reverse Transcription Kit (Qiagen). Quantitative gene expression analysis was performed using Mx3000PTM Real-Time PCR System with Brilliant� SYBR� Green QPCR Master Mix (Stratagene) and ROX as reference dye. Quantitative PCR reactions were performed under conditions standardized for each primer. To minimize variability, every time point was investigated with four replicates, and the amplification of three independent treatments was performed. The primers used for real-time qPCR analysis were the followings: C/EBP�for
(5’AAGAACAGCAACGAGTAC-3’), C/EBP�rev (5’-GTCATTGTCACTGGTCAG-3’), C/EBP�for (5’AGCGACGAGTACAAGATC-3’), C/EBP�rev (5’-TGCTCCACCTTCTTCTGC-3’), C/EBP�for (5’-CATGTACGACGAGAG-3’), C/EBP�rev (5’-GTGATTGCTGTTGAAGAGG-3’), PU.1for (5’-GACAGGCAGCAAGAAGAAG-3’), PU.1rev (5’-TTGGACGAGAACTGGAAGG-3’), c-junfor (5’-CTTATGGCTACAGTAACC-3’), c-junrev (5’-TTGCTGGACTGGATTATC-3’), M-CSF receptorfor (5’-GTTCTGCTGCTCCTGCTG-3’), M-CSF receptorrev (5’-CGTTGCTCCTGGCTTCAC-3’). Human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) mRNA was the house-keeping gene. The ∆∆Ct method was used to determine modulation of the gene of interest (46). Immunoblot analysis- U937 cells were cultured for 24 hours in standard conditions or with fatty acid (100 µM f.c.). For 5-aza-2’-deoxy-cytidine treatment, the compound was added every day for 2 and 5 days (1 µM, f.c.). Protein samples from total cell lysates (50 µg) were subjected to electrophoresis on a 12% SDS-polyacrilamide gel, electroblotted onto a nitrocellulose membrane (Protran, Schleicher and Schuell Italia S.r.l., Milan, Italy) and probed using the following antibodies: anti-C/EBPα (14AA) sc-61, anti-C/EBP� (C-19) sc-150, anti-C/EBPδ (C-22) sc-151, anti-c-jun (N) sc-45 (Santa Cruz Biotecnology Inc.), anti-PU.1 (9G7) #2258, and M-CSF receptor #3152 (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were visualized using the ECL assay (Amersham Pharmacia Biotech, Amersham,, U.K.), according to the manufacture’s instruction. An anti-�-tubulin antibody (Sigma-Aldrich) was used to normalize the amount of the samples analyzed. Images were acquired using the VersaDoc Imaging System and signals were quantified using Quantity One software (Bio-Rad, Milan, Italy). Chromatin immunoprecipitation- ChIP assays were performed on U937 cells grown in standard conditions, or with 100 µM OA, or with 100 µM EPA for 24 hours, using the EZ-Chip kit in accordance to the protocol provided by the manufacturer (Millipore-Upstate, USA). Briefly, after cross-linking with 1% formaldehyde and quenching with 125 mM glycine, cells were recovered, resuspended in SDS-lysis buffer in the presence of protease inhibitors, and sonicated
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to obtained 200-1000 bp chromatin fragments. Aliquots of the fragmented chromatin corresponding to about 106 cells were precleared with protein G-agarose. The precleared chromatin (1%) was kept as “input” and the residual was incubated overnight with the following antibodies: anti-C/EBP� (sc-61X), anti-CEBP� (sc-150X), anti-C/EBP� (sc-636X) and anti-PU.1(sc-352X). Five µg of each antibody and rabbit IgG (5 µg) were used. Antibody-DNA complexes were captured by incubation with protein G-agarose, eluted and subjected to cross-linking reversal. The M-CSF receptor sequence containing the C/EBPs and PU.1 binding sites was amplified using Mx3000PTM Real-Time PCR System with Brilliant� SYBR� Green QPCR Master Mix. The sequences of the primers used for ChIP were the followings: forward (-87/-66) 5’-GACTGCGACCCCTCCCTCTTG-3’, and reverse (+15/+36) 5’-CCTCCTCCTTGGGCTGATCCTC. The relative amount of immunoprecipitated DNA fragment (123 bp) was determined based on the threshold cycle (Ct) for each PCR product. Data were quantitatively analyzed according to the formula 2-�[C(IP)-C(input)]-2-�[C(control IgG)-C(input)] (47). DNA isolation and quantitative DNA methylation analysis of C/EBP� CpG island- Genomic DNA from un-supplemented U937 cells or U937 grown for 24 hours with 100 µM OA, or 100 µM EPA was extracted using FlexiGene DNA Kit in accordance to the protocol instructions (Qiagen). DNA methylation levels were quantified using Methyl-Profiler qPCR Primer Assay for human C/EBP� (MePH28341-1A) (Sabiosciences-Qiagen) in accordance to the protocol provided by the manufacturer. Primers are designed by optimized computer algorithm to insures that the amplicon contains cutting sites for both methyl sensitive and methyl dependent enzymes and are specifically designed for analyzing the DNA methylation status of CpG islands using restriction enzyme digestion (DNA Methylation Enzyme kit MeA-03 Sabiosciences–Qiagen) followed by SYBER green–based-real time PCR detection. Briefly, each genomic DNA was subjected to four separate treatments according to the enclosed instructions. i) Mock Digest (Mo). No enzymes were added in this reaction. The product of the mock digested (Mo) represented the total amount of input DNA for real-time PCR detection. ii) Methylation Sensitive Digest (Ms). Cleavage with a methylation-sensitive enzyme, which digested
unmethylated. The remaining hypermethylated DNA was detected by real-time PCR. iii) Methylation Dependent Digest (Md). Cleavage with a methylation-dependent enzyme, which digested methylated DNA. The remaining unmethylated DNA was detected by real-time PCR. iv) Double Digested (Msd). Both enzymes were added in the double digested, and all DNA molecules (both methylated and unmethylated) were digested. This reaction measures the background and the fraction of input DNA refractory to enzyme digestion. The four mixtures were incubated at 37°C overnight. The enzymes were inactivated at 65°C for 20 min. The resulting DNA was stored at -20°C or utilized for real-time PCR using as described above. The specific RT-PCR program was: 95°C 10 min; 40 cycles of (97°C, 15 s; 72°C, 60 s) as indicated in the manual instructions. The relative amount of each DNA fraction (methylated and unmethylated) was calculated using a standard �Ct method, normalizing the amount of DNA in each digestion against the total amount of input DNA in the Mo digest. The amount of hypermethylated target DNA copies (CHM) is defined as (2-�Ct(Ms-Mo)-CR)/(1-CR), where CR represents the amount of target DNA copies that are resistant to enzymes digestion and is defined as: 2-�Ct(Msd-Mo). The amount of unmethylated DNA (CUM) can be determined as (2- �Ct(Md-Mo)-CR)/(1-CR). Bisulfite modification of genomic DNA and Sequencing- Genomic DNA from un-supplemented U937 cells or U937 grown for 24 hours with 100 µM OA, or 100 µM EPA was extracted as described above. Bisulfite modification of genomic DNA was done as described (48, 49). Briefly, 1 µg of genomic DNA (50 µl volume) was denatured by using mild heat at alkaline pH by adding 3.5 µl of 3 M NaOH and incubating for 10 min at 37°C. Immediately, 10 µl of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (Sigma) were added and incubated for 12-16 h at 50°C. Bisulfite-treated DNA was purified and eluted in 50 µl of H2O. The conversion of uracil was completed by alkaline desulfonation, by adding 5 µl of 3 M NaOH and incubating at 37°C. The treated DNA was purified using Qiaquick gel extraction kit and eluted in 30 µl of H2O. Sequencing primers for bisulfite-modified DNA were designed by using the online program MethPrimer (available at the University of California San Diego Web site). Bisulfite-modified DNA was amplified using sequencing
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primers for the C/EBP� promoter. The primers for the first PCR were: forward primer, 5’-GATTTTATTTTTAATTTYGAGGAGY-3’ (Y=C/T); reverse primer, 5’-AAACTATCACCTCRCTAAACCCAACCC-3’ (R=C/T). Following the initial amplification, an aliquot of the initial PCR products was used as template DNA in a nested PCR. The primers for the nested PCR were: forward, 5’-GATTTTATTTTTAATTTYGAGGAGY-3’(Y=C/T); reverse primer, 5’-CCTTTTCTAACCCCRACTAARTA-3’ (R=C/T). Nested PCR conditions were as follows: 95°C for 5 min; 35 cycles of (94°C for 30 s, 50°C for 30 s, 72°C for 60 s), followed by 10 min at 72°C. The amplified products were confirmed by electrophoresis on agarose gel. The nested 350 bp PCR products (-370/-20) were sub-cloned into pCR 2.1 TA cloning vector (Invitrogen, Carlsbad CA). Single clones were selected and cultured, and plasmid DNA was isolated using GeneElute plasmid miniprep kit (Sigma) and sequenced using T7 primer at the Genechron-Ylichron srl Laboratory (Rome). Eight-ten clones were sequenced for each sample. Electrophoretic mobility shift assays (EMSAs) and antibody interference assays- Nuclear extracts from U937 cells were prepared and electrophoretic mobility shift/antibody interference assays were performed as previously described (50). Complementary pairs of site-specific methylated and unmethylated oligonucleotides were from IDT-TEMA Ricerca (Bologna, Italy). Oligonucleotides were annealed by boiling for 5 min at 95°C, followed by cooling, and end-labeled with [�-32P]-dATP (Amersham Biosciences Corp- Piscataway, NJ) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Labeled fragments were separated from residual [�-32P]-dATP using Quick Spin columns (Roche Molecular Biochemcals) according to the manufacturer’s instructions. The upper strand C/EBP� promoter oligonucleotide sequence used was 5’-CGGGCGGGGCGTGCACGTCAGCCGGGGCTAG-3’ (underlined C corresponds to the C demethylated after EPA treatment). Binding reactions were carried out by mixing 20 µg of nuclear extract with 4 µl of 5 x binding buffer (40 mM HEPES, pH 8.0, 200 mM KCl, 0.4 mM EDTA, 20 mM MgCl2, 40% glycerol, 20 mM dithiothreitol, 0.05% bromophenol blue) in a total volume of 18 µl and incubating at 4°C for 1 h. Approximately 90,000 cpm (2 µl of ~ 0.5
ng/µl) of �-32P-dATP labeled probe were added per reaction and incubated for 15 min at room temperature. The reaction mixtures were loaded onto a 6% polyacrilamide gel (0.75 mm thick) and separated for 3 h at 200 volts in 0.5 x TBE. The gel was dried for 30 min under vacuum and exposed to autoradiograph film or using an Instant Imager autoradiography system (Packard, BioScience Co.,IL). For antibody interference assays, 3.0 µl of anti-Sp1 antibody (Santa Cruz Biotechnology, Santa Cruz CA sc-59) was incubated with the nuclear extract and binding buffer for 1 h at 4°C in a total volume of 18 µl. After addition of the probe, subsequent steps were identical to those for the band shift assays. Statistical analyses- All average results are presented as mean ± S.D. One-way or two-way (where appropriate) ANOVA with Bonferroni’s post test were used. Values were considered significant at P<0.05.
RESULTS
Effects of PUFA on cell viability, proliferation and cellular morphology of U937 cells. Variable effects of PUFA on proliferation, differentiation, and apoptosis in leukemia cells have been reported, in relation to cell line and fatty acid concentration (9, 11, 12). To study the effects of fatty acids on the percentage distribution in the various phases of the cell cycle, U937 cells were analyzed by flow-cytometry after incubation with fatty acids of increasing carbon chain length and double bond number. Twenty-four hours treatment with LNA, AA, EPA, and DHA increased significantly the percentage in Go-G1 phase in a concentration-dependent manner, compared to untreated cells. EPA exhibited the highest effect when compared with LNA, AA and DHA (Fig.1A). Contrarily, SA, OA, and LA had no effect at any of the concentrations studied (Fig.1A). Untreated U937 cells were 43.4% in Go-G1 phase, 44.5% in S phase, and 11.9% in G2-M phase (Fig 1B). After 24 hours supplementation with LNA, AA, EPA, and DHA (100 µM f.c.), the percentage of cells in Go-G1 phase increased, (maximal effect 64% with EPA) paralleled by a decrease� in the percentage of cells in S and G2-M phases (33% and 2.5%, respectively, with EPA). Only minor changes were observed in SA, OA, and LA treated cells in the same experimental conditions (Fig 1B). No fatty acid treatment, except SA (51), induced U937 apoptosis up to 200 µM concentration for 24, 48, and 72 hours (not
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shown). When cells were exposed to 100 µM LNA, AA, EPA, and DHA for 24 hours a significant reduction in the cells number was observed, as compared with untreated cells (P<0.001), while SA, OA, and LA had no significant effect on cell proliferation (Fig 1C). The increased number of cells in Go-G1 phase and the decrease of total cell number induced by LNA, AA, EPA, and DHA treatments were in agreement with the reduced DNA synthesis. Indeed, LNA, AA, EPA, and DHA inhibited significantly [3H] thymidine incorporation (P<0.001). As expected, no effect on [3H] thymidine incorporation was found in SA, OA, and LA treatments (Fig 1D). EPA treatment produced the highest effect on reduction of cell cycle progression.
Flow-cytometry analyses, measuring forward and side scatter, was used to obtain information about morphology of U937 leukemia cells after fatty acid treatments. Analysis of the cells showed a significant increase both in the forward and in the side scatter of the U937 cells following 24 hours treatment with 100 µM AA, EPA, and DHA, indicating an increase in cell size and granularity. SA, OA, LA, and LNA addition resulted in lower increase in forward and side scatter (Fig. 1E)
EPA increases C/EBPs, PU.1, and c-jun expression. We examined whether the effects on cell viability, proliferation, and cellular morphology induced by PUFA conditioning could be related to enhanced expression of lineages specific transcription factors involved in cell cycle progression and differentiation process of myeloid cell lines. Fatty acids treatments (100 µM) had no effect on any of C/EBPα isoforms content (Fig.2A). LNA, AA, EPA, and DHA increased the protein content of C/EBPβ, C/EBPδ, PU.1, and c-jun, whereas SA, OA and LA had lesser or no effect and were unable to induce simultaneously all four transcription factors (Fig. 2A). The enhanced protein expression levels of C/EBPβ, C/EBPδ, c-jun, and PU.1 let us to suppose that U937 cells undergo the early phase of myeloid differentiation process. Indeed, C/EBPβ, C/EBPδ, c-jun, and PU.1 are involved in the transcriptional control of granulocyte and monocyte development (25).
To determine whether PUFA treatment affected C/EBPs, PU.1, and c-jun protein levels due to transcriptional events, RT-PCR was performed. We selected OA and EPA as
potential inactive and active inducers, respectively, of C/EBPα, C/EBPβ, C/EBPδ, PU.1, and c-jun mRNA levels. To assess the expression kinetic profile, we evaluated mRNA content in un-supplemented U937 cells and after 1 hour, 3 hours, and 24 hours treatment with 100 µM OA or EPA. A simultaneous and significant increase of C/EBP�, C/EBP�, PU.1, and c-jun mRNA levels was observed after 3 hours of EPA treatment, that further increased up to 24 hours (Fig. 2B). On the contrary, OA did not induce any significant change. In agreement with the unchanged protein content, OA and EPA conditioning had no effect on C/EBP� mRNA levels (Fig. 2B).
EPA enhances M-CSF receptor expression. The increased C/EBP�, C/EBP�, PU.1, and c-jun mRNA levels in response to EPA suggested a potential transcriptional effect on their target genes. M-CSF receptor is expressed very early during the monocyte differentiation process, PU.1, C/EBPs, and co-activator partner protein c-jun being their transcriptional activators (30, 32). The M-CSF receptor mRNA levels were evaluated in U937 cells after 1 hour, 3 hours, and 24 hours treatment with 100 µM OA or EPA. Similarly to C/EBP�, C/EBP�, PU.1, and c-jun (Fig. 2B), M-CSF receptor mRNA levels increased significantly at 3 and 24 hours EPA treatment (p < 0.001 versus control U937 untreated cell) (Fig. 3A), whereas OA had no effect. M-CSF receptor protein was not or barely detectable in U937 untreated cell line. Twenty four hours conditioning with 100 µM SA, OA, and LA did not produce significant increase. As expected, EPA, DHA, LNA, or AA addition resulted in evident protein increase (Fig. 3B).
C/EBP�, C/E�BP�, and PU.1 bind and activate M-CSF receptor promoter after EPA treatment. To investigate whether C/EBPs and PU.1 transcription factors induced by EPA were able to bind their consensus sequences on M-CSF receptor promoter (30, 32), ChIP analysis were performed in U937 cell after 24 hours of OA and EPA treatments, using C/EBPs and PU.1 antibodies. RT-PCR amplification of the -87/+36 (123 bp) promoter region, containing the elements under investigation (30, 32), was performed in the immunoprecipitated DNA. No amplification product was found with normal rabbit IgG as negative control for the antibodies utilized. The results of ChIP experiments demonstrated a significant ability of C/EBP�, C/EBP�, and PU.1 to bind the M-CSF receptor
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consensus sequences after EPA treatment, whereas C/EBP� was ineffective (Fig. 3C). No significant effect was exerted by OA. These results suggest that both C/EBP� and C/EBP� specifically bind to the same C/EBP consensus sequence, most likely as C/EBP�/C/EBP� heterodimer. As a consequence, their enhanced expression following EPA treatment is particularly relevant.
EPA induces C/EBP� expression acting as site-specific demethylating agent. C/EBP� is silenced by hypermethylation in U937 cells and the demethylating agent 5-aza-2’ -deoxy-cytidine is able to induce its expression (43). We verified the effect of 5-aza-2’ -deoxy-cytidine on C/EBP� protein levels in our experimental conditions and compared it with EPA. Western blot analysis of U937cells after 2 and 5 days treatment with 1 µM 5-aza-2’ -deoxy-cytidine, as well as after 24 hours 100 µM EPA treatment, showed increased protein levels (Fig. 4A). We hypothesized that EPA could exert its effets on protein expression by acting as a demethylating agent.
The analysis of C/EBPδ gene (-3000/+1269) using the EMBOSS (European Molecular Biology Open Software Suite) or MethPrimer online software programs retrieved six putative islands. To verify wether EPA treatment demethylates C/EBP� CpG islands, the percent content of CpG DNA methylation was quantified using Methyl-Profiler qPCR Primer Assay. Quantitative RT-PCR indicated that the amount of C/EBP� hypermethylated DNA copies decreased significantly (p < 0.001) after EPA conditioning compared to OA treatment or untreated cells (Fig. 4B). The 5’ -upstream promoter region containing a CpG island with high CG content and determinant for C/EBP� gene re-expression (43) (Fig. 4C) was analyzed in detail. We performed bisulfite sequencing of this region and found high methylation degree, in agreement with the low C/EBP� protein level. EPA treatment induced a site-specific CpG demethylation in all the sequenced clones (Fig. 4D). The same was not found for OA treatment, which induced random and low level CpG demethylation. Indeed the specific CpG dinucleotide demethylated after EPA treatment is located in a critical region of C/EBP� proximal promoter between the TATA box and a Sp1 binding site (Fig. 4C) and may be functional to C/EBP� expression.
To evaluate the influence of this specific CpG methylation on Sp1 binding activity to the C/EBP� gene promoter, the Sp1 site was
investigated by comparative electromobility shift assay (EMSA). C/EBP� gene promoter-specific oligos containing unmethylated or methylated specific CpG (Fig. 5) were incubated with U937 nuclear extracts and the binding activity was compared. Figure 5 shows that the signal of the shifted band is stronger with the unmethylated, compared to the methylated probe. Incubation of the EMSA reactions with an anti-Sp1 antibody supershifted the unmethylated probe, consistent with Sp1 specific binding to the probe. Overall, these experiments indicate that demethylation of a specific CpG can increase Sp1 binding to C/EBP� promoter.
DISCUSSION
In this study we evaluated the effects of PUFA, relative to saturated and monounsaturated fatty acids, on U937 promyelocytic leukemia cells. PUFA inhibited DNA synthesis and cell cycle progression, and promoted changes in cell morphology by increasing size and granularity of U937 cells. At the same time, M-CSF receptor expression increased. M-CSF receptor, specifically induced in the early phase of monocyte differentiation commitment, is expressed on the monocyte-macrophage cell lineage and, as such, it is a useful marker to discriminate between monocyte and granulocyte progenitor cells and their differentiated progeny (39). Expression of the M-CSF receptor gene is under stringent control of both extracellular and intracellular stimuli, and appears to occur primarily at the transcriptional level (40). M-CSF receptor mRNA levels are under the control of monocyte lineage-specific transcription factors, such as PU.1 and C/EBPs, being the activity and specificity of the M-CSF receptor promoter mediated by a small DNA fragment containing binding sites for PU.1 and C/EBPs (30, 32). All three CEBPα, C/EBPβ, and C/EBPδ can bind to and activate the M-CSF receptor promoter, a function that has been ascribed to their highly similar bZIP domains (52). Moreover, studies on differences in the binding affinities of recombinant C/EBPs to the M-CSF receptor promoter demonstrated that C/EBPβ binding is relatively weaker than CEBPα and C/EBPδ binding (32).
We found that EPA treatment induced the C/EBPβ and C/EBPδ expression and promoted C/EBPβ/C/EBPδ heterodimer binding to the same response element on the M-CSF receptor
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promoter, resulting in enhanced M-CSF expression. In addition, EPA treatment induced an increase on c-jun mRNA and protein levels. This last result is in agreement with previous data indicating that c-jun and M-CSF receptor mRNA increase during monocyte differentiation of U937 cells (53). Interestingly, the M-CSF receptor promoter has no consensus binding site for c-jun transcription factor (37). As a consequence, c-jun does not directly bind to M-CSF receptor promoter, but it associates with PU.1 and C/EBPβ via its basic domain (34, 37). Moreover, coactivator c-jun recruitment by C/EBPβ to DNA facilitates RNA polymerase II recruitment (34). C-jun, PU.1, and C/EBPβ have been shown to physically interact with each other and enhance the transcription of monocyte-specific genes via binding to their respective sites on DNA (33, 54). As it concerns c-jun expression, it has been shown to be differentially upregulated during monocyte, but not granulocyte, differentiation of myeloid cell lines, implying a specific role for c-jun expression in monocyte development (55). Our results confirm that there is not a single master myeloid transcription factor that alone governs myeloid lineage commitment, but multiple transcription factors work cooperatively and coordinately to regulate both temporal and lineage-specific genes. In this light, the simultaneous effects induced by EPA treatment on myeloid lineage-specific PU.1, C/EBPβ, C/EBPδ, and c-jun gene expression suggest a complex regulatory network.
Among PU.1, C/EBPβ, C/EBPδ, and c-jun transcription factors, we focused on the induction of the tumor suppressor C/EBPδ. Indeed, C/EBPδ is specifically inactivated by promoter CpG island hypermethylation in U937, as well as in other leukemia cell lines and in acute myeloid leukemia, being the treatment with a demethylating agent associated with gene re-expression (43). DNA hypermethylation is a common mechanism for tumor suppressor gene inactivation in haematopoietic malignancies (56). Methylation of promoter CpG islands is associated with a closed chromatin structure and transcriptional silencing of the associated genes. Aberrant methylation of normally unmethylated promoter 5’ -CpG-rich areas is the most commonly studied epigenetic mechanism associated with the transcriptional silencing of known and candidate tumor suppressor genes (57). The pattern of promoter methylation found
in haematopoietic malignancies can be considered to be aberrant and a cancer specific phenomenon, with disease-specific methylation pattern of key CpG islands found for particular genes (57).
In the present study, both 5-aza-2’ -deoxy-cytidine and EPA conditioning induced C/EBPδ protein expression (Ref. 43 and Fig.4A). C/EBPδ enhanced expression is ascribed to a demethylation process of a site-specific CpG downstream the Sp1 site in the C/EBPδ proximal promoter (Fig. 4C) required for C/EBPδ basal transcriptional activation (58). Indeed, methylatation of this specific CpG impairs the Sp1 binding to its consensus sequence (Fig 5). Our results are consistent with the site-specific CpG demethylation of C/EBPδ proximal promoter in primary breast cancer (59). Moreover, evidence has been reported demonstrating gene promoter site-specific methylation as a mechanism of tumor suppressor gene silencing in various types of cancer cells (60, 61). A single CpG demethylation appears to be the molecular event associated with a putative anti-tumor gene expression in prostate carcinogenesis (62). On this basis, we can assume that the CpG demethylation events are not all equal within a CpG island. Although it is generally accepted that high concentrations of demethylating agents induce the maximal gene reactivation (63), paradoxically, global demethylation may result in increased tumorigenicity (64). Moreover, a strong demethylating action prevents ascertaining wether any of the CpG dinucleotides exhibits differential susceptibility towards methylation-demethylation process and hinders from locating the critical regulatory sequences within CpG islands. We found a CpG dinucleotide in C/EBPδ proximal promoter as a key element that was proven useful in identifying hypersensitive regions essential for gene regulation. This site may serve as dynamic switch to activate C/EBPδ gene through a possible chromatin conformation change.
Surprisingly, even 3 hours EPA treatment was able to induce significantly C/EBPβ, C/EBPδ, c-jun, and Μ-CSF receptor mRNA level increase (Figs. 2B and 3A). These results let us to suppose that the site-specific C/EBPδ promoter demethylation occurred within 3 hours, suggesting the involvement of some active demethylation mechanism(s) occurring in the absence of DNA replication (cell
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cycle ~ 24 h in these cells). The existence of demethylating enzymes has been previously postulated when the rapid demethylation of genes occurs (65, 66).
The effects induced by EPA treatment on PU.1, C/EBPβ, C/EBPδ, c-jun, and M-CSF receptor protein levels are similar to LNA, AA, and DHA treatments. On this basis, a potential similar mechanism for all PUFA conditioning
cannot be excluded. Although the molecular mechanisms underlying the CpG site-specific demethylation process and the potential common PUFA mechanisms need to be investigated, our findings provide for the first time evidence of a tight correlation among C/EBPδ site-specific CpG demethylation, M-CSF receptor expression, and the begin of the differentiation program induced by EPA in U937 leukemia cells.
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FOOTNOTES
We thank Rita Roberti for critical reading of the manuscript and Davide Chiasserini for statistical analysis. The work was supported by grants from Ministero delle Politiche Agricole, Alimentari e Forestali to LB and the Italian Association for Cancer Research (AIRC) to FG. The abbreviations used are: PUFAs, polyunsaturated fatty acids; M-CSF, (macrophage colony-stimulating factor) receptor, C/EBP, CCAAT/enhancer-binding protein (�,β,δ); SA, Stearic acid (18:0); OA, oleic acid (18:1, n-9); LA, linoleic acid (18:2, n-6), LNA, α-linolenic acid (18:3, n-3); AA, arachidonic acid (20:4, n-6); EPA, eicosapentenoic acid (20:5, n-3); DHA, dochosaexaenoic acid (22-6, n-3).
FIGURE LEGENDS
Fig. 1. Effects of fatty acids on U937 cell viability and morphology. A. U937 cells were treated with 50, 100, or 200 µM fatty acids for 24 hours. Cells were harvested, stained with PI, and analyzed for cell cycle distribution using flow cytometry. Stearic acid (SA, open circle); oleic acid (OA, open triangle); linoleic acid (LA, open square); α-linolenic acid (LNA, solid square); arachidonic acid (AA, solid diamond); eicosapentaenoic ecid (EPA, solid circle); docosahexaenoic acid (DHA, solid triangle). The means ± SD of four separate experiments are shown. Two-way ANOVA was utilized to determine impact of fatty acid type and concentration (*P<0.001, LNA, AA, EPA, and DHA versus U937 untreated cells; #P< 0.001, LNA, AA, and DHA versus EPA). B. Cells were treated with 100 µM fatty acids as described in A. Percentage cell cycle distribution was evaluated. One representative of five experiments is shown. C. Cell proliferation was determined by counting triplicate samples for trypan bleu dye-exclusion after 100 µM fatty acid treatments. The means ± SD of five separate experiments are shown (*P < 0.001, versus U937 untreated cells). D. Cells were treated with 100 µM fatty acids and incubated for 4 hours with [3H]thymidine and the uptake was evaluated as described in Experimental Procedures. The means ± SD of three separate experiments are shown (*P < 0.001 versus U937 untreated cells). E. Morfological changes after 100 µM fatty acids treatment. Flow cytometry analyses for size (forward scatter-FSC) and granulariry (side scatter-SSC). One representative of five experiments is shown. FSC and SSC peak signals were collected from about 10.000 cells. Mean values of these scattering signals were calculated using SYSTEM II Software. Values shown (FSC, X axis and SSC, Y axis) are mean ± S.D. of five experiments (*P<0.05, #P<0.01, and **P<0.001 relative to U937 untreated cells)
Fig. 2. Effect of fatty acids on myeloid-lineage specific transcription factors. A . U937 cells were treated with 100 µM fatty acids for 24 hours. Total cell lysates (50 µg protein) were subject to Western blotting with the indicated antibodies as described in Experimental Procedures. For each protein, one representative out of four experiments is reported. Images of independent blots were acquired using the Versadoc Imaging System and signals were quantified using Quanty One Software. The fold change in each protein was compared with control U937 cells and was calculated after correction for �-tubulin loading differences. Data are the mean ± S.D. of four separate experiments (*P<0.01 versus U937 untreated cells) B. mRNA content was evaluated after 1, 3, and 24 hours treatment with 100 µM fatty acids, using qRT-PCR as described in Experimental Procedures. Unsupplemented U937 (white bar); OA (gray bar); EPA (black bar). Data are presented as relative expression by calculating 2-��Ct normalized to its untreated U937 cells. The means ± SD of three separate experiments are shown (*P<0.01 and **P<0.001 versus U937 untreated cells).
Fig. 3. Effect of fatty acids on M-CSF receptor expression. A. U937 cells were treated with 100 µM fatty acids for 1, 3, and 24 hours. mRNA content was evaluated using qRT-PCR as described in
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Experimental Procedures. Unsupplemented U937 (white bar); OA (gray bar); EPA (black bar). Data are presented as relative expression by calculating 2-��Ct normalized to its untreated U937 cells. The means ± SD of three separate experiments are shown (*P<0.001 versus U937 untreated cells). B. M-CSF receptor protein levels after 24 hours of 100 µM fatty acid conditioning. Total cell lysates (50 µg protein) were subject to Western blotting as described in Experimental Procedures. One representative out of four experiments is shown. C. Cells were treated with 100 µM fatty acids for 1 24 hours. Chromatin immunoprecipitations were performed in untreated (white bar), OA treated (gray bar), and EPA treated (black bar) U937 cells. Antibodies against C/EBPα, C/EBPβ, C/EBPδ, and PU.1 were used. Real-time PCR was performed using M-CSF receptor promoter-specific primers. The results shown are the mean ± SD of three independent experiments.
Fig. 4. Effect of EPA on C/EBPδ protein expression and methylation status in U937 cells. A. Western blot analysis of C/EBPδ in U937 untreated cells (lane 1), cells after 2 days (lane 2), and 5 days (lane 3) treatment with 1 µM 5-aza 2’ -deoxicytidine, and cells after 24 hours 100 µM EPA treatment (lane 4). Cell lysates (50 µg protein) were loaded. One representative out of three experiments is shown. Images of independent blots were acquired using the Versadoc Imaging System and signals were quantified using Quantity One software. The fold change of C/EBPδ protein was compared with control U937 cell and was calculated after correction for β-tubulin loading differences. Data are the mean ± SD of three separate experiments. B. Cells were treated with 100 µM fatty acids for 24 hours and the methylated DNA levels of C/EBPδ CpG islands were quantified using the Methyl-Profiler qPCR Primer Assay as described under Experimental Procedures. The means ± SD of three separate experiments are shown (*P<0.001 versus OA or U937 untreated cells). C. C/EBPδ gene proximal promoter DNA sequence contains a CpG island. The underlined sequences indicate the forward and reverse nested primers utilized for cloning and sequencing after bisulfite reaction. The cloned fragment (350 bp) contains 32 CpG (in bold). The arrow indicates the CpG demethylated nucleotide after EPA treatment. D. Sequencing of the individual clones generated by PCR after bisulfite reaction. Black and white circles represent methylated and unmethylated CpGs, respectively.
Fig. 5. Influence of a demethylated CpG on Sp1 binding to C/EBPδ gene promoter. Electromobility shift assay (EMSA). End labeled double-stranded unmethylated and methylated oligonucleotides were incubated with U937 nuclear extracts. Sequences in bold italics represent the Sp1 consensus sequence. Unmethylated oligonucleotides (U); methylated oligonucleotides (M); unmethylated oligonucleotides plus Sp1 antibody (Sp1-Ab).
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Grignani, Luciano Binaglia and Alba VecchiniKatia Fettucciari, Carlo Riccardi, Pierfrancesco Marconi, Paolo Di Nardo, Francesco Veronica Ceccarelli, Serena Racanicchi, Maria Paola Martelli, Giuseppe Nocentini,
tumor suppressor CCAAT/enhancer-binding protein delta in U937 leukemia cellsEicosapentaenoic acid demethylates a single CpG that mediates the expression of
published online June 9, 2011J. Biol. Chem.
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