RUNX3 Negatively Regulates CD36 Expression in Myeloid Cell Lines Amaya Puig-Kröger, Angeles Domínguez-Soto, Laura Martínez-Muñoz, Diego Serrano-Gómez, María This information is current as Lopez-Bravo, Elena Sierra-Filardi, Elena Fernández-Ruiz, of September 26, 2021. Natividad Ruiz-Velasco, Carlos Ardavín, Yoram Groner, Narendra Tandon, Angel L. Corbí and Miguel A. Vega J Immunol 2006; 177:2107-2114; ;

doi: 10.4049/jimmunol.177.4.2107 Downloaded from http://www.jimmunol.org/content/177/4/2107

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

RUNX3 Negatively Regulates CD36 Expression in Myeloid Cell Lines1

Amaya Puig-Kro¨ger,* Angeles Domı´nguez-Soto,* Laura Martı´nez-Mun˜oz,† Diego Serrano-Go´mez,* Marı´a Lopez-Bravo,¶ Elena Sierra-Filardi,* Elena Ferna´ndez-Ruiz,† Natividad Ruiz-Velasco,* Carlos Ardavı´n,¶ Yoram Groner,‡ Narendra Tandon,§ Angel L. Corbı´,2* and Miguel A. Vega2,3*

CD36 is a member of the scavenger type B family implicated in the binding of lipoproteins, phosphatidylserine, throm- bospondin-1, and the uptake of long-chain fatty acids. On mononuclear phagocytes, recognition of apoptotic cells by CD36 contributes to peripheral tolerance and prevention of autoimmunity by impairing dendritic cell (DC) maturation. Besides, CD36

acts as a coreceptor with TLR2/6 for sensing microbial diacylglycerides, and its deficiency leads to increased susceptibility to Downloaded from Staphylococcus aureus infections. The RUNX3 participates in reprogramming DC transcription after patho- gen recognition, and its defective expression leads to abnormally accelerated DC maturation. We present evidence that CD36 expression is negatively regulated by the RUNX3 transcription factor during myeloid cell differentiation and activation. In mo- lecular terms, RUNX3 impairs the activity of the proximal regulatory region of the CD36 in myeloid cells through in vitro recognition of two functional RUNX-binding elements. Moreover, RUNX3 occupies the CD36 gene proximal regulatory region in

vivo, and its overexpression in myeloid cells results in drastically diminished CD36 expression. The down-regulation of CD36 http://www.jimmunol.org/ expression by RUNX3 implies that this transcription factor could impair harmful autoimmune responses by contributing to the loss of pathogen- and apoptotic cell-recognition capabilities by mature DCs. The Journal of Immunology, 2006, 177: 2107–2114.

class B scavenger receptor, CD36 plays important phys- falciparum-infected erythrocytes, thrombospondin-1, or phospha- iological roles in the development of chronic inflamma- tidylserine) enhances IL-10 secretion, reduces the secretion of A tory diseases, such as atherosclerosis and Alzheimer’s TNF-␣ and IL-1␤ (7), and negatively regulates human DC func- disease, through the uptake of modified lipoproteins (1) and bind- tional maturation (8–10). Recently, the hypersusceptibility of ing to fibrillar ␤-amyloid (2), in angiogenesis via its interaction oblivious mutant mice to Staphylococcus aureus infection has led with thrombospondin-1 (3), and in energy consumption through its to the finding that CD36 participates in pathogen recognition by by guest on September 26, 2021 participation in transport of long-chain fatty acids (4). CD36 par- acting as a TLR2/6 coreceptor for sensing microbial diacylglycer- ticipates in the clearance of apoptotic cells by dendritic cells ides (11). (DCs)4 and macrophages (5, 6) through recognition of the anionic The human CD36 gene exhibits independently active proximal phospholipid phosphatidylserine in the outer leaflets of apoptotic (12) and distal promoters (13), and CD36 expression in monocytes cell membranes (6). Engagement of cell surface CD36 on mono- is regulated at the transcriptional level by numerous cytokines in- nuclear phagocytes by either Abs or specific ligands (Plasmodium cluding M-CSF, GM-CSF, IL-4, and TGF-␤1 (14). Human CD36 expression appears to be regulated by the nuclear hormone recep- tor peroxisome proliferator-activated receptor (PPAR-␥) (13, 15), *Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cienti- ficas (CSIC), Madrid, Spain; †Unidad de Biologı´a Molecular, Hospital Universitario and previous studies have shown that IL-4 induces CD36 in mac- de la Princesa, Madrid, Spain; ‡Department of Molecular Genetics, Weizmann Insti- rophages via generation of the PPAR-␥ ligand 15-deoxy-PGJ2 tute of Science, Rehovot, Israel; §Otsuka Maryland Medicinal Laboratories, Rock- ␤ ville, MD 20850; and ¶Department of Immunology and Oncology, Centro Nacional (16), whereas CD36 down-regulation by TGF- correlates with de Biotecnologia/CSIC, Madrid, Spain inactivation of PPAR-␥ (17). Received for publication June 24, 2005. Accepted for publication May 24, 2006. RUNX3 belongs to the RUNX family of context-dependent The costs of publication of this article were defrayed in part by the payment of page transcriptional regulators which control growth and differentiation charges. This article must therefore be hereby marked advertisement in accordance of hemopoietic cells and lineage-specific in major with 18 U.S.C. Section 1734 solely to indicate this fact. developmental pathways (18). RUNX3 contributes to neurogen- 1 This work was supported by the Ministerio de Educacio´n y Ciencia (Grant esis of the dorsal root ganglia and T cell differentiation, and its SAF2002-04615-C02-01, Grant GEN2003-20649-C06-01/NAC, and Grant AGL2004-02148-ALI) and Fundacio´n para la Investigacio´n y Prevencio´n del SIDA absence results in spontaneous inflammatory bowel disease, hy- en Espan˜a (FIPSE 36422/03) (to A.L.C.), and Ministerio de Educacio´n y Ciencia perplasia of the gastric mucosa, and tumorigenesis of gastric epi- (Grant GEN2003-20649-C06-06/NAC) (to M.A.V.). A.P.-K. was supported by an thelium (19–21). In the myeloid lineage, RUNX3 regulates TGF-␤ I3P-Consejo Superior de Investigaciones Cientificas postdoctoral contract. signaling in DC and is essential for generation of Langerhans cells 2 A.L.C. and M.A.V. contributed equally to this work. (22). Gene expression profiling has revealed that RUNX3 is con- 3 Address correspondence and reprint requests to Dr. Miguel A. Vega, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientificas, Ramiro sistently and transiently up-regulated in immature DC and macro- de Maeztu, 9, Madrid 28040, Spain. E-mail address: [email protected] phages exposed to a variety of “danger signals” (22–24). The find- Ϫ/Ϫ 4 Abbreviations used in this paper: DC, dendritic cell; MDM, monocyte-derived mac- ing that RUNX3 DC display accelerated phenotypic and rophage; AAM␾, alternatively activated macrophage; CAM␾, classically activated functional maturation has led to the suggestion that RUNX3 crit- macrophage; MDDC, monocyte-derived DC; MFI, mean fluorescence intensity; ChIP, chromatin immunoprecipitation assay; PPAR-␥, peroxisome proliferator-acti- ically contributes to the acquisition of the phenotypic and func- vated receptor. tional capabilities of activated/mature DC (22). In the present

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2108 RUNX3 REGULATES CD36 EXPRESSION study, we demonstrate that RUNX3 exerts a negative regulatory (5Ј-GAGGGGGTGTGGTTGCATAT-3Ј), which includes the wild-type effect on CD36 expression in myeloid cells through occupancy of CD36 promoter sequence between Ϫ45/Ϫ26, RUNXCONS (5Ј-GGATAT Ј Ј two functional RUNX-binding sites within the proximal promoter TTGCGGTTAGCA-3 ), and the CD11a promoter-based MS7 (5 -CTC CCTGAACCCCTGCGGTTTCACAACTCCTGC-3Ј). Double-stranded region of the CD36 gene. oligonucleotides used as competitors included CD36–33WT and CD36– 33mut (5Ј-GAGGGGGGAATTCTGCATAT-3Ј), which includes the Materials and Methods CD36 proximal promoter sequence between Ϫ45/Ϫ26 mutated at the Ϫ33 Cell culture RUNX-binding site. The human cell lines K562 (chronic myelogenous leukemia), U937 (his- Western blot analysis tiocytic lymphoma), Raji (Lymphoblastoid B), and THP-1 (monocytic leu- Total cell lysates were obtained in 50 mM HEPES (pH 7.5), 250 mM NaCl, kemia) were cultured in RPMI 1640 supplemented with 10% FCS, 25 mM 1 mM EDTA, 0.5% Triton X-100, 0.5 mM DTT, 10 mM NaF, 1 mM HEPES, and 2 mM glutamine (complete medium) at 37°C in a humidified Na VO , 20 mM Pefabloc, and 2 ␮g/ml aprotinin, antipain, leupeptin, and atmosphere with 5% CO . Induction of differentiation of THP-1 cells was 3 4 2 pepstatin. Ten micrograms of each lysate was subjected to SDS-PAGE accomplished in the presence of PMA at 10 ng/ml. When indicated, IL-4 under reducing conditions and transferred onto an Immobilon polyvinyl- (PeproTech) was added at 1000 U/ml. In differentiation experiments, cells idene difluoride membrane (Millipore). After blocking of the unoccupied were seeded at 5 ϫ 105 cells/ml in tissue culture dishes with no change of sites with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, the culture medium after addition of the differentiation inducer, and dif- 0.1% Tween 20, detection was performed using the Supersignal ferentiation allowed to proceed for 96 h. Individual clones of U937 cells West Pico Chemiluminescent system (Pierce). For reprobing, membranes stably transfected with RUNX3 (U937-RUNX3/p44) (25) were maintained were incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.7), 100 mM in complete medium with G418 (1 mg/ml). COS-7 cells were grown in 2-ME, 2% SDS) for 30 min at 50°C with occasional agitation. Detection of DMEM with 10% FCS. human RUNX3 was conducted using previously described polyclonal an- Human PBMC were isolated from buffy coats from normal donors over Downloaded from tisera (26), whereas CD36 was detected with a monospecific anti-CD36 a Lymphoprep (Nycomed Pharma) gradient according to standard proce- polyclonal Ab (27). For loading control, blots were reprobed with a mAb dures. Monocytes were purified from PBMC by a 1-h adherence step at against human ␤-actin (Sigma Immunochemicals). 37°C in complete medium or by magnetic cell sorting using CD14 mi- crobeads (Miltenyi Biotec). To generate monocyte-derived macrophages Flow cytometry and Abs (MDM), adherent or CD14ϩ cells (Ͼ95% purity) were cultured at 0.5–1 ϫ 106 cells/ml in complete medium containing 1000 U/ml GM-CSF (Leuco- Phenotypic analysis of U937-RUNX3/p44 transfectants, PMA-differenti- ; Schering-Plough) for 5–7 days, with cytokine addition every second ated THP-1 cells, MDDC, AAM␾, and CAM␾ was conducted by indirect http://www.jimmunol.org/ day. MDM were then treated with IL-4 (1000 U/ml) or IFN-␥ (500 U/ml) immunofluorescence, using unlabeled primary mAbs followed by incuba- ␾ Ј for 48 h to generate alternatively activated macrophages (AAM ) or clas- tion with FITC-labeled F(ab )2 goat anti-mouse IgG. mAbs used for cell sically activated macrophages (CAM␾), respectively. To generate imma- surface staining included T3b (anti-CD3) as a control, TS1/11 (anti- ture monocyte-derived DCs (MDDC), monocytes were cultured in com- CD11a), HC1/1 (anti-CD11c), W6/32 (anti-MHC class I), HB1/5 (anti- plete medium containing 1000 U/ml GM-CSF and 1000 U/ml IL-4, with CD83; Immunotech), and MR1 (anti-DC-SIGN, CD209). FITC-labeled cytokine addition every second day. For maturation, immature MDDC FA6–152 mAb (Immunotech) was used for CD36 detection, with an FITC- were treated with either TNF-␣ (20 ng/ml) or LPS from Escherichia coli labeled isotype-matched Ab as control. All incubations were done in the 055:B5 (10 ng/ml) for 24–48 h. presence of 50 ␮g/ml human IgG to prevent binding through the Fc portion of the Abs. Flow cytometry analysis was performed with an EPICS-CS Transfections, plasmids, and site-directed mutagenesis (Coulter Cientı´fica) using log amplifiers. Where indicated, results were

expressed as expression index: percentage of marker-positive cells multi- by guest on September 26, 2021 Transfection in COS-7 and K562 cells was performed with Superfect (Qia- plied by their mean fluorescence intensity (MFI). gen) according to manufacturer’s instructions. Transfections were con- ducted in 24-well plates using 1 ␮g of eukaryotic expression plasmid DNA Determination of CD36 mRNA by RT-PCR on 4 ϫ 104 (COS-7) or 1 ␮g of firefly luciferase-based reporter plasmid and 100–200 ng of eukaryotic expression plasmid DNA on 8–15 ϫ 105 (K562) Total cellular RNA was isolated with RNeasy columns (Qiagen) following cells. THP-1 cells were transfected using DEAE-dextran following stan- manufacturer’s recommendations and 2 ␮g of total RNA was reversed dard procedures. In reporter gene experiments, the amount of DNA in each transcribed using the First Strand cDNA Synthesis kit for RT-PCR (Roche transfection was normalized by using the corresponding insertless expres- Applied Science). Real-time PCR of CD36 mRNA was performed by am- sion vectors (CMV-0) as carrier. Transfection efficiencies were normalized plifying duplicate samples of target cDNA in a LyghtCycler (Roche Di- by cotransfection with the pCMV-␤gal plasmid, and ␤-galactosidase levels agnostics) using a SYBR Green kit (Roche Diagnostics) and a primer set were determined using the Galacto-Light kit (Tropix). The CD36-based specific for the CD36 mRNA region 597–939 (sense, 5Ј-AACAGAGG reporter gene constructs pCD36-158-luc (CD36Luc) and pCD36-158(m- CTGACAACTTCACA-3Ј; antisense, 5Ј-GCAGTGACTTTCCCAATAG 102/-98)-luc (represented as CD36Luc-98mut) have been previously de- GAC-3Ј). PCR amplification was normalized according to the level of am- scribed (20). CD36Luc contains the Ϫ158/ϩ43 fragment of the CD36 plification of the GAPDH mRNA in each sample. proximal promoter driving the expression of the firefly luciferase cDNA, while CD36Luc-98mut contains a mutated RUNX-binding site (at Ϫ98). Chromatin immunoprecipitation (ChIP) assays Site-directed mutagenesis was performed on the CD36 promoter constructs ChIP assays were performed using the ChIP assay kit (Upstate Biotech- CD36Luc and CD36Luc-98mut using the QuikChange System (Strat- nology) and according to manufacturer’s recommendations. Briefly, THP-1 agene). For mutation of the RUNX-binding site at position Ϫ33, the oli- cells were cross-linked with 1% formaldehyde for 30 min at 37°C. After gonucleotides Runx-33mutS (5Ј-GGGGGGGGGAGGGGGGGAATTCT washing with ice-cold PBS, cells were lysed in 200 ␮l of a solution con- GCATATTTAAACTCTCACG-3Ј) and Runx-33mutAS (5Ј-CGTGAGA taining 1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), and including GTTTAAATATGCAGAATTCCCCCCTCCCCCCCCC-3Ј) were used, 1 ␮g/ml aprotinin, leupeptin, and pepstatin and 1 mM PMSF. Chromatin and the resulting plasmids were termed CD36Luc-33mut and CD36Luc- samples were sonicated with three sets of 10-s pulses at 50% maximum 33/-98mut. DNA constructs and mutations were confirmed by DNA se- power in a Soniprep 150 MSE, to reduce DNA length to ϳ200–500 bp. quencing. The RUNX3 expression plasmids pCDNA3.1-RUNX3/p44 and Sonicated lysates were then diluted to 2 ml with 0.01% SDS, 1.1% Triton CDM8-CBF␤1 have been previously described. CDM8-CBF␤1 was pro- X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl (pH 8.1), and vided by Dr. S. Hiebert (Vanderbilt Cancer Center, Vanderbilt University 20 ␮l of this solution were removed for later PCR analysis (Input). After School of Medicine, Nashville, TN). preclearing with salmon sperm DNA/protein A agarose for1hat4°C, Abs EMSAs (1 ␮g) were added and the sonicated lysates were incubated overnight at 4°C in a rocking platform. Immune complexes were collected with 60 ␮l Nuclear extracts and EMSA were performed essentially as described (25). of salmon sperm DNA/protein A agarose (1 h at 4°C), and agarose beads For Ab inhibition/supershift experiments, 1 ␮l of anti-RUNX3 antiserum, washed with solutions of increasing ionic strength. After a final wash in 1 anti-RUNX1 (provided by Dr. N. A. Speck, Dartmouth Medical School, mM EDTA, 10 mM Tris-HCl (pH 8.0), bound immune complexes were Hanover, NH) or anti-Sp1 polyclonal antiserum (2892-E; provided by Dr. eluted in a freshly prepared solution of 1% SDS, 0.1 M NaHCO3, and S. Jackson, University of Zurich, Zurich, Switzerland), was incubated with cross-links were reversed in the samples (and the input from the sonicated the nuclear extracts at 4°C for 30 min before the addition of the probe. The lysates) by heating at 65°C for 4 h. Samples were then treated with pro- RUNX-binding oligonucleotide probes used for EMSA were CD36–33WT teinase K, and DNA was phenol-chloroform extracted and precipitated. The Journal of Immunology 2109

DNA was resuspended (10 ␮l) and 2 ␮l was used for detection of the CD36 exhibited higher CD36 expression than IFN-␥-activated macro- promoter by PCR using the oligonucleotides 5Ј-GTTGGTACCTCAGTA phages by both flow cytometry and Western blot. These experi- ATGTGCTGTGT-3Ј and 5Ј-GGTCTCGAGGATCAAATGGTATTCTG Ј ments indicate that CD36 is preferentially expressed on IL-4-ac- CAGG-3 , which together amplify a 201-bp region between positions ␾ Ϫ158 and ϩ43 (12). DNA from the input was resuspended in 20 ␮l, and tivated MDM and might constitute a marker for AAM . 1 ␮l was used for PCR. Immunoprecipitating Abs included rabbit poly- clonal antisera against human RUNX3 (26), C/EBP-␣ (sc-61X; Santa Cruz Biotechnology) and CD40 (sc-9096; Santa Cruz Biotechnology) as a RUNX3 binds the proximal regulatory region of the CD36 gene control. The above experiments indicated that CD36 is down-regulated during MDDC maturation and its expression is higher on AAM␾. Results The up-regulation of RUNX3 upon MDDC maturation (22, 23) CD36 is differentially regulated during DC maturation and (see Fig. 2C) and the slightly lower expression of RUNX3 in alternative macrophage activation AAM␾ (compare lanes 4 and 5 in Fig. 1E), together with the To determine the factors controlling CD36 gene transcription in presence of a RUNX-binding element within the CD36 gene reg- myeloid cells, we initially determined CD36 protein and RNA lev- ulatory region (12), prompted us to analyze whether RUNX3 reg- els during LPS- and TNF-␣-induced MDDC maturation. Consis- ulates the activity of the CD36 proximal promoter. Besides the tently with previous reports (5), LPS maturation led to a consid- reported RUNX-binding element at Ϫ98 (12), sequence analysis erably diminished cell surface expression of CD36 (Fig. 1A), a revealed the presence of another potential RUNX-binding element finding further confirmed in Western blot experiments on whole at Ϫ33 within the CD36 proximal promoter. EMSA experiments

cell lysates (data not shown). Although to a lower extent, TNF-␣ using cell extracts from COS-7 cells overexpressing RUNX1–3 Downloaded from also reduced CD36 cell surface expression, indicating that reduc- (Fig. 2A) demonstrated that the CD36-33 element is specifically tion of CD36 takes place with both maturation-inducing agents. In recognized by RUNX1 (Fig. 2A, left panel, and lane 1, middle agreement with the cell surface expression data, CD36 mRNA levels panel), RUNX2 (Fig. 2A, lane 3, middle panel), and RUNX3 (Fig. were also reduced in response to either LPS or TNF-␣ (Fig. 1B). The 2A, lanes 2 and 5–9, middle panel). The presence of cold RUNX- maturation-dependent decrease of CD36 mRNA was observed 24 and CONS oligonucleotide prevented recognition of the CD36-33 se-

48 h after addition of the maturation stimuli (Fig. 1B). quence by RUNX1 (Fig. 2A, lane 4, left panel) and RUNX3 (Fig. http://www.jimmunol.org/ To extend these findings to the macrophage activation process, 2A, lane 9, middle panel or lane 2, right panel). Similarly, RUNX and given the effects of IL-4 on CD36 expression (28, 29), CD36 binding to the CD36-33 sequence was prevented by an oligonu- protein and mRNA levels were examined during classical (treat- cleotide containing the RUNX-binding site from the CD11a prox- ment with IFN-␥) and alternative (treatment with IL-4) macro- imal promoter (25) (CD11a-MS7, Fig. 2A, lane 6, left panel and phage activation. As shown in Fig. 1, C and D, IL-4-treated MDM lane 8, middle panel). By contrast, no inhibition was observed by guest on September 26, 2021

FIGURE 1. Expression of CD36 during MDDC maturation and macrophage activation. A, Cell surface expression of CD209 (DC-SIGN), CD83, and CD36 on immature MDDC or MDDC matured for 72 h in the presence or either LPS or TNF-␣. Flow cytometry was performed with specific Abs and the supernatant of the murine myeloma P3X63Ag8 was used as control. The percentage of positive cells (upper number) and the MFI (lower number)is indicated in each case. B, Determination of CD36 mRNA on MDM or MDDC, either untreated or subjected to the indicated treatments, from two independent donors by means of real-time RT-PCR. Results are shown after normalization with GAPDH mRNA amplification. C, Cell surface expression of MHC class I, CD11a, CD11c, and CD36 on MDMs either untreated (Ϫ) or activated with IFN-␥ (CAM␾) or IL-4 (AAM␾) for 48 h. Flow cytometry was performed with specific Abs and the supernatant of the murine myeloma P3X63Ag8 was used as control. The data are presented as the MFI of the whole cell population in each case. D, Determination of CD36 expression by Western blot on extracts from MDM either untreated (Ϫ) or activated with IFN-␥ (CAM␾) or IL-4 (AAM␾) for 48 h. E, Determination of RUNX3 expression by Western blot on extracts from MDM either untreated (Ϫ) or activated with IFN-␥ (CAM␾) or IL-4 (AAM␾) for 48 h. Extracts from K562 and Raji cells were used as negative and positive controls, respectively. 2110 RUNX3 REGULATES CD36 EXPRESSION

FIGURE 2. Identification and character- ization of RUNX-binding elements within the CD36 gene proximal regulatory region. A, EMSA was performed on the CD36-33 oligo- nucleotide using nuclear extracts from the in- dicated COS-7 cells transfected with either RUNX1, RUNX2, or RUNX3 together with CBF-␤. The position of the RUNX1-, RUNX3-, and Sp1-containing complexes is shown. Where indicated, unlabeled competi- tor oligonucleotides (100-fold molar excess) or a polyclonal antisera against RUNX1, RUNX3, or Sp1 were added to the binding reaction. B, EMSA was performed on the CD36-33 oligonucleotide using nuclear ex- tracts from MDDC, either immature or mature (LPS for 24 h). Where indicated, unlabeled

competitor oligonucleotides (at 100-fold mo- Downloaded from lar excess) or specific polyclonal antisera against RUNX1 or RUNX3 were added to the binding reaction. The position of RUNX3- containing complexes is indicated. C, Deter- mination of RUNX3 expression by means of Western blot on nuclear extracts from MDDC during LPS-triggered maturation. http://www.jimmunol.org/

when the CD36–33 mut oligonucleotide was used as competitor in Ref. 18). To assay RUNX3 effect on the CD36 promoter activ- (Fig. 2A, lane 3, left panel; lane 7, middle panel; and lane 3, right ity, RUNX3 was cotransfected in K562 cells, which are devoid of panel). Moreover, specific polyclonal Abs also inhibited the rec- RUNX3 expression (26), together with CD36 promoter-based re- ognition by RUNX1 (Fig. 2A, lane 5, left panel) and RUNX3 (Fig. porters harboring either wild-type or mutated RUNX-binding sites 2A, lane 4, right panel). Altogether, these results demonstrate that (Fig. 3A). Transfection of RUNX3 led to a great increase (18.9- Ϯ by guest on September 26, 2021 the CD36 promoter includes two RUNX-binding elements at Ϫ33 5.6-fold) in the activity of the wild-type CD36 proximal regulatory and Ϫ98. In contrast, the CD36-33 element was also recognized by region (Fig. 3B). Mutation of either RUNX-binding site consider- the (Fig. 2A, middle and right panels), and ably reduced (60 and 85%) the transactivating capacity of RUNX3, inhibition of Sp1 binding to the CD36-33 element significantly demonstrating that RUNX3 increases the activity of the CD36 pro- increased the binding of RUNX3 (compare lanes 4 and 5 in Fig. moter through interaction with either RUNX-binding element (Fig. 2A, right panel). Therefore, occupancy of the CD36-33 element by 3B). In this regard, the Ϫ98 element appears to play a more rele- RUNX3 might be modulated by the relative level of Sp1, which is vant role in the RUNX3-dependent activity of the promoter, as its expressed at low levels in DCs (30). disruption virtually abolished RUNX3 transactivation, and muta- The involvement of RUNX3 on CD36 expression in DCs was tion of CD36-33 in the context of a mutated CD36-98 did not initially analyzed by EMSA experiments on the CD36-33 element, cause a further reduction in transactivation (Fig. 3B). Therefore, using immature and mature (LPS for 24 h) MDDC and an anti- RUNX3 regulates the activity of the CD36 promoter through rec- RUNX3 polyclonal antiserum. The EMSA complex observed with ognition of the RUNX-binding elements at positions Ϫ33 MDDC was inhibited in the presence of cold CD36-33 (Fig. 2B, and Ϫ98. lanes 2 and 7) but not by the mutated CD36-33 (Fig. 2B, lanes 3 and 8). More importantly, the complex was specifically inhibited In vivo occupancy and negative regulation of the CD36 in the presence of a polyclonal antiserum against RUNX3 (Fig. 2B, promoter by RUNX3 lanes 5 and 10), whereas an anti-RUNX1 antiserum had a minor The opposed regulation of RUNX3 and CD36 expression that we effect (Fig. 2B, lanes 4 and 9). Therefore, CD36-33 is primarily had observed during LPS-induced maturation (compare Figs. 1A recognized by RUNX3 in nuclear extracts from immature and ma- and 2C) and in IL-4- and IFN-␥-activated macrophages led us to ture MDDC, although its intensity is higher in mature MDDC hypothesize that RUNX3 negatively regulates CD36 expression. (compare lanes 1 and 6 in Fig. 2B). This result is in agreement with To further extend these observations, the changes in RUNX3 and the higher expression of RUNX3 in MDDC matured with LPS for CD36 expression were analyzed during macrophage differentiation 24 h (Fig. 2C). Altogether, these results indicate that RUNX3 binds of THP-1 cells in the presence or absence of IL-4 (31). This my- sequences within the CD36 proximal promoter, and suggests that eloid cell line was selected because its transfectability allowed us RUNX3 contributes to CD36 expression during MDDC maturation. to correlate protein expression data with promoter activity. In agreement with previous reports (28), flow cytometry revealed that Functional relevance of RUNX3 binding to the CD36 promoter the expression of CD36 in PMA-differentiated THP-1 cells is RUNX factors are well-known to be context-dependent transcrip- lower than the cell surface expression on THP-1 cells differentiated tional regulators, and their effect on a given regulatory region var- in the presence of PMA and IL-4 (Fig. 4A). By contrast, Western ies with the cell lineage and the cellular activation state (reviewed blot on both cell types revealed that THP-1 cells differentiated in The Journal of Immunology 2111

the presence of PMA ϩ IL-4 exhibit a lower level of RUNX3 than those differentiated only with phorbol ester (Fig. 4B). These results confirmed that, like in the case of maturing MDDC and activated macrophages, the expression of RUNX3 and CD36 are inversely correlated. To test whether RUNX3 negatively regulates CD36 promoter activity, the influence of mutations at the RUNX3-binding sites was evaluated in THP-1 cells. Disruption of the CD36-98 element produced a slight (40%) but significant ( p Ͻ 0.009) increase in the activity of the CD36 promoter (Fig. 4C), demonstrating that pre- venting RUNX binding to the CD36-98 element increases the ac- tivity of the CD36 promoter. Although to a lower extent, mutation of the CD36-33 element also resulted in increased promoter ac- tivity (20%, p Ͻ 0.02), and simultaneous mutation of both ele- ments have an effect similar to that observed after mutating CD36-98 (Fig. 4C). Because RUNX3 binds in vivo to the CD36 proximal promoter harboring both CD36-98 and CD36-33 ele- ments (Fig. 4D), these results demonstrate that RUNX3 binding to

both elements has a negative regulatory effect on the activity of the Downloaded from CD36 promoter in THP-1 myeloid cells. FIGURE 3. RUNX3 regulates the activity of the CD36 promoter through recognition of the CD36-98 and CD36-33 elements. A, Schematic RUNX3 inhibits CD36 cell surface expression in U937 cells representation of the proximal regulatory region of the CD36 gene, and reporter plasmids used for its functional dissection. B, K562 cells were To definitively prove the direct influence of RUNX3 on CD36 expression, we measured CD36 cell surface levels in U937 cells transfected with the indicated reporter plasmids in the presence of CMV-0 http://www.jimmunol.org/ (empty expression vector) or pCDNA3.1-RUNX3/p44, and luciferase ac- stably transfected with RUNX3 (U937-RUNX3/p44), whose gen- tivity was determined after 24 h. For each individual reporter construct, eration has been previously described (25). Unlike CD11a, whose fold induction represents the luciferase activity yielded by pCDNA3.1- expression increased in U937-RUNX3/p44 (25), flow cytometry RUNX3/p44 relative to the activity produced by the CMV-0 plasmid. Data analysis of two independent clones revealed that RUNX3 overex- Ϯ represent mean SD of three independent experiments using two different pression leads to greatly diminished/absent cell surface levels of .(p Ͻ 0.005 ,ءءء) DNA preparations CD36, affecting both the percentage of positive cells (Fig. 5A) and by guest on September 26, 2021

FIGURE 4. Functional relevance of RUNX-binding sites in THP-1 myeloid cells. A, Cell surface expression of CD209 (DC-SIGN) and CD36 on THP-1 cells differentiated in the presence of PMA or PMA and IL-4. Flow cytometry was performed with specific Abs and the supernatant of the murine myeloma P3X63Ag8 was used as control. The percentage of positive cells (upper number) and the MFI (lower number) is indicated in each case. B, Determination of RUNX3 expression by Western blot on nuclear extracts from THP-1 cells either untreated (Ϫ) or differentiated in the presence of PMA or PMA and IL-4. As a control, whole cell extracts from COS-7 cells transfected with RUNX3/p44 were analyzed in parallel. C, Disruption of the RUNX-binding elements leads to increased CD36 gene promoter activity in THP-1 cells. THP-1 were transfected with the indicated reporter plasmids and luciferase activity was determined after 24 h. Promoter activity is expressed relative to the activity produced by the wild-type CD36Luc reporter plasmid (arbitrarily set to 1) after normalization for transfection efficiency using CMV-␤-gal plasmid. Data represent mean Ϯ SD of triplicate determinations with two different DNA for (ءءء) preparations. Statistical significance of the comparison of the activity of each construct with the activity of the wild-type construct: p Ͻ 0.009 .for CD36Luc-33/-98mut. D, In vivo occupancy of the CD36 proximal promoter by RUNX3 (ءء) CD36Luc-33mut and CD36Luc-98mut, and p Ͻ 0.02 Chromatin immunoprecipitations on THP-1 cells was performed with affinity-purified polyclonal antisera specific for RUNX3, C/EBP␣, CD40, or no Ab. Immunoprecipitated chromatin was analyzed by PCR using a pair of CD36 promoter-specific primers that amplify a 201-bp fragment flanking the RUNX binding sites at Ϫ158 and ϩ43. “Input” lanes represent the PCR analysis performed on DNA from a 1/20 dilution of the starting sonicated lysate. For control purposes, PCR on a sample from THP-1 genomic DNA was analyzed in parallel. 2112 RUNX3 REGULATES CD36 EXPRESSION

FIGURE 5. Phenotypic analysis of U937 myeloid cells overexpressing RUNX3. A, Cell surface expression of CD36 on untransfected U937 cells or two independent clones of U937 cells stably transfected with RUNX3. Flow cytometry was performed with specific Abs against CD36, and an FITC-labeled isotype-matched Ab was used as control. The percentage of positive cells (upper num- ber) and the MFI (lower number) is indicated in each case. B, CD36 cell surface expression in untransfected U937 cells or in two independent clones of U937 cells stably transfected with RUNX3, and shown as expression index. C, Determination of the expression of CD36 in untrans- fected U937 cells or in two independent clones of U937 cells stably transfected with RUNX3. Ten micrograms of whole cell extracts from the indicated cells was subjected to Western blot using a polyclonal antiserum specific for human CD36 (upper panel), RUNX3 (middle panel), or ␤-actin (lower panel). D, Determination of the expression of murine RUNX3 in CD8ϩ and CD8Ϫ splenic DCs. Ten micrograms of whole cell extracts from the indicated cells was subjected to Western blot using a polyclonal anti- Downloaded from serum specific for human RUNX3 (upper panel)or␣-tu- bulin (lower panel). Whole cell extract from RUNX3- transfected COS cells (COS-RUNX3) was used as control to identify murine RUNX3. http://www.jimmunol.org/ the MFI (Fig. 5, A and B). By contrast, the expression of a large split, as well as histone deacetylases (34). However, other alter- panel of other cell surface molecules (including CD1a, CD4, native mechanisms appear to exist, because RUNX-mediated CD4 CD14, and CD56) was unaffected by RUNX3 overexpression (data repression is independent of association with the corepressors not shown). Furthermore, Western blot analysis of two inde- Groucho/transducin-like enhancer of split or Sin3 and, instead, re- pendent U937-RUNX3/p44 clones confirmed that CD36 protein quires the presence of the nuclear matrix targeting sequence (35). levels are greatly diminished in RUNX3-overexpressing cells The dissection of the molecular mechanism underlying the tran- (Fig. 5C). Therefore, overexpression of RUNX3 in U937 cells scriptional down-regulation of CD36 by RUNX3 is currently un- results in diminished CD36 cell surface levels, demonstrating der investigation. that RUNX3 negatively regulates the expression of CD36 in Considering the inhibitory effects of CD36 on DC maturation by guest on September 26, 2021 myeloid cells. and the repressive action of RUNX3 on the CD36 expression, the To find out whether the inverse correlation between CD36 and lack of RUNX3 would be expected to have a negative effect on DC RUNX3 expression could be extended to the murine system, we maturation. However, and in an apparent discrepancy with this determined the expression of RUNX3 in murine splenic DCs. ϩ Ϫ prediction, RUNX3 gene deletion results in murine bone marrow- CD8 and CD8 splenic DCs are known to differ in their expres- derived DCs with an accelerated maturation and enhanced T sion of CD36 (32, 33) and, consequently, were analyzed for their cell stimulatory activity (22). A possible explanation for this level of RUNX3. As shown in Fig. 5D, no significant levels of paradox stems from the fact that RUNX3 is an integral com- RUNX3 could be detected in CD8ϩ murine DCs, which express ponent of the TGF-␤-signaling cascade and mediates TGF-␤- CD36 (32, 33). On the contrary, RUNX3 was easily detected in Ϫ initiated responses (36, 37). In this regard, RUNX3 is required murine CD8 DCs, which are devoid of CD36 (32, 33). Therefore, for TGF-␤ responsiveness in gastric epithelial cells (19) and is the inverse correlation between CD36 and RUNX3 expression is induced by (38) and cooperates with (39) TGF-␤-activated observed in both murine and human myeloid cells. Smads, whereas TGF-␤ inhibits RUNX3 degradation by stim- Discussion ulating its p300-dependent acetylation (40). Therefore, it is con- ceivable that RUNX3Ϫ/Ϫ DCs become refractory to TGF-␤- The RUNX3 transcription factor and the CD36 scavenger receptor are capable of modulating the functional maturation of DCs (10, induced maturation inhibition, which would explain their 22). In this study, we present evidence that RUNX3 and CD36 acquisition of a somewhat “advanced” mature phenotype and ␤ expression are oppositely regulated during MDDC maturation, function (22). In addition, and in line with our results, TGF- during macrophage activation and upon myeloid cell line differ- increases RUNX3 expression whereas it down-regulates CD36 entiation, and that overexpression of RUNX3 results in down-reg- in myeloid cell lines (17). ulated expression of CD36. Structural and functional dissection of Regarding CD36 expression on macrophages, our results also the proximal regulatory region of the CD36 gene revealed the pres- constitute the first indication that CD36 is differentially expressed ence of two RUNX-binding sites preferentially occupied by on AAM␾ and CAM␾, with higher levels detected on IL-4-treated RUNX3 in DCs, and whose disruption leads to increased CD36 macrophages. This effect is also seen in THP-1 cells driven along promoter activity in THP-1 cells. Altogether, these observations their alternative differentiation pathway (31). The differential ex- indicate that CD36 expression is regulated by RUNX factors and pression of CD36 in CAM␾ and AAM␾ might be of relevance, that RUNX3 directly down-regulates CD36 expression in myeloid especially considering that CD36 has important roles in foam cell cells. RUNX factors are context-dependent transcriptional regula- formation by mediating oxidized low-density lipoprotein uptake tors, and transcriptional repression by RUNX involves recruitment (1) and in monocyte proliferation and recruitment through its in- of corepressors, such as mSin3a and transducin-like enhancer of teraction with ␤-amyloid (2). In this context, our results suggest The Journal of Immunology 2113 that RUNX3 expression might be a critical determinant in the ac- regulates the maturation of human dendritic cells. J. Immunol. 173: quisition of these capabilities by macrophages pushed along the 2985–2994. 10. Urban, B. C., N. Willcox, and D. J. Roberts. 2001. A role for CD36 in the alternative activation pathway. regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98: RUNX3 is highly expressed in monocytes and macrophages, 8750–8755. where several RUNX3 isoforms have been identified (41), and its 11. Hoebe, K., P. Georgel, S. Rutschmann, X. Du, S. Mudd, K. Crozat, S. Sovath, L. Shamel, T. Hartung, U. Zahringer, and B. Beutler. 2005. CD36 is a sensor of expression is regulated during differentiation and activation of my- diacylglycerides. Nature 433: 523–527. eloid cells (22, 26, 41). In DCs, RUNX3 expression is transiently 12. Armesilla, A. L., D. Calvo, and M. A. Vega. 1996. Structural and functional characterization of the human CD36 gene promoter: identification of a proximal up-regulated in response to all maturation-inducing pathogens and PEBP2/CBF site. J. Biol. Chem. 271: 7781–7787. pathogen-derived products, and is therefore considered as one of 13. Sato, O., C. Kuriki, Y. Fukui, and K. Motojima. 2002. 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Transforming growth factor-␤1 (TGF-␤1) and TGF-␤2 decrease expres- ulatory activity by mature DCs (10): once DCs have initiated their sion of CD36, the type B scavenger receptor, through mitogen-activated protein kinase phosphorylation of peroxisome proliferator-activated receptor-␥. J. Biol. maturation program, down-regulation of CD36 might prevent ma- Chem. 275: 1241–1246. turing cells from receiving maturation-inhibitory signals from the 18. Ito, Y. 2004. Oncogenic potential of the RUNX gene family: “overview”. On- binding/uptake of apoptotic cells. Moreover, the decrease in CD36 cogene 23: 4198–4208. 19. Li, Q. L., K. Ito, C. Sakakura, H. Fukamachi, K. Inoue, X. Z. Chi, K. Y. Lee, http://www.jimmunol.org/ expression during DC maturation, in the context of the reduced S. Nomura, C. W. Lee, S. B. Han, et al. 2002. Causal relationship between the turnover of peptides presented by MHC in mature DC (42), might loss of RUNX3 expression and gastric cancer. 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