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The Journal of Immunology, 2006, 177: 2107-2114.
Copyright © 2006 by The American Association of Immunologists

RUNX3 Negatively Regulates CD36 Expression in Myeloid Cell Lines1

Amaya Puig-Kröger*, Angeles Domínguez-Soto*, Laura Martínez-Muñoz{dagger}, Diego Serrano-Gómez*, María Lopez-Bravo, Elena Sierra-Filardi*, Elena Fernández-Ruiz{dagger}, Natividad Ruiz-Velasco*, Carlos Ardavín, Yoram Groner{ddagger}, Narendra Tandon§, Angel L. Corbí2,* and Miguel A. Vega2,3,*

* Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain; {dagger} Unidad de Biología Molecular, Hospital Universitario de la Princesa, Madrid, Spain; {ddagger} Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel; § Otsuka Maryland Medicinal Laboratories, Rockville, MD 20850; and Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD36 is a member of the scavenger receptor type B family implicated in the binding of lipoproteins, phosphatidylserine, thrombospondin-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 Staphylococcus aureus infections. The RUNX3 transcription factor participates in reprogramming DC transcription after pathogen 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 molecular terms, RUNX3 impairs the activity of the proximal regulatory region of the CD36 gene 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A class B scavenger receptor, CD36 plays important physiological roles in the development of chronic inflammatory diseases, such as atherosclerosis and Alzheimer’s disease, through the uptake of modified lipoproteins (1) and binding to fibrillar beta-amyloid (2), in angiogenesis via its interaction with thrombospondin-1 (3), and in energy consumption through its participation in transport of long-chain fatty acids (4). CD36 participates in the clearance of apoptotic cells by dendritic cells (DCs)4 and macrophages (5, 6) through recognition of the anionic phospholipid phosphatidylserine in the outer leaflets of apoptotic cell membranes (6). Engagement of cell surface CD36 on mononuclear phagocytes by either Abs or specific ligands (Plasmodium falciparum-infected erythrocytes, thrombospondin-1, or phosphatidylserine) enhances IL-10 secretion, reduces the secretion of TNF-{alpha} and IL-1beta (7), and negatively regulates human DC functional maturation (8, 9, 10). Recently, the hypersusceptibility of oblivious mutant mice to Staphylococcus aureus infection has led to the finding that CD36 participates in pathogen recognition by acting as a TLR2/6 coreceptor for sensing microbial diacylglycerides (11).

The human CD36 gene exhibits independently active proximal (12) and distal promoters (13), and CD36 expression in monocytes is regulated at the transcriptional level by numerous cytokines including M-CSF, GM-CSF, IL-4, and TGF-beta1 (14). Human CD36 expression appears to be regulated by the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR-{gamma}) (13, 15), and previous studies have shown that IL-4 induces CD36 in macrophages via generation of the PPAR-{gamma} ligand 15-deoxy-PGJ2 (16), whereas CD36 down-regulation by TGF-beta correlates with inactivation of PPAR-{gamma} (17).

RUNX3 belongs to the RUNX family of context-dependent transcriptional regulators which control growth and differentiation of hemopoietic cells and lineage-specific gene expression in major developmental pathways (18). RUNX3 contributes to neurogenesis of the dorsal root ganglia and T cell differentiation, and its absence results in spontaneous inflammatory bowel disease, hyperplasia of the gastric mucosa, and tumorigenesis of gastric epithelium (19, 20, 21). In the myeloid lineage, RUNX3 regulates TGF-beta signaling in DC and is essential for generation of Langerhans cells (22). Gene expression profiling has revealed that RUNX3 is consistently and transiently up-regulated in immature DC and macrophages exposed to a variety of "danger signals" (22, 23, 24). The finding that RUNX3–/– DC display accelerated phenotypic and functional maturation has led to the suggestion that RUNX3 critically contributes to the acquisition of the phenotypic and functional capabilities of activated/mature DC (22). In the present study, we demonstrate that RUNX3 exerts a negative regulatory effect on CD36 expression in myeloid cells through occupancy of two functional RUNX-binding sites within the proximal promoter region of the CD36 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

The human cell lines K562 (chronic myelogenous leukemia), U937 (histiocytic lymphoma), Raji (Lymphoblastoid B), and THP-1 (monocytic leukemia) were cultured in RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, and 2 mM glutamine (complete medium) at 37°C in a humidified atmosphere with 5% CO2. Induction of differentiation of THP-1 cells was accomplished in the presence of PMA at 10 ng/ml. When indicated, IL-4 (PeproTech) was added at 1000 U/ml. In differentiation experiments, cells were seeded at 5 x 105 cells/ml in tissue culture dishes with no change of the culture medium after addition of the differentiation inducer, and differentiation allowed to proceed for 96 h. Individual clones of U937 cells stably transfected with RUNX3 (U937-RUNX3/p44) (25) were maintained in complete medium with G418 (1 mg/ml). COS-7 cells were grown in DMEM with 10% FCS.

Human PBMC were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma) gradient according to standard procedures. Monocytes were purified from PBMC by a 1-h adherence step at 37°C in complete medium or by magnetic cell sorting using CD14 microbeads (Miltenyi Biotec). To generate monocyte-derived macrophages (MDM), adherent or CD14+ cells (>95% purity) were cultured at 0.5–1 x 106 cells/ml in complete medium containing 1000 U/ml GM-CSF (Leucomax; Schering-Plough) for 5–7 days, with cytokine addition every second day. MDM were then treated with IL-4 (1000 U/ml) or IFN-{gamma} (500 U/ml) for 48 h to generate alternatively activated macrophages (AAM{phi}) or classically activated macrophages (CAM{phi}), respectively. To generate immature monocyte-derived DCs (MDDC), monocytes were cultured in complete medium containing 1000 U/ml GM-CSF and 1000 U/ml IL-4, with cytokine addition every second day. For maturation, immature MDDC were treated with either TNF-{alpha} (20 ng/ml) or LPS from Escherichia coli 055:B5 (10 ng/ml) for 24–48 h.

Transfections, plasmids, and site-directed mutagenesis

Transfection in COS-7 and K562 cells was performed with Superfect (Qiagen) according to manufacturer’s instructions. Transfections were conducted in 24-well plates using 1 µg of eukaryotic expression plasmid DNA on 4 x 104 (COS-7) or 1 µg of firefly luciferase-based reporter plasmid and 100–200 ng of eukaryotic expression plasmid DNA on 8–15 x 105 (K562) cells. THP-1 cells were transfected using DEAE-dextran following standard procedures. In reporter gene experiments, the amount of DNA in each transfection was normalized by using the corresponding insertless expression vectors (CMV-0) as carrier. Transfection efficiencies were normalized by cotransfection with the pCMV-betagal plasmid, and beta-galactosidase levels were determined using the Galacto-Light kit (Tropix). The CD36-based reporter gene constructs pCD36-158-luc (CD36Luc) and pCD36-158(m-102/-98)-luc (represented as CD36Luc-98mut) have been previously described (20). CD36Luc contains the –158/+43 fragment of the CD36 proximal promoter driving the expression of the firefly luciferase cDNA, while CD36Luc-98mut contains a mutated RUNX-binding site (at –98). Site-directed mutagenesis was performed on the CD36 promoter constructs CD36Luc and CD36Luc-98mut using the QuikChange System (Stratagene). For mutation of the RUNX-binding site at position –33, the oligonucleotides Runx-33mutS (5'-GGGGGGGGGAGGGGGGGAATTCTGCATATTTAAACTCTCACG-3') and Runx-33mutAS (5'-CGTGAGAGTTTAAATATGCAGAATTCCCCCCTCCCCCCCCC-3') were used, and the resulting plasmids were termed CD36Luc-33mut and CD36Luc-33/-98mut. DNA constructs and mutations were confirmed by DNA sequencing. The RUNX3 expression plasmids pCDNA3.1-RUNX3/p44 and CDM8-CBFbeta1 have been previously described. CDM8-CBFbeta1 was provided by Dr. S. Hiebert (Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, TN).

EMSAs

Nuclear extracts and EMSA were performed essentially as described (25). For Ab inhibition/supershift experiments, 1 µl of anti-RUNX3 antiserum, anti-RUNX1 (provided by Dr. N. A. Speck, Dartmouth Medical School, Hanover, NH) or anti-Sp1 polyclonal antiserum (2892-E; provided by Dr. S. Jackson, University of Zurich, Zurich, Switzerland), was incubated with the nuclear extracts at 4°C for 30 min before the addition of the probe. The RUNX-binding oligonucleotide probes used for EMSA were CD36–33WT (5'-GAGGGGGTGTGGTTGCATAT-3'), which includes the wild-type CD36 promoter sequence between –45/–26, RUNXCONS (5'-GGATATTTGCGGTTAGCA-3'), and the CD11a promoter-based MS7 (5'-CTCCCTGAACCCCTGCGGTTTCACAACTCCTGC-3'). Double-stranded oligonucleotides used as competitors included CD36–33WT and CD36–33mut (5'-GAGGGGGGAATTCTGCATAT-3'), which includes the CD36 proximal promoter sequence between –45/–26 mutated at the –33 RUNX-binding site.

Western blot analysis

Total cell lysates were obtained in 50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5 mM DTT, 10 mM NaF, 1 mM Na3VO4, 20 mM Pefabloc, and 2 µg/ml aprotinin, antipain, leupeptin, and pepstatin. Ten micrograms of each lysate was subjected to SDS-PAGE under reducing conditions and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). After blocking of the unoccupied sites with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20, protein detection was performed using the Supersignal West Pico Chemiluminescent system (Pierce). For reprobing, membranes were incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, 2% SDS) for 30 min at 50°C with occasional agitation. Detection of human RUNX3 was conducted using previously described polyclonal antisera (26), whereas CD36 was detected with a monospecific anti-CD36 polyclonal Ab (27). For loading control, blots were reprobed with a mAb against human beta-actin (Sigma Immunochemicals).

Flow cytometry and Abs

Phenotypic analysis of U937-RUNX3/p44 transfectants, PMA-differentiated THP-1 cells, MDDC, AAM{phi}, and CAM{phi} was conducted by indirect immunofluorescence, using unlabeled primary mAbs followed by incubation with FITC-labeled F(ab')2 goat anti-mouse IgG. mAbs used for cell surface staining included T3b (anti-CD3) as a control, TS1/11 (anti-CD11a), HC1/1 (anti-CD11c), W6/32 (anti-MHC class I), HB1/5 (anti-CD83; Immunotech), and MR1 (anti-DC-SIGN, CD209). FITC-labeled FA6–152 mAb (Immunotech) was used for CD36 detection, with an FITC-labeled isotype-matched Ab as control. All incubations were done in the presence of 50 µg/ml human IgG to prevent binding through the Fc portion of the Abs. Flow cytometry analysis was performed with an EPICS-CS (Coulter Científica) using log amplifiers. Where indicated, results were expressed as expression index: percentage of marker-positive cells multiplied by their mean fluorescence intensity (MFI).

Determination of CD36 mRNA by RT-PCR

Total cellular RNA was isolated with RNeasy columns (Qiagen) following manufacturer’s recommendations and 2 µg of total RNA was reversed transcribed using the First Strand cDNA Synthesis kit for RT-PCR (Roche Applied Science). Real-time PCR of CD36 mRNA was performed by amplifying duplicate samples of target cDNA in a LyghtCycler (Roche Diagnostics) using a SYBR Green kit (Roche Diagnostics) and a primer set specific for the CD36 mRNA region 597–939 (sense, 5'-AACAGAGGCTGACAACTTCACA-3'; antisense, 5'-GCAGTGACTTTCCCAATAGGAC-3'). PCR amplification was normalized according to the level of amplification of the GAPDH mRNA in each sample.

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed using the ChIP assay kit (Upstate Biotechnology) and according to manufacturer’s recommendations. Briefly, THP-1 cells were cross-linked with 1% formaldehyde for 30 min at 37°C. After washing with ice-cold PBS, cells were lysed in 200 µl of a solution containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), and including 1 µg/ml aprotinin, leupeptin, and pepstatin and 1 mM PMSF. Chromatin samples were sonicated with three sets of 10-s pulses at 50% maximum power in a Soniprep 150 MSE, to reduce DNA length to ~200–500 bp. Sonicated lysates were then diluted to 2 ml with 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl (pH 8.1), and 20 µl of this solution were removed for later PCR analysis (Input). After preclearing with salmon sperm DNA/protein A agarose for 1 h at 4°C, Abs (1 µg) were added and the sonicated lysates were incubated overnight at 4°C in a rocking platform. Immune complexes were collected with 60 µl of salmon sperm DNA/protein A agarose (1 h at 4°C), and agarose beads washed with solutions of increasing ionic strength. After a final wash in 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), bound immune complexes were eluted in a freshly prepared solution of 1% SDS, 0.1 M NaHCO3, and cross-links were reversed in the samples (and the input from the sonicated lysates) by heating at 65°C for 4 h. Samples were then treated with proteinase K, and DNA was phenol-chloroform extracted and precipitated. DNA was resuspended (10 µl) and 2 µl was used for detection of the CD36 promoter by PCR using the oligonucleotides 5'-GTTGGTACCTCAGTAATGTGCTGTGT-3' and 5'-GGTCTCGAGGATCAAATGGTATTCTGCAGG-3', which together amplify a 201-bp region between positions –158 and +43 (12). DNA from the input was resuspended in 20 µl, and 1 µl was used for PCR. Immunoprecipitating Abs included rabbit polyclonal antisera against human RUNX3 (26), C/EBP-{alpha} (sc-61X; Santa Cruz Biotechnology) and CD40 (sc-9096; Santa Cruz Biotechnology) as a control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD36 is differentially regulated during DC maturation and alternative macrophage activation

To determine the factors controlling CD36 gene transcription in myeloid cells, we initially determined CD36 protein and RNA levels during LPS- and TNF-{alpha}-induced MDDC maturation. Consistently with previous reports (5), LPS maturation led to a considerably diminished cell surface expression of CD36 (Fig. 1A), a finding further confirmed in Western blot experiments on whole cell lysates (data not shown). Although to a lower extent, TNF-{alpha} also reduced CD36 cell surface expression, indicating that reduction of CD36 takes place with both maturation-inducing agents. In agreement with the cell surface expression data, CD36 mRNA levels were also reduced in response to either LPS or TNF-{alpha} (Fig. 1B). The maturation-dependent decrease of CD36 mRNA was observed 24 and 48 h after addition of the maturation stimuli (Fig. 1B).


Figure 1
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  		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-{alpha}. 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 (–) or activated with IFN-{gamma} (CAM{phi}) or IL-4 (AAM{phi}) 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 (–) or activated with IFN-{gamma} (CAM{phi}) or IL-4 (AAM{phi}) for 48 h. E, Determination of RUNX3 expression by Western blot on extracts from MDM either untreated (–) or activated with IFN-{gamma} (CAM{phi}) or IL-4 (AAM{phi}) for 48 h. Extracts from K562 and Raji cells were used as negative and positive controls, respectively.

 
To extend these findings to the macrophage activation process, and given the effects of IL-4 on CD36 expression (28, 29), CD36 protein and mRNA levels were examined during classical (treatment with IFN-{gamma}) and alternative (treatment with IL-4) macrophage activation. As shown in Fig. 1, C and D, IL-4-treated MDM exhibited higher CD36 expression than IFN-{gamma}-activated macrophages by both flow cytometry and Western blot. These experiments indicate that CD36 is preferentially expressed on IL-4-activated MDM and might constitute a marker for AAM{phi}.

RUNX3 binds the proximal regulatory region of the CD36 gene

The above experiments indicated that CD36 is down-regulated during MDDC maturation and its expression is higher on AAM{phi}. The up-regulation of RUNX3 upon MDDC maturation (22, 23) (see Fig. 2C) and the slightly lower expression of RUNX3 in AAM{phi} (compare lanes 4 and 5 in Fig. 1E), together with the presence of a RUNX-binding element within the CD36 gene regulatory region (12), prompted us to analyze whether RUNX3 regulates the activity of the CD36 proximal promoter. Besides the reported RUNX-binding element at –98 (12), sequence analysis revealed the presence of another potential RUNX-binding element at –33 within the CD36 proximal promoter. EMSA experiments using cell extracts from COS-7 cells overexpressing RUNX1–3 (Fig. 2A) demonstrated that the CD36-33 element is specifically recognized by RUNX1 (Fig. 2A, left panel, and lane 1, middle panel), RUNX2 (Fig. 2A, lane 3, middle panel), and RUNX3 (Fig. 2A, lanes 2 and 5–9, middle panel). The presence of cold RUNXCONS oligonucleotide prevented recognition of the CD36-33 sequence by RUNX1 (Fig. 2A, lane 4, left panel) and RUNX3 (Fig. 2A, lane 9, middle panel or lane 2, right panel). Similarly, RUNX binding to the CD36-33 sequence was prevented by an oligonucleotide containing the RUNX-binding site from the CD11a proximal promoter (25) (CD11a-MS7, Fig. 2A, lane 6, left panel and lane 8, middle panel). By contrast, no inhibition was observed when the CD36–33 mut oligonucleotide was used as competitor (Fig. 2A, lane 3, left panel; lane 7, middle panel; and lane 3, right panel). Moreover, specific polyclonal Abs also inhibited the recognition by RUNX1 (Fig. 2A, lane 5, left panel) and RUNX3 (Fig. 2A, lane 4, right panel). Altogether, these results demonstrate that the CD36 promoter includes two RUNX-binding elements at –33 and –98. In contrast, the CD36-33 element was also recognized by the Sp1 transcription factor (Fig. 2A, middle and right panels), and inhibition of Sp1 binding to the CD36-33 element significantly increased the binding of RUNX3 (compare lanes 4 and 5 in Fig. 2A, right panel). Therefore, occupancy of the CD36-33 element by RUNX3 might be modulated by the relative level of Sp1, which is expressed at low levels in DCs (30).


Figure 2
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  		FIGURE 2. Identification and characterization of RUNX-binding elements within the CD36 gene proximal regulatory region. A, EMSA was performed on the CD36-33 oligonucleotide using nuclear extracts from the indicated COS-7 cells transfected with either RUNX1, RUNX2, or RUNX3 together with CBF-beta. The position of the RUNX1-, RUNX3-, and Sp1-containing complexes is shown. Where indicated, unlabeled competitor 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 extracts from MDDC, either immature or mature (LPS for 24 h). Where indicated, unlabeled competitor oligonucleotides (at 100-fold molar excess) or specific polyclonal antisera against RUNX1 or RUNX3 were added to the binding reaction. The position of RUNX3-containing complexes is indicated. C, Determination of RUNX3 expression by means of Western blot on nuclear extracts from MDDC during LPS-triggered maturation.

 
The involvement of RUNX3 on CD36 expression in DCs was initially analyzed by EMSA experiments on the CD36-33 element, using immature and mature (LPS for 24 h) MDDC and an anti-RUNX3 polyclonal antiserum. The EMSA complex observed with MDDC was inhibited in the presence of cold CD36-33 (Fig. 2B, 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 the presence of a polyclonal antiserum against RUNX3 (Fig. 2B, lanes 5 and 10), whereas an anti-RUNX1 antiserum had a minor effect (Fig. 2B, lanes 4 and 9). Therefore, CD36-33 is primarily recognized by RUNX3 in nuclear extracts from immature and mature MDDC, although its intensity is higher in mature MDDC (compare lanes 1 and 6 in Fig. 2B). This result is in agreement with the higher expression of RUNX3 in MDDC matured with LPS for 24 h (Fig. 2C). Altogether, these results indicate that RUNX3 binds sequences within the CD36 proximal promoter, and suggests that RUNX3 contributes to CD36 expression during MDDC maturation.

Functional relevance of RUNX3 binding to the CD36 promoter

RUNX factors are well-known to be context-dependent transcriptional regulators, and their effect on a given regulatory region varies with the cell lineage and the cellular activation state (reviewed in Ref. 18). To assay RUNX3 effect on the CD36 promoter activity, RUNX3 was cotransfected in K562 cells, which are devoid of RUNX3 expression (26), together with CD36 promoter-based reporters harboring either wild-type or mutated RUNX-binding sites (Fig. 3A). Transfection of RUNX3 led to a great increase (18.9- ± 5.6-fold) in the activity of the wild-type CD36 proximal regulatory region (Fig. 3B). Mutation of either RUNX-binding site considerably reduced (60 and 85%) the transactivating capacity of RUNX3, demonstrating that RUNX3 increases the activity of the CD36 promoter through interaction with either RUNX-binding element (Fig. 3B). In this regard, the –98 element appears to play a more relevant role in the RUNX3-dependent activity of the promoter, as its disruption virtually abolished RUNX3 transactivation, and mutation of CD36-33 in the context of a mutated CD36-98 did not cause a further reduction in transactivation (Fig. 3B). Therefore, RUNX3 regulates the activity of the CD36 promoter through recognition of the RUNX-binding elements at positions –33 and –98.


Figure 3
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  		FIGURE 3. RUNX3 regulates the activity of the CD36 promoter through recognition of the CD36-98 and CD36-33 elements. A, Schematic representation of the proximal regulatory region of the CD36 gene, and reporter plasmids used for its functional dissection. B, K562 cells were transfected with the indicated reporter plasmids in the presence of CMV-0 (empty expression vector) or pCDNA3.1-RUNX3/p44, and luciferase activity was determined after 24 h. For each individual reporter construct, fold induction represents the luciferase activity yielded by pCDNA3.1-RUNX3/p44 relative to the activity produced by the CMV-0 plasmid. Data represent mean ± SD of three independent experiments using two different DNA preparations (***, p < 0.005).

 
In vivo occupancy and negative regulation of the CD36 promoter by RUNX3

The opposed regulation of RUNX3 and CD36 expression that we had observed during LPS-induced maturation (compare Figs. 1A and 2C) and in IL-4- and IFN-{gamma}-activated macrophages led us to hypothesize that RUNX3 negatively regulates CD36 expression. To further extend these observations, the changes in RUNX3 and CD36 expression were analyzed during macrophage differentiation of THP-1 cells in the presence or absence of IL-4 (31). This myeloid cell line was selected because its transfectability allowed us to correlate protein expression data with promoter activity. In agreement with previous reports (28), flow cytometry revealed that the expression of CD36 in PMA-differentiated THP-1 cells is lower than the cell surface expression on THP-1 cells differentiated in the presence of PMA and IL-4 (Fig. 4A). By contrast, Western blot on both cell types revealed that THP-1 cells differentiated in the presence of PMA + 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.


Figure 4
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  		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 (–) 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-beta-gal plasmid. Data represent mean ± SD of triplicate determinations with two different DNA preparations. Statistical significance of the comparison of the activity of each construct with the activity of the wild-type construct: p < 0.009 (***) for CD36Luc-33mut and CD36Luc-98mut, and p < 0.02 (**) for CD36Luc-33/-98mut. D, In vivo occupancy of the CD36 proximal promoter by RUNX3. Chromatin immunoprecipitations on THP-1 cells was performed with affinity-purified polyclonal antisera specific for RUNX3, C/EBP{alpha}, 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 –158 and +43. "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.

 
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 < 0.009) increase in the activity of the CD36 promoter (Fig. 4C), demonstrating that preventing RUNX binding to the CD36-98 element increases the activity of the CD36 promoter. Although to a lower extent, mutation of the CD36-33 element also resulted in increased promoter activity (20%, p < 0.02), and simultaneous mutation of both elements 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 elements (Fig. 4D), these results demonstrate that RUNX3 binding to both elements has a negative regulatory effect on the activity of the CD36 promoter in THP-1 myeloid cells.

RUNX3 inhibits CD36 cell surface expression in U937 cells

To definitively prove the direct influence of RUNX3 on CD36 expression, we measured CD36 cell surface levels in U937 cells stably transfected with RUNX3 (U937-RUNX3/p44), whose generation has been previously described (25). Unlike CD11a, whose expression increased in U937-RUNX3/p44 (25), flow cytometry analysis of two independent clones revealed that RUNX3 overexpression leads to greatly diminished/absent cell surface levels of CD36, affecting both the percentage of positive cells (Fig. 5A) and the MFI (Fig. 5, A and B). By contrast, the expression of a large panel of other cell surface molecules (including CD1a, CD4, CD14, and CD56) was unaffected by RUNX3 overexpression (data not shown). Furthermore, Western blot analysis of two independent U937-RUNX3/p44 clones confirmed that CD36 protein levels are greatly diminished in RUNX3-overexpressing cells (Fig. 5C). Therefore, overexpression of RUNX3 in U937 cells results in diminished CD36 cell surface levels, demonstrating that RUNX3 negatively regulates the expression of CD36 in myeloid cells.


Figure 5
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  		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 number) 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 untransfected 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 beta-actin (lower panel). D, Determination of the expression of murine RUNX3 in CD8+ and CD8– splenic DCs. Ten micrograms of whole cell extracts from the indicated cells was subjected to Western blot using a polyclonal antiserum specific for human RUNX3 (upper panel) or {alpha}-tubulin (lower panel). Whole cell extract from RUNX3-transfected COS cells (COS-RUNX3) was used as control to identify murine RUNX3.

 
To find out whether the inverse correlation between CD36 and RUNX3 expression could be extended to the murine system, we determined the expression of RUNX3 in murine splenic DCs. CD8+ and CD8– splenic DCs are known to differ in their expression of CD36 (32, 33) and, consequently, were analyzed for their level of RUNX3. As shown in Fig. 5D, no significant levels of RUNX3 could be detected in CD8+ murine DCs, which express CD36 (32, 33). On the contrary, RUNX3 was easily detected in murine CD8– DCs, which are devoid of CD36 (32, 33). Therefore, the inverse correlation between CD36 and RUNX3 expression is observed in both murine and human myeloid cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The RUNX3 transcription factor and the CD36 scavenger receptor are capable of modulating the functional maturation of DCs (10, 22). In this study, we present evidence that RUNX3 and CD36 expression are oppositely regulated during MDDC maturation, during macrophage activation and upon myeloid cell line differentiation, and that overexpression of RUNX3 results in down-regulated expression of CD36. Structural and functional dissection of the proximal regulatory region of the CD36 gene revealed the presence of two RUNX-binding sites preferentially occupied by RUNX3 in DCs, and whose disruption leads to increased CD36 promoter activity in THP-1 cells. Altogether, these observations indicate that CD36 expression is regulated by RUNX factors and that RUNX3 directly down-regulates CD36 expression in myeloid cells. RUNX factors are context-dependent transcriptional regulators, and transcriptional repression by RUNX involves recruitment of corepressors, such as mSin3a and transducin-like enhancer of split, as well as histone deacetylases (34). However, other alternative mechanisms appear to exist, because RUNX-mediated CD4 repression is independent of association with the corepressors Groucho/transducin-like enhancer of split or Sin3 and, instead, requires the presence of the nuclear matrix targeting sequence (35). The dissection of the molecular mechanism underlying the transcriptional down-regulation of CD36 by RUNX3 is currently under investigation.

Considering the inhibitory effects of CD36 on DC maturation and the repressive action of RUNX3 on the CD36 expression, the lack of RUNX3 would be expected to have a negative effect on DC maturation. However, and in an apparent discrepancy with this prediction, RUNX3 gene deletion results in murine bone marrow-derived DCs with an accelerated maturation and enhanced T cell stimulatory activity (22). A possible explanation for this paradox stems from the fact that RUNX3 is an integral component of the TGF-beta-signaling cascade and mediates TGF-beta-initiated responses (36, 37). In this regard, RUNX3 is required for TGF-beta responsiveness in gastric epithelial cells (19) and is induced by (38) and cooperates with (39) TGF-beta-activated Smads, whereas TGF-beta inhibits RUNX3 degradation by stimulating its p300-dependent acetylation (40). Therefore, it is conceivable that RUNX3–/– DCs become refractory to TGF-beta-induced maturation inhibition, which would explain their acquisition of a somewhat "advanced" mature phenotype and function (22). In addition, and in line with our results, TGF-beta increases RUNX3 expression whereas it down-regulates CD36 in myeloid cell lines (17).

Regarding CD36 expression on macrophages, our results also constitute the first indication that CD36 is differentially expressed on AAM{phi} and CAM{phi}, with higher levels detected on IL-4-treated macrophages. This effect is also seen in THP-1 cells driven along their alternative differentiation pathway (31). The differential expression of CD36 in CAM{phi} and AAM{phi} might be of relevance, especially considering that CD36 has important roles in foam cell formation by mediating oxidized low-density lipoprotein uptake (1) and in monocyte proliferation and recruitment through its interaction with beta-amyloid (2). In this context, our results suggest that RUNX3 expression might be a critical determinant in the acquisition of these capabilities by macrophages pushed along the alternative activation pathway.

RUNX3 is highly expressed in monocytes and macrophages, where several RUNX3 isoforms have been identified (41), and its expression is regulated during differentiation and activation of myeloid cells (22, 26, 41). In DCs, RUNX3 expression is transiently up-regulated in response to all maturation-inducing pathogens and pathogen-derived products, and is therefore considered as one of the DC maturation "core-response" genes (23). RUNX3 expression is also transiently up-regulated upon bacteria-induced macrophage activation (24). By contrast, DC maturation and bacteria-mediated macrophage activation result in diminished cell surface expression of CD36 (Ref. 5 and this report). The repressive activity of RUNX3 on CD36 expression reported here might constitute the molecular mechanism underlying the down-regulation of CD36 during both processes. The reduction in CD36 expression during DC maturation can be interpreted in the light of the signaling capabilities exhibited by this scavenger receptor, as its ligation on the cell surface impairs the acquisition of optimal T cell stimulatory activity by mature DCs (10): once DCs have initiated their maturation program, down-regulation of CD36 might prevent maturing cells from receiving maturation-inhibitory signals from the binding/uptake of apoptotic cells. Moreover, the decrease in CD36 expression during DC maturation, in the context of the reduced turnover of peptides presented by MHC in mature DC (42), might contribute to the diminished ability of primed DC to sample and present self Ags via uptake of apoptotic cells, which might avoid undesired autoimmune responses. Therefore, the role of RUNX3 in the down-regulation of CD36 expression 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.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Ministerio de Educación y Ciencia (Grant SAF2002-04615-C02-01, Grant GEN2003-20649-C06-01/NAC, and Grant AGL2004-02148-ALI) and Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36422/03) (to A.L.C.), and Ministerio de Educación y Ciencia (Grant GEN2003-20649-C06-06/NAC) (to M.A.V.). A.P.-K. was supported by an I3P-Consejo Superior de Investigaciones Cientificas postdoctoral contract. Back

2 A.L.C. and M.A.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Miguel A. Vega, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu, 9, Madrid 28040, Spain. E-mail address: mavega{at}cib.csic.es Back

4 Abbreviations used in this paper: DC, dendritic cell; MDM, monocyte-derived macrophage; AAM{phi}, alternatively activated macrophage; CAM{phi}, classically activated macrophage; MDDC, monocyte-derived DC; MFI, mean fluorescence intensity; ChIP, chromatin immunoprecipitation assay; PPAR-{gamma}, peroxisome proliferator-activated receptor. Back

Received for publication June 24, 2005. Accepted for publication May 24, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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