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Originally published online as doi:10.1189/jlb.0607349 on December 18, 2007

Published online before print December 18, 2007
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(Journal of Leukocyte Biology. 2008;83:648-662.)
© 2008 by Society for Leukocyte Biology

Hypoxia transcriptionally induces macrophage-inflammatory protein-3{alpha}/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-{kappa}B

Florinda Battaglia*, Silvana Delfino*, Elisa Merello{dagger}, Maura Puppo*, Roberto Piva{ddagger}, Luigi Varesio* and Maria Carla Bosco*,1

* Laboratories of Molecular Biology and
{dagger} Neurosurgery, G. Gaslini Institute, Genova, Italy; and
{ddagger} Department of Pathology and Center for Experimental Research and Medical Studies (CeRMS), University of Turin, Torino, Italy

1Correspondence: Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. E-mail: mcbosco1{at}virgilio.it


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ABSTRACT
 
Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-{kappa}B-binding site located at position –92/–82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-{kappa}B motif; 2) three copies of the wild-type NF-{kappa}B-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-{kappa}B binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-{kappa}B family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-{kappa}B complexes binding the cognate {kappa}B site on the CCL20 promoter upon hypoxia exposure, and NF-{kappa}Bp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-{kappa}Bp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the –92/–82 {kappa}B site. Moreover, nuclear expression of the other NF-{kappa}B family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-{kappa}B pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Key Words: monocytes • chemokines • gene regulation • transcription • inflammation


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INTRODUCTION
 
Peripheral blood monocytes (Mn) represent the early mononuclear phagocyte component of the leukocyte infiltrate at sites of inflammation and tumor growth, where they are recruited from the circulation along defined chemotactic gradients terminally differentiating into inflammatory and tumor-associated macrophages (Mf) [1 ]. Mn/Mf are highly versatile cells able to express different functional programs in response to signals of various nature, which include cytokines and growth factors [2 , 3 ], viral or bacterial products [4 ], cellular metabolites [2 , 5 ], and tissue-specific stimuli [6 ]. A common denominator of many pathological processes is represented by low partial oxygen pressure (pO2;<20 mm Hg; hypoxia) [7 8 9 ]. Mononuclear phagocytes accumulate and are retained in avascular and hypoxic/necrotic areas of solid tumors, wounds, atherosclerotic plaques, and arthritic joints, where they undergo marked changes in gene transcription that result in the onset of a unique "hypoxic" phenotype [1 , 10 ]. Hypoxia can profoundly modify Mn/Mf functional status by modulating the expression of genes with angiogenic, proinflammatory, and immunologic relevance [11 12 13 14 ], with important implications for the pathogenesis of inflammatory diseases and tumor progression [7 , 9 , 10 , 15 ]. Mononuclear phagocytes express a unique repertoire of chemokine receptors and are an important source of chemokines. Low O2 availability could finely tune the chemotactic network in cells of the Mn lineage by differentially modulating receptor, CXCR4 [16 ] and CCR5 [17 ], and ligand, CCL2 [18 ], CCL3 [18 ], CXCL2 [19 ], and CXCL8 [15 ], expression patterns, thus representing a key determinant for leukocyte recruitment and activation in pathological tissues [1 ].

Activation of gene transcription is the primary mechanism by which mammalian cells respond to decreased pO2 and is mediated primarily by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix–loop–helix NF composed of a constitutive β subunit (HIF-1β) and an oxygen-sensitive {alpha} subunit (HIF-1{alpha}, -2{alpha}, or -3{alpha}) [7 , 20 21 22 ]. The {alpha} subunits are post-translationally stabilized under hypoxia and translocate to the nucleus, where they heterodimerize with HIF-1β, transactivating the hypoxia responsive element (HRE) present in the promoter of many hypoxia-responsive genes (for a review, see refs. [7 , 20 ]). Transcriptional activation by hypoxia is a complex and tightly regulated process, which requires functional and/or physical HIF-1 interaction with various cofactors and other transcription factors [20 , 23 24 25 ], and HIF-independent pathways mediating gene transcription activation by hypoxia were also described [26 27 28 ]. Hypoxic up-regulation of HIF-1{alpha} and HIF-2{alpha} has been demonstrated in mononuclear phagocytes in vitro and in vivo [21 , 29 , 30 ], and Cramer et al. [31 ] have recently demonstrated the relevance of HIF-1{alpha} for Mn/Mf recruitment at hypoxic sites and for activation of their proinflammatory and immunoregulatory responses. Moreover, a diverse array of hypoxia-responsive genes endowed with transcriptional activity has been found in cells of the Mn lineage [14 , 32 ], and transcription factors other than HIFs were shown to be activated by hypoxia, directly or indirectly, and to contribute to cytokine and chemokine gene regulation in mononuclear phagocytes [15 , 19 , 33 ].

We have recently identified the CC-chemokine MIP-3{alpha} (also known as CCL20), a selective chemoattractant for immature dendritic cells (iDC), effector/memory T lymphocytes, and naive B cells [34 ], as a new hypoxia-inducible gene in human peripheral blood Mn [14 ]. Given its central role in the control of innate and acquired immunity [34 ], we were interested in further characterizing CCL20 regulation by hypoxia in mononuclear phagocytes. We show rapid and reversible hypoxic induction of CCL20 in primary human Mn and Mn-derived Mf (MDM) and demonstrate that the NF-{kappa}B pathway is directly responsible for CCL20 transcriptional activation. Furthermore, we provide the first evidence that hypoxia differentially modulates the NF-{kappa}B family members in primary human Mn and that NF-{kappa}Bp50 is the mediator of CCL20 promoter transactivation by hypoxia.


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MATERIALS AND METHODS
 
Cells and culture conditions
Human peripheral blood Mn were isolated from platelet-apheresis of healthy donors, obtained by the Blood Transfusion Center of the Gaslini Institute (Genova, Italy), by density gradient centrifugation on a Ficoll cushion (Ficoll-PaqueTM PLUS, Amersham, Milano, Italy), followed by counter-current centrifugal elutriation (JE-6 elutriation chamber and AvantiTM J-20XP rotor system, Beckman, Milano, Italy) and MACS magnetic bead separation (Human Monocyte Isolation Kit-II, Miltenyi Biotec, Bologna, Italy), as detailed in ref. [14 ]. MDM were obtained by Mn adherence to tissue-culture plates (Falcon, Sacco, Cadorago, CO, Italy) and culture for 7 days in RPMI 1640 (Euroclone, Celbio, Milano, Italy), supplemented with 2.5% human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Celbio). Mn and MDM purity was ≥95%, as assessed by immunostaining with anti-CD14 and anti-CD68 mAb (BD PharMingen, Milano, Italy), respectively. The human monocytic cell line U937 was purchased from American Type Culture Collection (Manassas, VA, USA). The mouse Mf cell line, ANA-1, was established by infecting bone marrow-derived cells from C57BL/6 mice with the J2 recombinant retrovirus carrying the v-raf/v-myc oncogenes [13 ]. Human cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (HyClone, Celbio), whereas ANA-1 Mf were grown in DMEM (Euroclone) supplemented with 5% FCS.

For experimental purpose, cells were maintained for the indicated time-points at 37°C in a humidified incubator containing 21% O2, 5% CO2, and 75% N2, referred to as normoxic conditions. Hypoxic conditions (i.e., 1% O2, equivalent to 7.1 mm Hg) [7 , 9 ] were achieved by cell incubation and handling at 37°C in a humidified anaerobic workstation incubator (BUG BOX, ALC International, Milano, Italy) flushed with 94% N2, 5% CO2, and 1% O2, as detailed [14 ]. The endotoxin content, determined by the Limulus amebocyte lysate test (QCL-1000, BioWhittaker, Walkersville, MD, USA), was <0.125 EU/ml in all reagents.

Real-time RT-PCR
Total RNA was purified using the RNeasy mini kit (Qiagen, Milano, Italy). The quality control of RNA integrity was carried out with a Bioanalyzer 2100 (Agilent, Waldbroon, Germany). RNA (1 µg) was reverse-transcribed into double-stranded cDNA on a GeneAmp PCR System 2700 (Applied Biosystems, Milano, Italy), using the Advantage RT-for-PCR kit (Becton Dickinson, San Jose, CA, USA), according to the manufacturer’s instructions. Real-time quantitative RT-PCR (qRT-PCR) was performed in triplicate for each target transcript on a 7500 Real-Time PCR system (Applied Biosystems), using SYBR Green PCR Master Mix and 300 nM sense and antisense oligonucleotide primers (TIBMolbiol, Genova, Italy; listed in Table 1 ), as detailed previously [14 ]. Expression data were normalized on the values obtained in parallel for the ARPC1B, LAPTM5, and THBS1 reference genes [14 ], using the Bestkeeper software [35 ]. Relative expression values were calculated using Q-gene software [36 ].


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  		Table 1. Primer Pairs Used for Real-Time qRT-PCRa

Northern blot analysis
Total RNA (10 µg/lane) was electrophoresed under denaturing conditions on a 1.2% agarose/2.2 M formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell Inc., Keen, NH, USA). Hybridization was carried out with [32P]dCTP (Amersham)-radiolabeled human CCL20 cDNA [37 ], kindly provided by Prof. Toshifumi Matsuyama (Nagasaki University Graduate School of Biomedical Sciences, Japan), as described [18 ]. Densitometric analysis of the autoradiographs and quantitative assessment of the band intensities were carried out with the VersaDoc Image Analyzer from BioRad Laboratories (Hercules, CA, USA). For each sample, expression values were normalized to the corresponding level of ethidium bromide-stained 18S rRNA.

ELISA
Secreted CCL20 was measured in cell-free supernatants using the Quantikine human CCL20 immunoassay kit from R&D Systems (Milano, Italy; Space Import Export S.r.l.), according to the manufacturer’s instructions. OD was determined by a Spectrafluor Plus plate reader (Tecan, Milano, Italy) at 450 nm. All assays were done in duplicate and repeated three times. Data were analyzed with the GraphPad Prism 3 software (GraphPad Software, San Diego, CA, USA).

Plasmids
The pGL2-MIP-3{alpha} reporter plasmid, containing the –871/+58 fragment of the human CCL20 promoter, cloned into the promoterless firefly luciferase reporter vector pGL2-basic, and the pGL2-MIP-3{alpha}/plasmid(p)m{kappa}B luciferase construct, with an inactivated NF-{kappa}B-binding site (nt –92 to –82) [38 ], were kindly provided by Prof. Toshifumi Matsuyama. To obtain deletion constructs, oligonucleotide primers bearing suitable restriction sites at the 5' ends (KpnI for forward primers; XhoI for the reverse primer; listed in Table 2 ) were synthesized (TIBMolbiol) and used for CCL20 promoter fragment amplification by PCR. Fragments of PCR were digested with the appropriate restriction enzymes and directionally subcloned into the KpnI/XhoI sites of the linearized pGL2-basic vector. Mutant reporter constructs containing targeted substitutions in the AP-1 and Ets-binding sites were prepared by in vitro mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene, Milano, Italy) and the following oligos (TIBMolbiol): mAP-1: 5'-ACTGGATGAAAGTCTTTTCTCACTCACAGGGCTGAGCTGCT-3'; mEts: 5'-CCCAATATGAGGAAAAAGCAGCCCGTTTTCCTTGCGGGTT-3' (mutations in bold). All constructs were bidirectionally sequenced using the CEQ DTCS Quick Start kit (Beckman) and a CEQ 2000 XL DNA automated sequence analyzer (Beckman) following purification with the GeneDia Seq-prep GD400 kit (GeneDia, Lammari, LU, Italy).


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  		Table 2. Primer Pairs Used for Serial 5' Deletions of the CCL20 Promotera

pGL3-3x-{kappa}B and pGL3-3x-m{kappa}B constructs were generated by subcloning three tandem copies of the double-strand oligos (see Fig. 5A ), encompassing the wild-type or the mutated CCL20 NF-{kappa}B-binding site, into the KpnI/XhoI restriction sites of the pGL3-promoter vector (Promega, Milano, Italy), 5' upstream of a minimal SV40 promoter ({Delta}SV40)-driven luciferase coding sequence.


Figure 5
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  		Figure 5. The NF-{kappa}B-binding element is sufficient to confer responsiveness to hypoxia. (A) Schematic representation of the pGL3 reporter constructs containing three tandem copies of the wild-type (pGL3-3x-{kappa}B) or the mutated (pGL3-3x-m{kappa}B) NF-{kappa}B site from the CCL20 promoter upstream of the minimal enhancerless SV40 promoter ({Delta}SV40). The transcription start site (+1) is indicated. The sequence of the oligomers subcloned into the pGL3 vector is shown. *, Mutation is in bold. (B) Enhancer activity of the NF-{kappa}B motif under hypoxia. ANA-1 and U937 cells were transiently transfected with the parental pGL3-promoter vector and the pGL3-3x-{kappa}B or the pGL3-3x-m{kappa}B reporter constructs and exposed to 21% (Normo) or 1% O2 (Hypo) for 16 h. Luciferase values were normalized to the expression of a cotransfected pRL-TK Renilla luciferase plasmid. The activity of each construct is presented as relative luciferase activity of hypoxic (solid bars) versus normoxic (open bars) cells (considered as equal to 1) after normalization for the activity of the parental vector and represent the mean ± SD of three independent experiments; *, P < 0.01.

pGL2-HRE construct, expressing the luciferase gene under control of a hypoxia responsive synthetic promoter, was generated by subcloning three copies of the HRE (5'-GTGACTACGTGCTGCCTAG-3') from the mouse inducible NO synthase (iNOS) promoter [13 ] into the pGL2 promoter vector (Promega) upstream of the {Delta}SV40 promoter.

The expression vector plasmid-respiratory syncytial virus (pRSV)-NF-{kappa}B1, encoding the NF-{kappa}Bp50 transcription factor driven by the RSV promoter [39 ], was a kind gift of Dr. Howard Young [National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, MD, USA].

Plasmid VEGF-P7, containing a 1005-bp fragment from the human VEGF 5' flanking sequence (–1005 to +306) upstream of the firefly luciferase gene, was described previously [40 ].

Cell transfection and luciferase assay
Transient transfection with luciferase reporter constructs (10 µg DNA/107 cells) was performed by a modification of the DEAE-dextran method, as described [13 ]. To correct for variations in DNA uptake, each construct was cotransfected with 1 µg pRL-TK luciferase control vector (Promega), containing the Renilla luciferase gene under control of the HSV-Tk promoter. Twenty-four hours after transfection, cells (1x106/ml) were exposed to hypoxia or normoxia for an additional 16 h and lysed with 1x luciferase assay passive lysis buffer (Promega). Lysates were adjusted for protein concentration, determined using the BioRad protein assay. Firefly and Renilla luciferase activities were then assayed with the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s recommendations using a TD 20/20 luminometer (Turner Biosystem, Promega). All assays were done in duplicate and repeated at least three times.

Construction of lentiviral vector expressing short hairprin (sh)RNA to NF-{kappa}Bp50 and cell transduction
shRNAs (five sequences) targeting the human NF{kappa}Bp50 (shp50) were obtained from the Lentiviral Expression Arrest-TRC Library (Open Biosystems, Celbio) [41 ]. pLKO1 lentivector expressing control shRNA (LV-shC) was modified by substitution of the puromycin-resistance gene with a KpnI-BamHI fragment from pCCL containing enhanced GFP (EGFP)-woodchuck post-transcriptional regulatory element. High-titer self-inactivating lentiviral particles carrying shp50 (LV-shp50) were produced as described previously [42 ], and efficacy of the constructs was tested by transduction into Sup-M2-TS cells and quantification of NF-{kappa}Bp50 transcript levels by qRT-PCR. The lentiviral construct with the highest knockdown performance (>85% relative to mock-infected cells) was used for infection of Mn cells. Briefly, exponentially growing U937 cells were seeded at a density of 2.5 x 105/ml/well in a six-well culture plate (Falcon) and infected with 200 µl unconcentrated LV-shp50-containing supernatants in the presence of 8 µg/ml polybrene. Fresh medium was added 24 h after infection, and stable transfectants were selected by treatment with 2 µg/ml puromycin (Sigma, Milano, Italy) for an additional 5 days. Infectivity was determined by FACS analysis of cells transduced with the LV-shC lentivector expressing control shRNA and EGFP, as detailed [43 ].

Preparation of nuclear extracts
Nuclear extracts were prepared by modification of a standard protocol [18 ]. Briefly, cells were washed twice with ice-cold PBS and centrifuged at 1200 rpm for 5 min at 4°C. Pellets were solubilized in a hypotonic buffer [15 mM KCl, 10 mM Hepes, pH 7.6, 2 mM MgCl2, 0.1mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, 1 mM Na Vanadate, 1 mM NaF, 1 mM aminoethyl-benzenesulfonylfluoride (AEBSF)] containing 10 µg/ml of the protease inhibitors leupeptin, aprotinin, and pepstatin for 10 min on ice, and cytosolic extracts were recovered by centrifugation at 1000 rpm for 1 min at 4°C. Nuclear pellets were solubilized in lysis buffer (2 M KCl, 25 mM Hepes, pH 7.6, 0.1 mM EDTA, 1 mM DTT, 1 mM Na Vanadate, 1 mM AEBSF, 10 µg/ml protease inhibitors) for 20 min on ice. Dilution buffer (25 mM Hepes, pH 7.6, 0.1 mM EDTA, 1 mM DTT, 20% glycerol, 1 mM Na Vanadate, 1 mM AEBSF, 10 µg/ml protease inhibitors) was then added to the suspension, debris pelletted by centrifugation at 15,000 rpm for 15 min, and nuclear extracts stored at –80°C.

Western blot analysis
Protein extracts (40 µg) were electrophoresed on 10% SDS-PAGE and electroblotted to Immobilon-P nitrocellulose membranes (Millipore, Bredford, MA, USA). A prestained protein marker (175–6.5 kDa; New England Biolabs) was run as a molecular size standard. Immunoblotting was performed as described previously [18 ] with anti-NF-{kappa}B (p50, p65, and c-Rel) rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-actin antibody (Santa Cruz Biotechnology) as an internal loading control. Chemiluminescence detection was carried out with peroxidase-conjugated goat anti-rabbit antibodies (Sigma) using an ECL kit (Pierce, Celbio) according to the manufacturer’s instructions. Densitometry and quantitative assessment of band intensities was carried out with the VersaDoc image analyzer.

EMSA and UV-cross-linking analysis
Double-strand oligonucleotides (TIBMolbiol) corresponding to –99/–69 bp of the CCL20 promoter sequence and encompassing the wild-type and mutated NF-{kappa}B site (see Fig. 6A ) were 5'-end-labeled by [{alpha}-32P]dCTP. EMSAs were performed as described [13 ]. Briefly, 5 µg nuclear extracts were incubated at room temperature for 30 min with radiolabeled probe (20,000 cpm/reaction) in a 25-µl reaction mixture containing 1x binding buffer (BB; 20 mM HEPES, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 5% glycerol) and 2 µg polyinosinic:polycytidylic acid (Amersham). Competition and supershift experiments were performed by preincubating nuclear extracts in BB in the presence of 50-fold molar excess of unlabeled {kappa}B wild-type ({kappa}B-wt) or {kappa}B mutant ({kappa}B-mut) oligo and 200 ng rabbit polyclonal antibodies specific for NF-{kappa}B p50, p65, or c-Rel, respectively, for 15 min at room temperature before addition of the radiolabeled probe. Nucleoprotein complexes were separated from free DNA probe by electrophoresis on a 4% nondenaturating poly/bisacrylamide gel for 3 h at 200 V and 4°C. Gels were vacuum-dried in a BioRad gel dryer and exposed to X-ray film at –80°C. For UV-cross-linking analysis, 40 µg nuclear extracts were incubated with the radiolabeled probe, and the mixture was irradiated with a 254-nM UV light source (UVGL-25 mineral-light lamp, UVP, Upland, CA, USA) for 20 min on ice. The UV-cross-linked complexes were resolved on 10% SDS-PAGE. After two washings in 10% acetic acid and 30% methyl alcohol, gels were vacuum-dried and exposed to X-ray films.


Figure 6
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  		Figure 6. Increased NF-{kappa}Bp50 binding to the CCL20 promoter in response to hypoxia. (A–C) EMSA analysis of NF-{kappa}B binding to the CCL20-{kappa}B consensus sequence. Nuclear extracts (5 µg) were prepared from fresh Mn (A and B) and U937 cells (C) incubated under normoxic (–) or hypoxic (+) conditions for 3 h and analyzed by EMSA. 32P-radiolabeled, double-stranded oligomers (upper sequence; A) encompassing the wild-type (A, Lanes 1 and 2; B and C) or the mutated (A, Lanes 3 and 4) NF-{kappa}B site were used. Competition experiments were performed using a 50-fold molar excess of unlabeled {kappa}B-wt (B, Lane 2) or {kappa}B-mut (B, Lane 3) oligomer. Supershift analysis was performed with 200 ng anti-NF-{kappa}Bp65, anti-NF-{kappa}Bp50, or anti-c-Rel antibodies (B and C), as detailed in Materials and Methods. DNA–protein binding is indicated by brackets, the free radiolabeled probe by a black arrow, and the slower-migrating, supershifted complex by open arrows. A representative of five experiments is shown. (D) UV-cross-linking analysis of NF-{kappa}B-binding proteins. 32P-radiolabeled {kappa}B-wt (Lanes 1 and 2) and {kappa}B-mut (Lanes 3 and 4) probes were incubated with nuclear extracts (40 µg) from fresh Mn cultured under normoxia (–) or hypoxia (+) for 3 h, and the reaction mixture was irradiated under a 254-nM UV light source. The size of UV-cross-linked protein-DNA adducts was determined by electrophoresis on a 10% SDS-PAGE. The position of size markers (kDa) is shown on the right. UV-cross-linked adducts are indicated by a bracket.

Statistical analysis
Data are the mean ± SE of at least three independent experiments, unless specified differently in the text. The Student’s t-test was used to determine the significance of the results (significance at P<0.01). Significance was only indicated when appropriate.


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RESULTS
 
Hypoxia induces CCL20 in human mononuclear phagocytes
Initial experiments were carried out to assess hypoxia effects on CCL20 expression in human Mn. Peripheral blood Mn purified from three different donors were cultured for 16 h in 1% O2, a condition that effectively modulates gene expression in these cells [12 , 14 , 16 ], and CCL20 mRNA levels were determined by Northern blot. As shown in Figure 1A , the CCL20 transcript was detectable in normoxic Mn and up-regulated upon hypoxia exposure. The magnitude of hypoxia effects was then evaluated by qRT-PCR in a panel of 14 different donors (Fig. 1B) . Expression of the classical hypoxia-inducible gene, VEGF [12 ], was measured in parallel as an index of Mn response to hypoxia. In agreement with previous findings [12 , 14 ], VEGF mRNA was strongly induced in all the donors tested, although with some degree of donor-to-donor variability. CCL20 mRNA up-regulation was also consistently triggered by hypoxia, ranging from 3.2- to 19.6-fold among individual donors (Fig. 1B) , confirming the general inducibility of the gene in Mn. Hypoxia stimulatory effects were exerted at early time-points, being detectable within 3 h of culture, with a maximal increase observed after 10 h (Fig. 1C) , and were reversible, as reoxygenation for 3 h restored CCL20 mRNA expression to normoxic levels (Fig. 1D) .


Figure 1
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  		Figure 1. CCL20 mRNA up-regulation by hypoxia in primary human Mn. Total RNA was isolated from different donor-derived human peripheral blood Mn cultured under normoxia or hypoxia. (A) Northern blot analysis. RNA from Donors 1–3 was assayed for CCL20 mRNA expression by Northern blot after 16 h culture. 28S/18S rRNA is shown as a loading control. CCL20 fold-increase, determined by densitometric analysis, is indicated for each donor as hypoxic (Hypo; +) relative to normoxic (–) values (defined as equal to 1) following normalization to the corresponding levels of 18S rRNA. (B) qRT-PCR analysis. CCL20 mRNA up-regulation by 16 h hypoxia (solid bars) was determined by qRT-PCR in Mn purified from 14 independent donors. Relative transcript expression was calculated in relation to the values obtained for three reference genes (Table 1) . VEGF mRNA expression was assessed in parallel (open bars). Data shown represent the mean of triplicate measurement for each single experiment/donor and are expressed as log2 ratio of fold increases (1% O2 relative to 21% O2). The number associated with each bar indicates the linear fold-change in hypoxic relative to normoxic cells (equal to 1). (C) Kinetics of CCL20 mRNA induction by hypoxia. Mn from two representative donors were incubated under hypoxia for the indicated time-points and tested for CCL20 mRNA levels by qRT-PCR. Data shown represent the mean of triplicate measurement for each single experiment/donor and are expressed as mRNA fold increase in hypoxic relative to normoxic cells. (D) Effect of reoxygenation (Reox) on CCL20 mRNA expression. Fresh Mn from two independent donors were exposed to hypoxia for 16 h and subsequently returned to normoxic conditions for 3 h. CCL20 mRNA levels were determined by qRT-PCR. Data are expressed as mean normalized gene expression values, calculated on the basis of triplicate measurements for each experiment/donor, relative to the values obtained for the reference genes. Error bars within triplicate measurements are shown. Fold induction relative to normoxic cells (Normo; equal to 1) is indicated.

Mn differentiate into Mf after tissue infiltration [1 ]. It was of interest to determine whether hypoxia induced CCL20 in differentiated Mf. CCL20 mRNA expression and protein secretion under normoxic and hypoxic conditions were compared in Mn and matched MDM preparations. As depicted in Figure 2 , MDM expressed constitutively low levels of CCL20 mRNA, relative to Mn, and exposure to 1% O2 for 16 h resulted in a three- to fourfold up-regulation of transcript expression (Fig. 2A) . A parallel, significant increase in chemokine secretion was measured by ELISA (Fig. 2B) . A similar pattern of results, but a higher extent of induction, was obtained in fresh Mn (Fig. 2) . It can be concluded that hypoxia is active on different types of mononuclear phagocytes as a CCL20 inducer and that the magnitude of response differs depending on the cell differentiation stage.


Figure 2
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  		Figure 2. Hypoxic induction of CCL20 in matched Mn and MDM. CCL20 mRNA expression (A) and protein secretion (B) were compared in Mn isolated from two representative donors and in matched MDM cultured under normoxia (open bars) or hypoxia (solid bars). (A) qRT-PCR mRNA levels were determined after 16 h culture. Data are expressed as in the legend to Figure 1D . Error bars within triplicate measurements for each single experiment/donor are shown. Fold induction values of hypoxic versus normoxic cells are indicated. (B) ELISA. Conditioned medium from normoxic and hypoxic cultures was harvested after 24 h and assayed for secreted CCL20. Results are expressed as pg per 1 x 106 cells/ml and represent the mean ± SD of three independent experiments/donor; *, P < 0.01.

Hypoxia transcriptionally activates the CCL20 gene
CCL20 gene transactivation in response to other stimuli has been previously reported [37 , 44 ]. We analyzed hypoxia effects on CCL20 promoter activity by transient transfection using the pGL2-MIP-3{alpha} reporter construct, which contains a 929-bp fragment from the human CCL20 5'-flanking region (–871/+ 58) linked upstream to the firefly luciferase coding sequence [38 ]. As primary Mn/Mf are difficult to transfect, we used for these experiments the ANA-1 murine Mf cell line that we have shown to be susceptible to transfection and to hypoxia stimulation [12 , 13 , 18 ]. Inducibility of the CCL20 promoter construct by hypoxia was compared with that of a luciferase reporter construct driven by 1005 bp of the human VEGF promoter (VEGF- p7), which contains a functional HIF-1{alpha}-binding site and is transactivated by hypoxia in ANA-1 [12 ]. The pGL2-HRE construct, encoding the luciferase gene driven by three tandem copies of the HRE from the mouse iNOS promoter upstream of the minimal SV40 promoter, and the pGL2 promoter vector were used as a positive and a negative control, respectively. Cotransfection with the Renilla luciferase control plasmid was carried out to normalize for transfection efficiency. Luciferase activity of each plasmid under normoxia (data not shown) was taken as a reference to assess hypoxia effects. The pGL2-MIP-3{alpha} reporter construct showed low basal activity, and exposure to hypoxia for 16 h resulted in a median threefold luciferase increase above the baseline in eight independent experiments (Fig. 3A ). A similar magnitude of response was detectable in cells transfected with the VEGF-p7 construct, suggesting that hypoxia is a comparable stimulus for CCL20 and VEGF gene transactivation in ANA-1 Mf. Luciferase activity was substantially increased (by approximately sevenfold in hypoxic relative to normoxic cells) upon transfection with the pGL2-HRE construct, whereas it was not affected in cells transfected with the pGL2-promoter vector (Fig. 3A) .


Figure 3
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  		Figure 3. CCL20 promoter activation by hypoxia in Mn cell lines. (A) The mouse Mf cell line ANA-1 and (B) the human Mn cell line U937 were transiently transfected with the indicated Firefly luciferase reporter constructs. The pRL-TK Renilla luciferase control vector was cotransfected as an internal control, as described in Materials and Methods. Luciferase activity was measured after exposure to normoxia or hypoxia for 16 h. Results are expressed as relative Firefly luciferase activity of hypoxic (solid bars) versus normoxic (open bars) cells (defined as equal to 1), determined after normalization for protein content and to the respective Renilla luciferase activity. Means ± SD of the results obtained in eight independent experiments are shown. Fold increase values are indicated; *, P < 0.01; **, P < 0.001.

To confirm CCL20 promoter inducibility by hypoxia in human cells, we performed transfection experiments in the human Mn cell line U937, which expresses CCL20 mRNA after hypoxia stimulation, consistent with the data obtained in primary Mn (data not shown). Despite lower transfection efficiency with respect to ANA-1 cells, we detected a comparable, significant increase of CCL20 promoter transactivation in response to hypoxia upon transfection with the pGL2-MIP-3{alpha} plasmid. Transcription of the VEGF promoter (VEGF-p7 construct) was also increased by hypoxia, whereas no substantial change in luciferase activity was observed with the pGL2-promoter vector (Fig. 3B) . These results clearly indicate that the CCL20 gene is transcriptionally induced by hypoxia in cells of the Mn lineage and that the –871/+58 promoter region contains cis-acting regulatory elements targeted by hypoxia.

A NF-{kappa}B-binding element is required for CCL20 promoter transactivation by hypoxia
To identify the HRE(s) involved in CCL20 gene activation, we generated a series of deletion mutants in the 5'-flanking region of the CCL20 gene linked to the luciferase reporter gene and analyzed luciferase activity under hypoxia (Fig. 4A ). Deletion of the region from –871 to –626 (plasmid pGL2-MIP-3{alpha}/{Delta}626) reduced promoter transactivation to ~50% of that seen with the full-length plasmid in ANA-1 (upper panel) and U937 (lower panel) cells. Further deletion from –626 to –358 (pGL2-MIP-3{alpha}/{Delta}358 construct) did not substantially affect luciferase activity, which was increased with respect to the full-length construct after promoter truncation to nt –288 (pGL2-MIP-3{alpha}/{Delta}288 construct). Interestingly, deletion to nt –77 (pGL2-MIP-3{alpha}/{Delta}77 construct) completely abrogated luciferase activity induced by hypoxia in both cell lines (Fig. 4A) . Taken together, these findings suggest that CCL20 gene regulation by hypoxia in Mn cells is controlled through positive and negative cis-acting elements present in the –871/–77 promoter fragment and that the region comprised between nt –288 and –77 is essential for CCL20 promoter activation. We, thus, focused on this region for the following studies.


Figure 4
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  		Figure 4. Identification of the HREs in the CCL20 promoter. (A) Characterization of the promoter regions involved in CCL20 transactivation by hypoxia. The full-length pGL2-MIP-3{alpha} plasmid or serially deleted CCL20 promoter constructs were transiently transfected into ANA-1 or U937 cells along with the pRL-TK Renilla luciferase (LUC) control vector. Cells were exposed to normoxia or hypoxia for 16 h before luciferase activity was measured. Results are expressed as relative luciferase activity of hypoxic versus normoxic (equal to 1) cells and are the mean ± SD of five independent experiments. Fold induction values are indicated; *, P < 0.01, relative to cells transfected with the full-length construct. (B) Schematic representation of mutated CCL20 promoter reporter constructs. *, Substituted base pairs in the AP-1, Ets, and NF-{kappa}B-binding sites are in bold. The transcription start site (+1) and the TATA box are indicated. (C) Requirement of the NF-{kappa}B-binding site for CCL20 promoter transactivation by hypoxia. ANA-1 and U937 cells were cotransfected with the wild-type pGL2-MIP-3{alpha} or the indicated mutant constructs and the pRL-TK Renilla luciferase control vector. Relative luciferase activities were measured after 16 h of exposure to 21% and 1% O2 and normalized for protein content and to the respective Renilla luciferase values. Hypoxic induction of luciferase activity is presented relative to normoxic conditions (equal to 1). Data are the mean ± SD of four independent experiments, in which each sample was tested in duplicate; *, P < 0.01, relative to cells transfected with the wild-type construct.

No HRE was found in the –288/–77 CCL20 promoter sequence, which, however, contained several putative-binding sites for various transcription factors previously shown to be involved in CCL20 gene transactivation by inflammatory cytokines in other cell systems, including AP-1, c-Ets, and NF-{kappa}B [37 , 38 , 44 , 45 ]. To determine whether these motifs played any role in the hypoxic activation of the CCL20 promoter, we generated by site-directed mutagenesis a series of constructs containing a 3-bp substitution of each of the consensus sequences in the pGL2-MIP-3{alpha} (Fig. 4B and ref. [38 ]), and the effect of mutations was analyzed in ANA-1 (Fig. 4C , upper panel) and U937 (Fig. 4C , lower panel) cells. Mutation of the AP-1 motif (nt –272 to –266; pGL2-MIP-3{alpha}/mAP1 construct) did not substantially affect pGL2-MIP-3{alpha} inducibility by hypoxia, whereas a 40–45% reduction in promoter activation was detected upon transfection with the pGL2-MIP-3{alpha}/mEts construct, containing substitutions in the Ets-binding site (nt –154 to –143), suggesting a role for the Ets motif in CCL20 gene activation by hypoxia. Interestingly, disruption of the putative NF-{kappa}B-binding site (nt –92 to –82; pGL2-MIP-3{alpha}/m{kappa}B construct) completely abrogated luciferase activation by hypoxia, suggesting the requirement of this element for CCL20 promoter transactivation by hypoxia in mononuclear phagocytes.

The ability of the NF-{kappa}B motif to confer hypoxia inducibility in a different promoter context was then investigated. We subcloned three tandem copies of the CCL20 NF-{kappa}B-binding site, wild-type (pGL3-3x-{kappa}B construct) or mutated (pGL3-3x-m{kappa}B construct), into the pGL3-promoter luciferase vector upstream of the minimal SV40 promoter (Fig. 5A ) and analyzed their enhancer activity on the luciferase gene in transient transfection assays (Fig. 5B) . The pGL3-promoter plasmid was transfected in parallel as a control. A mean 3.8-fold increase in luciferase activity was attained after 16 h exposure to hypoxia in ANA-1 cells transfected with the pGL3-3x-{kappa}B construct (Fig. 5B , upper panel). Mutation in the NF-{kappa}B-binding site completely abrogated responsiveness to hypoxia. Comparable results were obtained in U937 cells, confirming the enhancer activity of the NF-{kappa}B motif under hypoxia (Fig. 5B , lower panel). We conclude that the NF-{kappa}B-binding site is essential in driving CCL20 transcription in response to hypoxia.

Hypoxia induces DNA-binding activity to the CCL20 NF-{kappa}B motif in Mn
To confirm the role of NF-{kappa}B in CCL20 transcriptional activation by hypoxia, nuclear extracts from fresh Mn exposed to hypoxia for 3 h were assayed for binding activity to a 31-bp 32P-labeled, double-strand oligonucleotide encompassing the NF-{kappa}B consensus sequence of the CCL20 promoter ({kappa}B-wt; Fig. 6A ) by EMSA. As depicted in Figure 6A , nuclear extracts from normoxic Mn bound the {kappa}B-wt oligomer (Fig. 6A , Lane 1). Binding activity was markedly increased in response to hypoxia (Fig. 6A , Lane 2), ranging from 2.6- to 4.5-fold in five different experiments (data not shown). Binding required the integrity of the NF-{kappa}B motif, as it did not occur when EMSA was performed using a mutated oligonucleotide ({kappa}B-mut) containing the same 3-bp substitution in the NF-{kappa}B site that abolished functional activity in transfection assays (Fig. 6A , Lanes 3 and 4). Competition experiments demonstrated specificity of the DNA–protein interaction. Binding of nuclear extracts from hypoxic Mn (Fig. 6B , Lane 1) was specifically competed for by a 50-fold molar excess of a cold wild-type NF-{kappa}B element (Fig. 6B , Lane 2), whereas the mutated form of the consensus oligomer failed to compete for binding (Fig. 6B , Lane 3). To identify the NF-{kappa}B-binding subunit(s), we performed supershift analysis using antibodies directed to NF-{kappa}Bp65, NF-{kappa}Bp50, or cRel proteins (Fig. 6B , Lanes 4–6). The binding complex was completely supershifted by the anti-NF-{kappa}Bp50 antibody (Fig. 6B , Lane 5), whereas it was not affected by antibodies to NF-{kappa}Bp65 (Fig. 6B , Lane 4) or cRel (Fig. 6B , Lane 6). Similar EMSA and supershift results were observed with nuclear extracts from U937 cells (Fig. 6C) , suggesting that p50 was the only NF-{kappa}B family member binding to the {kappa}B element of the CCL20 promoter in Mn-lineage cells under hypoxia.

As shown by UV-cross-linking studies performed with the {kappa}B-wt oligomer on the same nuclear extracts analyzed by EMSA (Fig. 6D) , DNA–protein adducts of 50–55 kDa molecular weight were cross-linked in normoxic Mn (Fig. 6D , Lane 1), and their amounts were increased upon hypoxia exposure (Fig. 6D Lane 2) . Formation of cross-linked adducts required the integrity of the {kappa}B site, as it did not occurr when the {kappa}B-mut oligo was used as a probe (Fig. 6D , Lanes 3 and 4). These findings indicate that p50/50 homodimers are the only detectable NF-{kappa}B protein complexes binding to the functional –92/–82 NF-{kappa}B site in the CCL20 promoter in hypoxic Mn.

NF-{kappa}Bp50 transactivates the CCL20 promoter in Mn cells
The ability of NF-{kappa}Bp50 protein to functionally transactivate the CCL20 promoter was then investigated. The pRSV-NF-{kappa}B1 expression plasmid encoding the full-length NF-{kappa}Bp50 cDNA or the control empty vector pRSV was cotransfected into ANA-1 (Fig. 7A ) and U937 (Fig. 7B) cells with the pGL2-MIP-3{alpha} or the pGL2-MIP-3{alpha}/m{kappa}B reporter constructs. Cell extracts were assayed for luciferase activity after 16 h. Overexpression of NF-{kappa}Bp50 significantly increased CCL20 promoter-driven luciferase activity relative to cells cotransfected with the empty vector. CCL20 promoter transactivation was dependent on the NF-{kappa}B site, as overexpressed NF-{kappa}Bp50 was unable to transactivate the mutated pGL2-MIP-3{alpha}/m{kappa}B construct. A similar pattern of results was obtained when the pRSV-NF-{kappa}B1 vector was cotransfected with the pGL3-3x-{kappa}B reporter construct, containing the CCL20-{kappa}B trimer or the corresponding mutated construct (pGL3-3x-m{kappa}B). The NF-{kappa}B motif was able to efficiently drive p50-induced luciferase activity, which was increased by 5.3- and 4.7-fold in ANA-1 (Fig. 7A) and U937 (Fig. 7B) cells, respectively, compared with cells cotransfected with the control pRSV plasmid, and reporter gene activation was repressed by mutation of the NF-{kappa}B site. We conclude that NF-{kappa}Bp50 not only binds to the –92/–82 {kappa}B site in the CCL20 promoter but is also able to drive CCL20 promoter transactivation in Mn cells.


Figure 7
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  		Figure 7. Transactivation of the CCL20 promoter by NF-{kappa}Bp50. ANA-1 (A) and U937 (B) cells were transiently cotransfected with the indicated luciferase constructs and with the pRSV control vector or the pRSV-NF-{kappa}B1 expression plasmid. Luciferase activity is presented relative to cells cotransfected with control pRSV (arbitrarily defined as 1) after normalization for protein content and to the levels of cotransfected pRL-TK-Renilla luciferase plasmid. The mean ± SD of three independent experiments is shown. Fold increase values are indicated; *, P < 0.01; **, P < 0.001, versus control pRSV-cotransfected cells.

Hypoxia differentially modulates the expression of the NF-{kappa}B family members in Mn
NF-{kappa}B-dependent gene transcriptional activation is regulated by proinflammatory stimuli in a cell type-specific manner through several mechanisms, including control of the relative amounts of the different NF-{kappa}B subunits [46 , 47 ]. Western blot analysis was carried out to establish whether hypoxia could differentially regulate NF-{kappa}Bp50, NF-{kappa}Bp65, and cRel expression and/or nuclear translocation in Mn. As shown in Figure 8 , expression of all three subunits was detectable in cytoplasmic extracts from normoxic Mn, and exposure to hypoxia induced an increase of NF-{kappa}Bp50 levels within 3 h culture (Fig. 8 , Lane 4), without substantially affecting the expression of the other two subunits. In contrast, NF-{kappa}Bp65 expression was abrogated, whereas that of cRel was reduced by hypoxia in nuclear extracts. Hypoxia inhibitory effects were exerted at early time-points, being detectable already within the first hour of culture (Fig. 8 , Lane 6), and were maintained at least for 3 h. These data suggest that hypoxia selectively inhibits NF-{kappa}Bp65 and to a lesser extent, c-Rel nuclear translocation in primary Mn.


Figure 8
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  		Figure 8. Western blot analysis of NF-{kappa}B proteins in primary Mn. Cytosolic and nuclear extracts (40 µg) were prepared from fresh Mn exposed to normoxia (–) or hypoxia (+) for 1 and 3 h. Proteins were resolved on a 10% SDS-PAGE, and the blots were hybridized with antibodies directed to NF-{kappa}Bp50, NF-{kappa}Bp65, c-Rel, and actin as a loading control. A representative Western blot of three performed with Mn from different donors is shown.

NF-{kappa}Bp50 knockdown results in the abrogation of hypoxia-induced binding to the CCL20 NF-{kappa}B motif
Finally, to confirm the specific role of NF-{kappa}Bp50 in mediating binding activity to the CCL20-{kappa}B motif in hypoxic Mn cells, we induced targeted knockdown of the endogenous NF-{kappa}Bp50 protein by lentiviral-mediated RNA interference. Because of low lentivirus transduction efficiency in primary Mn (approximately 5%), we used for these experiments, the U937 cell line, whose transduction efficiency was >50%, as assessed by infection with a control EGFP-expressing LV-shC lentivector (data not shown). Specificity and efficiency of the LV-shp50 lentiviral vector, containing shRNA for the human NF-{kappa}Bp50 gene, as well as its optimal concentration and kinetics, were determined experimentally by immunoblotting (data not shown). As depicted in Figure 9A , which shows a representative Western blot of three performed, U937 cells constitutively expressed high nuclear levels of NF-{kappa}Bp50 (Fig. 9A , Lane 1), which were substantially unaffected by exposure to hypoxia for 3 h (Fig. 9A , Lane 2), similarly to what was shown for primary Mn. Complete suppression of NF-{kappa}Bp50 expression was achieved in cells infected with LV-shp50 (Fig. 9A , Lanes 3 and 4), whereas p50 levels were not affected by infection with control LV-shC (Fig. 9A , Lanes 5 and 6). LV-shp50 transduction did not modify the expression of actin used as an internal control (Fig. 9A , Lanes 3 and 4). Binding activity to the {kappa}B-wt oligomer, encompassing the CCL20-{kappa}B consensus sequence, was then assessed by EMSA in U937 cells, mock-transduced or transduced with LV-shp50 or LV-shC lentivectors (Fig. 9B) . NF-{kappa}B-binding activity was markedly increased (by an average of 3.5-fold) in response to hypoxia in uninfected cells (Fig. 9B , Lane 2). NF-{kappa}Bp50 silencing resulted in the abrogation of constitutive (Fig. 9B , Lane 3) and hypoxia-induced (Fig. 9B , Lane 4)-binding activity, demonstrating complete binding dependency on the NF-{kappa}Bp50 subunit. No changes were observed in a control LV-shC vector-infected population (Fig. 9B , Lanes 5 and 6) relative to mock-infected cells (Fig. 9B , Lanes 1 and 2). These data complement the results obtained by supershift UV cross-linking and promoter-driven reporter experiments, providing further evidence that CCL20 transactivation by hypoxia in Mn cells is mediated by NF-{kappa}Bp50.


Figure 9
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  		Figure 9. Inhibition of hypoxia-induced binding to the CCL20-{kappa}B site by NF-{kappa}Bp50 knockdown. (A) Western blot analysis. U937 cells mock-transduced (–) or transduced with the LV-shp50 or the control LV-shC lentivector were exposed to normoxia (–) or hypoxia (+) for 3 h, as described in Materials and Methods, and then lysed. Nuclear extracts (40 µg) were immunoblotted with antibodies to NF-{kappa}Bp50 and actin (see legend to Fig. 8 ). A representative Western blot of three performed is shown. (B) EMSA analysis. Nuclear extracts (5 µg) from U937 cells mock-transduced (–) or transduced with the control LV-shC or the LV-shp50 vectors and exposed to normoxia (–) or hypoxia (+) for 3 h were analyzed by EMSA with 32P-radiolabeled {kappa}B-wt oligomer, as detailed in the legend to Figure 6 , A–C. The results shown are representative of three independent experiments.


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DISCUSSION
 
Regulation of leukocyte recruitment and activation by changes in O2 tension is a central event during the organization of host responses in inflammatory and neoplastic tissues [1 ]. The influence of the hypoxic microenvironment on the chemotactic network in cells of the Mn lineage has been previously reported [1 , 14 15 16 17 18 19 ]. In this study, we have characterized a previously unrecognized role for hypoxia as a transcriptional inducer of the CCL20 chemokine in primary Mn, MDM, and Mn cell lines. Moreover, our results clearly establish the direct involvement of the NF-{kappa}B pathway in promoting CCL20 transcription by hypoxia and identify a single NF-{kappa}B family member, NF-{kappa}Bp50, as a transcriptional activator of the CCL20 gene in hypoxic Mn.

Previous reports have shown that CCL20 is inducible by various mediators of inflammation, such as proinflammatory cytokines, bacterial and viral infections, and plant products, in several cell lines and primary cells, with the extent of induction highly dependent on the cell background and the nature of the inflammatory stimulus [37 , 38 , 44 ]. Expression of CCL20 in human Mn was found up-regulated in response to LPS [48 ]. Here, we show that hypoxia is a new stimulus for CCL20 production by mononuclear phagocytes. Exposure to hypoxia consistently up-regulated CCL20 mRNA levels in primary Mn, which were paralleled by a rise in chemokine secretion. Interestingly, the amounts of CCL20 secreted by hypoxic Mn (~4 ng/ml) were in the range of those triggered by IL-1β or measle virus in fibroblasts, epithelial, and tumor cell lines [44 , 48 ] and by LPS or PMA in fresh PBMC and in myelomonocytic THP-1 cells [48 ], suggesting that the potency of hypoxia as a CCL20 inducer is comparable with that of classical, proinflammatory stimuli. Approximately 20- to 35-fold lower constitutive levels of CCL20 were detectable in MDM, relative to matched, fresh Mn. However, similarly to Mn, MDM were susceptible to hypoxia stimulatory effects. A three- to fourfold CCL20 mRNA up-regulation was detectable upon MDM exposure to 1% O2 and was paralleled by increased amounts of secreted chemokine (an average of 200 pg/ml), which were in the range of those produced by human T cell lymphotropic virus-1-infected T cell lines [38 ]. These results demonstrate that mononuclear phagocytes are an important source of CCL20 under hypoxic conditions, although the magnitude of the response varies depending on their differentiation stage.

Induction of CCL20 expression was dependent, not only on the O2 concentration but also on the extent and duration of the hypoxic exposure. We observed a time-dependent increase of CCL20 mRNA, which occurred rapidly, being detectable within 3 h of culture and was maximal after 10 h, suggesting a direct response to hypoxia. This effect was fully reversible, as cell reoxygenation restored mRNA expression to levels comparable with those detected in normoxic cells, suggesting that CCL20 expression in Mn in vivo may vary dynamically with the degree of local oxygenation, which is quite heterogeneous and rapidly fluctuating within inflammatory and tumor lesions [49 ], thus resulting in shifting gradients of secreted CCL20. These findings may explain, at least in part, the high levels of CCL20 present in vivo in areas of inflammation [34 ] and in various chronic inflammatory conditions, such as rheumatoid arthritis, inflammatory skin disorders, and malignant tumors (for a review, see ref. [34 ]), which are characterized by areas of hypoxia and heavy mononuclear phagocyte infiltration [1 , 7 8 9 ]. Given the central role of CCL20 in the recruitment of iDC, effector/memory T lymphocytes, and naive B cells, which express the CCL20-specific receptor, CCR6, modulation of its production is critical for the regulation of innate and acquired immunity [34 ]. Hence, hypoxic induction of CCL20 in mononuclear phagocytes is likely to represent an important mechanism to control the kinetics and composition of the cellular infiltrate in pathological tissues. Whether cells other than those of the Mn lineage are susceptible to hypoxia stimulatory effects on CCL20 production still remains to be determined.

Chemokine production is primarily regulated at the level of gene transcription [5 , 18 , 19 , 27 ]. Activation of CCL20 transcription in response to proinflammatory cytokines has been shown to depend on various cis-acting regulatory elements present in the 5'-flanking region of the gene, exhibiting cell-type and stimulus-specific patterns [37 , 38 , 44 , 45 ]. Our data show inducibility by hypoxia of a luciferase construct containing a CCL20 promoter fragment comprised between nt –871 and + 58 in the mouse Mf line, ANA-1, and in the human Mn cell line, U937, providing the first evidence that hypoxia can exert transcriptional control on CCL20 gene expression in mononuclear phagocytes. The increase in transcription was comparable with that detectable in cells transfected with the VEGF-p7 construct, suggesting that hypoxia is a comparable stimulus for CCL20 and VEGF gene transactivation and raising the issue of the regulatory pathway(s) involved. No region of homology with HRE was found by inspection of the –871/+ 58 CCL20 promoter sequence (GenBank Accession Number AY150053; data not shown), suggesting the involvement of other putative, cis-acting sequences. Signaling pathways involving a number of transcription factors and regulatory elements other than HIF/HRE can be activated by hypoxia, directly or indirectly, and mediate gene transcription [26 ]. Deletion experiments revealed that transcription factor-binding sites within the first 288 nt of the CCL20 promoter were necessary and sufficient for CCL20 transcriptional activation in hypoxic Mn, and site-directed mutagenesis identified a NF-{kappa}B-binding motif, located at position –92/–82, relative to the putative transcription start site, as the primary hypoxia target. The requirement for the NF-{kappa}B element was demonstrated by the following evidences: 1) A 3-bp mutation in the NF-{kappa}B-binding site completely abrogated CCL20 promoter inducibility by hypoxia, indicating that the integrity of this motif was critical for transcription; 2) three copies of the wild-type, but not the mutated, NF-{kappa}B motif were sufficient to confer 3) hypoxia responsiveness to a minimal heterologous promoter; and hypoxia increased a specific DNA-binding activity to an oligomer encompassing the CCL20-{kappa}B-binding site but not to an oligomer carrying the same NF-{kappa}B site mutation that abrogated hypoxia inducibility in functional assays. It is important to acknowledge that other binding elements, in addition to NF-{kappa}B, may be involved in the regulation of CCL20 inducibility by hypoxia, as shown by functional assays with mutation/deletion reporter constructs. Specifically, we found that deletion of the promoter region, comprised from nt –358 to –288, increased gene transcription by hypoxia, suggesting that this sequence binds to a putative hypoxia-inducible transcription repressor(s), similarly to what has been reported for other genes [18 , 50 ]. On the other hand, the putative c-Ets-binding site (nt –154/–143) seemed to possess an additional, although not essential, role in CCL20 transcription activation, in agreement with previous findings showing the involvement of the c-Ets-binding factor, ETS-1, in a hypoxia-transcriptional response [25 , 26 ]. Activation of the NF-{kappa}B pathway by hypoxia was previously shown to mediate the induction of genes coding for IL-6 [28 ], erythropoietin [24 ], CXCL2 [19 ], CXCL8 [27 , 51 ], TNF-{alpha} [26 ], and cyclooxygenase-2 [52 ]. This report extends the list of target genes to include CCL20.

NF-{kappa}B/Rel transcription factors are key regulators of innate and adaptive immunity, driving transcription of a variety of inflammatory genes [53 ]. Various homo- and heterodimeric NF-{kappa}B complexes with different DNA sequence specificities and DNA-binding affinities can form in response to stimulation in a cell type-specific manner, contributing to the specificity of NF-{kappa}B signaling and NF-{kappa}B-mediated gene transcription [46 , 54 , 55 ]. Previous studies about hypoxic NF-{kappa}B activation demonstrated that target gene induction was dependent on NF-{kappa}Bp65 in various types of cells [19 , 28 , 33 , 52 , 56 ]. Furthermore, the involvement of NF-{kappa}Bp65 homodimers and/or NF-{kappa}Bp50/p65 heterodimers in CCL20 induction by proinflammatory cytokines was reported [37 , 38 , 44 , 45 ]. Conversely, this study provides the first evidence of a role for the single NF-{kappa}Bp50 family member in mediating CCL20 gene transactivation by hypoxia. Constitutive binding of NF-{kappa}Bp50, but not of the other NF-{kappa}B subunits, to the –92/–82 {kappa}B consensus sequence in the CCL20 promoter was detected in the nucleus of primary Mn and Mn cells under normoxia by cellular fractionation, followed by immunoblotting, EMSA, and UV cross-linking, consistent with previous data showing the constitutive presence of p50 homodimers in unstimulated Mn cell lines and their binding to a number of gene promoters [46 , 54 , 57 ]. Hypoxia exposure increased NF-{kappa}B-binding activity, and addition of the anti-NF-{kappa}Bp50 antibody completely supershifted the binding complex. In contrast, antibodies directed to NF-{kappa}Bp65 or cRel, as well as to RelB or NF-{kappa}Bp52 (data not shown), failed to supershift, suggesting that p50 was the only NF-{kappa}B family member binding the functional CCL20-{kappa}B site in hypoxic Mn cells. Results from UV cross-linking experiments were consistent with p50/p50 homodimer binding. The specific role of p50 was further confirmed by binding experiments performed in NF-{kappa}Bp50 knockdown Mn cells, which demonstrated complete inhibition of hypoxia-induced binding to the CCL20-{kappa}B motif upon p50 silencing. These results, together with the demonstration of the ability of NF-{kappa}Bp50 to transactivate the full-length CCL20 promoter construct through the {kappa}B site and induce the enhancer activity of the CCL20-{kappa}B trimer in transient transfection assays, support the conclusion that CCL20 induction by hypoxia is fully dependent on p50 action in Mn cells.

NF-{kappa}Bp50 homodimers are known to act predominantly as gene repressors, and several studies demonstrated that p50 overexpression or enhanced DNA-binding activity can decrease the expression of several NF-{kappa}B-dependent genes in different cell types [47 , 55 , 58 ]. However, in vitro studies have shown that p50/p50 homodimers may also be endowed with transactivating properties toward specific NF-{kappa}B consensus sequences [46 , 59 , 60 ] and that they can mediate cytokine-induced transactivation of a restricted number of genes [60 61 62 ], suggesting a dual role for NF-{kappa}Bp50 on gene transcription, depending on the promoter involved. This study together with a recent report by Cao et al. [62 ], showing that NF-{kappa}Bp50 alone can promote transcription of IL-10 by LPS in Mf, raise the possibility that p50/p50 homodimers can act as transcriptional activators in cells of the Mn lineage in response to various stimuli. Additional support for a role of p50 in gene induction in Mn cells is provided by our previous observations that transactivation of the IL-2R{alpha} promoter by IFN-{gamma} is associated with increased NF-{kappa}Bp50 binding in primary human Mn (unpublished results). Studies are currently underway to investigate whether stimuli other than hypoxia can transcriptionally activate CCL20 in human Mn through NF-{kappa}Bp50 homodimers.

The mechanisms leading to the selective formation of p50 homodimers in response to hypoxia remain to be characterized. Modulation of the relative amounts of the NF-{kappa}B family members in the cell nucleus may influence the formation of the different homo- and heterodimeric complexes [46 , 47 ]. NF-{kappa}B proteins in resting cells are sequestered in the cytoplasm as preformed factors by inhibitory proteins (I{kappa}B), which become phosphorylated and subsequently, degraded after cell activation, allowing NF-{kappa}B subunits to translocate to the nucleus and bind their cognate DNA-binding sites [46 , 47 ]. Interestingly, we found that hypoxia rapidly (within 1 h) and selectively abrogated NF-{kappa}Bp65 and reduced c-Rel nuclear expression in Mn, without modifying their cytoplasmic levels, suggesting an inhibitory effect on p65 and c-Rel nuclear translocation and providing a possible explanation for the detection of p50 homodimers as the only NF-{kappa}B complexes binding to the CCL20 promoter in hypoxic cells. Whether hypoxia may act by inhibiting phosphorylation-dependent I{kappa}B degradation or by up-regulating I{kappa}B protein expression still remains to be assessed. Inhibition of p65 nuclear translocation associated with increased p50 homodimer binding was previously observed in human Mn cells in response to IL-10 [63 ]. On the other hand, the reason why the increase of NF-{kappa}Bp50 cytoplasmic levels following 3 h exposure to hypoxia was not associated with concomitant nuclear up-regulation is unclear. Previous studies have shown that the kinetics of NF-{kappa}Bp50 increase in the nucleus of Mn cells differs depending on the stimulatory agent, ranging from a few minutes to several hours, presumably reflecting a stimulus-specific ability to mobilize the preformed pool of NF-{kappa}Bp50 sequestered in the cytoplasm [54 ]. A possible interpretation of our results is that hypoxia, like PMA [54 ], is inefficient in releasing p50 from inhibitory I{kappa}B for transport to the nucleus but that it induces de novo NF-{kappa}Bp50 synthesis, as suggested by the observed increase of this subunit in the cytoplasm, thus resulting in delayed NF-{kappa}Bp50 nuclear up-regulation. Hence, it is likely that the increased NF-{kappa}Bp50 homodimer binding detectable in Mn in response to 3 h hypoxia may be accounted for by post-translational modifications of the preformed NF-{kappa}Bp50 subunit present in the nucleus, which are required to generate active DNA-binding proteins [60 ]. Another possible explanation is that hypoxia may enhance p50 binding indirectly, by affecting the expression and/or functional activity of known p50-interacting, transcriptional coactivator(s), such as Bcl-3 [59 ], C/EBP [61 ], p300 [60 ], CREB-binding protein [62 ], and epithelium-specific ETS-1 (ESE-1) [64 ]. The observation that the 5'-flanking region of the human CCL20 gene contains putative-binding sites for ESE-1 (nt –154/–143) and C/EBP (nt –101/–93), adjacent to the NF-{kappa}B motif [45 ], is consistent with the possibility that these two transcription factors may cooperate with NF-{kappa}Bp50, and future studies to address this possibility are warranted.

In conclusion, this is the first report showing a role for the single NF-{kappa}Bp50 subunit in gene transcription activation by hypoxia. These results add new insights to our current understanding of gene regulation by hypoxia and to the molecular mechanisms linking low pO2 to the control of inflammatory responses and inflammation-associated tumorigenesis. Strong evidence suggests that inflammation and cancer are causally linked and that the degree of inflammation and the type of inflammatory/immune cells present at tumor sites are responsible for titling the balance between tumor progression and regression [65 ]. Tumor-infiltrating mononuclear phagocytes are a major component of the inflammatory circuit that promotes tumor progression [10 ], and NF-{kappa}Bp50 was recently shown to account for their protumoral phenotype by promoting IL-10 and inhibiting IL-12 and TNF-{alpha} production [58 , 62 ]. Hence, it is tempting to speculate that increased NF-{kappa}Bp50 activity may represent one of the molecular mechanisms responsible for the functional alterations of Mn-infiltrating hypoxic tumor areas [10 ]. Recent findings implicating CCL20 overexpression in the progression and metastatic spread of several types of tumors [66 ] are in line with this hypothesis. Inhibition of NF-{kappa}Bp50 activity in tumor-associated mononuclear phagocytes may thus represent a potential anti-cancer strategy.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Italian Association for Cancer Research (AIRC), Fondazione Italiana per la Lotta al Neuroblastoma, San Paolo Company, Italian Health Ministry, and Ministero Istruzione Universitá e Ricerca (MIUR-FIRB). The authors thank Prof. T. Matsuyama (Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan) for kindly providing the human CCL20 cDNA, the pGL2-MIP-3{alpha}, and the pGL2-MIP-3{alpha}/m{kappa}B luciferase construct and Dr. H. Young (NCI-FCRDC, Frederick, MD, USA) for kindly providing the pRSV-NF-{kappa}B1 construct.

Received May 29, 2007; revised November 13, 2007; accepted November 14, 2007.


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REFERENCES
 
    1
  1. Murdoch, C., Giannoudis, A., Lewis, C. E. (2004) Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues Blood 104,2224-2234[Abstract/Free Full Text]
    2. 2
  2. Varesio, L., Espinoza-Delgado, I., Gusella, G., Cox, G., Melillo, G., Musso, T., Bosco, M. C. (1995) Role of cytokines in the activation of monocytes Aggarwal, B. B. Puri, R. K. eds. Human Cytokines: Their Role in Disease and Therapy ,55-70 Blackwell Scientific Cambridge, MA, USA.
    3. 3
  3. Hamilton, T. A., Ohmori, Y., Tebo, J. M., Kishore, R. (1999) Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines Pathobiology 67,241-244[CrossRef][Medline]
    4. 4
  4. Kopydlowski, K. M., Salkowski, C. A., Cody, M. J., Van Rooijen, N., Major, J., Hamilton, T. A., Vogel, S. N. (1999) Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo J. Immunol. 163,1537-1544[Abstract/Free Full Text]
    5. 5
  5. Bosco, M. C., Rapisarda, A., Massazza, S., Melillo, G., Young, H., Varesio, L. (2000) The tryptophan catabolite picolinic acid selectively induces the chemokines macrophage-inflammatory protein-1{alpha} and -1β in macrophages J. Immunol. 164,3283-3291[Abstract/Free Full Text]
    6. 6
  6. Stout, R. D., Jiang, C., Matta, B., Tietzel, I., Watkins, S. K., Suttles, J. (2005) Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences J. Immunol. 175,342-349[Abstract/Free Full Text]
    7. 7
  7. Semenza, G. L. (2001) Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology Trends Mol. Med. 7,345-350[CrossRef][Medline]
    8. 8
  8. Le, Q. T., Denko, N. C., Giaccia, A. J. (2004) Hypoxic gene expression and metastasis Cancer Metastasis Rev. 23,293-310[CrossRef][Medline]
    9. 9
  9. Distler, J. H., Wenger, R. H., Gassmann, M., Kurowska, M., Hirth, A., Gay, S., Distler, O. (2004) Physiologic responses to hypoxia and implications for hypoxia-inducible factors in the pathogenesis of rheumatoid arthritis Arthritis Rheum. 50,10-23[CrossRef][Medline]
    10. 10
  10. Pollard, J. W. (2004) Tumor-educated macrophages promote tumor progression and metastasis Nat. Rev. Cancer 4,71-78[CrossRef][Medline]
    11. 11
  11. White, J. R., Harris, R. A., Lee, S. R., Craigon, M. H., Binley, K., Price, T., Beard, G. L., Mundy, C. R., Naylor, S. (2004) Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis Genomics 83,1-8[CrossRef][Medline]
    12. 12
  12. Melillo, G., Sausville, E. A., Cloud, K., Lahusen, T., Varesio, L., Senderovicz, A. M. (1999) Flavopiridol, a protein kinase inhibitor, down-regulates hypoxic induction of vascular endothelial growth factor expression in human monocytes Cancer Res. 59,5433-5437[Abstract/Free Full Text]
    13. 13
  13. Melillo, G., Musso, T., Sica, A., Taylor, L. S., Cox, G., Varesio, L. (1995) A hypoxia responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter J. Exp. Med. 182,1683-1693[Abstract/Free Full Text]
    14. 14
  14. Bosco, M. C., Puppo, M., Santangelo, C., Anfosso, L., Pfeffer, U., Fardin, P., Battaglia, F., Varesio, L. (2006) Hypoxia modifies the transcriptome of primary human monocytes: modulation of novel immune-related genes and identification of CC-chemokine ligand 20 as a new hypoxia-inducible gene J. Immunol. 177,1941-1955[Abstract/Free Full Text]
    15. 15
  15. Hirani, N., Antonicelli, F., Strieter, R. M., Wiesener, M. S., Haslett, C., Donnelly, S. C. (2001) The regulation of interleukin-8 by hypoxia in human macrophages. A potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS) Mol. Med. 7,685-697[Medline]
    16. 16
  16. Schioppa, T., Uranchimeg, B., Saccani, A., Biswas, S. K., Doni, A., Rapisarda, A., Bernasconi, S., Saccani, S., Nebuloni, M., Vago, L., Mantovani, A., Melillo, G., Sica, A. (2003) Regulation of the chemokine receptor CXCR4 by hypoxia J. Exp. Med. 198,1391-1402[Abstract/Free Full Text]
    17. 17
  17. Bosco, M. C., Reffo, G., Puppo, M., Varesio, L. (2004) Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages Cell. Immunol. 228,1-7[CrossRef][Medline]
    18. 18
  18. Bosco, M. C., Puppo, M., Pastorino, S., Mi, Z., Melillo, G., Massazza, S., Rapisarda, A., Varesio, L. (2004) Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages J. Immunol. 172,1681-1690[Abstract/Free Full Text]
    19. 19
  19. Zampetaki, A., Mitsialis, S. A., Pfeilschifter, J., Kourembanas, S. (2004) Hypoxia induces macrophage inflammatory protein-2 (MIP-2) gene expression in murine macrophages via NF-{kappa}B: the prominent role of p42/ p44 and PI3 kinase pathways FASEB J. 18,1090-1092[Abstract/Free Full Text]
    20. 20
  20. Wenger, R. H., Stiehl, D. P., Camenisch, G. (2005) Integration of oxygen signaling at the consensus HRE Sci. STKE 2005,re12[Abstract/Free Full Text]
    21. 21
  21. Talks, K. L., Turley, H., Gatter, K. C., Maxwell, P. H., Pugh, C. W., Ratcliffe, P. J. (2000) The expression and distribution of the hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} in normal human tissues, cancers, and tumor-associated macrophages Am. J. Pathol. 157,411-421[Abstract/Free Full Text]
    22. 22
  22. Sowter, H. M., Raval, R. R., Moore, J. W., Ratcliffe, P. J., Harris, A. L. (2003) Predominant role of hypoxia-inducible transcription factor (Hif)-1{alpha} versus Hif-2{alpha} in regulation of the transcriptional response to hypoxia Cancer Res. 63,6130-6134[Abstract/Free Full Text]
    23. 23
  23. Miki, N., Ikuta, M., Matsui, T. (2004) Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor {alpha}4 gene by an interaction between hypoxia-inducible factor-1 and Sp1 J. Biol. Chem. 279,15025-15031[Abstract/Free Full Text]
    24. 24
  24. Figueroa, Y. G., Chan, A. K., Ibrahim, R., Tang, Y., Burow, M. E., Alam, J., Scandurro, A. B., Beckman, B. S. (2002) NF-{kappa}B plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression Exp. Hematol. 30,1419-1427[CrossRef][Medline]
    25. 25
  25. Elvert, G., Kappel, A., Heidenreich, R., Englmeier, U., Lanz, S., Acker, T., Rauter, M., Plate, K., Sieweke, M., Breier, G., Flamme, I. (2003) Cooperative interaction of hypoxia-inducible factor-2{alpha} (HIF-2{alpha}) and Ets-1 in the transcriptional activation of vascular endothelial growth factor receptor-2 (Flk-1) J. Biol. Chem. 278,7520-7530[Abstract/Free Full Text]
    26. 26
  26. Cummins, E. P., Taylor, C. T. (2005) Hypoxia-responsive transcription factors Pflugers Arch. 450,363-371[CrossRef][Medline]
    27. 27
  27. Shi, Q., Le, X., Abbruzzese, J. L., Wang, B., Mujaida, N., Matsushima, K., Huang, S., Xiong, Q., Xie, K. (1999) Cooperation between transcription factor AP-1 and NF-{kappa}B in the induction of interleukin-8 in human pancreatic adenocarcinoma cells by hypoxia J. Interferon Cytokine Res. 19,1363-1371[CrossRef][Medline]
    28. 28
  28. Matsui, H., Ihara, Y., Fujio, Y., Kunisada, K., Akira, S., Kishimoto, T., Yamauchi-Takihara, K. (1999) Induction of interleukin (IL)-6 by hypoxia is mediated by nuclear factor (NF)-{kappa} B and NF-IL6 in cardiac myocytes Cardiovasc. Res. 42,104-112[Abstract/Free Full Text]
    29. 29
  29. Burke, B., Tang, N., Corke, K. P., Tazzyman, D., Ameri, K., Wells, M., Lewis, C. E. (2002) Expression of HIF-1{alpha} by human macrophages: implications for the use of macrophages in hypoxia-regulated cancer gene therapy J. Pathol. 196,204-212[CrossRef][Medline]
    30. 30
  30. Leek, R. D., Talks, K. L., Pezzella, F., Turley, H., Campo, L., Brown, N. S., Bicknell, R., Taylor, M., Gatter, K. C., Harris, A. L. (2002) Relation of hypoxia-inducible factor-2 {alpha} (HIF-2 {alpha}) expression in tumor-infiltrative macrophages to tumor angiogenesis and the oxidative thymidine phosphorylase pathway in human breast cancer Cancer Res. 62,1326-1329[Abstract/Free Full Text]
    31. 31
  31. Cramer, T., Yamanishi, Y., Clausen, B. E., Förster, I., Pawlinski, R., Mackman, N., Haase, V. H., Jaenisch, R., Corr, M., Nizet, V., Firestein, G. S., Gerber, H-P., Ferrara, N., Johnson, R. S. (2003) HIF-1{alpha} is essential for myeloid cell-mediated inflammation Cell 112,645-657[CrossRef][Medline]
    32. 32
  32. Yan, S. F., Lu, J., Zou, Y. S., Soh-Won, J., Cohen, D. M., Buttrick, P. M., Cooper, D. R., Steinberg, S. F., Mackman, N., Pinsky, D. J., Stern, D. M. (1999) Hypoxia-associated induction of early growth response-1 gene expression J. Biol. Chem. 274,15030-15040[Abstract/Free Full Text]
    33. 33
  33. Chandel, N. S., Trzyna, W. C., McClintock, D. S., Schumacker, P. T. (2000) Role of oxidants in NF-{kappa} B activation and TNF-{alpha} gene transcription induced by hypoxia and endotoxin J. Immunol. 165,1013-1021[Abstract/Free Full Text]
    34. 34
  34. Schutyser, E., Struyf, S., Van Damme, J. (2003) The CC chemokine CCL20 and its receptor CCR6 Cytokine Growth Factor Rev. 14,409-426[CrossRef][Medline]
    35. 35
  35. Pfaffl, M. W., Tichopad, A., Prgomet, C., Neuvians, T. P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations Biotechnol. Lett. 26,509-515[CrossRef][Medline]
    36. 36
  36. Muller, P. Y., Janovjak, H., Miserez, A. R., Dobbie, Z. (2002) Processing of gene expression data generated by quantitative real-time RT-PCR Biotechniques 32,1372-1379[Medline]
    37. 37
  37. Sugita, S., Kohno, T., Yamamoto, K., Imaizumi, Y., Nakajima, H., Ishimaru, T., Matsuyama, T. (2002) Induction of macrophage-inflammatory protein-3{alpha} gene expression by TNF-dependent NF-{kappa}B activation J. Immunol. 168,5621-5628[Abstract/Free Full Text]
    38. 38
  38. Imaizumi, Y., Sugita, S., Yamamoto, K., Imanishi, D., Kohno, T., Tomonaga, M., Matsuyama, T. (2002) Human T cell leukemia virus type-I Tax activates human macrophage inflammatory protein-3 {alpha}/CCL20 gene transcription via the NF-{kappa} B pathway Int. Immunol. 14,147-155[Abstract/Free Full Text]
    39. 39
  39. Duckett, C. S., Perkins, N. D., Kowalik, T. F., Schmid, R. M., Huang, E. S., Baldwin, A. S., Jr, Nabel, G. J. (1993) Dimerization of NF-{kappa}B2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I {kappa} B-{alpha} (MAD-3) Mol. Cell. Biol. 13,1315-1322[Abstract/Free Full Text]
    40. 40
  40. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., Semenza, G. L. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 Mol. Cell. Biol. 16,4604-4613[Abstract/Free Full Text]
    41. 41
  41. Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., et al (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen Cell 124,1283-1298[CrossRef][Medline]
    42. 42
  42. Piva, R., Pellegrino, E., Mattioli, M., Agnelli, L., Lombardi, L., Boccalatte, F., Costa, G., Ruggeri, B. A., Cheng, M., Chiarle, R., et al (2006) Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and BCL2A1 as critical target genes J. Clin. Invest. 116,3171-3182[CrossRef][Medline]
    43. 43
  43. Puppo, M., Bosco, M. C., Federico, M., Pastorino, S., Varesio, L. (2007) Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages J. Leukoc. Biol. 81,528-538[Abstract/Free Full Text]
    44. 44
  44. Fujiie, S., Hieshima, K., Izawa, D., Nakayama, T., Fujisawa, R., Ohyanagi, H., Yoshie, O. (2001) Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3(/CCL20 in mucosal epithelial cells through NF-(B Int. Immunol. 13,1255-1263[Abstract/Free Full Text]
    45. 45
  45. Kwon, J. H., Keates, S., Simeonidis, S., Grall, F., Libermann, T. A., Keates, A. C. (2003) ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3( gene expression in Caco-2 human colonic epithelial cells J. Biol. Chem. 278,875-884[Abstract/Free Full Text]
    46. 46
  46. Lin, R., Gewert, D., Hiscott, J. (1995) Differential transcriptional activation in vitro by NF-{kappa} B/Rel proteins J. Biol. Chem. 270,3123-3131[Abstract/Free Full Text]
    47. 47
  47. Ghosh, S., Karin, M. (2002) Missing pieces in the NF-{kappa}B puzzle Cell 109(Suppl.),S81-S96[CrossRef][Medline]
    48. 48
  48. Schutyser, E., Struyf, S., Menten, P., Lenaerts, J. P., Conings, R., Put, W., Wuyts, A., Proost, P., Van Damme, J. (2000) Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 {alpha} from normal and transformed cells J. Immunol. 165,4470-4477[Abstract/Free Full Text]
    49. 49
  49. Evans, S. M., Hahn, S. M., Magarelli, D. P., Koch, C. J. (2001) Hypoxic heterogeneity in human tumors: EF5 binding, vasculature, necrosis, and proliferation Am. J. Clin. Oncol. 24,467-472[CrossRef][Medline]
    50. 50
  50. Kitamuro, T., Takahashi, K., Ogawa, K., Udono-Fujimori, R., Takeda, K., Furujama, K., Nakayama, M., Sun, J., Fujita, H., Hida, W., et al (2003) Bach 1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells J. Biol. Chem. 278,9125-9133[Abstract/Free Full Text]
    51. 51
  51. Mizukami, Y., Jo, W. S., Duerr, E. M., Gala, M., Li, J., Zhang, X., Zimmer, M. A., Iliopoulos, O., Zukerberg, L. R., Kohgo, Y., et al (2005) Induction of interleukin-8 preserves the angiogenic response in HIF-1{alpha}-deficient colon cancer cells Nat. Med. 11,992-997[Medline]
    52. 52
  52. Schmedtje, J. F., Jr, Ji, Y. S., Liu, W. L., DuBois, R. N., Runge, M. S. (1997) Hypoxia induces cyclooxygenase-2 via the NF-{kappa}B p65 transcription factor in human vascular endothelial cells J. Biol. Chem. 272,601-608[Abstract/Free Full Text]
    53. 53
  53. Hayden, M. S., West, A. P., Ghosh, S. (2006) NF-{kappa}B and the immune response Oncogene 25,6758-6780[CrossRef][Medline]
    54. 54
  54. Kaufman, P. A., Weinberg, J. B., Greene, W. C. (1992) Nuclear expression of the 50- and 65-kD Rel-related subunits of nuclear factor-{kappa} B is differentially regulated in human monocytic cells J. Clin. Invest. 90,121-129[Medline]
    55. 55
  55. Chen, F. E., Ghosh, G. (1999) Regulation of DNA binding by Rel/NF-{kappa}B transcription factors: structural views Oncogene 18,6845-6852[CrossRef][Medline]
    56. 56
  56. Leeper-Woodford, S. K., Detmer, K. (1999) Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-{kappa}B expression Am. J. Physiol. 276,L909-L916[Medline]
    57. 57
  57. Schreiber, J., Jenner, R. G., Murray, H. L., Gerber, G. K., Gifford, D. K., Young, R. A. (2006) Coordinated binding of NF-{kappa}B family members in the response of human cells to lipopolysaccharide Proc. Natl. Acad. Sci. USA 103,5899-5904[Abstract/Free Full Text]
    58. 58
  58. Saccani, A., Schioppa, T., Porta, C., Biswas, S. K., Nebuloni, M., Vago, L., Bottazzi, B., Colombo, M. P., Mantovani, A., Sica, A. (2006) p50 Nuclear factor-{kappa}B overexpression in tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance Cancer Res. 66,11432-11440[Abstract/Free Full Text]
    59. 59
  59. Watanabe, N., Iwamura, T., Shinoda, T., Fujita, T. (1997) Regulation of NF{kappa}B1 proteins by the candidate oncoprotein BCL-3: generation of NF-{kappa}B homodimers from the cytoplasmic pool of p50–p105 and nuclear translocation EMBO J. 16,3609-3620[CrossRef][Medline]
    60. 60
  60. Deng, W. G., Wu, K. K. (2003) Regulation of inducible nitric oxide synthase expression by p300 and p50 acetylation J. Immunol. 171,6581-6588[Abstract/Free Full Text]
    61. 61
  61. Agrawal, A., Cha-Molstad, H., Samols, D., Kushner, I. (2001) Transactivation of C-reactive protein by IL-6 requires synergistic interaction of CCAAT/enhancer binding protein β (C/EBP β) and Rel p50 J. Immunol. 166,2378-2384[Abstract/Free Full Text]
    62. 62
  62. Cao, S., Zhang, X., Edwards, J. P., Mosser, D. M. (2006) NF-{kappa}B1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages J. Biol. Chem. 281,26041-26050[Abstract/Free Full Text]
    63. 63
  63. Driessler, F., Venstrom, K., Sabat, R., Asadullah, K., Schottelius, A. J. (2004) Molecular mechanisms of interleukin-10-mediated inhibition of NF-{kappa}B activity: a role for p50 Clin. Exp. Immunol. 135,64-73[CrossRef][Medline]
    64. 64
  64. Rudders, S., Gaspar, J., Madore, R., Voland, C., Grall, F., Patel, A., Pellacani, A., Perrella, M. A., Libermann, T. A., Oettgen, P. (2001) ESE-1 is a novel transcriptional mediator of inflammation that interacts with NF-{kappa} B to regulate the inducible nitric-oxide synthase gene J. Biol. Chem. 276,3302-3309[Abstract/Free Full Text]
    65. 65
  65. Ben Baruch, A. (2006) Inflammation-associated immune suppression in cancer: the roles played by cytokines, chemokines and additional mediators Semin. Cancer Biol. 16,38-52[CrossRef][Medline]
    66. 66
  66. Rubie, C., Oliveira-Frick, V., Rau, B., Schilling, M., Wagner, M. (2006) Chemokine receptor CCR6 expression in colorectal liver metastasis J. Clin. Oncol. 24,5173-5174[Free Full Text]



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