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Mice.
CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007). Cd45.1 and Rag2-/- mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively. For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice. 5 x 106 cells were used per transfer. 6 wk later, TCRbeta+CD4+ gated cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine were analyzed for Foxp3-GFP expression. All analyses and experiments were performed on animals at 6-8 wk of age. Animals were housed under specific pathogen-free conditions at the animal facility of the Skirball Institute, and experiments were performed in accordance with approved protocols for the New York University Institutional Animal Care and Usage Committee.
Isolation of PBMCs, CD4+ T cells, and culture conditions.
Human PBMCs were isolated by Ficoll (Biochrom) density gradient centrifugation and CD4+ T cells were then isolated using the Dynal CD4+ Isolation kit (Invitrogen) according to the manufacturer's instructions. The purity of CD4+ T cells was initially tested by flow cytometry and was >/=95%. Cells were stimulated with the following combination of mAbs to T cell surface molecules (Meiler et al., 2008): anti-CD2 (clone 4B2 and 6G4; 0.5 microg/ml), anti-CD3 (clone OKT3; 0.5 microg/ml), and anti-CD28 mAb (clone B7G5; 0.5 microg/ml; all from Sanquin) and cultured in serum-free AIM -V medium (Life Technologies) with the addition of 1 nmol/liter IL-2 (Roche). TGF-beta (R&D Systems) was used at 5 ng/ml, if not stated otherwise. A combination of PMA (25 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) was used.
Human CD4+ CD127- CD25high and CD4+ CD127+ CD25neg cells were purified by flow cytometry using anti-CD127, anti-CD25-PC5 (Beckman Coulter), and anti-CD4-FITC antibodies (Dako).
Mouse naive (CD62Lhi44lo25-) CD4+ T cells were purified by flow cytometry and activated in vitro with 5 microg/ml plate-bound anti-CD3 and 1 microg/ml soluble anti-CD28 antibodies (eBioscience) in RPMI supplemented with 10% FCS, 5 mM beta-mercaptoethanol, and antibiotics. Neutralizing anti-IFN-gamma and anti-IL-4 mAbs (BD) were used at 1 microg/ml concentrations when indicated.
In vitro T cell differentiation.
CD4+ CD45RA+ magnetically sorted (CD45RO depletion with AutoMACS; Miltenyi Biotec) cells were stimulated with immobilized plate-bound anti-CD3 (1 microg/ml; OKT3; IgG1) and anti-CD28 (2 microg/ml). For Th1 differentiation conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 microg/ml anti-IL-4, and 25 ng/ml IL-12 (R&D Systems). For Th2 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 25 ng/ml IL-4, and 5 microg/ml anti-IL-12 (R&D Systems). For T reg cell conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 ng/ml TGF-beta, 5 microg/ml anti-IL-12, 5 microg/ml anti-IL-4. For Th17 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 20 ng/ml IL-6, 5 ng/ml TGF-beta, 20 ng/ml IL-23 (Alexis Biochemicals Corp.), 10 ng/ml IL-1beta, 5 microg/ml anti-IL-4, and 5 microg/ml anti-IL-12 were used. Proliferating cells were expanded in medium containing IL-2. The cytokine profile of these cells demonstrated that IFN-gamma is the predominant cytokine in Th1 cells, IL-4 and IL-13 in Th2 cells, and IL-17 in Th17 cells (Akdis et al., 2000; Burgler et al., 2009).
Immunohistochemistry.
Human tonsils were obtained from tonsillectomy samples of hypertrophic and obstructive tonsils without a current infection. Ethical permission was obtained from Cantonal Ethics Commission, and informed consent was obtained from patients. Paraformaldehyde-fixed tonsil cryosections were stained with unconjugated rabbit IgG polyclonal antibody to human RUNX1 (Santa Cruz Biotechnology, Inc.) or unconjugated mouse IgG1 mAb to human RUNX3 (Abcam). After a washing step, the sections were stained with the corresponding secondary antibodies. RUNX1-binding antibodies were detected by using Alexa Fluor 633-conjugated goat anti-rabbit IgG and RUNX3-binding antibodies were detected by using Alexa Fluor 532-conjugated goat anti-mouse IgG1. Afterward, the sections were washed and in the case of RUNX3 staining a blocking step with an unconjugated mouse IgG1 mAb was used. Finally, the sections were stained with Alexa Fluor 488-conjugated mouse IgG1 mAb to human FOXP3 (eBioscience) or the corresponding isotype control. Tissue sections were stained with DAPI for the demonstration of nuclei and mounted with Prolong antifade (Invitrogen). Images were acquired and analyzed using the confocal microscope DMI 4000B and the TCS SPE system (both from Leica).
In vitro suppression assays.
Mouse Foxp3+ CD4+ CD8- T cells were FACS purified based on GFP expressed from a Foxp3-IRES-GFP knock-in allele (Foxp3GFP). Naive (CD62Lhi44lo25-) CD4+ T cells (effectors) were FACS-purified from Cd45.1 mice, then loaded with 5 microM CFSE (Invitrogen). Total splenocytes from C57BL/6 mice inactivated with 50 microg/ml mitomycin C (Sigma-Aldrich) for 45 min were used as APCs. A total of 4 x 105 CD4+ cells (CFSE-loaded CD25- plus Foxp3-GFP+) was mixed with 105 APCs + 1 microg/ml anti-CD3 mAb per well of a 96 well round bottom plate. Proliferation of the effector cells was analyzed by CFSE dilution. Apoptosis of the cells was investigated by annexin V staining and flow cytometry. Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.
Human naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA. Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 x 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells. T reg cell to responder cell ratio was 1:20, 1:10, and 1:5. To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group. Cells were stimulated with 2.5 microg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture. Gating on the CD4+CFSE+ T cells enabled the exclusion of APCs and T reg cells.
Cloning of the FOXP3 promoter, construction of mutant FOXP3 promoter, and RUNX expression plasmids.
The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate pGL3 FOXP3 -511/+176 (Mantel et al., 2006). Site-directed mutagenesis for the three putative RUNX binding sites in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions and confirmed by sequencing the DNA. The following primers and their complementary strands were used: foxp runx-333, forward 5'-CACTTTTGTTTTAAAAACTGTCCTTTCTCATGAGCCCTATTATC-3'; foxp runx-333 reverse 5'-GATAATAGGGCTCATGAGAAAGGACAGTTTTTAAAACAAAAGTG-3'; foxp runx-287 forward 5'-CCTCTCACCTCTGTCCTGAGGGGAAGAAATC-3'; foxp runx-287 reverse 5'-GATTTCTTCCCCTCAGGACAGAGGTGAGAGG-3'; foxp runx-53 forward 5'-GCTTCCACACCGTACAGCGTCCTTTTTCTTCTCGGTATAAAAG-3'; foxp runx-53 reverse 5'-CTTTTATACCGAGAAGAAAAAGGACGCTGTACGGTGTGGAAGC-3'.
The human RUNX1 fragment from the Addgene plasmid 12504 (Biggs et al., 2006) pFlagCMV2-AML1B was sub-cloned in the pEGFPN1 vector (Clontech Laboratories). The RUNX3 vector pCMV human RUNX3, which was a gift from K. Ito (Institute of Molecular and Cell Biology, Proteos, Singapore) was subcloned into pEGFPN1 vector (Clontech Laboratories; Yamamura et al., 2006).
Transfections and reporter gene assays.
T cells were rested in serum-free AIM-V medium overnight. 3.5 microg of the FOXP3 promoter luciferase reporter vector or a combination together with the RUNX1, RUNX3 pEGFPN1 vector, and 0.5 microg phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated using the program U-15. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega) according to the manufacturer's instructions. PMA/ionomycin was used to stimulate the cells, because the transfection was only transient and the luciferase assay required a strong and fast stimulation of the cells. To evaluate the effect of overexpression of RUNX1 or RUNX3 on FOXP3 protein levels, CD4+ T cells were preactivated with 2 microg/ml phytohemagglutinin (Sigma-Aldrich) in serum-free AIM-V medium in the presence of 1 nmol/liter IL-2 (Roche) for 12 h, and then transfected with the vector pEGFPN1 containing the RUNX1 or RUNX3 fragment using the Nucleofector system (Amaxa Biosystems) and the program T-23. FOXP3 expression was evaluated by flow cytometry after 48 h of culture in AIM-V medium containing 1 nmol/liter IL-2.
RNA interference.
CD4+ or naive CD4+ T cells were resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated with 2 microM siRNA using the Nucleofector technology program U-14 (Lonza). Five different Silencer or Silencer Select Pre-designed siRNAs for RUNX1 (Applied Biosystems) and three Silencer Pre-designed siRNAs for RUNX3 (Applied Biosystems) were tested, and the best was selected for all further experiments. The Silencer Negative Control #1 siRNA (Applied Biosystems) was used for normalization. Cells were then left unstimulated or were stimulated after 12 h with anti-CD2, anti-CD3, and anti-CD28. Cells were cultured in serum-free AIM-V medium with the addition of 1 nmol/l IL-2 (Roche). Cells were harvested for mRNA detection of the target genes after 24 h and for protein detection after 48 h.
RNA isolation and cDNA synthesis.
RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription of human samples was performed with reverse-transcription reagents (Fermentas) with random hexamers according to the manufacturer's protocol.
Real-time PCR.
PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer, 5'-GAAACAGCACATTCCCAGAGTTC-3'; FOXP3 reverse primer, 5'-ATGGCCCAGCGGATGAG-3'; EF-1a forward primer, 5'-CTGAACCATCCAGGCCAAAT-3'; and EF-1a reverse primer, 5'-GCCGTGTGGCAATCCAAT-3', as previously described (Mantel et al., 2007). GATA3 forward primer, 5'-GCGGGCTCTATCACAAAATGA-3'; and GATA3 reverse primer 5'-GCTCTCCTGGCTGCAGACAGC-3' (Mantel et al., 2007). T-bet forward primer, 5'-GATGCGCCAGGAAGTTTCAT-3'; T-bet reverse primer, 5'-GCACAATCATCTGGGTCACATT-3'; RORC2 forward primer, 5'-CAGTCATGAGAACACAAATTGAAGTG-3'; and RORC2 reverse primer 5'-CAGGTGATAACCCCGTAGTGGAT-3'. The prepared cDNAs were amplified using SYBR green PCR master mix (Fermentas) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
RUNX1 and RUNX3 mRNA was detected by using TaqMan Gene Expression Assays from Applied Biosystems and used according to the manufacturer's instruction using TaqMan master mix using a 7000 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding elongation factor (EF)-1alpha or by using the 18S rRNA Gene Expression Assay (Applied Biosystems) was performed to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative DeltaDeltaCT method (Applied Biosystems). The percentage of FOXP3 mRNA in siRNA-mediated RUNX knockdown cells was calculated in relation to cells, which were transfected with scrambled control siRNA. Arbitrary units show the 2-(Deltact) values multiplied by 10,000 incorporating the ct values of the gene of interest and the housekeeping gene.
Flow cytometry.
For analysis of human FOXP3 expression on the single-cell level, cells were first stained with the monoclonal CD4 mAb (Beckman Coulter), and after fixation and permeabilization, they were incubated with anti-human Foxp3-Alexa Fluor 488 antibody (BioLegend) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL; Beckman Coulter). A mouse IgG1 antibody (BioLegend) was used as an isotype control. Data were analyzed with the CXP software (Beckman Coulter). Cells were cultured with IL-2, and then left unstimulated or stimulated with anti-CD2/-CD3/-CD28 mAb.
Flow cytometry analyses of the mouse cells were performed on an LSRII (BD) and cell sorting was performed on a FACSAria (BD). All antibodies for these experiments were purchased from eBioscience or BD.
Western blotting.
For human RUNX1 and RUNX3 analysis on the protein level, 106 cells were lysed and loaded next to a protein-mass ladder (Invitrogen) on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (GE Healthcare). Unspecific binding was blocked with 3% milk in TBS Tween, and the membranes were subsequently incubated with a 1:1,000 dilution of rabbit anti-RUNX1 (ab11903; Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50; Santa Cruz Biotechnology, Inc.) in blocking buffer containing 3% milk in TBS Tween overnight at 4degreesC. The blots were developed using an anti-rabbit IgG HRP-labeled mAb (Cell Signaling Technology) and visualized with a LAS-1000 gel documentation system (Fujifilm). To confirm sample loading and transfer membranes were incubated in stripping buffer and reblocked for 1 h and reprobed using anti-GAPDH (6C5; Ambion) and developed using an anti-mouse IgG HRP-labeled mAb (Cell Signaling Technology).
Pull-down assay.
HEK293T cells were transfected with RUNX1 or RUNX3 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Cells were lysed by sonication in HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche Diagnostics). The cell lysate was precleared using streptavidin-agarose beads (GE Healthcare), incubated with biotinylated double-stranded oligonucleotides containing the wild-type or mutated RUNX binding sites, and polydeoxyinosinicdeoxycytidylic acid (Sigma-Aldrich). A combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used in the assay. DNA-bound proteins were collected with streptavidin-agarose beads, washed with HKMG buffer, and finally resuspended in NuPAGE loading buffer (Invitrogen Life Technologies), heated to 70degreesC for 10 min, and separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen Life Technologies). The proteins were electroblotted onto a PVDF membrane (GE Healthcare) and detected using RUNX1 or RUNX3 antibodies described in the previous section.
Promoter enzyme immuno assay.
As performed in the pull-down assay, HEK293T cells were transfected with RUNX1 or RUNX3 and subsequently lysed. Insoluble material was removed by centrifugation. 384-well plates, precoated with streptavidin (Thermo Fisher Scientific) were washed 3 times with washing buffer (PBS and 0.05% Tween 20). Biotinylated FOXP3 promoter/oligonucleotides probes containing the RUNX binding sites were added (1 pmol per well; 50 fmol/microl) and incubated for 1 h at room temperature. Either a combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used or single oligonucleotides were used in the assay. After 3 washing steps with washing buffer, the nuclear extract was added (concentration > 0.2 microg/microl) and incubated overnight at 4degreesC. The lysates were incubated with 10 microg of poly-deoxyinosinic-deoxycytidylic acid (Sigma-Aldrich). The plate was washed with HKMG buffer and incubated with a 1:1000 dilution of rabbit anti-RUNX1 (ab11903, Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50, Santa Cruz Biotechnology, Inc.) at 4degreesC for 2 h. After three washing steps with HKMG buffer, a secondary antibody (anti-rabbit IgG-HRP, 1:3,000 in HKMG buffer, Cell Signaling Technology) was added, and the plate was incubated for 1 h at 4degreesC. The wells were washed 4 times with HKMG buffer before adding the substrate reagent (R&D Systems). The colorimetric reaction was stopped by adding 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader (Berthold Technologies).
ChIP.
Human naive CD4+ T cells were cultured either with IL-2 only or with IL-2, anti-CD2/3/28, and TGF-beta for 72 h, and protein-DNA complexes were fixed by cross-linking with formaldehyde in a final concentration of 1.42% for 15 min. Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were subsequently harvested. The ChIP assay was performed as described in the fast chromatin immunoprecipitation method (Nelson et al., 2006). Cells were lysed with immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, NP-40 [0.5% vol/vol]) containing phosphatase (Roche) and protease inhibitors cocktails (Roche), the nuclear pellet was washed, the chromatin was sheared by sonication and incubated with antibodies for RUNX1 (H-65 X; Santa Cruz Biotechnology, Inc.), RUNX3 (H-50 X; Santa Cruz Biotechnology, Inc.), CBFbeta (PEBP2beta; FL-182 X; Santa Cruz Biotechnology, Inc.), and as controls normal rabbit IgG (Santa Cruz Biotechnology, Inc.), anti-human RNA polymerase II antibody, and mouse control IgG (both from SA Biosciences). The cleared chromatin was incubated with protein A agarose beads and, after several washing steps, DNA was isolated with 10% (wt/vol) Chelex 100 resin. Samples were treated with proteinase K at 55degreesC for 30 min. The proteinase K was then inactivated by boiling the samples for 10 min. The purified DNA was used in a real-time PCR reaction. Specific primers for the FOXP3 promoter, spanning the region from -87 to -3, FOXP3 promoter forward primer 5'-AGAGGTCTGCGGCTTCCA-3', FOXP3 promoter reverse primer 5'-GGAAACTGTCACGTATCAAAAACAA-3', or control GAPDH primer (SA Biosciences) for the RNA polymerase II were used. A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting ORF-free intergenic DNA (SA Biosciences) was used. The fold enrichment in site occupancy was calculated incorporating IgG control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).
Quantification of cytokine levels.
IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-gamma secretion was assessed using fluorescent bead-based technology. The Bio-Plex-hu Cytokine Panel, 17-Plex Group 1 was used according to the manufacturer's instructions (Bio-Rad Laboratories). Fluorescent signals were read and analyzed using the Bio-Plex 200 System (Bio-Rad Laboratories).
Online supplemental material.
Fig. S1 shows the induction of RUNX1, RUNX3, and FOXP3 mRNA in human CD4+ T cells after anti-CD2/3/28 mAb and TGF-beta stimulation. Fig. S2 shows decreased RUNX1 and RUNX3 mRNA and protein expression after siRNA-mediated knockdown and decreased FOXP3 expression in human CD4+ T cells after RUNX1 and RUNX3 knockdown. Fig. S3 shows the quantification of IL-4, IL-5, IL-10, IL-13, and IFN-gamma levels in control siRNA transfected or RUNX1 and RUNX3 siRNA transfected human CD4+ T cells. Fig. S4 shows the putative RUNX binding sites in the FOXP3 core promoter sequence of human, mouse, and rat. Fig. S5 shows the induction of FOXP3 protein after overexpression of RUNX1 and RUNX3 in human CD4+ T cells. Fig. S6 shows that endogenous IL-4 and IFN-gamma do not effect Foxp3 expression in naive CD4+ T cells of CbfbF/F CD4-cre and CbfbF/F control mice, which were stimulated with anti-CD3 and anti-CD28 mAbs, IL-2 and TGF-beta in the absence or presence of anti-IL-4 and anti-IFN-gamma neutralizing mAbs. Fig. S7 shows similar cell death (A) and proliferation (B) of Foxp3+ and Foxp3- cells in CbfbF/F CD4-cre and CbfbF/F control mice cultures. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090596/DC1.

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Supplementary Material
[Supplemental Material]


Input: RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-beta stimulation in the presence of IL-2. Real-time PCR of human naive CD4+ T cells after 48 h of culture. Bars show the mean +/- SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-beta in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-beta and IL-2. Bars show the mean +/- SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean +/- SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-beta in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

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Input: RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127-, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127-, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-beta, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25- cells. Bars show the mean +/- SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy. Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls. HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining. Data shown are representative from one of the three tissue samples with similar results. Bars, 5 microm. Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

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Input: Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide-transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean +/- SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFbeta to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-beta. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean +/- SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P < 0.05

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Input: Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector. Bars show the mean luciferase activity +/- SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene. Bars show the mean luciferase activity +/- SD measured as arbitrary light units of three independent experiments.

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Input: Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, +/- 10 nM retinoic acid (RA), and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-gamma expression. One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 x 106 cells per transfer). 6 wk later, TCRbeta+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.

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Input: CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8- T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-beta. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown.

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Input:
Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1.
Our previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation.

bound

main NF-kappaB-binding site 2

production

production

production

suppressed

induced

prevented

induction

activation

up-regulation

stimulates

production

induced

up-regulation

up-regulation

mediated

induced

induced

production

induction

production

induced

up-regulation

up-regulation

operate

induced

Involvement

activation

up-regulation


Input:
Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells.
Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation.

expressed

Tax

expression

Tax

increased

degradation

enhanced

degradation

degradation

IkappaBalpha

degradation

IkappaBalpha


Input:
Apoptosis-resistant T cells have a deficiency in NF-kappaB-mediated induction of Fas ligand transcription.
Apoptosis induced through the TCR in CD4+ T cells is mostly mediated by the inducible expression of Fas ligand (FasL) as a primary event leading to the commitment to death. To gain a better understanding of the transcriptional events that regulate this expression, we took advantage of our previously described mutant Jurkat cells. These cells are deficient in FasL expression and apoptosis induced upon TCR triggering, although their cytokine (IL-2 and IFN-gamma) production is normal. Here we show that both a FasL- and a consensus NF-kappaB- reporter construct are inefficiently induced in these cells compared to wild-type cells. In addition, we demonstrate that the inducible transcriptional activity of the FasL reporter is abolished by specific inhibitors of NF-kappaB activation. Thus, we could trace the deficit of the mutant cells to an inefficient NF-kappaB activation, evidencing a relevant role for NF-kappaB in the regulation of FasL expression in activated T cells. Furthermore, our results suggest that the induction of FasL versus cytokine gene expression is differentially sensitive to NF-kappaB deprivation.

expression

FasL

expression

FasL

production

production

IFN-gamma

expression

FasL

expression

FasL

deficiency

induction

deficient

induced

normal

production

induction

transcription

inducible

expression

induced

expression

induction

expression

role

regulation

regulation

expression

transcription

Fas ligand


Input:
NF-kappaB regulates Fas/APO-1/CD95- and TCR- mediated apoptosis of T lymphocytes.
The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-kappaB has been shown to play a role in protecting cells against death mediated by TNF We show here that NF-kappaB also has a role in regulating Fas/APO-1/CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-kappaB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-kappaB activation by a soluble peptide inhibitor or a dominant form of the NF-kappaB inhibitor, IkappaB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IkappaB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-kappaB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-kappaB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-kappaB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance.

Transfection

Transfection

transfection

transfection

Transfection

Transfection

transfection

transfection


Input:
Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model.
Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

expression

expression

expression

expression

RelB

expression

expression

TNF receptors 1

expression

expression

PKC isoforms alpha

expression

beta I

expression

beta II

expression

mu

expression

RelB

important

expression

TNF receptors 1

important

expression

important

expression

PKC isoforms alpha

important

expression

beta I

important

expression

beta II

important

expression

mu

important

expression

RelB


Input:
IL-2-mediated cell cycle progression and inhibition of apoptosis does not require NF-kappa B or activating protein-1 activation in primary human T cells.
The IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-kappa B and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-kappa B and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce I kappa B alpha degradation or NF-kappa B activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-kappa B nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells.

induce

degradation

induce

activation

activation

degradation

I kappa B alpha


Input:
Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation.
We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development.

express

mut-I kappa B

expression

TCR beta-chain

appeared normal

expression


Input:
Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation.
Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.

decreases

phosphorylation

results

activation

activation

increases

regulation

regulation

regulation

regulation

affecting

phosphorylation


Input:
Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis.
Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs.

ligation

ligation

ligation

interactions

expression

expression

expression

negative

expression

expression

intercellular adhesion molecule-1

secretion

granulocyte macrophage colony-stimulating factor

secretion

secretion

secretion

tumor necrosis factor-a

down-regulated

expression

Stimulation

enhanced

expression

IFN-gamma

enhanced

expression

tumor necrosis factor-alpha

enhanced

expression

interleukin (IL)-1beta

enhanced

expression

up-regulated

ligation

expression

up-regulated

ligation

secretion


Input:
Essential role of alveolar macrophages in intrapulmonary activation of NF-kappaB.
Acute inflammatory injury in rat lung induced by deposition of immunoglobulin G immune complexes requires expression of cytokines and chemokines as well as activation of the transcription factor nuclear factor (NF)-kappaB. There is little direct evidence regarding the role of alveolar macrophages in these activation events. In the present studies, rat lungs were depleted of alveolar macrophages by airway instillation of liposome-encapsulated dichloromethylene diphosphonate. These procedures, which greatly reduced the number of retrievable alveolar macrophages, suppressed activation of lung NF-kappaB in the inflammatory model. In addition, bronchoalveolar lavage levels of tumor necrosis factor-alpha (TNF-alpha) and the CXC chemokine, macrophage inflammatory protein-2, were substantially reduced. In parallel, upregulation of the lung vascular adhesion molecule, intercellular adhesion molecule-1, was greatly reduced by intrapulmonary instillation of phosphonate-containing liposomes. Neutrophil accumulation and development of lung injury were also substantially diminished. Lung instillation of TNF-alpha in alveolar macrophage-depleted rats restored the NF-kappaB activation response in whole lung. These data suggest that, in this inflammatory model, initial activation of NF-kappaB occurs in alveolar macrophages and the ensuing production of TNF-alpha may propagate NF-kappaB activation to other cell types in the lung.

production

TNF-alpha

reduced

TNF-alpha

reduced

macrophage inflammatory protein-2

reduced

upregulation

upregulation

intercellular adhesion molecule-1


Input:
Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP.
The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10-->R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10-->R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.

associated

tumor necrosis factor receptor-associated factor (TRAF) 1

associated

associated

association

association

associate

transformation

associated


Input:
Regulation of Fas ligand expression and cell death by apoptosis-linked gene 4.
Programmed cell death is a process required for the normal development of an organism. One of the best understood apoptotic pathways occurs in T lymphocytes and is mediated by Fas/Fas ligand (FasL) interaction. During studies of apoptosis induced by T cell-receptor engagement, we identified ALG-4F, a truncated transcript that prevents T cell-receptor-induced FasL upregulation and cell death. Overexpression of full-length ALG-4 induced transcription of FasL and, consequently, apoptosis. These results indicate that ALG-4 is necessary and sufficient for FasL expression. Fas/FasL interaction initiates cell death in many other systems, and its dysregulation is a mechanism by which several pathologic conditions arise. Understanding the molecular mechanisms of FasL regulation could be very useful in elucidating how these diseases develop and in identifying potential therapeutic targets.

interaction

Fas

FasL

interaction

Fas

FasL

expression

Fas ligand

Overexpression

expression

FasL

prevents

induced

induced

upregulation

upregulation

FasL

Overexpression

Overexpression

induced

Overexpression

transcription

necessary and sufficient

expression

Regulation

expression

apoptosis-linked gene 4

dysregulation

interaction

transcription

FasL


Input:
High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages.
It is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages.

expression

increased

expression


Input:
Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3.
NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.

activation

activation


Input:
Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.
Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.

associates

recruit

form complexes

coexpressed

NF-kappaB-inducing kinase

coexpressed

coexpressed

TNF receptor-associated factor 6

inhibition

activated

toll-like receptor 2

responds


Input:
Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells.
CD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported.

interactions

CD40 ligand

associated

Janus kinase 3

binding

phosphorylation

Janus kinase 3

induced

induced

induction

phosphorylation

induction

binding


Input:
Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas.
T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity.

binding

RelA

localization

nuclear

localization

RelA

nuclear

suppress

localization

activation

binding

following

degraded

degraded

IkappaBalpha

alter

alter

RelA


Input:
Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels.
OBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis.

expression

vWF

expression

intercellular adhesion molecule 1

expression

E-selectin

secretion

secretion

vWF

secretion

secretion

vWF

increased

secretion


Input:
The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation.
A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.

engages

latent membrane protein 1

TRADD

engages

latent membrane protein 1

associates

TRADD

interact

TRADD

interact

associates

associates

association

TRADD

association


Input:
Regulatory effects of interleukin-11 during acute lung inflammatory injury.
The role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1.

production

TNF-alpha

reduced levels

TNF-alpha

diminished

up-regulation

diminished

production

reduced

up-regulation

up-regulated

up-regulation

up-regulation

affect

affect


Input:
ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha.
We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha.

bound

GABP

activity

TNF-alpha

production

TNF-alpha

eliminated

resulted in an enhancement

when

activity

resulted in an enhancement

CAT

inducible

bound

interferon-gamma

regulate

TNF-alpha

role

production


Input:
Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression.
Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

decreased or absent

transcriptionally active

transcriptionally active


Input:
Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds.
Different glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor.

production

production

production

TNF-alpha

production

effects

production


Input:
The p53 paradox in the pathogenesis of tumor progression.
Recent evidence suggests that the p53 molecule appears in two different forms: the mutant p53 that stimulates tumor progression, and wild type p53 that inhibits tumor progression. In addition, it has been established that tumor necrosis factor-alpha (TNF-alpha) can activate the expression of wild type p53 in concert with the nuclear transcription factor, NF-kappa B. Both TNF-alpha and NF-kappa B are also involved in the stimulation of the pathway that leads to the expression of major histocompatibility complex (MHC) class I molecules and, hence, antigen presentation to the T cells. In this paper we shall advance the hypothesis that: (i) TNF-alpha indirectly controls immune surveillance; and (ii) TNF-alpha controls DNA repair and tumor suppression through the regulation of wild type p53. Thus, it is hypothesized that elevated TNF-alpha is primarily responsible for promoting tumor progression.

expression

activate

TNF-alpha

expression

elevated

TNF-alpha

regulation

TNF-alpha


Input:
Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis.
The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease.

association

NIK

association

IkappaB-alpha

association

-epsilon

disappearance

IkappaB-alpha

disappearance

IkappaB-epsilon

reassembly

IkappaB-alpha

reassembly

IkappaB-epsilon

associated

IKK-alpha

associated

IKK-beta

associated

NIK

phosphorylate

IkappaB-alpha

stimulated

IKK-beta

increased

stimulated

stimulation

-beta

stimulation

IKK-alpha

led

disappearance

led

reassembly

phosphorylate

NIK

phosphorylate

effect

IKK-alpha

effect

IKK-beta

remained

associated


Input:
Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells.
We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription.

decrease

Jun

interact synergistically to raise

c-Fos

stimulates

PKC

levels

c-Fos

levels

Jun

increase

immunoreactivity

had only a slight effect

immunoreactivity

increase

c-Fos

activation

PKC

effect

levels

immunoreactivity

c-Fos

immunoreactivity

Jun


Input:
Functional interaction between the two zinc finger domains of the v-erb A oncoprotein.
The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function.

acts as a dominant negative inhibitor

retinoic acid receptor

rendered defective

v-erb A

restore function

v-erb A

crucial

v-erb A

important

v-erb A

role

v-erb A


Input:
Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant.
The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.

binds

enhancer A

binds as a homodimer

binds as a homodimer

form heterodimers

c-rel

form heterodimers

c-rel

form heterodimers

v-rel

form heterodimers

v-rel

bind

bind

form homo-

form homo-

heterodimers

c-rel

heterodimers

v-rel

heterodimers

c-rel

heterodimers

v-rel

binding

binding

binding

c-

binding

v-rel

reduces or abolishes

binding


Input:
NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I.
The effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters.

expressing

Tax

expression

Tax

expressing

Tax

expressing

Tax

Expression

Tax

expression

Tax

resulted

Expression

dependent

dependent

transcription

transcription

granulocyte-macrophage colony stimulating factor


Input:
Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes.
The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression.

interaction


Input:
v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.
The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein.

bind

v-erbA

CAII

bind

c-erbA

CAII

overexpression

v-erbA

coexpressing

v-erbA

coexpressing

c-erbA

overexpression

v-erbA

extinguish

overexpression

regulation

suppressed

transcription

v-erbA

down-regulation

transcription

activated

transcription

overexpression

overexpression

required

overexpression

v-erbA

regulation

c-erbA

CAII

target

down-regulation

v-erbA

transcription

CAII

transcription

CAII

transcription

CAII


Input:
NF-X2 that binds to the DRA X2-box is activator protein 1. Expression cloning of c-Jun.
Human class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression.

complex

c-Jun

c-Fos

form

c-Fos

c-Jun

bind

c-Fos

bind

c-Jun

exist

c-Fos

exist

c-Jun

expression

DRA

repression

c-Fos

expression

repression

c-Jun

expression


Input:
Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor.
The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation.

gene expression

jun

gene expression

fos

expression

jun

expression

fos

expression

c-jun

expression

c-jun

expression

jun-B

expression

expression

c-fos

expression

fos-B

detectable

transcripts

increases

levels

increase

transcription

prolongation in the half-life

c-jun

induction

jun-B

prolonged

expression

increases

c-fos

induction

c-fos

induction

fos-B

induced

expression

Regulation

gene expression

effects

involving

involving

expression

regulates

expression

transcripts

c-jun

levels

c-jun

transcription

c-jun


Input:
cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins.
We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.

bind

JunD

bind

JunB

binding

JunB

in the presence of

c-Fos

binding


Input:
Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells.
The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression.

interacted

symmetric NF-kappa B site

interferon beta

increased

transcription

increased

Inducibility

transcription

c-fms

Inducibility

IFN-beta

Inducibility

IFN-alpha


Input:
The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter.
Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.

induction

expression

c-fos

expression

c-jun

abrogates

induction

abolishes

induction

abrogate

induction

induction

chloramphenicol acetyltransferase

inducibility

promoter

induction

promoter

enhance

expression

induction

induction

promoter

role

induction

effect

inducibility

induction


Input:
The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation.
Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process.

expression

c-jun

expression

c-jun


Input:
Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein.
Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex.

bind

DCoH

complex

DCoH

HNF-1 alpha

binding

HNF-1 alpha

distribution

DCoH

stabilized

DCoH

HNF-1 alpha

required

complex

change

complex

binding


Input:
Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes.
Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.

homodimer

binds

B2 site

proenkephalin

heterotetramer

abolish

activation

activates

transcription

activation

proenkephalin

promoter

activation

proenkephalin

promoter

mediated

activation

transcription

proenkephalin


Input:
ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.
Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.

interact

interact

binding

specific

GM-CSF

promoter

expression

GM-CSF

expressed

expressed

production

GM-CSF

exception

binding

transactivates

GM-CSF

promoter

increased

expression

increases

increases

transcription

transactivate

GM-CSF

transactivate

GM-CSF

induction

GM-CSF

promoter

transactivation

mediating

increased

increased

production

involved

mediating

transcription

GM-CSF


Input:
Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer.
The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors.

expressed

lox

down-regulate

expressed

regulation


Input:
Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.
Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

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