Molecular Regulation of Monocyte Chemoattractant Protein-1 Expression in Pancreatic ß-Cells
1 Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium}l, http://www.100md.com
2 Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium}l, http://www.100md.com
ABSTRACT}l, http://www.100md.com
Pancreatic ß-cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine-induced MCP-1 gene expression in human and rat pancreatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluorescence-activated cell sorting-purified rat ß-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1ß-responsive enhancer region between -2,180 bp and -2,478 bp. Mutation of either of the two nuclear factor (NF)-{kappa} B sites present in this region abrogated IL-1ß-induced MCP-1 promoter activity. Binding of NF-{kappa} B to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/p50 heterodimers and p65 homodimers. In vivo binding of NF-B was confirmed by chromatin immunoprecipitation assay. Blocking of NF-B activation in cytokine-exposed primary ß-cells by an adenovirus overexpressing a nondegradable form of IB or by pyrrolidine dithiocarbamate decreased IL-1ß-induced MCP-1 mRNA expression. We conclude that NF-{kappa} B plays an important role for MCP-1 expression in ß-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.
There is strong evidence that type 1 diabetes develops as a consequence of autoimmunity, leading to ß-cell destruction (1). In the early stages of insulitis, activated macrophages and T-cells are attracted to the islets and produce cytokines and free radicals, which contribute to ß-cell dysfunction and death (2,3). The mechanisms regulating the attraction of monocytes and T-cells to the islets remain to be clarified. We have recently described that human and rat islets, and islets isolated from prediabetic NOD mice, express the chemokine MCP-1 (4,5). Subsequent studies by other groups confirmed that human islets produce and secrete biologically active MCP-1 (6). Importantly, MCP-1 released by pancreatic islets plays a relevant role for the clinical outcome of islet allografts in type 1 diabetic patients, as suggested by the observation that high MCP-1 secretion is negatively correlated with successful engraftment and long-lasting insulin independence (6). These intriguing observations suggest that MCP-1 and other chemokines produced by the cytokine-exposed islet cells (7,8) are involved in the recruitment and activation of immune cells during both insulitis and islet graft destruction.
MCP-1 belongs to the C-C family of chemokines (9–11) and is produced by diverse cell types in response to cytokines, oxidized LDL, and bacterial lipopolysaccharide (12–14). The differential induction and binding of activated transcription factors to the promoter region of the MCP-1 gene provides a critical regulatory step, allowing expression of the chemokine in a cell- and stimulus-specific manner (15). MCP-1 attracts monocytes (16), T-cells (17), and natural killer cells (18) to the site of inflammation, and it also regulates the release of arachidonic acid and the respiratory burst by monocytes (19,20). Local release of MCP-1 probably contributes to the inflammatory response in rheumatoid arthritis, atherosclerosis, glomerular diseases, and pulmonary granulomatous vasculitis (21–27). Of note, transgenic mice expressing MCP-1 under control of the insulin promoter develop an intense insulitis (28).7y|4*, http://www.100md.com
In vivo footprinting in murine fibroblasts showed that tumor necrosis factor (TNF)-{alpha} induces site occupancy in both proximal and distal regions of the MCP-1 promoter (29). The distal region is located more than 2.2 kb upstream of the transcription start site and is required for induction of MCP-1 expression by TNF-{alpha} and IL-1ß in mouse fibroblasts (30) and in human mesangial cells (31). This enhancer region contains two binding sites for NF-{kappa} B that are conserved in the human, mouse, and rat MCP-1 genes (30,32). Moreover, in the human gene a proximal NF-{kappa} B site cooperates with an adjacent AP-1 site for induction by TNF-{alpha} or IL-1ß (33). In human osteoblastic cells interferon (IFN)- stimulates MCP-1 transcription through a complex element in the promoter region, namely an IFN response-inhibitory sequence (IRIS) adjacent to an IFN- activation site (-activated sequence [GAS]) (34). This element is conserved in the human, mouse, and rat genes (35). It is noteworthy that IFN- does not stimulate MCP-1 expression in rat ß-cells or human pancreatic islets (5), suggesting that regulation of this gene in ß-cells differs from other cell types and is mostly dependent on IL-1ß. Unfortunately, there are no available data on the promoter regulation of MCP-1 gene expression in insulin-producing cells.
In the present study, we investigated the transcriptional regulation by cytokines of the rat MCP-1 gene in ß-cells. To achieve this goal, we carried out transfection experiments in an insulinoma cell line, INS-1E, and in fluorescence-activated cell sorting (FACS)-purified rat primary ß-cells. We demonstrated by site-directed mutagenesis and gel-shift assays that NF-B is involved in the induced activity of the MCP-1 promoter in vitro. In vivo binding of NF-{kappa} B to the enhancer region in the rat MCP-1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, followed by real-time PCR quantification. Finally, by using an adenovirus expressing a nondegradable form of IB, or the NF-B inhibitor pyrrolidine dithiocarbamate (PDTC), we showed that NF-B translocation to the nucleus is necessary for MCP-1 mRNA induction in response to cytokines.m74[jg&, http://www.100md.com
RESEARCH DESIGN AND METHODSm74[jg&, http://www.100md.com
Plasmid constructs and mutagenesis.m74[jg&, http://www.100md.com
The MCP-1 promoter and enhancer region were isolated by PCR using the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Mannheim, Germany) on 0.5 µg rat genomic DNA prepared from liver as described (36). The primers used were based on the Genbank sequence (accession number AF079313). For the promoter region, the forward primer (position -515 to -499) was 5'-CGAATTCAAGCTCTTCGGTTTGG with an added EcoRI site, and the reverse primer (position +31 to +53) was 5'-CGGGATCCGGCTTCAGTGAGAG with an added BamHI site. For the enhancer region, the forward primer (position -2,478 to -2,459) was 5'-GCTCTAGACCAGTAGAGGCTCAATCC with an added XbaI site, and the reverse primer (position -2,464 to -2,180) was 5'-CGGAATTCACTTTACTCTGTCAAC with an added EcoRI site. The amplified products were cloned into pBluescript KS (Stratagene, La Jolla, CA). Clones were sequenced and shown to be 99% identical to the published sequence. The promoter region (-514 to +53) was isolated from pBluescript by digestion with EcoRV (5' end) and BamHI (3' end). It was later cloned into the SmaI-BglII sites of pGL3-Basic (Promega, Madison, WI) to obtain the pMCP-1-514 luc construct. To build the pMCP-1-514(enh) luc construct, the promoter region was isolated by EcoRI (5' end)-BamHI (3' end) digestion, and the enhancer region was isolated by XbaI (5' end)-EcoRI (3' end) digestion from the pBluescript. The promoter and the enhancer fragments were ligated into the BglII and NheI sites of pGL3-Basic. Mutations for the two NF-B sites at positions -2,250 and -2,276 were generated on pMCP-1-514(enh) luc by PCR (37) using the Expand High Fidelity PCR system. The pairs of primers were as follows: 5'-CTGAAGGGTCTGAACTTCCAATAC and 5'-GAGAGATATTTAGCTGGAATATGAGC for mutation in NF-B site A and 5'-CAATACTGCCTCAGAATGAATTTCCACAC and 5'-GAAGTTCCCAGACCCTTCAGGAGAG for mutation in NF-{kappa} B site B (mutated bases are underlined). All mutations were confirmed by sequencing.
Cell culture, transient transfection, and luciferase assay.#:'c@, 百拇医药
Insulin-producing INS-1E cells, a kind gift from Prof. C. Wollheim, Center Medical Universitaire, Geneva, Switzerland, were cultured in RPMI-1640 (with Glutamax-1) supplemented with 10 mmol/l HEPES, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mmol/l sodium pyruvate, and 50 µmol/l 2-mercaptoethanol (38). RINm5F insulinoma cells were cultured in RPMI-1640 medium with Glutamax-1 supplemented with 10% FCS. Islets were isolated from the pancreas of male Wistar rats by collagenase digestion in cold Hank’s salt solution followed by hand picking under a stereomicroscope. Rat ß-cells were FACS purified from the islets, as previously described (39). These preparations contained >95% viable ß-cells and were cultured in Ham’s F-10 medium supplemented with 10 mmol/l glucose, 2 mmol/l glutamine, 0.5% BSA, and 50 µmol/l 3-isobutyl-1-methylxanthine. The presence of 3-isobutyl-1-methylxanthine at this concentration preserves ß-cell survival in culture (40) with minimal effects on cAMP formation in the absence of adenylate cyclase activators (41). INS-1E and primary ß-cells were cotransfected with the luciferase test plasmids, and pRL-CMV (Promega) as an internal control, by lipofection with lipofectAMINE (Invitrogen, Baesley, Scotland), as previously described (42), and were exposed for 16 h to recombinant human IL-1ß (10–50 units/ml; kindly provided by Dr. C. W. Reynolds, National Cancer Institute, Bethesda, MD) and recombinant murine IFN- (100–1,000 units/ml; Invitrogen) in various combinations. The concentrations of cytokines used in the experiments with primary ß-cells and INS-1E cells were selected based on dose-response studies (2 and B.K. and D.L.E., unpublished data). Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega). Values obtained for the test plasmids in unstimulated cell extracts were at least 90-fold higher than values for the promoterless vector pGL3-Basic (0.1 light unit). Test values were corrected for the luciferase activity value of the internal control plasmid, pRL-CMV. The results for cytokine-exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in control condition, taking control (no cytokine added) value as 1.
Electrophoretic mobility shift assays.&hz{k&, 百拇医药
Nuclear extracts were prepared from INS-1E cells as described (43). Nuclear proteins (4 µg) were preincubated with 1 µg poly(dIdC) in 20 µl containing 10 mmol/l HEPES (pH 7.9), 50 mmol/l KCl, 5 mmol/l MgCl2, 0.05 mmol/l EDTA, 0.5 mmol/l dithiothreitol, and 10% glycerol for 10 min at 4°C before addition of 50 molar excess of competing oligonucleotide, as indicated, and radiolabeled probe (40,000 cpm). The incubation was continued for 20 min at 4°C. Where indicated, 1 µl antibodies specific for p50, p65, or Stat1 (Santa Cruz Biotechnology, Santa Cruz, CA) were added for 20 min at room temperature. Stat1 antibody was used as a control for nonspecific binding. The samples were electrophoresed on 5% polyacrylamide gels in 25 mmol/l Tris, 25 mmol/l boric acid, and 0.5 mmol/l EDTA. Oligonucleotides for electrophoretic mobility shift assays (EMSAs) were as follows (upper strand shown): MCP-1 enhancer NF-{kappa} B site A, 5'-agAAGGGTCTGGGAACTTCCAATA; MCP-1 enhancer NF-{kappa} B site B, 5'-agAGAATGGGAATTTCCACACTCTT; and NF-B consensus, 5'-agCTTCAGAGGGGACTTTCCGAGA. The lowercase letters represent added nucleotides to allow end labeling with the Klenow fragment.
ChIP assays.9a8f}/}, 百拇医药
Cross-linked chromatin from 5.107 RINm5F cells, left untreated or exposed to IL-1ß for 30 min, 2 h, or 4 h, was prepared as described (44), with the following modifications: ß-glycerophosphate and sodium butyrate were omitted and 1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin were added as protease inhibitors. Samples were sonicated with a Branson Sonifier 250 for four 20-s pulses. SDS was added at a final concentration of 1%, and samples were rotated for 1 h at room temperature and centrifuged at 12,000 rpm for 15 min. Supernatants were further sonicated for two 20-s pulses to an average size of 500–1,500 bp. For immununoprecipitation, 60 µl chromatin corresponding to 5.106 cells was diluted 10-fold in 0.01% SDS, 1.1% Triton X-100, 155 mmol/l NaCl, 1 mmol/l EDTA, and 10 mmol/l Tris-HCl (pH 8) with protease inhibitors. The chromatin solution was precleared with 20 µl 50% protein A-Sepharose containing 250 µg/ml BSA and 100 µg/ml sonicated herring sperm DNA for 45 min at 4°C. Precleared chromatin (20 µl saved as total input chromatin) was rotated without or with 3 µg rabbit polyclonal antibody overnight at 4°C. The antibody was directed against p65. As a negative control, cross-linked chromatin was incubated overnight in the absence of antibody. Protein A-Sepharose (50 µl) containing BSA and herring sperm DNA was added, and the incubation was continued for 3 h at 4°C. Immune complexes were washed twice with each of the following buffers: low salt buffer (0.1% SDS, 1% Triton X-100, 150 mmol/l NaCl, 2 mmol/l EDTA, and 20 mmol/l Tris-HCl, pH 8), high-salt buffer (0.1% SDS, 1% Triton X-100, 500 mmol/l NaCl, 2 mmol/l EDTA, and 20 mmol/l Tris-HCl, pH 8), LiCl buffer (0.25 mol/l LiCl, 1% Igepal, 1% deoxycholate, 1 mmol/l EDTA, and 10 mmol/l Tris-HCl, pH 8), TE buffer (1 mmol/l EDTA and 10 mmol/l Tris-HCl, pH 8). Immune complexes were eluted with two times 200 µl 0.1 mol/l NaHCO3, 1% SDS for 15 min. Cross-links were reversed by addition of NaCl at a final concentration of 200 mmol/l and incubation overnight at 65°C. Samples were treated with 40 µg/ml proteinase K for 1 h at 42°C, extracted with phenol-chloroform and chloroform, and ethanol-precipitated. Pellets were resuspended in 25–30 µl H2O.
For quantification of the precipitated sequences, real-time PCR of MCP-1 enhancer sequence was performed with a single-step PCR on a LightCycler instrument (Roche Diagnostics, Mannheim, Germany) as described previously (45,46). The rat MCP-1 enhancer region was specifically detected using the same primers as those used for the plasmid construction (see above). PCR amplification using a LightCycler instrument was carried out in 20 µl reaction containing 3.5 mmol/l MgCl2, 0.5 µmol/l forward and reverse primers, 2 µl FastStart SYBR Green 1 mix (Roche), and 2 µl template genomic DNA/plasmid in a glass capillary tube. The amplification of the MCP-1 enhancer was started with an initial activation step at 95°C (10 min) followed by 45 rounds of amplification at 95°C (10 s) for denaturation, 58°C (10 s) for annealing, and 72°C (14 s) for extension.49d2w/, 百拇医药
External standards for MCP-1 enhancer sequence were prepared by 10-fold serial dilutions of a plasmid containing the rat MCP-1 enhancer region; each dilution was ascribed an arbitrary copy number. Each experimental run consisted of six dilutions of the external standard, a negative control without a template, and the samples, containing total input DNA or immunoprecipitated products. The real-time PCR assays were repeated twice. The MCP-1 enhancer content of the immunoprecipitated reactions were determined as follows: the values obtained for each p65 antibody immunoprecipitation were corrected by substracting their corresponding background values (no Ab added) and further normalized by their individual input chromatin values to equalize 100%. The results were expressed in arbitrary units, defined by comparisons with the serial dilutions of the standard plasmid preparation (see above).
Infection with recombinant adenoviruses and treatment with PDTC.4, 百拇医药
ß-Cells were cultured overnight, as described above, and then infected with control virus AdGFP, encoding the green fluorescence protein gene (47), or with AdI{kappa} B(SA)2, expressing a NF-B-super-repressor (see below). As an additional control, a group of ß-cells was left noninfected. The recombinant replicative-deficient adenovirus containing a mutated nondegradable I{kappa} B{alpha} (AdI{kappa} B[SA]2) was prepared as previously reported (48) and was a kind gift from Dr. C. Jobin, Division of Digestive Diseases and Nutrition, University of North Carolina, Chapel Hill, North Carolina. ß-cells were infected at a multiplicity of infection 7.5 for 2 h at 37°C. We have previously demonstrated that AdIB(SA)2, at this multiplicity of infection, prevents cytokine and double-stranded RNA- induced NF-{kappa} B activation, inducible nitric oxide synthase and Fas mRNA expression, and ß-cell death (47,49) without affecting ß-cell insulin content, glucose-induced insulin release (47), or the expression of genes involved in differentiated ß-cell functions (8). In additional experiments, rat islets were pretreated with 100 µmol/l PDTC (Sigma, Belgium) for 1 h and then exposed for 6 or 24 h to IL-1ß plus IFN- in the continuous presence of PDTC (same concentrations and culture conditions as above). We have previously shown that 100 µmol/l PDTC prevents cytokine-induced NF-B activation in both human and rat pancreatic islets (50), a finding confirmed in the present series of experiments (data not shown).
RT-PCR analysis.m, 百拇医药
RT-PCR was performed on poly(A)+ RNA as described (8). Primers for MCP-1 were 5'-ACCTGCTGCTACTCATTCAC-3' and 5'-CTACAGAAGTGCTTGAGGTG-3' (325 bp). The primers for glyceraldehyde 3-phosphate dehydrogenase were described previously (7). The ethidium bromide-stained agarose gels were photographed under ultraviolet transillumination using a Kodak Digital Science DC 120 camera (Eastman Kodak, Brussels, Belgium). Abundance of the PCR products were assessed by Biomax one-dimensional image analysis software (Kodak), and MCP-1 mRNA contents are expressed as optical densities corrected for GAPDH. Cytokines do not modify GAPDH expression in primary ß-cells (8,51 and present data).m, 百拇医药
Statistical analysis.m, 百拇医药
Results are given as means ± SE. Comparisons versus the respective control groups were performed using the Student’s paired t test.m, 百拇医药
RESULTSm, 百拇医药
Cytokine regulation of MCP-1 expression in INS-1E cells.
To determine whether cytokines induce MCP-1 mRNA expression in INS-1E cells, as previously described for primary ß-cells and RINm5F cells (4,5), these cells were exposed for 6 or 24 h to IL-1ß, IFN-, or a combination of both cytokines, and MCP-1 mRNA content was measured by RT-PCR. There was no detectable MCP-1 expression in unstimulated cells or cells exposed to IFN- alone, but MCP-1 expression was induced in cells exposed to IL-1ß for 6 and 24 h, with higher value at 6 h. This increase was not potentiated by IFN-. These findings are similar to our previous observations indicating that IL-1ß-induced MCP-1 expression in rat ß-cells and human islets is not modified by IFN- (4,5). Thus, most of the subsequent experiments on MCP-1 promoter regulation were performed in the presence of IL-1ß alone.]*, http://www.100md.com
fig.ommtted]*, http://www.100md.com
MCP-1 mRNA expression in INS-1E cells. INS-1E cells were cultured for 6 and 24 h in control conditions (no cytokine added) or with IL-1ß (10 units/ml [IL]), IFN- (100 units/ml [IFN]), or IL-1ß plus IFN-. The concentration of cytokines was determined by dose-response studies (data not shown). MCP-1 and GAPDH mRNA content were analyzed by RT-PCR. The MCP-1 mRNA contents are expressed as ODs corrected for GAPDH. The results are the means ± SE of three independent experiments. P < 0.05, P < 0.01, P < 0.001 vs. control.
Identification of the cytokine-responsive element in the MCP-1 promoter.c\a.[, 百拇医药
To determine the MCP-1 promoter regions responsive to IL-1ß, the promoter fragment from nucleotides -514 to +53 relative to the transcription start site, with or without the enhancer fragment between -2,180 to -2,478 relative to the transcription start site, were cloned into the luciferase reporter plasmid pGL3 and transfected into FACS-purified ß-cells treated or not with IL-1ß. In IL-1ß-treated cells, the luciferase activity of the constructs pMCP-1-514(enh) luc was induced threefold, whereas pMCP-1-514 luc did not significantly respond to IL-1ß . This lack of response of the promoter region alone, even though there is a potential NF-{kappa} B binding site in this region, indicates a role for the potential NF-{kappa} B binding sites in the enhancer region . Similar results were observed in INS-1E cells transfected with the same constructs. Thus, IL-1ß induced a significant increase in the activity of pMCP-1-514[enh] but not of pMCP-1-514 luc. Due to the large number of cells required, the subsequent experiments to elucidate the regulatory elements in the MCP-1 enhancer were performed in INS-1E cells.
fig.ommtted4q, 百拇医药
. Schematic representation of the rat MCP-1 promoter. The arrow indicates the transcriptional start site. Putative binding sites for transcription factors are indicated by boxes. The promoter and the NF-B mutants in the enhancer region cloned upstream of the luciferase reporter gene are indicated in the lower part of the figure. mutA, mutated NF-B site at -2,265; mutB, mutated NF-B site at -2,291; pMCP-1-514[enh], MCP-1 promoter sequence including the enhancer region.4q, 百拇医药
fig.ommtted4q, 百拇医药
MCP-1 promoter activity after cytokine treatment of primary rat ß-cells. ß-cells were transfected with the wild-type constructs shown in and either left untreated (control [C]) or treated with IL-1ß (IL) for 16 h. Relative luciferase activity values are expressed as % of control. The data shown are means ± SE of four independent experiments. *P < 0.05 vs. respective control.4q, 百拇医药
fig.ommtted4q, 百拇医药
Identification of the cytokine-responsive elements in the MCP-1 promoter in INS-1E cells. INS-1E cells were transfected with the same wild-type and mutant constructs shown in and left untreated (control [C]) or treated with IL-1ß (IL) for 16 h. Relative luciferase activity values are expressed as percent of control. The data shown are means ± SE of four independent experiments. ***P < 0.001 vs. respective control.
The enhancer region contains two potential NF-{kappa} B binding sites . Therefore, we mutated each NF-{kappa} B site separately and denoted the mutated constructs as pMCP-1-514[enh]mutA luc and pMCP-1-514[enh]mutB luc. The fold induction of MCP-1 promoter activity in response to IL-1ß was reduced by 75 and 80% for mutA and mutB, respectively . These results indicate that both binding sites are important for IL-1ß-induced MCP-1 promoter activity.&x, 百拇医药
NF-{kappa} B binding to the MCP-1 promoter in vitro.&x, 百拇医药
To characterize the factors binding to the IL-1ß-responsive elements in the enhancer region, we performed EMSAs using probes spanning the NF-{kappa} B sites from nucleotides -2,272 to -2,297 containing the first NF-{kappa} B motif of the enhancer (site A) and from -2,242 to -2,266 containing the second NF-{kappa} B motif of the enhancer (site B). Based on our previous observation that the maximal IL-1ß-induced NF-B binding activity in vitro in insulin-producing cells takes place after 30 min (42,52), this time point was selected for in vitro determination of nuclear factor binding. Two complexes, denoted by "a" and "b," were induced in cells stimulated with IL-1ß, with a higher affinity for the probe of site B . The nature of complexes a and b was further identified by competition and supershift experiments. The two complexes were specifically competed by an excess of unlabelled probe (, lane 3, and , lane 10) and by a consensus NF-{kappa} B oligonucleotide (, lane 4, and , lane 11). An antibody directed against the NF-{kappa} B family member p50 supershifted complex b (, lane 5, and , lane 12), whereas an antibody directed against the NF-B family member p65 supershifted both complexes a and b (, lane 6, and , lane 13). An unrelated antibody against Stat1 had no effect on the migration of the two complexes (, lane 7, and , lane 14). These results suggest that complex a contains p65 homodimers, whereas complex b consists of p65 and p50 heterodimers.
fig.ommttedj, 百拇医药
NF-B binding to the MCP-1 promoter. A: EMSAs were performed in electrophoresis buffer Tris-borate with an oligonucleotide spanning from nucleotides -2,272 to -2,297 containing the first NF-B motif of the enhancer region (site A). B: EMSAs were performed with an oligonucleotide spanning from -2,242 to -2,266 containing the second NF-{kappa} B motif of the enhancer region (site B). Experiments were done with nuclear extracts from INS-1E cells untreated (control [C]) or treated with IL-1ß (IL) for 30 min or with nuclear extracts treated with IL-1ß and competing oligonucleotides (lanes 3–4 and 10–11) or with specific antibodies against p50 or p65 (lanes 5–6 and 12–13). a, p65 homodimers; b, p65/p50 complexes. The figure is representative of two to three similar experiments.j, 百拇医药
NF-{kappa} B binding to the MCP-1 promoter in vivo.j, 百拇医药
To confirm in vivo our in vitro observations indicating NF-{kappa} B binding to the enhancer region, we performed ChIP assays with an antibody directed against the NF-{kappa} B p65 protein. ChIP assay allows for detailed analyses of protein-DNA interactions in a native chromatin environment (53), and the detection of association of rare transcription factor complexes can be achieved by real-time PCR quantification (45). We currently observed IL-1ß-induced NF-{kappa} B binding in vivo by quantification of the rat MCP-1 enhancer region with real-time PCR (see RESEARCH DESIGN AND METHODS). NF-{kappa} B binding was detected as early as 30 min following IL-1ß exposure, with continuously increasing values at 2 and 4 h. The rat MCP-1 enhancer was undetectable in unstimulated control cells.
fig.ommtted71z#g, 百拇医药
In vivo binding of NF-B to the MCP-1 promoter in insulin-producing cells as evaluated by chromatin immunoprecipitation assay followed by real-time PCR analysis. RINm5F cells were either exposed to IL-1ß for 30 min, 2 h, or 4 h or left untreated (control). Real-time PCR analyses were performed in duplicate for the MCP-1 enhancer region and results are expressed in arbitrary units (AU) as detailed in RESEARCH DESIGN AND METHODS. The figure shows data from two independent experiments.71z#g, 百拇医药
Effect of an I{kappa} B{alpha} super-repressor protein on cytokine-mediated MCP-1 gene expression.71z#g, 百拇医药
Overexpression of the I{kappa} B super-repressor by an adenovirus, AdI{kappa} B(SA)2, with serine 32 and 36 substituted by alanine residues, specifically blocks NF-{kappa} B activation without interfering with other transcription factors (47,48). We currently observed that AdI{kappa} B(SA)2 partially blocked cytokine-induced MCP-1 mRNA expression (Fig 7A). Thus, the optical density (OD) values of AdI{kappa} B(SA)2 plus cytokines were 55 ± 9% of the values observed in noninfected cells exposed to the same cytokines (n = 3; P < 0.05), while infection with the control virus AdGFP did not modify the effects of cytokines (; OD 86 ± 33% of the values observed in noninfected cells exposed to cytokines; n = 3). To further evaluate the role of NF-{kappa} B for cytokine-induced MCP-1 expression, rat islets were exposed to cytokines in the presence of the NF-{kappa} B blocker PDTC (50; ). There was no detectable MCP-1 expression in unstimulated cells or cells exposed to PDTC alone . MCP-1 mRNA was induced by cytokines after 6 and 24 h, and this induction was, to a major extent, prevented by PDTC. The results, expressed in OD after correction for GAPDH mRNA, for experiments 1 and 2, respectively, were as follows: cytokines-6 h, 1.96 and 1.61; PDTC plus cytokines-6 h, 0.54 and 0.47 (inhibition 72 and 71%, respectively); cytokines-24 h, 1.09 and 1.17; and PDTC plus cytokines-24 h, below detection limit .
fig.ommtted&jtusx0, http://www.100md.com
The effect of IB(SA)2 expression and PDTC exposure on cytokine-induced transcription of MCP-1. A: Rat ß-cells were infected with the recombinant adenoviruses expressing GFP (AdGFP), the IB super-repressor protein (AdIB[SA]2), or left uninfected (no virus). Twenty-four hours after infection, cells were exposed to IL-1ß (50 units/ml) plus IFN- (1,000 units/ml) for 24 h. B: Rat islets were exposed to PDTC for 1 h before treatment with cytokines (same concentrations as in ) for 6 or 24 h. MCP-1 and GAPDH mRNA contents were analyzed by RT-PCR. The figures shown are representative for three and two similar experiments, respectively. The observed ODs are provided in RESULTS.&jtusx0, http://www.100md.com
DISCUSSION&jtusx0, http://www.100md.com
MCP-1 expression is induced by cytokines in pancreatic ß-cells and probably plays a role in attracting monocytes and T-cells to the site of insulitis (5,7,54). Indeed, the temporal pattern of MCP-1 expression, along with other CC chemokines, in the islets of NOD mice is well correlated with the progression of insulitis and ß-cell destruction (5,54), and transgenic MCP-1 expression in ß-cells induces mononuclear cell infiltration (28). In this context, it is of relevance to understand the molecular regulation of MCP-1 expression in the ß-cells.
In the present study, we identified the transcription factor NF-{kappa} B as a key regulator of MCP-1 promoter activity. Mutation of either of the two NF-{kappa} B binding sites, located in the enhancer region at -2,242 and at -2,272, respectively, abrogated IL-1ß-induced MCP-1 promoter activity. Moreover, we detected the binding of NF-{kappa} B p50 and p65 factors to these two sites by EMSAs and supershift experiments. In vivo binding of NF-{kappa} B to the enhancer region was confirmed by ChIP assays. Finally, overexpression of an I{kappa} B super-repressor, AdI{kappa} B(SA)2, or preculture in the presence of the NF-{kappa} B blocker PDTC decreased cytokine-induced MCP-1 mRNA expression. The proximal promoter region by itself does not respond to cytokines, suggesting no role in insulin-producing cells for neither the putative NF-{kappa} B and AP1 binding sites (33), nor for the IRIS and GAS elements (34) located in this region.65osvm, 百拇医药
Stereospecific alignment between adjacent cis elements has been observed in several genes, including those containing {kappa} B motifs (55). The two NF-{kappa} B binding sites in the enhancer region of MCP-1 promoter, site A and site B, are not identical in sequence, suggesting that they may serve distinct functions. Although higher affinity of NF-{kappa} B binding was observed for site B than A (Fig. 5), the fold induction of MCP-1 promoter activity in response to IL-1ß was reduced by 75 and 80% for mutated site A and B, respectively (present data). The presence of these sites was also described as necessary to maintain TNF-induced MCP-1 gene promoter activity in mouse fibroblasts (30). On the other hand, insertion of a half or full helical turn between the {kappa} B sites, or site reversion between the two NF-{kappa} B binding sites in the enhancer region, did not affect TNF-induced MCP-1 gene promoter activity in these cells (30). Taken together with our findings, these results suggest that both binding sites for NF-{kappa} B are important for the IL-1ß-induced MCP-1 promoter activity in insulin-producing cells.
Mammalian cells contain five NF-{kappa} B family members: p50, p52, p65, c-Rel, and RelB. Interactions between these proteins have been characterized in detail (56). P65/p50 heterodimers, the most frequent dimers, and p65 homodimers are activators of gene transcription, whereas p50 homodimers, which lack a transactivation domain, are transcriptionally inactive (57). Our supershift experiments with antibodies directed against p65 and p50 proteins suggest a role for p65/p50 and p65/p65 dimers in the regulation of MCP-1 in insulin-producing cells. The differential induction and binding of activated transcription factors to the promoter region of the MCP-1 gene provides a critical regulatory step, allowing expression of the chemokine in a cell- and stimulus-specific manner (15). Of note, we have observed the presence of the same active dimers of NF-{kappa} B in the context of cytokine regulation of Fas expression in primary ß-cells (42).^ln+kp^, 百拇医药
In vitro determination of transcription factor binding to a promoter does not necessarily reflect the complex environment and the three-dimensional conformation of chromatin in a living cell. Indeed, it has been shown that the compact nature of chromatin can modulate accessibility to some DNA binding sites (45,53). Therefore, to confirm in vivo our observations of NF-{kappa} B binding to the rat MCP-1 promoter, we have performed ChIP assay, a novel method that allows detection of transcription factor binding in situ (53). We detected NF-{kappa} B binding in vivo at 30 min after IL-1ß stimulation, with subsequent increases at 2 and 4 h (present data). Interestingly, it has been previously reported the presence of two distinct waves of early and late NF-{kappa} B recruitment to target promoters in a murine macrophage cell line, depending on the acetylation state of chromatin (58). NF-{kappa} B binding to the MCP-1 enhancer was detected only after 90 min of LPS exposure to macrophage cells (58). Our data suggest an early and sustained IL-1ß-induced binding of NF-{kappa} B to the MCP-1 promoter in ß-cells , helping to explain the long-lasting effect of IL-1ß on MCP-1 mRNA expression .
The results described above suggest that NF-{kappa} B translocation to the nucleus is necessary for MCP-1 mRNA induction in ß-cells exposed to cytokines. Indeed, rat ß-cells infected with an adenovirus overexpressing the nondegradable I{kappa} B{alpha} super-repressor protein (AdI{kappa} B[SA]2) have a 50% lower induction of MCP-1 mRNA expression in response to IL-1ß. The partial blocking observed may be due to the limited efficiency of infection by the adenoviral system used, which is ~ 70–80% (47). These data are in good agreement with our previous microarray results, showing a 50–60% inhibition of cytokine-induced MCP-1 mRNA expression in ß-cells previously infected with the AdIB(SA)2 (8) and with the present observations that PDTC, a potent inhibitor of NF-B, blocked cytokine- induced MCP-1 mRNA expression >70% in cytokine-exposed islets. However, the fact that the observed inhibitory effect of NF-{kappa} B blockers on MCP-1 expression in vitro is not complete suggests that other transcription factors may also contribute to MCP-1 regulation in ß-cells.
As a whole, these findings suggest an essential role for NF-{kappa} B in the regulation of MCP-1 in ß-cells. Of note, in vivo overexpression of I{kappa} B{alpha} , a natural inhibitor of NF-{kappa} B, significantly decreases local MCP-1 and ICAM-1 expression, as well as recruitment of macrophages following iliac artery balloon angioplasty in rabbits, thus reducing arterial restenosis (59). This, and the fact that high basal MCP-1 production by human islets correlates with a poor clinical outcome following islet transplantation in patients with type 1 diabetes (6), suggests that adenovirus- or lentivirus-mediated NF-{kappa} B inhibition might be a useful approach to decrease mononuclear cell infiltration and graft dysfunction/destruction following islet transplantation.t}i+ivo, 百拇医药
ACKNOWLEDGMENTSt}i+ivo, 百拇医药
This work was supported by grants from the Juvenile Diabetes Foundation International, the Funds voor Wetenschappelijk Onderzoek (FWO), and the Fonds National de la Recherche Scientifique (FNRS), Belgium.
We thank the personnel from the Diabetes Research Center, VUB, involved in ß-cell purification; M.A. Neef, J. Schoonheydt, and M. Urbain, Laboratory of Experimental Medicine, ULB, for islet isolation; and E. Quartier for sequencing.x);+1l#, 百拇医药
REFERENCESx);+1l#, 百拇医药
Schranz DB, Lernmark A: Immunology in diabetes: an update. Diabete Metab Rev14 :3 –29,1998x);+1l#, 百拇医药
Eizirik DL, Mandrup-Poulsen T: A choice of death: the signal-transduction of immune-mediated ß-cell apoptosis. Diabetologia44 :2115 –2133,2001x);+1l#, 百拇医药
Mandrup-Poulsen T: The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia39 :1005 –1029,1996x);+1l#, 百拇医药
Chen MC, Schuit F, Eizirik DL: Identification of IL-1ß-induced messenger RNAs in rat pancreatic beta cells by differential display of messenger RNA. Diabetologia42 :1199 –1203,1999x);+1l#, 百拇医药
Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL: Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 ß-exposed human and rat islet cells. Diabetologia44 :325 –332,2001
Piemonti L, Leone BE, Nano R, Saccani A, Monti P, Maffi P, Bianchi G, Sica A, Peri G, Melzi R, Aldrighetti L, Secchi A, Di Carlo V, Allavena P, Bertuzzi F: Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation. Diabetes51 :55 –65,2002rl.{y^3, http://www.100md.com
Cardozo AK, Kruhoffer M, Leeman R, Orntoft T, Eizirik DL: Identification of novel cytokine-induced genes in pancreatic ß-cells by high-density oligonucleotide arrays. Diabetes50 :909 –920,2001rl.{y^3, http://www.100md.com
Cardozo AK, Heimberg H, Heremans Y, Leeman R, Kutlu B, Kruhoffer M, Orntoft T, Eizirik DL: A comprehensive analysis of cytokine-induced and nuclear factor-{kappa} B-dependent genes in primary rat pancreatic ß-cells. J Biol Chem276 :48879 –48886,2001rl.{y^3, http://www.100md.com
Luster AD: Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med338 :436 –445,1998rl.{y^3, http://www.100md.com
Baggiolini M, Dewald B, Moser B: Human chemokines: an update. Annu Rev Immunol15 :675 –705,1997rl.{y^3, http://www.100md.com
Rollins BJ: Chemokines. Blood90 :909 –928,1997rl.{y^3, http://www.100md.com
Arimilli S, Ferlin W, Solvason N, Deshpande S, Howard M, Mocci S: Chemokines in autoimmune diseases. Immunol Rev177 :43 –51,2000
Han KH, Quehenberger O: Ligands for peroxisome proliferator-activated receptor inhibit monocyte CCR2 expression stimulated by plasma lipoproteins. Trends Cardiovasc Med10 :209 –216,2000wxv!j, 百拇医药
Mackman N: Lipopolysaccharide induction of gene expression in human monocytic cells. Immunol Res21 :247 –251,2000wxv!j, 百拇医药
Roebuck KA, Carpenter LR, Lakshminarayanan V, Page SM, Moy JN, Thomas LL: Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-{kappa} B. J Leukoc Biol65 :291 –298,1999wxv!j, 百拇医药
Leonard EJ, Yoshimura T: Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today11 :97 –101,1990wxv!j, 百拇医药
Carr MW, Roth SJ, Luther E, Rose SS, Springer TA: Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A91 :3652 –3656,1994wxv!j, 百拇医药
Maghazachi AA, al-Aoukaty A, Schall TJ: C-C chemokines induce the chemotaxis of NK and IL-2-activated NK cells: role for G proteins. J Immunol153 :4969 –4977,1994
Stanimirovic D, Satoh K: Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation. Brain Pathol10 :113 –126,2000pp*vyp?, 百拇医药
Gu L, Tseng SC, Rollins BJ: Monocyte chemoattractant protein-1. Chem Immunol72 :7 –29,1999pp*vyp?, 百拇医药
Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D: Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A88 :5252 –5256,1991pp*vyp?, 百拇医药
Koch AE, Kunkel SL, Harlow LA, Johnson B, Evanoff HL, Haines GK, Burdick MD, Pope RM, Strieter RM: Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis. J Clin Invest90 :772 –779,1992pp*vyp?, 百拇医药
Brown Z, Robson RL, Westwick J: Regulation and expression of chemokines: potential role in glomerulonephritis. J Leukoc Biol59 :75 –80,1996pp*vyp?, 百拇医药
Lloyd CM, Dorf ME, Proudfoot A, Salant DJ, Gutierrez-Ramos JC: Role of MCP-1 and RANTES in inflammation and progression to fibrosis during murine crescentic nephritis. J Leukoc Biol62 :676 –680,1997
Boring L, Gosling J, Cleary M, Charo IF: Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature394 :894 –897,1998$, 百拇医药
Gosling J, Slaymaker S, Gu L, Tseng S, Zlot CH, Young SG, Rollins BJ, Charo IF: MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B. J Clin Invest103 :773 –778,1999$, 百拇医药
Flory CM, Jones ML, Warren JS: Pulmonary granuloma formation in the rat is partially dependent on monocyte chemoattractant protein 1. Lab Invest69 :396 –404,1993$, 百拇医药
Grewal IS, Rutledge BJ, Fiorillo JA, Gu L, Gladue RP, Flavell RA, Rollins BJ: Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1. J Immunol159 :401 –408,1997$, 百拇医药
Ping D, Jones PL, Boss JM: TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene. Immunity4 :455 –469,1996$, 百拇医药
Ping D, Boekhoudt GH, Rogers EM, Boss JM: Nuclear factor-{kappa} B p65 mediates the assembly and activation of the TNF-responsive element of the murine monocyte chemoattractant-1 gene. J Immunol162 :727 –734,1999
Stylianou E, Nie M, Ueda A, Zhao L: c-Rel and p65 trans-activate the monocyte chemoattractant protein-1 gene in interleukin-1 stimulated mesangial cells. Kidney Int56 :873 –882,1999s({!!, 百拇医药
Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T: NF-{kappa} B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol153 :2052 –2063,1994s({!!, 百拇医药
Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR: Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-{kappa} B and AP-1. Eur J Immunol27 :1091 –1097,1997s({!!, 百拇医药
Valente AJ, Xie JF, Abramova MA, Wenzel UO, Abboud HE, Graves DT: A complex element regulates IFN-{gamma} -stimulated monocyte chemoattractant protein-1 gene transcription. J Immunol161 :3719 –3728,1998s({!!, 百拇医药
Freter RR, Alberta JA, Hwang GY, Wrentmore AL, Stiles CD: Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-{kappa} B and a 90-kDa phosphoprotein coactivator. J Biol Chem271 :17417 –17424,1996
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. New York, Wiley & Sons,1994 , p.2.2.1 –2.2.32h/c|g@, 百拇医药
Coolidge CJ, Patton JG: Run-around PCR: a novel way to create duplications using polymerase chain reaction. Biotechniques18 :762 –764,19952h/c|g@, 百拇医药
Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology130 :167 –178,19922h/c|g@, 百拇医药
Pipeleers DG, in’t Veld PA, Van de Winkel M, Maes E, Schuit FC, Gepts W: A new in vitro model for the study of pancreatic A and B cells. Endocrinology117 :806 –816,19852h/c|g@, 百拇医药
Ling Z, Hannaert JC, Pipeleers D: Effect of nutrients, hormones and serum on survival of rat islet ß-cells in culture. Diabetologia37 :15 –21,19942h/c|g@, 百拇医药
Schuit FC, Pipeleers DG: Regulation of adenosine 3',5'-monophosphate levels in the pancreatic B cell. Endocrinology117 :834 –840,19852h/c|g@, 百拇医药
Darville MI, Eizirik DL: Cytokine induction of Fas gene expression in insulin-producing cells requires the transcription factors NF-{kappa} B and C/EBP. Diabetes50 :1741 –1748,2001
Schreiber E, Matthias P, Muller MM, Schaffner W: Rapid detection of octamer binding proteins with ’mini-extracts’, prepared from a small number of cells. Nucleic Acids Res17 :6419 ,1989r|(a, 百拇医药
Clayton AL, Rose S, Barratt MJ, Mahadevan LC: Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J19 :3714 –3726,2000r|(a, 百拇医药
Chakrabarti SK, James JC, Mirmira RG: Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1: importance of chromatin structure in directing promoter binding. J Biol Chem277 :13286 –13293,2002r|(a, 百拇医药
Ito S, Nakanishi H, Hirai T, Kato T, Kodera Y, Feng Z, Kasai Y, Ito K, Akiyama S, Nakao A, Tatematsu M: Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler. Cancer Lett183 :195 –203,2002r|(a, 百拇医药
Heimberg H, Heremans Y, Jobin C, Leemans R, Cardozo AK, Darville M, Eizirik DL: Inhibition of cytokine-induced nuclear factor-{kappa} B activation by adenovirus-mediated expression of a NF-{kappa} B super-repressor prevents ß-cell apoptosis. Diabetes50 :2219 –2224,2001
Jobin C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, Brenner DA, Sartor RB: Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor {kappa} B super-repressor in human intestinal epithelial cells. J Immunol160 :410 –418,19986k, 百拇医药
Liu D, Cardozo AK, Darville MI, Eizirik DL: Double-stranded RNA cooperates with interferon- and IL-1ß to induce both chemokine expression and nuclear factor-{kappa} B-dependent apoptosis in pancreatic ß-cells: potential mechanisms for viral-induced insulitis and ß-cell death in type 1 diabetes mellitus. Endocrinology143 :1225 –1234,20026k, 百拇医药
Flodstrom M, Welsh N, Eizirik DL: Cytokines activate the nuclear factor kappa B (NF-{kappa} B) and induce nitric oxide production in human pancreatic islets. FEBS Lett385 :4 –6,19966k, 百拇医药
Liu D, Darville M, Eizirik DL: Double-stranded ribonucleic acid (RNA) induces ß-cell Fas messenger RNA expression and increases cytokine-induced ß-cell apoptosis. Endocrinology142 :2593 –2599,20016k, 百拇医药
Darville MI, Ho YS, Eizirik DL: NF-{kappa} B is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells. Endocrinology141 :153 –162,2000
Kuo MH, Allis CD: In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment. Methods19 :425 –433,19997, http://www.100md.com
Cameron MJ, Arreaza GA, Grattan M, Meagher C, Sharif S, Burdick MD, Strieter RM, Cook DN, Delovitch TL: Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes. J Immunol165 :1102 –1110,20007, http://www.100md.com
Thanos D, Maniatis T: Virus induction of human IFN ß gene expression requires the assembly of an enhanceosome. Cell83 :1091 –1100,19957, http://www.100md.com
Baldwin AS Jr: The NF-{kappa} B and I{kappa} B proteins: new discoveries and insights. Annu Rev Immunol14 :649 –683,19967, http://www.100md.com
Ballard DW, Dixon EP, Peffer NJ, Bogerd H, Doerre S, Stein B, Greene WC: The 65-kDa subunit of human NF-{kappa} B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci U S A89 :1875 –1879,19927, http://www.100md.com
Saccani S, Pantano S, Natoli G: Two waves of nuclear factor {kappa} B recruitment to target promoters. J Exp Med193 :1351 –1359,20017, http://www.100md.com
Breuss JM, Cejna M, Bergmeister H, Kadl A, Baumgartl G, Steurer S, Xu Z, Koshelnick Y, Lipp J, De Martin R, Losert U, Lammer J, Binder BR: Activation of nuclear factor-{kappa} B significantly contributes to lumen loss in a rabbit iliac artery balloon angioplasty model. Circulation105 :633 –638,2002(Burak Kutlu Martine I. Darville Alessandra K. Cardozo and Décio L. Eizirik)
2 Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium}l, http://www.100md.com
ABSTRACT}l, http://www.100md.com
Pancreatic ß-cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine-induced MCP-1 gene expression in human and rat pancreatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluorescence-activated cell sorting-purified rat ß-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1ß-responsive enhancer region between -2,180 bp and -2,478 bp. Mutation of either of the two nuclear factor (NF)-{kappa} B sites present in this region abrogated IL-1ß-induced MCP-1 promoter activity. Binding of NF-{kappa} B to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/p50 heterodimers and p65 homodimers. In vivo binding of NF-B was confirmed by chromatin immunoprecipitation assay. Blocking of NF-B activation in cytokine-exposed primary ß-cells by an adenovirus overexpressing a nondegradable form of IB or by pyrrolidine dithiocarbamate decreased IL-1ß-induced MCP-1 mRNA expression. We conclude that NF-{kappa} B plays an important role for MCP-1 expression in ß-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.
There is strong evidence that type 1 diabetes develops as a consequence of autoimmunity, leading to ß-cell destruction (1). In the early stages of insulitis, activated macrophages and T-cells are attracted to the islets and produce cytokines and free radicals, which contribute to ß-cell dysfunction and death (2,3). The mechanisms regulating the attraction of monocytes and T-cells to the islets remain to be clarified. We have recently described that human and rat islets, and islets isolated from prediabetic NOD mice, express the chemokine MCP-1 (4,5). Subsequent studies by other groups confirmed that human islets produce and secrete biologically active MCP-1 (6). Importantly, MCP-1 released by pancreatic islets plays a relevant role for the clinical outcome of islet allografts in type 1 diabetic patients, as suggested by the observation that high MCP-1 secretion is negatively correlated with successful engraftment and long-lasting insulin independence (6). These intriguing observations suggest that MCP-1 and other chemokines produced by the cytokine-exposed islet cells (7,8) are involved in the recruitment and activation of immune cells during both insulitis and islet graft destruction.
MCP-1 belongs to the C-C family of chemokines (9–11) and is produced by diverse cell types in response to cytokines, oxidized LDL, and bacterial lipopolysaccharide (12–14). The differential induction and binding of activated transcription factors to the promoter region of the MCP-1 gene provides a critical regulatory step, allowing expression of the chemokine in a cell- and stimulus-specific manner (15). MCP-1 attracts monocytes (16), T-cells (17), and natural killer cells (18) to the site of inflammation, and it also regulates the release of arachidonic acid and the respiratory burst by monocytes (19,20). Local release of MCP-1 probably contributes to the inflammatory response in rheumatoid arthritis, atherosclerosis, glomerular diseases, and pulmonary granulomatous vasculitis (21–27). Of note, transgenic mice expressing MCP-1 under control of the insulin promoter develop an intense insulitis (28).7y|4*, http://www.100md.com
In vivo footprinting in murine fibroblasts showed that tumor necrosis factor (TNF)-{alpha} induces site occupancy in both proximal and distal regions of the MCP-1 promoter (29). The distal region is located more than 2.2 kb upstream of the transcription start site and is required for induction of MCP-1 expression by TNF-{alpha} and IL-1ß in mouse fibroblasts (30) and in human mesangial cells (31). This enhancer region contains two binding sites for NF-{kappa} B that are conserved in the human, mouse, and rat MCP-1 genes (30,32). Moreover, in the human gene a proximal NF-{kappa} B site cooperates with an adjacent AP-1 site for induction by TNF-{alpha} or IL-1ß (33). In human osteoblastic cells interferon (IFN)- stimulates MCP-1 transcription through a complex element in the promoter region, namely an IFN response-inhibitory sequence (IRIS) adjacent to an IFN- activation site (-activated sequence [GAS]) (34). This element is conserved in the human, mouse, and rat genes (35). It is noteworthy that IFN- does not stimulate MCP-1 expression in rat ß-cells or human pancreatic islets (5), suggesting that regulation of this gene in ß-cells differs from other cell types and is mostly dependent on IL-1ß. Unfortunately, there are no available data on the promoter regulation of MCP-1 gene expression in insulin-producing cells.
In the present study, we investigated the transcriptional regulation by cytokines of the rat MCP-1 gene in ß-cells. To achieve this goal, we carried out transfection experiments in an insulinoma cell line, INS-1E, and in fluorescence-activated cell sorting (FACS)-purified rat primary ß-cells. We demonstrated by site-directed mutagenesis and gel-shift assays that NF-B is involved in the induced activity of the MCP-1 promoter in vitro. In vivo binding of NF-{kappa} B to the enhancer region in the rat MCP-1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assay, followed by real-time PCR quantification. Finally, by using an adenovirus expressing a nondegradable form of IB, or the NF-B inhibitor pyrrolidine dithiocarbamate (PDTC), we showed that NF-B translocation to the nucleus is necessary for MCP-1 mRNA induction in response to cytokines.m74[jg&, http://www.100md.com
RESEARCH DESIGN AND METHODSm74[jg&, http://www.100md.com
Plasmid constructs and mutagenesis.m74[jg&, http://www.100md.com
The MCP-1 promoter and enhancer region were isolated by PCR using the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Mannheim, Germany) on 0.5 µg rat genomic DNA prepared from liver as described (36). The primers used were based on the Genbank sequence (accession number AF079313). For the promoter region, the forward primer (position -515 to -499) was 5'-CGAATTCAAGCTCTTCGGTTTGG with an added EcoRI site, and the reverse primer (position +31 to +53) was 5'-CGGGATCCGGCTTCAGTGAGAG with an added BamHI site. For the enhancer region, the forward primer (position -2,478 to -2,459) was 5'-GCTCTAGACCAGTAGAGGCTCAATCC with an added XbaI site, and the reverse primer (position -2,464 to -2,180) was 5'-CGGAATTCACTTTACTCTGTCAAC with an added EcoRI site. The amplified products were cloned into pBluescript KS (Stratagene, La Jolla, CA). Clones were sequenced and shown to be 99% identical to the published sequence. The promoter region (-514 to +53) was isolated from pBluescript by digestion with EcoRV (5' end) and BamHI (3' end). It was later cloned into the SmaI-BglII sites of pGL3-Basic (Promega, Madison, WI) to obtain the pMCP-1-514 luc construct. To build the pMCP-1-514(enh) luc construct, the promoter region was isolated by EcoRI (5' end)-BamHI (3' end) digestion, and the enhancer region was isolated by XbaI (5' end)-EcoRI (3' end) digestion from the pBluescript. The promoter and the enhancer fragments were ligated into the BglII and NheI sites of pGL3-Basic. Mutations for the two NF-B sites at positions -2,250 and -2,276 were generated on pMCP-1-514(enh) luc by PCR (37) using the Expand High Fidelity PCR system. The pairs of primers were as follows: 5'-CTGAAGGGTCTGAACTTCCAATAC and 5'-GAGAGATATTTAGCTGGAATATGAGC for mutation in NF-B site A and 5'-CAATACTGCCTCAGAATGAATTTCCACAC and 5'-GAAGTTCCCAGACCCTTCAGGAGAG for mutation in NF-{kappa} B site B (mutated bases are underlined). All mutations were confirmed by sequencing.
Cell culture, transient transfection, and luciferase assay.#:'c@, 百拇医药
Insulin-producing INS-1E cells, a kind gift from Prof. C. Wollheim, Center Medical Universitaire, Geneva, Switzerland, were cultured in RPMI-1640 (with Glutamax-1) supplemented with 10 mmol/l HEPES, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mmol/l sodium pyruvate, and 50 µmol/l 2-mercaptoethanol (38). RINm5F insulinoma cells were cultured in RPMI-1640 medium with Glutamax-1 supplemented with 10% FCS. Islets were isolated from the pancreas of male Wistar rats by collagenase digestion in cold Hank’s salt solution followed by hand picking under a stereomicroscope. Rat ß-cells were FACS purified from the islets, as previously described (39). These preparations contained >95% viable ß-cells and were cultured in Ham’s F-10 medium supplemented with 10 mmol/l glucose, 2 mmol/l glutamine, 0.5% BSA, and 50 µmol/l 3-isobutyl-1-methylxanthine. The presence of 3-isobutyl-1-methylxanthine at this concentration preserves ß-cell survival in culture (40) with minimal effects on cAMP formation in the absence of adenylate cyclase activators (41). INS-1E and primary ß-cells were cotransfected with the luciferase test plasmids, and pRL-CMV (Promega) as an internal control, by lipofection with lipofectAMINE (Invitrogen, Baesley, Scotland), as previously described (42), and were exposed for 16 h to recombinant human IL-1ß (10–50 units/ml; kindly provided by Dr. C. W. Reynolds, National Cancer Institute, Bethesda, MD) and recombinant murine IFN- (100–1,000 units/ml; Invitrogen) in various combinations. The concentrations of cytokines used in the experiments with primary ß-cells and INS-1E cells were selected based on dose-response studies (2 and B.K. and D.L.E., unpublished data). Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega). Values obtained for the test plasmids in unstimulated cell extracts were at least 90-fold higher than values for the promoterless vector pGL3-Basic (0.1 light unit). Test values were corrected for the luciferase activity value of the internal control plasmid, pRL-CMV. The results for cytokine-exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in control condition, taking control (no cytokine added) value as 1.
Electrophoretic mobility shift assays.&hz{k&, 百拇医药
Nuclear extracts were prepared from INS-1E cells as described (43). Nuclear proteins (4 µg) were preincubated with 1 µg poly(dIdC) in 20 µl containing 10 mmol/l HEPES (pH 7.9), 50 mmol/l KCl, 5 mmol/l MgCl2, 0.05 mmol/l EDTA, 0.5 mmol/l dithiothreitol, and 10% glycerol for 10 min at 4°C before addition of 50 molar excess of competing oligonucleotide, as indicated, and radiolabeled probe (40,000 cpm). The incubation was continued for 20 min at 4°C. Where indicated, 1 µl antibodies specific for p50, p65, or Stat1 (Santa Cruz Biotechnology, Santa Cruz, CA) were added for 20 min at room temperature. Stat1 antibody was used as a control for nonspecific binding. The samples were electrophoresed on 5% polyacrylamide gels in 25 mmol/l Tris, 25 mmol/l boric acid, and 0.5 mmol/l EDTA. Oligonucleotides for electrophoretic mobility shift assays (EMSAs) were as follows (upper strand shown): MCP-1 enhancer NF-{kappa} B site A, 5'-agAAGGGTCTGGGAACTTCCAATA; MCP-1 enhancer NF-{kappa} B site B, 5'-agAGAATGGGAATTTCCACACTCTT; and NF-B consensus, 5'-agCTTCAGAGGGGACTTTCCGAGA. The lowercase letters represent added nucleotides to allow end labeling with the Klenow fragment.
ChIP assays.9a8f}/}, 百拇医药
Cross-linked chromatin from 5.107 RINm5F cells, left untreated or exposed to IL-1ß for 30 min, 2 h, or 4 h, was prepared as described (44), with the following modifications: ß-glycerophosphate and sodium butyrate were omitted and 1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin were added as protease inhibitors. Samples were sonicated with a Branson Sonifier 250 for four 20-s pulses. SDS was added at a final concentration of 1%, and samples were rotated for 1 h at room temperature and centrifuged at 12,000 rpm for 15 min. Supernatants were further sonicated for two 20-s pulses to an average size of 500–1,500 bp. For immununoprecipitation, 60 µl chromatin corresponding to 5.106 cells was diluted 10-fold in 0.01% SDS, 1.1% Triton X-100, 155 mmol/l NaCl, 1 mmol/l EDTA, and 10 mmol/l Tris-HCl (pH 8) with protease inhibitors. The chromatin solution was precleared with 20 µl 50% protein A-Sepharose containing 250 µg/ml BSA and 100 µg/ml sonicated herring sperm DNA for 45 min at 4°C. Precleared chromatin (20 µl saved as total input chromatin) was rotated without or with 3 µg rabbit polyclonal antibody overnight at 4°C. The antibody was directed against p65. As a negative control, cross-linked chromatin was incubated overnight in the absence of antibody. Protein A-Sepharose (50 µl) containing BSA and herring sperm DNA was added, and the incubation was continued for 3 h at 4°C. Immune complexes were washed twice with each of the following buffers: low salt buffer (0.1% SDS, 1% Triton X-100, 150 mmol/l NaCl, 2 mmol/l EDTA, and 20 mmol/l Tris-HCl, pH 8), high-salt buffer (0.1% SDS, 1% Triton X-100, 500 mmol/l NaCl, 2 mmol/l EDTA, and 20 mmol/l Tris-HCl, pH 8), LiCl buffer (0.25 mol/l LiCl, 1% Igepal, 1% deoxycholate, 1 mmol/l EDTA, and 10 mmol/l Tris-HCl, pH 8), TE buffer (1 mmol/l EDTA and 10 mmol/l Tris-HCl, pH 8). Immune complexes were eluted with two times 200 µl 0.1 mol/l NaHCO3, 1% SDS for 15 min. Cross-links were reversed by addition of NaCl at a final concentration of 200 mmol/l and incubation overnight at 65°C. Samples were treated with 40 µg/ml proteinase K for 1 h at 42°C, extracted with phenol-chloroform and chloroform, and ethanol-precipitated. Pellets were resuspended in 25–30 µl H2O.
For quantification of the precipitated sequences, real-time PCR of MCP-1 enhancer sequence was performed with a single-step PCR on a LightCycler instrument (Roche Diagnostics, Mannheim, Germany) as described previously (45,46). The rat MCP-1 enhancer region was specifically detected using the same primers as those used for the plasmid construction (see above). PCR amplification using a LightCycler instrument was carried out in 20 µl reaction containing 3.5 mmol/l MgCl2, 0.5 µmol/l forward and reverse primers, 2 µl FastStart SYBR Green 1 mix (Roche), and 2 µl template genomic DNA/plasmid in a glass capillary tube. The amplification of the MCP-1 enhancer was started with an initial activation step at 95°C (10 min) followed by 45 rounds of amplification at 95°C (10 s) for denaturation, 58°C (10 s) for annealing, and 72°C (14 s) for extension.49d2w/, 百拇医药
External standards for MCP-1 enhancer sequence were prepared by 10-fold serial dilutions of a plasmid containing the rat MCP-1 enhancer region; each dilution was ascribed an arbitrary copy number. Each experimental run consisted of six dilutions of the external standard, a negative control without a template, and the samples, containing total input DNA or immunoprecipitated products. The real-time PCR assays were repeated twice. The MCP-1 enhancer content of the immunoprecipitated reactions were determined as follows: the values obtained for each p65 antibody immunoprecipitation were corrected by substracting their corresponding background values (no Ab added) and further normalized by their individual input chromatin values to equalize 100%. The results were expressed in arbitrary units, defined by comparisons with the serial dilutions of the standard plasmid preparation (see above).
Infection with recombinant adenoviruses and treatment with PDTC.4, 百拇医药
ß-Cells were cultured overnight, as described above, and then infected with control virus AdGFP, encoding the green fluorescence protein gene (47), or with AdI{kappa} B(SA)2, expressing a NF-B-super-repressor (see below). As an additional control, a group of ß-cells was left noninfected. The recombinant replicative-deficient adenovirus containing a mutated nondegradable I{kappa} B{alpha} (AdI{kappa} B[SA]2) was prepared as previously reported (48) and was a kind gift from Dr. C. Jobin, Division of Digestive Diseases and Nutrition, University of North Carolina, Chapel Hill, North Carolina. ß-cells were infected at a multiplicity of infection 7.5 for 2 h at 37°C. We have previously demonstrated that AdIB(SA)2, at this multiplicity of infection, prevents cytokine and double-stranded RNA- induced NF-{kappa} B activation, inducible nitric oxide synthase and Fas mRNA expression, and ß-cell death (47,49) without affecting ß-cell insulin content, glucose-induced insulin release (47), or the expression of genes involved in differentiated ß-cell functions (8). In additional experiments, rat islets were pretreated with 100 µmol/l PDTC (Sigma, Belgium) for 1 h and then exposed for 6 or 24 h to IL-1ß plus IFN- in the continuous presence of PDTC (same concentrations and culture conditions as above). We have previously shown that 100 µmol/l PDTC prevents cytokine-induced NF-B activation in both human and rat pancreatic islets (50), a finding confirmed in the present series of experiments (data not shown).
RT-PCR analysis.m, 百拇医药
RT-PCR was performed on poly(A)+ RNA as described (8). Primers for MCP-1 were 5'-ACCTGCTGCTACTCATTCAC-3' and 5'-CTACAGAAGTGCTTGAGGTG-3' (325 bp). The primers for glyceraldehyde 3-phosphate dehydrogenase were described previously (7). The ethidium bromide-stained agarose gels were photographed under ultraviolet transillumination using a Kodak Digital Science DC 120 camera (Eastman Kodak, Brussels, Belgium). Abundance of the PCR products were assessed by Biomax one-dimensional image analysis software (Kodak), and MCP-1 mRNA contents are expressed as optical densities corrected for GAPDH. Cytokines do not modify GAPDH expression in primary ß-cells (8,51 and present data).m, 百拇医药
Statistical analysis.m, 百拇医药
Results are given as means ± SE. Comparisons versus the respective control groups were performed using the Student’s paired t test.m, 百拇医药
RESULTSm, 百拇医药
Cytokine regulation of MCP-1 expression in INS-1E cells.
To determine whether cytokines induce MCP-1 mRNA expression in INS-1E cells, as previously described for primary ß-cells and RINm5F cells (4,5), these cells were exposed for 6 or 24 h to IL-1ß, IFN-, or a combination of both cytokines, and MCP-1 mRNA content was measured by RT-PCR. There was no detectable MCP-1 expression in unstimulated cells or cells exposed to IFN- alone, but MCP-1 expression was induced in cells exposed to IL-1ß for 6 and 24 h, with higher value at 6 h. This increase was not potentiated by IFN-. These findings are similar to our previous observations indicating that IL-1ß-induced MCP-1 expression in rat ß-cells and human islets is not modified by IFN- (4,5). Thus, most of the subsequent experiments on MCP-1 promoter regulation were performed in the presence of IL-1ß alone.]*, http://www.100md.com
fig.ommtted]*, http://www.100md.com
MCP-1 mRNA expression in INS-1E cells. INS-1E cells were cultured for 6 and 24 h in control conditions (no cytokine added) or with IL-1ß (10 units/ml [IL]), IFN- (100 units/ml [IFN]), or IL-1ß plus IFN-. The concentration of cytokines was determined by dose-response studies (data not shown). MCP-1 and GAPDH mRNA content were analyzed by RT-PCR. The MCP-1 mRNA contents are expressed as ODs corrected for GAPDH. The results are the means ± SE of three independent experiments. P < 0.05, P < 0.01, P < 0.001 vs. control.
Identification of the cytokine-responsive element in the MCP-1 promoter.c\a.[, 百拇医药
To determine the MCP-1 promoter regions responsive to IL-1ß, the promoter fragment from nucleotides -514 to +53 relative to the transcription start site, with or without the enhancer fragment between -2,180 to -2,478 relative to the transcription start site, were cloned into the luciferase reporter plasmid pGL3 and transfected into FACS-purified ß-cells treated or not with IL-1ß. In IL-1ß-treated cells, the luciferase activity of the constructs pMCP-1-514(enh) luc was induced threefold, whereas pMCP-1-514 luc did not significantly respond to IL-1ß . This lack of response of the promoter region alone, even though there is a potential NF-{kappa} B binding site in this region, indicates a role for the potential NF-{kappa} B binding sites in the enhancer region . Similar results were observed in INS-1E cells transfected with the same constructs. Thus, IL-1ß induced a significant increase in the activity of pMCP-1-514[enh] but not of pMCP-1-514 luc. Due to the large number of cells required, the subsequent experiments to elucidate the regulatory elements in the MCP-1 enhancer were performed in INS-1E cells.
fig.ommtted4q, 百拇医药
. Schematic representation of the rat MCP-1 promoter. The arrow indicates the transcriptional start site. Putative binding sites for transcription factors are indicated by boxes. The promoter and the NF-B mutants in the enhancer region cloned upstream of the luciferase reporter gene are indicated in the lower part of the figure. mutA, mutated NF-B site at -2,265; mutB, mutated NF-B site at -2,291; pMCP-1-514[enh], MCP-1 promoter sequence including the enhancer region.4q, 百拇医药
fig.ommtted4q, 百拇医药
MCP-1 promoter activity after cytokine treatment of primary rat ß-cells. ß-cells were transfected with the wild-type constructs shown in and either left untreated (control [C]) or treated with IL-1ß (IL) for 16 h. Relative luciferase activity values are expressed as % of control. The data shown are means ± SE of four independent experiments. *P < 0.05 vs. respective control.4q, 百拇医药
fig.ommtted4q, 百拇医药
Identification of the cytokine-responsive elements in the MCP-1 promoter in INS-1E cells. INS-1E cells were transfected with the same wild-type and mutant constructs shown in and left untreated (control [C]) or treated with IL-1ß (IL) for 16 h. Relative luciferase activity values are expressed as percent of control. The data shown are means ± SE of four independent experiments. ***P < 0.001 vs. respective control.
The enhancer region contains two potential NF-{kappa} B binding sites . Therefore, we mutated each NF-{kappa} B site separately and denoted the mutated constructs as pMCP-1-514[enh]mutA luc and pMCP-1-514[enh]mutB luc. The fold induction of MCP-1 promoter activity in response to IL-1ß was reduced by 75 and 80% for mutA and mutB, respectively . These results indicate that both binding sites are important for IL-1ß-induced MCP-1 promoter activity.&x, 百拇医药
NF-{kappa} B binding to the MCP-1 promoter in vitro.&x, 百拇医药
To characterize the factors binding to the IL-1ß-responsive elements in the enhancer region, we performed EMSAs using probes spanning the NF-{kappa} B sites from nucleotides -2,272 to -2,297 containing the first NF-{kappa} B motif of the enhancer (site A) and from -2,242 to -2,266 containing the second NF-{kappa} B motif of the enhancer (site B). Based on our previous observation that the maximal IL-1ß-induced NF-B binding activity in vitro in insulin-producing cells takes place after 30 min (42,52), this time point was selected for in vitro determination of nuclear factor binding. Two complexes, denoted by "a" and "b," were induced in cells stimulated with IL-1ß, with a higher affinity for the probe of site B . The nature of complexes a and b was further identified by competition and supershift experiments. The two complexes were specifically competed by an excess of unlabelled probe (, lane 3, and , lane 10) and by a consensus NF-{kappa} B oligonucleotide (, lane 4, and , lane 11). An antibody directed against the NF-{kappa} B family member p50 supershifted complex b (, lane 5, and , lane 12), whereas an antibody directed against the NF-B family member p65 supershifted both complexes a and b (, lane 6, and , lane 13). An unrelated antibody against Stat1 had no effect on the migration of the two complexes (, lane 7, and , lane 14). These results suggest that complex a contains p65 homodimers, whereas complex b consists of p65 and p50 heterodimers.
fig.ommttedj, 百拇医药
NF-B binding to the MCP-1 promoter. A: EMSAs were performed in electrophoresis buffer Tris-borate with an oligonucleotide spanning from nucleotides -2,272 to -2,297 containing the first NF-B motif of the enhancer region (site A). B: EMSAs were performed with an oligonucleotide spanning from -2,242 to -2,266 containing the second NF-{kappa} B motif of the enhancer region (site B). Experiments were done with nuclear extracts from INS-1E cells untreated (control [C]) or treated with IL-1ß (IL) for 30 min or with nuclear extracts treated with IL-1ß and competing oligonucleotides (lanes 3–4 and 10–11) or with specific antibodies against p50 or p65 (lanes 5–6 and 12–13). a, p65 homodimers; b, p65/p50 complexes. The figure is representative of two to three similar experiments.j, 百拇医药
NF-{kappa} B binding to the MCP-1 promoter in vivo.j, 百拇医药
To confirm in vivo our in vitro observations indicating NF-{kappa} B binding to the enhancer region, we performed ChIP assays with an antibody directed against the NF-{kappa} B p65 protein. ChIP assay allows for detailed analyses of protein-DNA interactions in a native chromatin environment (53), and the detection of association of rare transcription factor complexes can be achieved by real-time PCR quantification (45). We currently observed IL-1ß-induced NF-{kappa} B binding in vivo by quantification of the rat MCP-1 enhancer region with real-time PCR (see RESEARCH DESIGN AND METHODS). NF-{kappa} B binding was detected as early as 30 min following IL-1ß exposure, with continuously increasing values at 2 and 4 h. The rat MCP-1 enhancer was undetectable in unstimulated control cells.
fig.ommtted71z#g, 百拇医药
In vivo binding of NF-B to the MCP-1 promoter in insulin-producing cells as evaluated by chromatin immunoprecipitation assay followed by real-time PCR analysis. RINm5F cells were either exposed to IL-1ß for 30 min, 2 h, or 4 h or left untreated (control). Real-time PCR analyses were performed in duplicate for the MCP-1 enhancer region and results are expressed in arbitrary units (AU) as detailed in RESEARCH DESIGN AND METHODS. The figure shows data from two independent experiments.71z#g, 百拇医药
Effect of an I{kappa} B{alpha} super-repressor protein on cytokine-mediated MCP-1 gene expression.71z#g, 百拇医药
Overexpression of the I{kappa} B super-repressor by an adenovirus, AdI{kappa} B(SA)2, with serine 32 and 36 substituted by alanine residues, specifically blocks NF-{kappa} B activation without interfering with other transcription factors (47,48). We currently observed that AdI{kappa} B(SA)2 partially blocked cytokine-induced MCP-1 mRNA expression (Fig 7A). Thus, the optical density (OD) values of AdI{kappa} B(SA)2 plus cytokines were 55 ± 9% of the values observed in noninfected cells exposed to the same cytokines (n = 3; P < 0.05), while infection with the control virus AdGFP did not modify the effects of cytokines (; OD 86 ± 33% of the values observed in noninfected cells exposed to cytokines; n = 3). To further evaluate the role of NF-{kappa} B for cytokine-induced MCP-1 expression, rat islets were exposed to cytokines in the presence of the NF-{kappa} B blocker PDTC (50; ). There was no detectable MCP-1 expression in unstimulated cells or cells exposed to PDTC alone . MCP-1 mRNA was induced by cytokines after 6 and 24 h, and this induction was, to a major extent, prevented by PDTC. The results, expressed in OD after correction for GAPDH mRNA, for experiments 1 and 2, respectively, were as follows: cytokines-6 h, 1.96 and 1.61; PDTC plus cytokines-6 h, 0.54 and 0.47 (inhibition 72 and 71%, respectively); cytokines-24 h, 1.09 and 1.17; and PDTC plus cytokines-24 h, below detection limit .
fig.ommtted&jtusx0, http://www.100md.com
The effect of IB(SA)2 expression and PDTC exposure on cytokine-induced transcription of MCP-1. A: Rat ß-cells were infected with the recombinant adenoviruses expressing GFP (AdGFP), the IB super-repressor protein (AdIB[SA]2), or left uninfected (no virus). Twenty-four hours after infection, cells were exposed to IL-1ß (50 units/ml) plus IFN- (1,000 units/ml) for 24 h. B: Rat islets were exposed to PDTC for 1 h before treatment with cytokines (same concentrations as in ) for 6 or 24 h. MCP-1 and GAPDH mRNA contents were analyzed by RT-PCR. The figures shown are representative for three and two similar experiments, respectively. The observed ODs are provided in RESULTS.&jtusx0, http://www.100md.com
DISCUSSION&jtusx0, http://www.100md.com
MCP-1 expression is induced by cytokines in pancreatic ß-cells and probably plays a role in attracting monocytes and T-cells to the site of insulitis (5,7,54). Indeed, the temporal pattern of MCP-1 expression, along with other CC chemokines, in the islets of NOD mice is well correlated with the progression of insulitis and ß-cell destruction (5,54), and transgenic MCP-1 expression in ß-cells induces mononuclear cell infiltration (28). In this context, it is of relevance to understand the molecular regulation of MCP-1 expression in the ß-cells.
In the present study, we identified the transcription factor NF-{kappa} B as a key regulator of MCP-1 promoter activity. Mutation of either of the two NF-{kappa} B binding sites, located in the enhancer region at -2,242 and at -2,272, respectively, abrogated IL-1ß-induced MCP-1 promoter activity. Moreover, we detected the binding of NF-{kappa} B p50 and p65 factors to these two sites by EMSAs and supershift experiments. In vivo binding of NF-{kappa} B to the enhancer region was confirmed by ChIP assays. Finally, overexpression of an I{kappa} B super-repressor, AdI{kappa} B(SA)2, or preculture in the presence of the NF-{kappa} B blocker PDTC decreased cytokine-induced MCP-1 mRNA expression. The proximal promoter region by itself does not respond to cytokines, suggesting no role in insulin-producing cells for neither the putative NF-{kappa} B and AP1 binding sites (33), nor for the IRIS and GAS elements (34) located in this region.65osvm, 百拇医药
Stereospecific alignment between adjacent cis elements has been observed in several genes, including those containing {kappa} B motifs (55). The two NF-{kappa} B binding sites in the enhancer region of MCP-1 promoter, site A and site B, are not identical in sequence, suggesting that they may serve distinct functions. Although higher affinity of NF-{kappa} B binding was observed for site B than A (Fig. 5), the fold induction of MCP-1 promoter activity in response to IL-1ß was reduced by 75 and 80% for mutated site A and B, respectively (present data). The presence of these sites was also described as necessary to maintain TNF-induced MCP-1 gene promoter activity in mouse fibroblasts (30). On the other hand, insertion of a half or full helical turn between the {kappa} B sites, or site reversion between the two NF-{kappa} B binding sites in the enhancer region, did not affect TNF-induced MCP-1 gene promoter activity in these cells (30). Taken together with our findings, these results suggest that both binding sites for NF-{kappa} B are important for the IL-1ß-induced MCP-1 promoter activity in insulin-producing cells.
Mammalian cells contain five NF-{kappa} B family members: p50, p52, p65, c-Rel, and RelB. Interactions between these proteins have been characterized in detail (56). P65/p50 heterodimers, the most frequent dimers, and p65 homodimers are activators of gene transcription, whereas p50 homodimers, which lack a transactivation domain, are transcriptionally inactive (57). Our supershift experiments with antibodies directed against p65 and p50 proteins suggest a role for p65/p50 and p65/p65 dimers in the regulation of MCP-1 in insulin-producing cells. The differential induction and binding of activated transcription factors to the promoter region of the MCP-1 gene provides a critical regulatory step, allowing expression of the chemokine in a cell- and stimulus-specific manner (15). Of note, we have observed the presence of the same active dimers of NF-{kappa} B in the context of cytokine regulation of Fas expression in primary ß-cells (42).^ln+kp^, 百拇医药
In vitro determination of transcription factor binding to a promoter does not necessarily reflect the complex environment and the three-dimensional conformation of chromatin in a living cell. Indeed, it has been shown that the compact nature of chromatin can modulate accessibility to some DNA binding sites (45,53). Therefore, to confirm in vivo our observations of NF-{kappa} B binding to the rat MCP-1 promoter, we have performed ChIP assay, a novel method that allows detection of transcription factor binding in situ (53). We detected NF-{kappa} B binding in vivo at 30 min after IL-1ß stimulation, with subsequent increases at 2 and 4 h (present data). Interestingly, it has been previously reported the presence of two distinct waves of early and late NF-{kappa} B recruitment to target promoters in a murine macrophage cell line, depending on the acetylation state of chromatin (58). NF-{kappa} B binding to the MCP-1 enhancer was detected only after 90 min of LPS exposure to macrophage cells (58). Our data suggest an early and sustained IL-1ß-induced binding of NF-{kappa} B to the MCP-1 promoter in ß-cells , helping to explain the long-lasting effect of IL-1ß on MCP-1 mRNA expression .
The results described above suggest that NF-{kappa} B translocation to the nucleus is necessary for MCP-1 mRNA induction in ß-cells exposed to cytokines. Indeed, rat ß-cells infected with an adenovirus overexpressing the nondegradable I{kappa} B{alpha} super-repressor protein (AdI{kappa} B[SA]2) have a 50% lower induction of MCP-1 mRNA expression in response to IL-1ß. The partial blocking observed may be due to the limited efficiency of infection by the adenoviral system used, which is ~ 70–80% (47). These data are in good agreement with our previous microarray results, showing a 50–60% inhibition of cytokine-induced MCP-1 mRNA expression in ß-cells previously infected with the AdIB(SA)2 (8) and with the present observations that PDTC, a potent inhibitor of NF-B, blocked cytokine- induced MCP-1 mRNA expression >70% in cytokine-exposed islets. However, the fact that the observed inhibitory effect of NF-{kappa} B blockers on MCP-1 expression in vitro is not complete suggests that other transcription factors may also contribute to MCP-1 regulation in ß-cells.
As a whole, these findings suggest an essential role for NF-{kappa} B in the regulation of MCP-1 in ß-cells. Of note, in vivo overexpression of I{kappa} B{alpha} , a natural inhibitor of NF-{kappa} B, significantly decreases local MCP-1 and ICAM-1 expression, as well as recruitment of macrophages following iliac artery balloon angioplasty in rabbits, thus reducing arterial restenosis (59). This, and the fact that high basal MCP-1 production by human islets correlates with a poor clinical outcome following islet transplantation in patients with type 1 diabetes (6), suggests that adenovirus- or lentivirus-mediated NF-{kappa} B inhibition might be a useful approach to decrease mononuclear cell infiltration and graft dysfunction/destruction following islet transplantation.t}i+ivo, 百拇医药
ACKNOWLEDGMENTSt}i+ivo, 百拇医药
This work was supported by grants from the Juvenile Diabetes Foundation International, the Funds voor Wetenschappelijk Onderzoek (FWO), and the Fonds National de la Recherche Scientifique (FNRS), Belgium.
We thank the personnel from the Diabetes Research Center, VUB, involved in ß-cell purification; M.A. Neef, J. Schoonheydt, and M. Urbain, Laboratory of Experimental Medicine, ULB, for islet isolation; and E. Quartier for sequencing.x);+1l#, 百拇医药
REFERENCESx);+1l#, 百拇医药
Schranz DB, Lernmark A: Immunology in diabetes: an update. Diabete Metab Rev14 :3 –29,1998x);+1l#, 百拇医药
Eizirik DL, Mandrup-Poulsen T: A choice of death: the signal-transduction of immune-mediated ß-cell apoptosis. Diabetologia44 :2115 –2133,2001x);+1l#, 百拇医药
Mandrup-Poulsen T: The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia39 :1005 –1029,1996x);+1l#, 百拇医药
Chen MC, Schuit F, Eizirik DL: Identification of IL-1ß-induced messenger RNAs in rat pancreatic beta cells by differential display of messenger RNA. Diabetologia42 :1199 –1203,1999x);+1l#, 百拇医药
Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL: Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 ß-exposed human and rat islet cells. Diabetologia44 :325 –332,2001
Piemonti L, Leone BE, Nano R, Saccani A, Monti P, Maffi P, Bianchi G, Sica A, Peri G, Melzi R, Aldrighetti L, Secchi A, Di Carlo V, Allavena P, Bertuzzi F: Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation. Diabetes51 :55 –65,2002rl.{y^3, http://www.100md.com
Cardozo AK, Kruhoffer M, Leeman R, Orntoft T, Eizirik DL: Identification of novel cytokine-induced genes in pancreatic ß-cells by high-density oligonucleotide arrays. Diabetes50 :909 –920,2001rl.{y^3, http://www.100md.com
Cardozo AK, Heimberg H, Heremans Y, Leeman R, Kutlu B, Kruhoffer M, Orntoft T, Eizirik DL: A comprehensive analysis of cytokine-induced and nuclear factor-{kappa} B-dependent genes in primary rat pancreatic ß-cells. J Biol Chem276 :48879 –48886,2001rl.{y^3, http://www.100md.com
Luster AD: Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med338 :436 –445,1998rl.{y^3, http://www.100md.com
Baggiolini M, Dewald B, Moser B: Human chemokines: an update. Annu Rev Immunol15 :675 –705,1997rl.{y^3, http://www.100md.com
Rollins BJ: Chemokines. Blood90 :909 –928,1997rl.{y^3, http://www.100md.com
Arimilli S, Ferlin W, Solvason N, Deshpande S, Howard M, Mocci S: Chemokines in autoimmune diseases. Immunol Rev177 :43 –51,2000
Han KH, Quehenberger O: Ligands for peroxisome proliferator-activated receptor inhibit monocyte CCR2 expression stimulated by plasma lipoproteins. Trends Cardiovasc Med10 :209 –216,2000wxv!j, 百拇医药
Mackman N: Lipopolysaccharide induction of gene expression in human monocytic cells. Immunol Res21 :247 –251,2000wxv!j, 百拇医药
Roebuck KA, Carpenter LR, Lakshminarayanan V, Page SM, Moy JN, Thomas LL: Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-{kappa} B. J Leukoc Biol65 :291 –298,1999wxv!j, 百拇医药
Leonard EJ, Yoshimura T: Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today11 :97 –101,1990wxv!j, 百拇医药
Carr MW, Roth SJ, Luther E, Rose SS, Springer TA: Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A91 :3652 –3656,1994wxv!j, 百拇医药
Maghazachi AA, al-Aoukaty A, Schall TJ: C-C chemokines induce the chemotaxis of NK and IL-2-activated NK cells: role for G proteins. J Immunol153 :4969 –4977,1994
Stanimirovic D, Satoh K: Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation. Brain Pathol10 :113 –126,2000pp*vyp?, 百拇医药
Gu L, Tseng SC, Rollins BJ: Monocyte chemoattractant protein-1. Chem Immunol72 :7 –29,1999pp*vyp?, 百拇医药
Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D: Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A88 :5252 –5256,1991pp*vyp?, 百拇医药
Koch AE, Kunkel SL, Harlow LA, Johnson B, Evanoff HL, Haines GK, Burdick MD, Pope RM, Strieter RM: Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis. J Clin Invest90 :772 –779,1992pp*vyp?, 百拇医药
Brown Z, Robson RL, Westwick J: Regulation and expression of chemokines: potential role in glomerulonephritis. J Leukoc Biol59 :75 –80,1996pp*vyp?, 百拇医药
Lloyd CM, Dorf ME, Proudfoot A, Salant DJ, Gutierrez-Ramos JC: Role of MCP-1 and RANTES in inflammation and progression to fibrosis during murine crescentic nephritis. J Leukoc Biol62 :676 –680,1997
Boring L, Gosling J, Cleary M, Charo IF: Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature394 :894 –897,1998$, 百拇医药
Gosling J, Slaymaker S, Gu L, Tseng S, Zlot CH, Young SG, Rollins BJ, Charo IF: MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B. J Clin Invest103 :773 –778,1999$, 百拇医药
Flory CM, Jones ML, Warren JS: Pulmonary granuloma formation in the rat is partially dependent on monocyte chemoattractant protein 1. Lab Invest69 :396 –404,1993$, 百拇医药
Grewal IS, Rutledge BJ, Fiorillo JA, Gu L, Gladue RP, Flavell RA, Rollins BJ: Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1. J Immunol159 :401 –408,1997$, 百拇医药
Ping D, Jones PL, Boss JM: TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene. Immunity4 :455 –469,1996$, 百拇医药
Ping D, Boekhoudt GH, Rogers EM, Boss JM: Nuclear factor-{kappa} B p65 mediates the assembly and activation of the TNF-responsive element of the murine monocyte chemoattractant-1 gene. J Immunol162 :727 –734,1999
Stylianou E, Nie M, Ueda A, Zhao L: c-Rel and p65 trans-activate the monocyte chemoattractant protein-1 gene in interleukin-1 stimulated mesangial cells. Kidney Int56 :873 –882,1999s({!!, 百拇医药
Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T: NF-{kappa} B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol153 :2052 –2063,1994s({!!, 百拇医药
Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR: Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-{kappa} B and AP-1. Eur J Immunol27 :1091 –1097,1997s({!!, 百拇医药
Valente AJ, Xie JF, Abramova MA, Wenzel UO, Abboud HE, Graves DT: A complex element regulates IFN-{gamma} -stimulated monocyte chemoattractant protein-1 gene transcription. J Immunol161 :3719 –3728,1998s({!!, 百拇医药
Freter RR, Alberta JA, Hwang GY, Wrentmore AL, Stiles CD: Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-{kappa} B and a 90-kDa phosphoprotein coactivator. J Biol Chem271 :17417 –17424,1996
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. New York, Wiley & Sons,1994 , p.2.2.1 –2.2.32h/c|g@, 百拇医药
Coolidge CJ, Patton JG: Run-around PCR: a novel way to create duplications using polymerase chain reaction. Biotechniques18 :762 –764,19952h/c|g@, 百拇医药
Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology130 :167 –178,19922h/c|g@, 百拇医药
Pipeleers DG, in’t Veld PA, Van de Winkel M, Maes E, Schuit FC, Gepts W: A new in vitro model for the study of pancreatic A and B cells. Endocrinology117 :806 –816,19852h/c|g@, 百拇医药
Ling Z, Hannaert JC, Pipeleers D: Effect of nutrients, hormones and serum on survival of rat islet ß-cells in culture. Diabetologia37 :15 –21,19942h/c|g@, 百拇医药
Schuit FC, Pipeleers DG: Regulation of adenosine 3',5'-monophosphate levels in the pancreatic B cell. Endocrinology117 :834 –840,19852h/c|g@, 百拇医药
Darville MI, Eizirik DL: Cytokine induction of Fas gene expression in insulin-producing cells requires the transcription factors NF-{kappa} B and C/EBP. Diabetes50 :1741 –1748,2001
Schreiber E, Matthias P, Muller MM, Schaffner W: Rapid detection of octamer binding proteins with ’mini-extracts’, prepared from a small number of cells. Nucleic Acids Res17 :6419 ,1989r|(a, 百拇医药
Clayton AL, Rose S, Barratt MJ, Mahadevan LC: Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J19 :3714 –3726,2000r|(a, 百拇医药
Chakrabarti SK, James JC, Mirmira RG: Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1: importance of chromatin structure in directing promoter binding. J Biol Chem277 :13286 –13293,2002r|(a, 百拇医药
Ito S, Nakanishi H, Hirai T, Kato T, Kodera Y, Feng Z, Kasai Y, Ito K, Akiyama S, Nakao A, Tatematsu M: Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler. Cancer Lett183 :195 –203,2002r|(a, 百拇医药
Heimberg H, Heremans Y, Jobin C, Leemans R, Cardozo AK, Darville M, Eizirik DL: Inhibition of cytokine-induced nuclear factor-{kappa} B activation by adenovirus-mediated expression of a NF-{kappa} B super-repressor prevents ß-cell apoptosis. Diabetes50 :2219 –2224,2001
Jobin C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, Brenner DA, Sartor RB: Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor {kappa} B super-repressor in human intestinal epithelial cells. J Immunol160 :410 –418,19986k, 百拇医药
Liu D, Cardozo AK, Darville MI, Eizirik DL: Double-stranded RNA cooperates with interferon- and IL-1ß to induce both chemokine expression and nuclear factor-{kappa} B-dependent apoptosis in pancreatic ß-cells: potential mechanisms for viral-induced insulitis and ß-cell death in type 1 diabetes mellitus. Endocrinology143 :1225 –1234,20026k, 百拇医药
Flodstrom M, Welsh N, Eizirik DL: Cytokines activate the nuclear factor kappa B (NF-{kappa} B) and induce nitric oxide production in human pancreatic islets. FEBS Lett385 :4 –6,19966k, 百拇医药
Liu D, Darville M, Eizirik DL: Double-stranded ribonucleic acid (RNA) induces ß-cell Fas messenger RNA expression and increases cytokine-induced ß-cell apoptosis. Endocrinology142 :2593 –2599,20016k, 百拇医药
Darville MI, Ho YS, Eizirik DL: NF-{kappa} B is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells. Endocrinology141 :153 –162,2000
Kuo MH, Allis CD: In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment. Methods19 :425 –433,19997, http://www.100md.com
Cameron MJ, Arreaza GA, Grattan M, Meagher C, Sharif S, Burdick MD, Strieter RM, Cook DN, Delovitch TL: Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes. J Immunol165 :1102 –1110,20007, http://www.100md.com
Thanos D, Maniatis T: Virus induction of human IFN ß gene expression requires the assembly of an enhanceosome. Cell83 :1091 –1100,19957, http://www.100md.com
Baldwin AS Jr: The NF-{kappa} B and I{kappa} B proteins: new discoveries and insights. Annu Rev Immunol14 :649 –683,19967, http://www.100md.com
Ballard DW, Dixon EP, Peffer NJ, Bogerd H, Doerre S, Stein B, Greene WC: The 65-kDa subunit of human NF-{kappa} B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci U S A89 :1875 –1879,19927, http://www.100md.com
Saccani S, Pantano S, Natoli G: Two waves of nuclear factor {kappa} B recruitment to target promoters. J Exp Med193 :1351 –1359,20017, http://www.100md.com
Breuss JM, Cejna M, Bergmeister H, Kadl A, Baumgartl G, Steurer S, Xu Z, Koshelnick Y, Lipp J, De Martin R, Losert U, Lammer J, Binder BR: Activation of nuclear factor-{kappa} B significantly contributes to lumen loss in a rabbit iliac artery balloon angioplasty model. Circulation105 :633 –638,2002(Burak Kutlu Martine I. Darville Alessandra K. Cardozo and Décio L. Eizirik)