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The Journal of Immunology, 2007, 179, 7605 -7613
Copyright © 2007 by The American Association of Immunologists, Inc.
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Peptide-Mediated Disruption of NF{kappa}B/NRF Interaction Inhibits IL-8 Gene Activation by IL-1 or Helicobacter pylori1

Myriam Bartels*, Aike Torben Schweda*, Ursula Dreikhausen*, Ronald Frank{dagger}, Klaus Resch*, Winfried Beil* and Mahtab Nourbakhsh2,*

* Institute of Pharmacology, Hannover Medical School, Hannover, Germany; and {dagger} Department of Chemical Biology Helmholtz, Centre for Infection Research, Braunschweig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NF{kappa}B repressing factor (NRF) and NF{kappa}B play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NF{kappa}B p65 in cells. We identified a synthetic peptide corresponding to aa 223–238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NF{kappa}B interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NF{kappa}B interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokines are a superfamily of chemotactic cytokines divided into CXC and CC subfamilies (1, 2). IL-8 is the prototype of the CXC chemokines, which acts as a major mediator of acute neutrophil-mediated inflammation (3, 4, 5). In response to inflammatory stimuli, IL-8 expression is induced in numerous types of cells. Among them, epithelial cells are the typical target cells for a variety of pathogens and represent the major site of infection associated inflammation. Depending on the source of epithelial cells, two specific stimuli, IL-1 or Helicobacter pylori (H. pylori) were preferentially used to induce IL-8 gene transcription (6, 7, 8). IL-1 and H. pylori, both activate p65 subunit of the NF{kappa}B transcription factor family (9, 10). The NF{kappa}B subunit p65 is well known as a key molecule in the transcriptional regulation of genes relevant to inflammation (11, 12). In almost all cases, p65 does not act alone to enhance promoter activity. Instead, p65 often physically associates with other DNA-binding factors and cooperatively acts to regulate transcription of target genes.

NF{kappa}B repressing factor (NRF)3 is a constitutively expressed nuclear transcription factor that binds to IFN-β, IL-8, and inducible NO synthase (iNOS) promoters (13, 14, 15). Initial reporter experiments have shown that the N-terminal domain of NRF spanning amino acids 1–296 inhibits the transcription activity of NF{kappa}B proteins in non stimulated cells (13). This led to the initial determination of NRF as a transcriptional silencer. An inducible NRF antisense expression approach in human epithelial cells was previously used to investigate the role of the endogenous NRF protein in transcription regulation of cytokine genes (15). In IL-8 gene expression, exclusively, NRF was found to play a dual function. It represses the basal transcription of IL-8 gene in unstimulated cells (15). Following IL-1 stimulation, however, NRF is required for the transcriptional activation of IL-8 gene. Together with NF{kappa}B p65, NRF acts as a coactivator to stimulate the IL-8 gene expression in IL-1 treated epithelial human cervical (HeLa) cells (15).

Considering the exceptional and important role of NRF in IL-8 expression, we studied the physical interaction of NRF with endogenous NF{kappa}B p65. Our data demonstrate that a central domain of NRF (aa 204–308) binds to the Rel homology domain of p65. This was confirmed by the use of synthetic peptides corresponding to aa 204–308 of NRF which compete with NRF/NF{kappa}B interaction in vitro. This led to the proposal that these peptides might interfere with NRF/NF{kappa}B interaction in the nucleus of living cells and decrease IL-8 transcription. This possibility was approved by nucleofection experiments which directly introduce synthetic peptides into the nucleus. In fact, specific NRF-derived inhibitory peptides, but not control peptides, are able to reduce the IL-8 gene transcription in epithelial cells following IL-1 stimulation or H. pylori infection. This study provides a new strategy to modulate cytokine gene expression via targeting interaction of transcription factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Plasmid constructions

The IL8-LUC reporter plasmid contains a –1481/+40-bp region of the IL-8 gene driving firefly coding region (16). Mutant reporter plasmids were constructed by Quick-Change mutagenesis (Stratagene) using complimentary synthetic oligonucleotides: NF{kappa}B mutation (sense): 5'-CCATCAGTTGCAAATCGATAACTTTCCTCTGACATAATG-3' NF{kappa}B mutation (antisense): 5'-CATTATGTCAGAGGAAAGTTATCGATTTGCAACTGATGG-3' NRF mutation (sense): 5'-GCAAATCGTGGAATTTCCCCCGGGATAATGAAAAGATG-3' NRF mutation (antisense): 5'-CCATGGCAATTCCCCCGACATG-3' NF{kappa}B/NRF mutation (sense): 5'-CCATCAGTTGCAAATCGATAACTTTCCCCCGGGATAATG-3' NF{kappa}B/NRF mutation (antisense): 5'-CATTATCCCGGGGGAAAGTTATCGATTTGCAACTG ATGG-3'.

p65 expression plasmid was described earlier (13). NdeI/BamHI fragment of p65 expression plasmid was removed to create p65 Rel homology domain (RHD) expression plasmid. Tandem affinity purification (TAP) expression plasmid was constructed by insertion of TAP cassette into BamHI/EcoRI of pCDNA3.1 (Invitrogen Life Technologies). For construction of expression plasmids encoding NRF-TAP fusion proteins, NRF cDNA fragments were amplified by PCR using complimentary oligonucleotides corresponding to amino acids as mentioned in text. PCR fragments were inserted into HindIII/BamHI of TAP expression plasmid.

Peptide mapping

p65 expression plasmid (13) was added to in vitro expression kit TNT obtained from Promega to express 35S labeled proteins. NRF peptide array was generated by the SPOT-synthesis technique described earlier (17). One hundred nanograms of 35S labeled protein p65 was added to NRF peptide array for 30 min at 30°C and washed three times with 10-fold volume of PBS (0.02 M sodium phosphate buffer with 0.15 M sodium chloride (pH 7.4)). Bound p65 protein was visualized by direct autoradiography.

Synthetic peptides

Purified synthetic peptides were obtained from Eurogentec. The quality of peptides was determined by mass spectrometry (MALDI-TOF).

Cell lines, gene transfer, and induction

HeLa (CCL-2, LGC Promochem) cells were maintained in DMEM with 5% FCS (FCS). Helicobacter pylori infected gastric epithelial (AGS) cells (CRL-1739, LGC Promochem, Middlesex, U.K.) were maintained in RPMI 1640 medium with 10% FCS. For reporter assays, cells were transfected by calcium phosphate coprecipitation (18). A total of 5 µg of each reporter plasmid together with 1 µg of a Renilla expression plasmid were transfected per 7.5 x 104 cells. After 48 h, HeLa cells were treated with IL-1β (10 ng/ml) and AGS cells were infected with 50 CFU of a cagA-positive H. pylori (HP87) per cell for 18 hours (19). Cells were harvested after 24 h and pooled for reporter gene assays. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase reporter system (Promega Corporation) as described in the manufacturer’s protocol. The amount of protein was determined using bicinchoninic acid reagent (Sigma-Aldrich) as described in the manufacturer’s protocol. Luciferase activity was measured in a Lumat LB 9501 luminometer (Berthold).

For TAP-purification, HeLa cells were transfected with indicated NRF-TAP expression plasmids by calcium phosphate precipitation. For nucleofection, HeLa or AGS cells were maintained in DMEM or RPMI 1640 medium, respectively, plus 10% FCS and transfected with nucleofector technology according to the manufacturer’s instructions (Amaxa Biosystems). In brief, for each nucleofection 106 cells were harvested and resuspended in 100 µl nucleofector solution with 20 µg of synthetic peptides. The cell-peptide mixtures were nucleofected and cells were immediately transferred into 3 ml medium with 10% FCS for 3 h at 37°C. Immediately following transfection or nucleofection, HeLa cells were treated with IL-1β (10 ng/ml) and AGS cells were infected with 50 CFU of a cagA-positive H. pylori (HP87) per cell for following 3 h (19).

Imaging of cells

Cells were placed into plates immediately after nucleofection with N-terminal fluorescein-labeled peptide obtained from Eurogentec. After 5 h, imaging was performed with inverted microscope (Carl Zeiss) and emission collected using a 450–490 nm bandpass filter. Images were obtained with a Axiocam HRC Zeiss with CCD-sensor.

FACS analysis

Quantification of fluorescein labeled peptides was performed with FACSCalibur (BD Biosciences) according to standard protocols. The data were visualized and analyzed with CellQuest Pro software (BD Biosciences).

TAP purification

TAP purification was essentially performed as described (20) with the following modifications. HeLa cells were transfected with either TAP or NRF-TAP fusion proteins encoding plasmids. Forty-eight hours later, cells were stimulated with IL-1β (10 ng/ml) for 3 h where indicated. Approximately 108 cells were lysed in buffer A (10 mM HEPES/KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 5 mM DTT, 0.1% Nonidet P-40, 0,3 mM Na3VO3, 20 mM glycerol-2-phosphat, 10 µM protease inhibitor E64, 2.5 µg/ml leupeptin, 0.3 mM PMSF, 1 µM pepstatin, 400 µM okadaic acid (pH 7.9). The amount of total protein was determined using bicinchoninic acid reagent (Sigma-Aldrich) as described in the manufacturer’s protocol. Equal aliquots of cellular extracts were used for Western blot analysis using {alpha}-p65 Ab (sc8008, Santa Cruz Biotechnology) and peroxidase anti-peroxidase soluble complex Ab produced in rabbit (PAP, Sigma-Aldrich) to confirm equal expression of TAP fusion proteins. Equal amounts of cellular extracts were incubated with IgG-agarose (Sigma-Aldrich Inc., Munich, Germany) in salt-adjusted buffer A (150 mM NaCl) at 4°C overnight to bind TAP-tagged proteins. Where indicated, 20 µg synthetic peptide was added prior overnight incubation. Following three times washing steps with wash buffer (10 mM Tris (pH 8.0), 150 mM NaCl and 0.1% Nonidet P-40), binding of TAP fusion proteins to equal aliquots of IgG-agarose (10%) was examined by Western blot analysis using peroxidase anti-peroxidase soluble complex Ab produced in rabbit (PAP) (Sigma-Aldrich). Complexes were released from IgG-agarose by tobacco etch virus (TEV)-protease (30 U; Invitrogen Life Technologies) in TEV buffer (10 mM Tris-HCl (pH 8.0); 150 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, 0.5 mM EDTA, 1 µM protease inhibitor E64). Complexes containing calmodulin binding peptide (CBP)-tagged proteins were immobilized on a calmodulin affinity resin (Stratagene) for 4 h at 4°C. Complexes were washed five times with CBP buffer (50 mM Tris-HCl (pH 8.0); 150 mM NaCl, 1 mM Mg-acetate, 1 mM imidazole, 10 mM 2-ME, 2 mM CaCl2). Finally, 20 µl of gel loading buffer (21) was added to the bound protein complexes and incubated for 10 min. at 65°C prior Western blot analysis (21) using a mouse mAb against p65 (sc8008) and secondary goat anti-mouse Ab (sc2005) from Santa Cruz Biotechnology.

Western blot analysis

Western blots were performed as described earlier (21). TAP fusion proteins were detected using peroxidase anti-peroxidase soluble complex Ab produced in rabbit (PAP) (Sigma-Aldrich). Abs against p65 (sc8008) were obtained from Santa Cruz Biotechnology.

Chromatin immunoprecipitation (ChIP) experiments

ChIP experiments were performed as described earlier (21). Transfected cells were cross-linked in vivo with 1% formaldehyde for 10 min at 37°C. Crosslink reaction was stopped by adding 125 mM glycin and cells were washed in PBS and then incubated in RIPA lysis buffer (40 mM Tris-HCl (pH 7.05), 120 mM NaPPi, 200 mM NaCl, 1% Triton, 8 mM Na3VO3, 2 mM NaF, 80 mM β-glycerolphosphat, 10 µg/ml Leupeptin, 1 mM PMSF, and 10 µM Pepstatin) on ice for 10 min. After sonication and centrifugation, 5 µl of the soluble extract was analyzed as total control. DNA fragments bound to various proteins were immunoprecipitated using IgG-agarose (Sigma-Aldrich). IL-8 promoter was detected by PCR using following primers: sense: 5'-GGGCCATCAGTTGCAAAT-3'and antisense 5'-TTCCTTCCGGTGGTTTCTTC-3'. PCR were performed in1 µM dNTP, 2 mM MgCl2, 2.5 U Taq-DNA-Polymerase in a final volume of 50 µl by single cycle of 5 min at 95 °C following by 30 amplification cycles of 0.5 min at 95°C, 0.5 min at 60°C, and final 1 min at 72°C.

RNA isolation and RT-PCR

Total RNA from HeLa cells were isolated using TRIzol reagent from Invitrogen Life Technologies according to the manufacturer’s protocol. RT-PCR were performed using 3 µg total RNA and MMLV reverse transcriptase from Invitrogen Life Technologies in total volume of 40 µl. For PCR, 2.5 µl of reversed transcribed cDNA was amplified using Taq polymerase from Invitrogen Life Technologies and following primers for IL-8: 5'-ACATACTCCAAACCTTTCCACCC-3' and 5'-CAACCCTCTGCACCCAGTTTTC-3'; GAPDH: 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; JunB: 5'-CCACGACGACTCATACACAGCTACGGGA-3' and 5'-TGACCAGAAAAGTAGCTGCCGCCACCG-3'. PCR were performed in 400 µM dNTP, 2.1 mM MgCl2, 2.5 U Taq-DNA-Polymerase in a final volume of 50 µl) by a single cycle of 5 min at 95°C followed by 30 cycles of 0.5 min at 95°C, 1 min at 66°C and final 2 min at 68°C.

Nuclease protection assay

Cells were nucleofected and stimulated or infected as described above. Total RNA was extracted from cells using TRIzol-reagent (BD Biosciences) and then treated with 10 U of RNase-free DNase (Roche Pharma AG). RNA was then extracted with phenol-chloroform-isoamyl alcohol (Carl Roth GmbH) and precipitated in ethanol and 0.6 M LiCl. Antisense probes were prepared using in vitro T7 transcription system (Promega) and the following templates: For the synthesis of IL-8 antisense RNA, pBLKS-IL-8 was linearized with Nhe enzyme, for the synthesis of transferrin recepror probe pT7AS-TFR was linearized with HindII enzyme. Each protection was performed on equal amounts of RNA (1 µg) and equal cpm of G50 purified in vitro transcribed antisense RNA as described earlier (21). The specific activity of IL-8 probe was ~10–6 cpm/copy and that of transferrin receptor probe 0.5 x 10–5 cpm/copy. Protected fragments were analyzed by electrophoresis on 6% polyacrylamide gels containing 6 M urea. For quantification, detected fragments were cut off from the gel and measured in an automatic scintillation counter (Beckman Coulter).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NRF is involved in activation of IL-8 gene transcription by H. pylori

The p65 subunit of NF{kappa}B transcription factors was previously shown to play a crucial role in the transcriptional activation of IL-8 gene in response to IL-1, viral infection and H. pylori (22, 23, 24). We have reported that NRF acts as a transcriptional coactivator of IL-8 gene in response to IL-1, but not to viral infection (15). This led to the suggestion that specifically IL-1 induces the coactivating function of NRF (13, 15). In this study, to study a possible role of NRF in H. pylori induced IL-8 expression, we first determined the time course of IL-8 mRNA expression in IL-1 stimulated HeLa cells and H. pylori infected human gastric AGS cells. Two forms of IL-1, IL-1{alpha} and IL-1β, are known. Both are structurally related and exert identical actions through binding to the IL-1 type I receptor (25). In all experiments of this study, we used IL-1β (in the following referred to as IL-1) and a previously described and well characterized wild type (wt) H. pylori strain (26). Similar to IL-1 stimulated HeLa cells, H. pylori infected AGS cells show a rapid increase of IL-8 mRNA within 2 h as determined by RT-PCR analysis (Fig. 1A). To estimate the impact of NRF and p65 proteins in H. pylori induced IL-8 expression, we used a luciferase reporter gene under the control of IL-8 promoter (16). Parallel experiments were conducted with wt or mutant promoter containing inactive p65 (nfkb), NRF (nrf) binding sites, or both (nfkb/nrf) (Fig. 1B) in HeLa and AGS cells (13, 16). As internal control, a Renilla luciferase expression plasmid was cotransfected. The reporter activities in IL-1 stimulated HeLa and AGS cells infected with H. pylori are shown in Fig. 1B. As expected, the wt IL-8 promoter is highly active in HeLa ({square}) and AGS cells (Formula ) following IL-1 treatment or H. pylori infection, respectively (Fig. 1B). In both cell lines, IL-8 promoter activity is clearly reduced by mutation of either NRF or NF{kappa}B binding sites compared with wt (nrf or nfkb, respectively). Mutations of NRF and NF{kappa}B binding sites together (nfkb/nrf) resulted in significant reduction of IL-8 promoter activity. These results are consistent with the previously reported crucial role of NRF in the IL-1 induced IL-8 expression (15). Most importantly, the data show for the first time that NRF is required for the full induction of IL-8 promoter in response to H. pylori infection in AGS cells.


Figure 1
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  		FIGURE 1. Activation of IL-8 expression by IL-1 and H. pylori infection. A, HeLa or AGS cells were stimulated with IL-1 or infected with H. pylori for indicated times, respectively. After treatment of cells, RNA was isolated and subjected to RT-PCR using IL-8 or GAPDH specific primers. As control, parallel reactions were performed without adding reverse transcriptase (Ø RT). Data are representative for at least five independent experiments. IL-8 and GAPDH signals are indicated by arrows. B, HeLa and AGS cells were transfected with 1 µg Renilla luciferase expression plasmid and 5 µg of reporter plasmids containing firefly luciferase coding sequences derived by wild type (wt), NF{kappa}B mutant (nfkb), NRF mutant (nrf), or NRF and NF{kappa}B mutant (nfkb/nrf) IL-8 promoter. Wild type IL-8 promoter and mutant sequences are shown at the top. After 48 h, cells were stimulated with IL-1 or H. pylori for 18 h. Cells were harvested and extracts were prepared and subjected to luciferase assay. Firefly luciferase light units were normalized to Renilla luciferase activity to obtain the relative firefly light units of each reporter plasmid. Mean values of relative firefly light units are shown ± SEM from three independent transfection experiments in HeLa cells ({square}) and AGS cells (Figure 1).

 
NRF aa 204–308 are sufficient for direct binding to endogenous p65 protein

We have previously shown that a recombinant bacterial GST-NRF fusion protein containing aa 1–380 of NRF directly interacts with an in vitro expressed p65 protein (13). However, the precise boundaries of NRF interaction domain were not known yet. To identify peptide sequences of the N-terminal domain of NRF containing aa 1–380 possibly involved in the interaction with p65, we used a peptide array exposing 15mer peptides derived from the N-terminal domain of NRF protein (17). The peptide array was incubated with the in vitro expressed and radioactive-labeled p65 protein. Following stringent wash steps, we detected p65 bound to distinct frames of overlapping peptides in the N-terminal domain of NRF, in particular, aa 19–60, 103–129, 169–186, 196–222, 220–258, and 265–312 (Fig. 2). This led to the suggestion that the identified peptide sequences might also be essential for direct interaction of NRF with endogenous p65 in cells.


Figure 2
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  		FIGURE 2. Mapping of p65 binding sequence in NRF protein. 500 ng of labeled p65 was incubated with array of 15-mer peptides corresponding to NRF protein sequences coupled to a cellulose membrane. Membrane was washed three times and binding of p65 protein was detected by autoradiography. Only bound peptides and corresponding amino acids of NRF protein sequence are indicated at the top or the bottom of array strips.

 
Next, we attempted to study the interaction of NRF and p65 expressed in cells. Because available anti-NRF Abs were inefficient to study the interaction of endogenous NRF protein with p65 by coimmunprecipitation, we used NRF-TAP fusion proteins (Fig. 3A). This TAP involves two affinity tags, CBP and IgG binding domains of protein A, separated by a specific TEV protease cleavage site (20). These two consecutive affinity purification steps allow much higher purification than could be achieved by a single tag (20). We transiently expressed four different NRF-TAP fusion proteins and the TAP tag as a control in HeLa cells. NRF1–690TAP fusion protein consists of the full-length NRF protein and a C-terminal TAP domain. NRF1–380TAP, NRF1–149TAP, and NRF 204–308TAP contain the N-terminal aa 1–380, aa 1–149, and 204–308, respectively. In a parallel set of experiments, transfected cells were additionally stimulated by IL-1. Cellular extracts were prepared from transfected cells and equal aliquots were used for Western blot analysis to control equal amounts of p65 protein in each experiment (Fig. 3A, p65 total). Simultaneously, equal expression of TAP fusion proteins was controlled using a peroxidase anti-peroxidase soluble complex Ab produced in rabbit (PAP) (data not shown). For purification, cellular extracts were incubated with IgG-agarose. Following binding of TAP fusion proteins to IgG-agarose, unbound proteins were removed by several wash steps. Next, equal aliquots of IgG-agarose were analyzed by Western blot using peroxidase anti-peroxidase Ab (PAP) to determine the amounts of bound TAP fusion proteins (Fig. 3A, total TAP fusion protein). Exceptionally, NRF1–149TAP was most efficiently expressed and binds to IgG-agarose. In NRF204–308TAP expressing cells, a 27 kDa product (lower band) is detectable in addition to the full length NRF204–308TAP protein (upper band). As detected by peroxidase anti-peroxidase Ab (PAP), the 27 kDa product is a TAP fusion peptide possibly generated by an endogenous protease. Precise calculation of molecular weights revealed that the 27 kDa product corresponds to the C-terminal TAP domain (25.66 kDa), additional linker sequences (30 aa), and C-terminal 5 aa of NRF204–308. Therefore, it is most unlikely that this proteolytic product is capable of p65 binding. Following the first purification step, IgG-agarose bound protein A domains were removed by TEV digestion (Fig. 3A). The supernatants were added to calmodulin beads for the second purification step. Finally, the amount of p65 was detected in each bound protein fraction by Western blot analysis (Fig. 3A, p65 bound). The data in Fig. 3A show that NRF1–690TAP, NRF1–380TAP, and NRF204–308TAP are able to interact with endogenous p65 protein (Fig. 3A, p65 bound). As expected, TAP tag alone and surprisingly, NRF1–149TAP fail to interact with p65 even at much higher concentrations. IL-1 stimulated cells (Fig. 3A, +IL-1) revealed no significant effect on p65 binding to NRF1–690TAP, NRF1–380TAP or NRF204–308TAP fusion proteins (Fig. 3A, p65 bound). These results led us assume that aa 204–308 of NRF are sufficient for the interaction between endogenous NRF and p65 proteins.


Figure 3
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  		FIGURE 3. Endogenous p65 binds to a central domain of NRF in cellular extracts. A, A schematic description shows successive steps of TAP purification experiments and the samples that were elected for Western blot analysis. HeLa cells were transfected with equal amounts of expression plasmids encoding NRF1–609TAP, NRF1–380TAP, NRF1–149TAP, NRF204–308TAP, or TAP domain alone. 48 h after transfection cells were stimulated with IL-1 (10 ng/ml) for 3 h where indicated. Cellular extracts were prepared and the amount of p65 (total p65) was detected by Western blot using {alpha}-p65 Ab (sc8008, Santa Cruz Biotechnology). Next, equal aliquots of cellular extracts were incubated with IgG-agarose. After several washing steps, 10% of the bound fraction of TAP fusion proteins were monitored by Western blot using peroxidase anti-peroxidase soluble complex Ab produced in rabbit (PAP). Bound NRF-TAP fusion proteins and TAP domain are indicated by arrowheads (total TAP fusion protein). Following TEV digestion and the subsequent second purification step, the amount of p65 was monitored by Western blotting using {alpha}-p65 Ab (sc8008, Santa Cruz Biotechnology) and marked as p65 bound. Data are representative for five independent experiments in unstimulated and three independent experiments in IL-1 stimulated cells. B, HeLa cells were cotransfected with equal amounts of expression plasmids encoding p65RHD either with NRF204–308TAP or with TAP domain alone. TAP analysis and detection were performed as described in A. Data are representative for three independent experiments. C, ChIP assay was conducted using soluble chromatin extracts from untransfected or transfected HeLa cells with NRF-TAP expressing plasmids as indicated. The extracts were precipitated using IgG-agarose or left untreated as an internal quality control (total). The precipitated DNAs were used for PCR with primers spanning the core region of IL-8 promoter. Results are representative for four independent ChIP experiments.

 
To identify the NRF binding domain of p65, cells were simultaneously transfected with plasmids encoding NRF204–308TAP or RHD of p65. Total of TAP fusion proteins, p65 and p65 RHD are shown in Fig. 3B (total). The results demonstrate that both endogenous p65 and exogenous RHD of p65 interacts with aa 204–308 of NRF. Control protein TAP domain binds to neither p65 nor p65 RHD. In summary, the data show that aa 204–308 of NRF and RHD domain of p65 are sufficient for a direct physical interaction between p65 and NRF.

Previous studies have shown, that a bacterial recombinant protein containing aa 297–380 of NRF is able to bind to an 11 bp DNA element in IL-8 promoter in vitro (13). Thus, we attempted to examine whether NRFTAP fusion proteins are able to bind to the IL-8 promoter. We used ChIP assay, which provides more considerable proof for the binding activity of a protein in an endogenous promoter. Cells were transfected with respective TAP fusion expression plasmids. Following transfection, chromatin was cross-linked in cells and total chromatin was isolated and sheared by sonification. Equal amounts of the total chromatin in each preparation was controlled by PCR using specific primers which amplify a 180 bp fragment from –100 to +80 bp with respect to the transcription start site of IL-8 gene (Fig. 3C, total chromatin). IgG-agarose was added into equal aliquots of total chromatin to purify NRFTAP binding complexes. Following purification, the binding of TAP fusion proteins to the endogenous IL-8 promoter was detected by PCR as described above. Nontransfected cells served as a negative control. We note that a negligible background signal was barely detectable in nontransfected, NRF1–380TAP, NRF1–149TAP, or NRF204–308TAP expressing cells (Fig. 3C). This was most likely due to the precipitation of unshared chromatin in each reaction (Fig. 3C). By comparison, however, NRF1–690TAP-expressing cells reveal a significantly high IL-8 signal indicating the capability of NRF1–690TAP protein to bind to the IL-8 promoter (Fig. 3C). Importantly, these data demonstrate that aa 1–380 and 204–308 of NRF do not bind to the IL-8 promoter. Thus, the observed interaction between these NRF domains and p65 or p65 RHD is independent of DNA binding.

Synthetic peptides derived from NRF aa 204–308 disrupt NF{kappa}B/NRF interaction

An initial goal was to inhibit NRF/p65 cooperation by targeting the identified interaction domains. For this approach, we used synthetic peptides exposing aa 203–218 (A), aa 223–238 (B) and aa 282–297 (C) of NRF which were derived from detected peptide sequences in peptide array analysis described above. In addition, a peptide exposing aa 94–109 (X) was used as a control which did not bind to p65 in peptide array analysis. Cells were transfected with the NRF1–308TAP expression plasmid. Equal aliquots of cellular extracts were added to IgG-agarose together with indicated peptides. One representative experiment is shown in the top of Fig. 4B. The total amounts of p65 and IgG bound NRF1–308TAP were detected by Western blot as described for Fig. 3A. The data of three independent experiments are summarized in the diagram (bottom of Fig. 4B). The binding of p65 to NRF aa 1–308 is enhanced by addition of peptide X compared with the binding reaction without peptide or with peptide A (p65 bound). In contrast, peptide B and peptide C significantly compete with the binding of NRF aa 1–308 to p65 showing its capability to interfere with NRF/p65 interaction in vitro. Note, a lower concentration of peptides B showed no significant effect on p65 binding (10 µg, data not shown). Concerning SEM, however, peptide B shows the most prominent inhibitory effect on NRF/p65 interaction. Thus, we focused on the effect of peptide B in the following experiments.


Figure 4
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  		FIGURE 4. Synthetic peptides corresponding to the minimal NRF interaction domain interfere with p65 binding in vitro. A, Sequence of synthetic peptides (X, A, B, C) and corresponding amino acid residues of NRF interaction domain (aa 204–308) is schematically presented. B, HeLa cells were transfected with NRF1–380 encoding plasmid. Forty-eight hours after transfection cellular extracts were prepared and bound to IgG-agarose as described in the legend of Fig. 3. Fractions of protein-IgG-agarose complex were left untreated (Ø) or incubated with either peptide X, A, B, or C. Purification and detection of bound p65 were performed as described in the legend of Fig. 3. Data are representative for three independent experiments. In diagram, densitometric analysis of detected bands from three independent experiments is shown ± SEM; *, p < 0.01; **, p < 0.05; NS, not significant.

 
Nuclear transfer of a synthetic peptide corresponding to aa 223–238 of NRF inhibits IL-8 gene activation by IL-1 or H. pylori

In mammalian cells, nucleofection was frequently used for efficient transfer of nucleic acids directly into the nucleus (27, 28, 29). We adopted this method for transfer of synthetic peptides into the nucleus to test the effect of inhibitory peptides on the endogenous IL-8 expression in vivo after IL-1 stimulation in HeLa or H. pylori infection of AGS cells. Therefore, peptide B was fluorescein labeled and delivered into the nucleus of HeLa cells. Approximately 1–2% of cells achieve strikingly high level of fluorescein signal. A fluorescence image of these cells is shown at low light intensity to visualize the predominant localization of nucleofected peptide in the nucleus (Fig. 5A). In nucleofected cells, fluorescein signal was detectable immediately after nucleofection and up to following 5 h. Similar results were obtained in nucleofected AGS cells (data not shown). The efficiency of nucleofection was estimated using FACS analysis in three independent experiments. The results of a typical experiment are demonstrated in Fig. 5B. Approximately 93% of AGS and HeLa cells nucleofected with fluorescein labeled peptide B display positive signals compared with the cells nucleofected with nonlabeled peptide. However, AGS cells achieved a higher fluorescent signal compared with HeLa cells.


Figure 5
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  		FIGURE 5. Peptide B is efficiently transduced into HeLa and AGS cells. A, HeLa cells were nucleofected with the fluorescein labeled synthetic peptide B. After 3 h, cells were visualized by phase-contrast (I) or fluorescence microscopy at low light intensity (II). B, Results from FACS analysis of HeLa (dotted lines) and AGS cells (continuous line) nucleofected with nonlabeled scramble peptide (green) or with fluorescein-labeled synthetic peptide B (black) are presented.

 
In the ultimate experiments, unlabeled peptides X, B and an additional synthetic peptide with scramble sequence of peptide B were nucleofected into HeLa and AGS cells to confirm the specificity of peptide sequence. As controls, cells were left untreated or were nucleofected without addition of synthetic peptides to nucleofection reagent. The initial experiments shown in Fig. 1A revealed that IL-8 mRNAs achieve the maximum level between 2 and 4 h after IL-1 stimulation. Immediately after nucleofection, therefore, cells were stimulated with IL-1 or infected with H. pylori for 3 hours followed by RNA isolation and RT-PCR using IL-8 specific primers. RT-PCR using GAPDH specific primers served as a control for equivalent cDNA synthesis and PCR in each experiment. Genomic DNA contamination was excluded by omitting of reverse transcriptase enzyme in reactions (data not shown). The results of a typical experiment are demonstrated in Fig. 6A. For a better reproducibility, we performed at least three independent nucleofection experiments. We observed that the nucleofection procedure itself slightly enhances the IL-8 gene transcription (Fig. 6A, lane 2). The IL-8 signal from IL-1 stimulated or H. pylori infected cells were densitometrically analyzed and summarized in Fig. 6B. The level of IL-1 stimulated IL-8 mRNA expression in nonnucleofected HeLa cells was set to 100%. IL-1 stimulated cells nucleofected with peptide X, S, or without addition of synthetic peptides show an indistinguishable level of IL-8 transcription compared with nonnucleofected cells. In contrast, peptide B decreases the endogenous IL-8 mRNA expression achieving an average reduction of IL-8 mRNA to 60%. Together, these results demonstrate potency of the synthetic peptide B to disturb NRF/NF{kappa}B interaction and thereby to decrease IL-8 gene transcription in response to IL-1 or H. pylori infection in HeLa or AGS cells, respectively.


Figure 6
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  		FIGURE 6. IL-8 mRNA expression is inhibited by synthetic peptides interfering with p65 binding in vivo. A, HeLa and AGS cells were left untreated or were nucleofected without or with 20 µg of synthetic peptides X, B, and S (containing scramble sequence of peptide B) indicated at the top. Immediately following nucleofection, cells were treated with IL-1 or H. pylori for 3 h and then harvested for isolation of total RNA. Equal amounts of isolated total RNA were subjected to RT-PCR using IL-8, JunB, and GAPDH specific primers. Obtained fragments were analyzed on 2% agarose gel and ethidium bromide staining. Data are representative for at least three independent experiments. B, Densitometric analysis of detected bands is shown ± SEM from three independent nucleofection experiments. IL-8 signals were quantified and normalized to corresponding GAPDH signals in each experiment. IL-8 expression in non nucleofected cells was set to 100% in each experiment. Inhibition by peptide B was calculated to be significantly higher compared with peptide S. *, p < 0.001. C, Densitometric analysis of detected bands is shown ± SEM from three independent nucleofection experiments. JunB signals were quantified and normalized to corresponding GAPDH signals in each experiment. IL-8 expression in non nucleofected cells was set to 100% in each experiment. *, p < 0.01; NS, not significant.

 
We further tested whether the inhibitory function of peptide B is restricted to NRF regulated genes. JunB is an IL-1 stimulated gene regulated by a well-characterized 100 bp promoter containing binding sites for p65, leukemia inhibitory factor, Smad, and STAT proteins. In contrast to IL-8 promoter, however, JunB promoter contains no NRF binding sites or related sequences (30, 31, 32). To confirm the specificity of the NRF-derived peptides, we tested whether endogenous JunB expression remains unaffected in IL-1 stimulated and nucleofected cells. Therefore, cDNAs analyzed in Fig. 6A were subjected to additional PCR using JunB specific primers. As shown in Fig. 6C, the mRNA levels of JunB are entirely constant in all experiments. In contrast to IL-8, JunB mRNA expression is insignificantly affected by peptide B. The scramble peptide shows no effect on JunB mRNA expression.

Compared with the RT-PCR including amplification steps, nuclease protection assays allow the direct detection of absolute mRNA copy number in cells. In fact, this technique is sensitive enough to enable quantitation of RNA from a small sample avoiding additional amplification steps. We performed nuclease protection experiments using mRNAs analyzed in Fig. 6A. We used two different probes (207 nt or 340 nt) able to separately monitor either IL-8 or transferrin receptor transcripts as internal control. Following nuclease digestion, as illustrated in Fig. 7A, hybridization to the proximal probe allows the detection of IL-8 transcripts by a 135 nt long protected fragment. Transferrin receptor transcripts are detected by a protected 179 nt long fragment. The number of detected specific transcripts was calculated from cpm obtained in three independent experiments and summarized in Fig. 7B. The data clearly confirm that peptide B, but not the scramble peptide (S), is able to inhibit the endogenous expression of IL-8 gene in response to IL-1 or H. pylori. Compared with the results from RT-PCR experiments, peptide B decreases the endogenous IL-8 mRNA expression to ~30%. These data demonstrate that peptide B, which acts as a potential inhibitor of NRF and p65 interaction is able to suppress IL-8 mRNA expression in response to inflammatory and infectious signals.


Figure 7
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  		FIGURE 7. IL-8 mRNA expression is selectively inhibited by synthetic peptide B. A, IL-8 antisense expression plasmid, pT7-anti-IL8, and antisense probe corresponding to IL-8 mRNA are schematically presented at the top. Length of protected region is indicated. HeLa and AGS cells were nucleofected without or with 20 µg of synthetic peptide B or S containing scramble sequence of peptide B (top of Fig. 6A). Cells were treated with IL-1 or H. pylori for 3 h and then harvested for isolation of total RNA. Equal amounts of isolated RNAs were subjected to nuclease protection analysis using IL-8 and transferrin receptor antisense RNA probes (lane 1). Following nuclease treatment, obtained fragments were analyzed on 6% denaturating PAGE. The autoradiography of protected transcripts is shown. Data are representative for three independent experiments. B, Following electrophoresis the total amounts of protected fragments were measured in cpm. The number of transcripts per 20 µg of total RNA is shown ± SEM from three independent experiments. The number of transcripts of stimulated HeLa or AGS cells nucleofected without peptide was set to 100% in each experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A number of NF{kappa}B inhibitors were identified with promising properties for therapeutic suppression of inflammatory response (7). Most of them decline the expression level of IL-8 which is the most potent chemotactic factor for neutrophils because of its high affinity for CXCR1 and CXCR2 chemokine receptors expressed on neutrophils (33). However, the use of NF{kappa}B inhibitors are supposed to have adverse side effects based on their comprehensive action on >400 known NF{kappa}B target genes involved in other cellular processes, like proliferation, angiogenesis, metastasis, and viral replication (11, 34).

In the current study, we focused on the identification of a more specific inhibitor targeting IL-8 mRNA expression. A distinctive feature of IL-8 promoter is the cooperative interaction of NF{kappa}B p65 with NRF to activate transcription in response to IL-1. By comparison, NRF was found to be redundant for activation of iNOS or IFN-β promoters, both containing NRF and NF{kappa}B binding sites (14, 35). Recently, transgenic mice lacking a major part of NRF gene exhibited no significant difference in IFN-β and iNOS expression pattern as compared with wt mice (35). Because IL-8 gene is completely absent in muroid rodent lineage, the role of NRF in IL-8 expression cannot be investigated in transgenic mice. In human cells, however, NRF and p65 interaction is apparently involved in regulatory mechanisms which specifically mediate IL-8 gene activation. In this study, we have taken a successive approach that isolate the interaction domain of NRF with NF{kappa}B and leads to the design of a more specific inhibitor of IL-8 expression. We used two different human epithelial cell lines and respective stimuli, IL-1 and H. pylori, to generalize the observed inhibitory effects on IL-8 expression. IL-1 is one of the most important immune response modifying interleukins produced primarily by macrophages during inflammation (33). H. pylori colonize the human stomach and increases the risk for gastric disease, including peptic ulceration and adenocarcinoma of the stomach (24, 36, 37, 38). Previous studies demonstrated that binding of NRF protein is required for the full induction of IL-8 promoter by IL-1 stimulation (15). Accordingly, we showed that NRF and p65 binding sites either apart or together are crucial for the activation of IL-8 promoter by IL-1 or H. pylori in HeLa or AGS cells, respectively (Fig. 1B). Previous studies using EMSA and ChIP experiments have established that NRF and p65 independently bind to their specific elements (21). Interestingly, the NRF binding sequences in IL-8 and IFN-β promoters are identical whereas the NF{kappa}B binding sequences differ by a single nucleotide mismatch. Moreover, studies of IFN-β promoter revealed that IFN-β mRNA expression is highly inducible by virus, but unresponsive to IL-1. It was previously shown that single nucleotide changes in NF{kappa}B binding sequence alter the requirement of specific coactivator proteins and resulted in loss of response to viral infections (39, 40). These observations suggest that IL-8 promoter sequences can be important components of the regulatory complexes that impart IL-1 or H. pylori specific activation of transcription.

Having established the cooperative action of NRF and p65 in the activation of IL-8 promoter by IL-1 or H. pylori, we systematically analyzed the physical interaction of NRF and p65 proteins. Peptide array was successfully used to define multiple peptide sequences within the NRF protein that can be potentially bound by p65. Because peptide arrays lack crucial features of a native protein structure, this experiment detects also peptide sequences that are probably hidden within the native NRF structure. The additional use of NRF-TAP fusion proteins which allow a higher order of protein structure exclusively defines aa 204–308 as a central p65 interaction domain of NRF. Considering the results from peptide array in Fig. 2, we suggest that this domain expose two interacting modules (aa 202–258 and aa 265–312) separated by a noninteracting spacer (aa 247–276). Based upon these results, we synthesized two peptides B and C representing the interacting modules. Consistently, the competition experiments shown in Fig. 4B confirm that these two peptides are able to inhibit NRF/p65 interaction. Peptide B revealed the most prominent inhibitory effect in these competition experiments. We note that simultaneous addition of peptide B and C showed no further inhibitory effect (data not shown). Remarkably, the control peptide X (aa 94–109) enhances the binding of endogenous p65 to NRFTAP fusion protein. Recently, we found that the amino acids 97, 100, and 106 of NRF are phosphorylated in cellular extracts (our unpublished data). Although not substantiated experimentally yet, it is conceivable that peptide X interferes with the phosphorylation of NRF which could possibly affect the physical interaction of NRF and p65.

Finally, we examined the capacity of synthetic peptides B and C to inhibit the transcriptional activation of IL-8 gene in vivo. As a prerequisite, the peptides have to be present in the nucleus of the cells. To accomplish this condition, we used the nucleofection method. In the past, nucleofection was used to introduce nucleic acids into the nucleus of mammalian cells which cannot be transfected by conventional methods (27, 29). In this study, for the first time, we successfully used this method to introduce inhibitory peptides into the nucleus of nucleofected cells. Essentially, we observed that the fluorescent signal of nucleofected peptides remained stable up to 5 h within the nucleus of HeLa and AGS cells (Fig. 5A). Under these experimental conditions, a significant reduction of IL-8 mRNA expression was achieved by peptide B in IL-1 or H. pylori treated HeLa or AGS cells, respectively. Most important, this inhibitory effect is highly specific because peptide X and the scrambled peptide failed to interfere with IL-8 expression (Fig. 6, A and B). Additionally, peptide B shows no effect on endogenous expression of JunB gene lacking a NRF binding site. Statistical picture of amino acids involved in protein-protein interactions indicated that proteins interact through specialized interface residues Pro, Ile, Asp, Arg, Tyr, and Try (41). Strikingly, peptide B contains three Pro, two Asp, and a single Ile residues. However, the sequence of these residues is presumably important as well, because scramble peptide conveys the same amino acid residues but shows no effect on IL-8 transcription.

Although NRF and NF{kappa}B interaction possibly occurs in the promoters of IFN-β and iNOS genes, activation of these genes were shown to be entirely independent of NRF protein. Neither deletion of NRF binding site nor down-regulation of NRF expression by antisense RNA affected the induction of iNOS and IFN-β promoters (13, 14, 21). In contrast, NRF was shown to be involved in the constitutive silencing of target promoters in nonstimulated cells (14, 15, 21, 42). In this study, we could not examine the effect of peptide B on unstimulated IL-8 promoter because nucleofection procedure itself slightly enhances IL-8 gene transcription (Figs. 6A and 7A). Therefore, it might be important to find another experimental approach to examine the effect of peptide B on IL-8 gene in nonstimulated cells. Nevertheless, the data presented in this study confirm the biological importance of NRF and p65 interaction in IL-8 gene activation.

There exist many inflammatory and infection diseases in which the inhibition of IL-8 expression would provide therapeutic benefits (43, 44, 45). On the one hand, this study provides new insights into the molecular interaction of transcription factors NRF and p65 by the induction of IL-8 promoter. On the other hand, peptide B or the isolated p65 interaction domain of NRF can serve as lead structure of drug development or, respectively, for screening of anti-inflammatory drugs.


   Acknowledgments
 
We thank H. F. Luecke for providing IL8-LUC reporter plasmid. We thank Birgit Ritter, Petra Killian, and Annette Garbe for the excellent technical assistance and Detlef Neumann for discussions.


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


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

1 This work was supported by grants from "Deutsche Forschungsgemeinschaft", DFG457 and SFB566. Back

2 Address correspondence and reprint requests to Dr. Mahtab Nourbakhsh, Institute of Pharmacology, Hannover Medical School, OE 5320, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. E-mail address: nourbakhsh.mahtab{at}mh-hannover.de Back

3 Abbreviations used in this paper: NRF, NF{kappa}B repressing factor; iNOS, inducible NO synthase; HeLa, human cervical cell; TAP, tandem affinity purification; AGS, gastric epithelial cell; CBP, calmodulin binding peptide; TEV, tobacco etch virus; RHD, Rel homology domain; ChIP, chromatin immunoprecipitation; wt, wild type. Back

Received for publication May 15, 2007. Accepted for publication September 5, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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