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The Journal of Immunology, 2006, 176: 4173-4181.
Copyright © 2006 by The American Association of Immunologists

A Novel NF-{kappa}B Binding Site Controls Human Granzyme B Gene Transcription1

Chunjian Huang*, Enguang Bi*, Yu Hu*, Weiwen Deng*, Zhigang Tian{dagger}, Chen Dong{ddagger}, Yuanjie Hu§ and Bing Sun2,*,¶

* Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; {dagger} School of Life Sciences, University of Science and Technology of China, Anhui, China; {ddagger} Department of Immunology, M. D. Anderson Cancer Center, Houston, TX 77030; § Shanghai Biochip, Shanghai, China; and Immunology Division, E-Institutes of Shanghai Universities, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-{kappa}B-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-{kappa}B binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-{kappa}B binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-{kappa}B binding site, which plays a pivotal role in controlling human granzyme B gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are an essential component of the innate and adaptive immune system. They provide a first line of defense against infections by viral and bacterial pathogens and play important roles in the immunosurveillance of tumors (1, 2, 3, 4). NK cells recognize different types of cells and have various effects via activating or inhibiting cell surface receptors (5, 6). Their cytolytic activity can be triggered by several distinct pathways (2, 4). The main pathway is dependent on perforin and granzymes. Other mechanisms, such as Fas ligand and TRAIL-dependent pathways, have also been described (2, 4, 7, 8). After NK cells conjugate with target cells, they impair the target cells by secreting cytotoxic granules (7, 9). Perforin and granzyme B are the two most essential molecules involved in this process. Evidence for the importance of perforin is indicated by the finding that perforin-deficient mice have profound defects in their clearance of viral infections, delayed type hypersensitivity, and tumor rejection (10, 11). Similarly, NK cells, lymphokine-activated killer (LAK)3 cells and CTL from granzyme B-deficient mice showed a rapid decrease in induction of DNA fragmentation and apoptosis on their target cells (12).

Though granzyme B plays a pivotal role for NK cell-mediated cytotoxicity, its transcriptional regulation is not well understood. The human and mouse granzyme B gene promoters have been cloned and some binding sites of transcription factors have been defined, such as Ikaros and AP-1/core-binding factor (CBF) (13, 14, 15, 16). Recently, Zhou et al. (17) demonstrated that IL-2 could up-regulate perforin and granzyme B expression in enriched mouse NK cells and this process seemed to be sensitive to N{alpha}-tosyl-Phe chloromethyl ketone (TPCK) and ammonium pyrrolidinedithiocarbamate (PDTC). However, in that report, the decreased mRNA expression of granzyme B by TPCK and PDTC was very modest, though TPCK and PDTC dramatically inhibited perforin gene expression. As these two inhibitors could also block other transcription factors besides NF-{kappa}B, it is still unclear whether NF-{kappa}B signaling is really involved in the transcriptional regulation of the granzyme B gene. In this report, we used two NF-{kappa}B-specific inhibitors, SN50 and sulfasalazine (SSZ), to block NF-{kappa}B activation. These inhibitors dramatically suppressed human granzyme B gene expression induced by IL-2 treatment both in an NK cell line–NK92–and in primary human NK cells. Furthermore, a novel NF-{kappa}B binding site (GGAGATTCCC) was identified downstream from the human granzyme B gene. EMSA, luciferase, and chromatin immunoprecipitation (ChIP) assays indicated that the NF-{kappa}B complex could bind to this {kappa}B site to induce granzyme B gene transcription. In summary, we demonstrated for the first time that NF-{kappa}B could interact with a downstream {kappa}B site (as an enhancer element) to regulate human granzyme B gene transcription.


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

The Annexin-V-FLUOS staining kit was purchased from Roche. Anti-granzyme B (N-19) polyclonal Ab was obtained from Santa Cruz Biotechnology. Anti-actin polyclonal Ab, TPCK, PDTC, and SSZ were purchased from Sigma-Aldrich. NF-{kappa}B translocation inhibitor SN50 and the control peptide SN50M were purchased from Calbiochem. Cell line nucleofector kits were obtained from Amaxa Biosystems. TaqDNA polymerase, gel-shift binding 5x buffer, luciferase reporter vectors-pGL3 vectors, phRL-SV40 vector, and the dual-luciferase reporter assay system were obtained from Promega. The NF-{kappa}B test kit containing anti-p50, anti-p65, anti-c-Rel, and anti-I{kappa}B{alpha} Abs were purchased from Oncogene. The human NK cell isolation kit was obtained from Miltenyi Biotec. The BCA protein assay reagent kit was purchased from Pierce. MicroSpin G-25 columns and [{gamma}-32P]ATP (~3000 Ci/mmol) were obtained from Amersham Biosciences.

Cell culture

Jurkat T cells and NK92 cells were obtained from American Type Culture Collection. Jurkat T cells were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. NK92 cells were cultured in RPMI 1640 medium containing 15% FCS, 2 mM L-glutamine, 5 x 10–5 M 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 U/ml recombinant human IL-2. To test for granzyme B gene expression, NK92 cells were starved overnight without IL-2 and then were stimulated with 200 U/ml IL-2 for 4 h. In inhibitor-treated groups, various inhibitors were added to the culture 30 min before stimulation with IL-2 and then the cells were cultured in a humidified incubator at 37°C with 5% CO2.

Primary human NK cell isolation

PBMC from healthy donors were purchased from Shanghai Blood Center and were purified by Ficoll-Hypaque gradient centrifugation separation. Primary NK cells were enriched from PBMC by negative selection using the human NK cell isolation kit according to the manufacturer’s instructions. The PBMC were resuspended in PBS buffer containing 0.1% BSA, and exposed to hapten-conjugated IgG Abs including anti-CD3, anti-CD4, anti-CD19, anti-CD36 and anti-IgE at 6–12°C for 15 min. The PBMC were then washed twice with PBS buffer and treated with anti-hapten microbeads for 15 min at 6–12°C. NK cells were then negatively selected using the MidiMACS cell separator system (Miltenyi Biotec). The purity of the enriched human NK cells was >90% CD3–CD56+ NK cells as determined by flow cytometry. These enriched human primary NK cells were cultured in the same condition as NK92 cells.

RT-PCR

Total RNA was extracted from Jurkat T cells, NK92 cells, or primary human NK cells using TRIzol reagent. The first-strand cDNA was synthesized from total RNA using oligo d(T)18 and the M-MLV preamplification system (Invitrogen Life Technologies). For PCR amplification, 2 µl of cDNA product was used in each reaction. The oligonucleotide primers used for PCR were: human granzyme B, sense primer: 5'-aaccaatcctgcttctgc-3' and antisense primer: 5'-actgtcgtaataatggcgta-3'; human GAPDH, sense primer: 5'-ggatttggtcgtattggg-3' and antisense primer: 5'-ggaagatggtgatgggatt-3'. Amplified products were 545 and 205 bp, respectively.

Cell viability assay

The potential cytotoxicity of NF-{kappa}B inhibitors toward NK92 cells and primary NK cells was assessed with annexin V and propidium iodide (PI) staining. NK cells were washed with ice-cold PBS buffer and then resuspended in HEPES buffer containing annexin V-fluorescein and propidium iodide for 15 min at room temperature in the dark and subsequently analyzed by flow cytometry.

Western blot

NK92 cells were starved overnight without IL-2. Then NK92 cells were stimulated with 200 U/ml IL-2 in the presence or absence of TPCK and PDTC. Whole cell lysates were prepared by sonication. Protein concentrations were determined using the BCA protein assay reagent kit according to the manufacturer’s instructions. A total amount of 20 µg of proteins was loaded onto 10% SDS-PAGE, and then were transferred to nitrocellulose membranes. The membranes were immunoblotted with primary anti-granzyme B or anti-actin Abs at 4°C overnight. After three washes, the membranes were further incubated with HRP-conjugated anti-goat or anti-rabbit IgG Abs for 1 h at room temperature, respectively. After washes, granzyme B and actin proteins were detected by chemiluminescence according to the manufacturer’s instructions (Pierce). Actin served as a loading control.

EMSA and supershift assays

NK92 or primary human NK cells were stimulated with 200 U/ml IL-2 in the presence of PDTC and SSZ. After 4 h, cells were harvested and washed with PBS. Cells were lysed with buffer A, which contained 10 mM HEPES, 1.5 mM MgCl2 · 6 H2O, 10 mM KCl (pH 7.9). Before lysis, it was supplemented with 0.5 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin and aprotinin containing 0.1% Nonidet P-40. Lysate was placed on ice for 10 min and centrifuged at 10,000 rpm for 5 min at 4°C to remove cytoplasmic proteins. Nuclear proteins were extracted from the pellet in ice-cold buffer C (20 mM HEPES, 1.5 mM MgCl2 · 6 H2O, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol (pH 7.9); before its use, 0.5 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin and aprotinin were added). Insoluble material was removed by centrifugation at 10,000 rpm for 5 min at 4°C. Protein concentration was measured with the BCA protein assay reagent kit according to the manufacturer’s instructions. Samples were stored at –70°C until use. Oligonucleotides used as probe or competitor in EMSA assays were synthesized as: NF-{kappa}B consensus, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; NF-{kappa}B consensus mutant, 5'-AGTTGAGGCGACTTTCCCAGGC-3'; potential 1, 5'-AGGGAGTGGGGGGCCTCCATTTCCC-3'; potential 1 mutant, 5'-AGGGAGTGGCGGGCCTGCATTTCCC-3'; potential 2, 5'-AGACCAGGAGATTCCCTTGTGT-3'; potential 2 mutant, 5'-AGACCAGCAGATTCGCTTGTGT-3'. The altered nucleotides are underlined. Oligonucleotides were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. The unincorporated radionucleotides were removed from the probe using the MicroSpin G-25 columns. Then, a probe (10,000 cpm) was incubated in a 20-µl binding reaction mixture containing 4 µl of gel-shift binding 5x buffer, 0.5 µg/µl BSA, and 5 µg of nuclear extract for 30 min at room temperature. For DNA competition experiments, a 100-fold molar excess of unlabeled oligonucleotide was added before incubation. For supershift experiments, 1–2 µl of Abs was added to the completed binding reaction mixture. These Abs included anti-p50, anti-p65, and anti-c-Rel Abs. The DNA-protein complexes were separated on 6% native polyacrylamide gels in 0.5x Tris-borate buffer.

Construction of reporter plasmids

A series of nucleotide sequences upstream or downstream from the transcriptional start point of human granzyme B gene was subcloned into the pGL3 promoter vector as shown in Fig. 3A. The oligonucleotide primers used for PCR amplification are shown in Table I.


Figure 3
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  		FIGURE 3. A reporter gene containing potential NF-{kappa}B binding sites was identified. A, The names of reporter genes and the position of nucleotides (relative to the transcription start point at +1) are indicated. B, The strategy for testing the reporter genes containing potential NF-{kappa}B binding sites. C, Luciferase activity of a cloned hgrB P7 reporter gene. NK92 cells were cotransfected with the hgrB P7 reporter gene, phRL-SV40 vector, and expression constructs as mentioned in Materials and Methods. The expression constructs included pcDNA3 vector (control), pcDNA3-I{kappa}B{alpha} AA vector (I{kappa}B{alpha} AA), pcDNA3-p50 vector (p50), pcDNA3-p65 vector (p65), and pcDNA3-p50 vector plus pcDNA3-p65 vector (p50+p65). Luciferase activity is shown as a fold induction relative to the control vector.

 

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  		Table I. PCR primers used for amplifying reporter genesa

 
Transient and stable transfections

Transient transfections were performed using the cell line nucleofector kit according to the manufacturer’s instructions. Approximately 4~5 x 106 NK92 cells were resuspended in 100 µl of Nucleofector solution T. For cotransfection experiments, NK92 cells received 2 µg of reporter gene construct, 0.2 µg of phRL-SV40, and 4 µg of the indicated expression plasmids and carrier DNA. Expression plasmids included pcDNA3-I{kappa}B{alpha} AA, pcDNA3-p50, and pcDNA3-p65 vectors. Jurkat T cells were transfected using Nucleofector technology according to the recommendations of the manufacturer. After transfection, cells were incubated in a humidified incubator at 37°C and 5% CO2 for 40–48 h and then were evaluated with the luciferase assay. NK92 cells were also transfected with pcDNA3-I{kappa}B{alpha} AA plasmid by electroporation and then stable transfectants were selected in culture medium containing 0.5 mg/ml G-418 (Invitrogen Life Technologies).

Luciferase assay

Cells were harvested and washed once with PBS. Then, they were lysed in passive lysis buffer (Promega). A clear lysate was obtained by centrifugation at 12,000 rpm. Luciferase activity was measured in 20 µl of the supernatant using a dual luciferase reporter assay system according to the manufacturer’s instructions.

ChIP assay

The ChIP assays were conducted using a commercial kit following the manufacturer’s instructions (Upstate Biotechnology). Briefly, 2 x 106 NK92 cells were starved without IL-2 overnight and then were stimulated with 200 U/ml IL-2 for 2 h. After cross-linking with 1% formaldehyde, the cell pellet was lysed with SDS lysis buffer. Chromatin was sheared by sonication. The cell lysate was then centrifuged, and the nucleosome-containing supernatant was diluted with 10-fold excess ChIP dilution buffer. An aliquot (1% volume) of the diluted supernatant was saved to control for the input DNA present in each strain. Nucleosome fractions were isolated by adding anti-p50, anti-p65, or rabbit IgG (as a negative control), and incubating overnight at 4°C with rotation. Salmon sperm DNA/protein A agarose was then used to immunoprecipitate the Ab-bound nucleosomes. After washing, the immunoprecipitated nucleosomes as well as the saved reference aliquot were incubated at 65°C for 4 h to preserve DNA/protein cross-linking. The immunoprecipitated DNA was purified by standard phenol-chloroform extraction and ethanol precipitation. Then the DNA was dissolved in 20 µl of H2O, and 2 µl of this DNA solution was used as a template for PCR amplification. The following oligonucleotide sequences were used as primers to PCR amplify 205-bp products spanning the hgrB NF-{kappa}B binding site: sense primer, 5'-gttgcctcacccagaaagt-3' and antisense primer, 5'-tggtgtctgcccaaatagc-3'. PCR products were resolved on 2% agarose gels and visualized by ethidium bromide staining.

Reproducibility and data presentation

Experiments were repeated at least twice, and usually three or more times. Figures show data compiled from several experiments, or from a representative experiment, as specified. Results represent the mean ± SD where applicable. Statistical significance of differences was analyzed using the independent Student t test. Probability values of ≤0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NF-{kappa}B-signaling pathway involvement in IL-2 induced human granzyme B gene expression in NK92 cell line

The NK92 cell line is an IL-2-dependent cell line. It was derived from PBMC from a 50-year-old Caucasian male with rapidly progressive non-Hodgkin’s lymphoma (18). This cell line has been widely used in functional studies (19, 20, 21). It has been demonstrated that IL-2 up-regulates granzyme B gene expression in primary human NK cells (22, 23). In this study, we initially confirmed in the NK92 cell line that IL-2 could up-regulate granzyme B gene expression. The NK92 cells were starved without IL-2 overnight and were subsequently treated with or without IL-2 (200 U/ml) for 4 h as mentioned in Materials and Methods. Upon stimulation, IL-2 up-regulated granzyme B mRNA and protein expression (Fig. 1, B and C), indicating that this cell line is a suitable model to study the transcriptional regulation of human granzyme B gene.


Figure 1
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  		FIGURE 1. Regulation of human granzyme B gene expression in NK92 cells. Cells were IL-2 starved overnight, and then stimulated with 200 U/ml IL-2 in the presence of various inhibitors. DMSO was used as a solution control. A, Cell viability assessed by annexin V and PI staining after 4 h. B, RT-PCR analysis of granzyme B and GAPDH mRNA expression after 4 h of IL-2 stimulation in the presence of 20 µM TPCK and 50 µM PDTC. C, Western blot analysis of granzyme B and actin protein levels after 8 h stimulation in the presence of TPCK or PDTC. D, NK92 cells were IL-2 starved overnight. After being pretreated with 100 µg/ml SN50 or SN50M or 2 mM SSZ for 0.5 h, NK92 cells were stimulated with 200 U/ml IL-2 for 4 h. RT-PCR and Western blot assays were then performed. E, NK92 cells were stably transfected with pcDNA3-I{kappa}B{alpha} AA vector. After being IL-2 starved overnight, these cells were stimulated with 200 U/ml IL-2. Granzyme B expression was analyzed by RT-PCR and Western blot. Densitometric analysis was performed using FURI SmartView 2000 software. The ratios of granzyme B-GAPDH or granzyme B-actins are shown as indicated, and the ratio of the starved group was assigned a value of 1.0 and the values from other groups were normalized based on this value.

 
NF-{kappa}B signaling is involved in the expression control of a variety of genes, including perforin, Fas ligand, Fas, TRAIL, et al. (24, 25, 26, 27, 28). To address whether NF-{kappa}B activation is also crucial for granzyme B gene expression, two distinct inhibitors (TPCK and PDTC) known to block NF-{kappa}B activation were selected to make this assessment. TPCK is the protease inhibitor which prevents the degradation of I{kappa}B proteins (29, 30) and PDTC is an antioxidant inhibitor of NF-{kappa}B activation which does not interfere with proteasome activities, but prevents de novo phosphorylation of I{kappa}B{alpha} as well as its subsequent degradation (31, 32). To determine whether these inhibitors are toxic to NK92 cells under our experimental conditions, cell viability was assessed with annexin V and PI staining. These inhibitors did not affect cell viability (Fig. 1A). Under the same conditions, in IL-2-treated NK92 cells, both inhibitors strongly suppressed granzyme B mRNA and protein expression (Fig. 1, B and C). These data are consistent with the results in mouse NK cells. Zhou and Meadows (17) demonstrated that IL-2 could up-regulate perforin and granzyme B expression in enriched mouse NK cells and this process seemed to be sensitive to TPCK and PDTC. But in that report, the decreased mRNA expression of granzyme B by TPCK and PDTC was very modest, so the conclusion is questionable. In our report, we demonstrated that TPCK and PDTC could dramatically inhibit human granzyme B gene expression in IL-2-stimulated NK cells. However, it has been reported that TPCK and PDTC not only inhibited NF-{kappa}B activation, but also suppressed other transcription factors in some conditions, such as AP-1 and Sp-1 (17, 33). We then used two other NF-{kappa}B-specific inhibitors: SN50 and SSZ. As shown in Fig. 1D, these two inhibitors dramatically suppressed granzyme B expression both in mRNA and protein level induced by IL-2 in NK92 cells, but a control peptide SN50M did not. Because SN50 and SSZ have been documented to specifically inhibit NF-{kappa}B activation (34, 35), our observation suggested that human granzyme B transcription depends on the activation of NF-{kappa}B signaling. We also made stable transfected NK92 cells with pcDNA3-I{kappa}B{alpha} AA plasmid. This plasmid encoded a dominant-negative form of I{kappa}B{alpha} protein which has mutations at aa 32 and 36 residues (serine substituted by alanine). Stable expression of this dominant-negative plasmid in NK92 cells completely abolished signal-induced phosphorylation and degradation of I{kappa}B{alpha} and consequently blocked NF-{kappa}B pathway activation (36, 37, 38). As shown in Fig. 1E, IL-2-induced granzyme B expression was significantly reduced in NK92 cells that were transfected with pcDNA3-I{kappa}B{alpha} AA plasmid as compared with control group. These data show that the NF-{kappa}B-signaling pathway is involved in IL-2-induced human granzyme B gene expression in the NK92 cell line.

Both p50 and p65 components of NF-{kappa}B subfamily contribute to the activation of NF-{kappa}B-signaling pathway in NK92 cells

A broad range of stimuli can activate the NF-{kappa}B-signaling pathway. Upon stimulation, I{kappa}B proteins become ubiquitinated and then degraded. NF-{kappa}B complexes are then released and translocated into nuclei where they activate target genes. To confirm whether the NF-{kappa}B-signaling pathway could be activated by IL-2R signaling in NK92 cells, EMSA was performed. As shown in Fig. 2A, NF-{kappa}B complexes were present in the IL-2-stimulated group, but not in the starved group, indicating that IL-2 treatment in NK92 cells can activate NF-{kappa}B. To further confirm whether the inducible band in EMSA is specific for the NF-{kappa}B complex, a competition assay was performed. As shown in Fig. 2A, the inducible band could be shifted off completely by an unlabeled NF-{kappa}B consensus motif, but not by an unlabeled mutant NF-{kappa}B consensus motif. These data indicate that the inducible DNA binding is specific for the NF-{kappa}B. Moreover, supershift experiments indicated that the NF-{kappa}B complex contained p50 and p65 components of the NF-{kappa}B subfamily (Fig. 2B). Taken together, these results suggest that IL-2 stimulation in NK92 cells leads to an activation of the NF-{kappa}B-signaling pathway through p50 and p65 components.


Figure 2
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  		FIGURE 2. IL-2 activated the NF-{kappa}B-signaling pathway in NK92 cells. A, EMSA of NF-{kappa}B DNA-binding activity induced by IL-2 in NK92 cell nuclear extracts. NK92 cells were IL-2 starved overnight. Then the cells were stimulated with 200 U/ml IL-2 in the presence of 50 µM PDTC or 2 mM SSZ. Cold competition was performed with a 100-fold molar excess of unlabeled NF-{kappa}B consensus wild type (WT) or mutant motif. B, Supershift assay performed with indicated Abs to investigate the IL-2-induced NF-{kappa}B binding complex. The arrows indicate the NF-{kappa}B complex containing p50 and p65 subunits, respectively; n.s. designates a nonspecific band. Free labeled probes are also indicated.

 
A novel NF-{kappa}B binding site was identified downstream from human granzyme B gene

As a group of transcription factors, NF-{kappa}B could directly activate gene expression by binding to the regulatory domains, such as promoter and enhancer sequences. So, we first analyzed the known promoter of the human granzyme B gene. Based on the analysis of the NF-{kappa}B consensus motif, we did not find any NF-{kappa}B binding sites in the promoter sequence. EMSA experiments also indicated that this granzyme B gene promoter could not directly bind to the NF-{kappa}B complex present in IL-2-treated NK92 cells (data not shown). In regard to the evidence for interaction between NF-{kappa}B and the granzyme B gene, we assumed that there are some unidentified NF-{kappa}B-binding sequences within the human granzyme B genome DNA sequence.

Then to locate these NF-{kappa}B binding sites, we subcloned a series of human granzyme B gene sequences into the pGL3 vector (Fig. 3A). Those reporter plasmids were then transfected into NK92 cells. In parallel, either pcDNA3 control or pcDNA3-I{kappa}B{alpha} AA vectors were cotransfected with reporter genes into NK92 cells. A phRL-SV40 vector was also cotransfected into NK92 cells in all groups as an internal control. As mentioned above, overexpression of pcDNA3-I{kappa}B{alpha} AA plasmid in NK92 cells completely abolished signal-induced phosphorylation and degradation of I{kappa}B{alpha} and consequently blocked NF-{kappa}B pathway activation. When pcDNA3-I{kappa}B{alpha} AA was introduced into NK92 cells, the luciferase activity from the reporter gene containing the NF-{kappa}B binding site was suppressed. When there was no NF-{kappa}B binding site in the cloned reporter gene, the luciferase activity was unchanged from the control (Fig. 3B). With this strategy, a reporter gene named hgrB P7 was identified (Table II and Fig. 3C). The results suggest that the hgrB P7 reporter gene contained a good candidate site for NF-{kappa}B binding.


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  		Table II. Selection for reporter genes containing potential NF-{kappa}B-binding sitesa

 
To further confirm this observation, the hgrB P7 reporter gene was cotransfected into NK92 cells with expression plasmids. As shown in Fig. 3C, cotransfection of the p50 and p65 expression vectors with the hgrB P7 reporter gene resulted in significant induction of luciferase activity. These data indicate that the hgrB P7 reporter gene indeed contains an NF-{kappa}B binding site.

To probe for the NF-{kappa}B binding sites within the hgrB P7 sequence, two potential NF-{kappa}B binding sites were predicted by bioinformatics analysis based on the consensus NF-{kappa}B-binding motif (GGGRNNYYCC). The results predicted two potential NF-{kappa}B-binding motifs designated potential 1 and potential 2, respectively (Fig. 4A). Then these two motifs and their mutants were synthesized and were used as competitors in EMSA. As shown in Fig. 4B, when the NF-{kappa}B consensus motif was used as a probe, potential 2 but not potential 1 competed for specific DNA binding. When potential 2 was mutated, its competitive inhibitory effect was eliminated, indicating that the NF-{kappa}B complex specifically binds to the potential 2 motif. When potential 2 was [{gamma}-32P]ATP labeled as a probe, it directly bound to the NF-{kappa}B complex present in IL-2-treated NK92 cells (Fig. 4C). Taken together, a novel NF-{kappa}B-binding motif (GGAGATTCCC) was identified within the hgrB P7 sequence.


Figure 4
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  		FIGURE 4. A novel NF-{kappa}B binding site was identified downstream from the human granzyme B gene. A, The nucleotide sequence downstream from the human granzyme B gene (relative to the transcription start point at +1). The location of potential 1 and potential 2 NF-{kappa}B binding sites are indicated. B, EMSA using NF-{kappa}B consensus motif as a probe. Cold competition was performed using different unlabeled probes as indicated. C, EMSA using potential 2 as a probe. Cold competition was performed using different unlabeled probes as indicated.

 
Putative {kappa}B site function as an enhancer element in NK92 cells and NF-{kappa}B p50 and p65 bind to this site in vivo

Because the identified NF-{kappa}B-binding motif (GGAGATTCCC) is a putative {kappa}B site, we tested for its functionality in NK92 cells. To test this possibility, this motif was subcloned into the pGL3 vector designated as phgrB-{kappa}B vector, and its mutant form (GCAGATTCGC) was also constructed (referred to as phgrB-{kappa}B mutant vector, in which the altered nucleotides are underlined). It was expected that the luciferase expression driven by the putative {kappa}B binding site would be affected by cotransfection of constructs that either increase or inhibit NF-{kappa}B activity. NK92 cells were cotransfected with 2 µg of reporter gene construct, 0.2 µg of phRL-SV40, and 4 µg of the indicated expression plasmids. These expression plasmids included pcDNA3-I{kappa}B{alpha} AA, pcDNA3-p50, and pcDNA3-p65 vectors. As expected, overexpression of pcDNA3-I{kappa}B{alpha} AA vector impaired luciferase activity in the phgrB-{kappa}B vector whereas cotransfection of the p50 and p65 expression vectors with the phgrB-{kappa}B resulted in significant induction of luciferase activity (Fig. 5A). In contrast, if the mutant was instead used, the expression of p50 and p65 had little effect on the phgrB-{kappa}B mutant reporter construct (Fig. 5B). These data further indicate that there is a functional {kappa}B binding site located downstream from the human granzyme B gene.


Figure 5
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  		FIGURE 5. The newly identified NF-{kappa}B binding site is functional in NK92 cells. Luciferase activity of the following reporter genes: A, phgrB-{kappa}B vector and, B, phgrB-{kappa}B mutant vector. NK92 cells were cotransfected with reporter gene, phRL-SV40 vector and expression constructs as mentioned in Fig. 3. Luciferase activities were normalized to the Renilla luciferase activity and are expressed relative to the pcDNA3 control.

 
The identified NF-{kappa}B binding site in this report seemed to be an enhancer element. First, it resided ~10 kb downstream from the human granzyme B gene’s transcription start point. Second, it functioned unrelated to its position and orientation (Fig. 6, A–C). When the {kappa}B site (GGAGATTCCC) was subcloned into the pGL3 vector either upstream or downstream of the luciferase gene, it worked well to increase luciferase activity in the presence of p50 and p65 expression constructs.


Figure 6
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  		FIGURE 6. The functional {kappa}B site serves as an enhancer element and is functional in vivo. The identified {kappa}B site (GGAGATTCCC) was inserted upstream of the SV40 promoter into the pGL3 vector in the natural orientation (referred to as phgrB-{kappa}B1) or in the opposite orientation (referred to as phgrB-{kappa}B2) or downstream of the luciferase gene (referred to as phgrB-{kappa}B3). These plasmids were used as reporter genes: A, phgrB-{kappa}B1; B, phgrB-{kappa}B2; C, phgrB-{kappa}B3. NK92 cells were cotransfected with reporter gene, phRL-SV40 vector, and expression constructs as mentioned in Fig. 3. Luciferase activities were normalized to the Renilla luciferase activity and are expressed relative to the pcDNA3 control. D, NF-{kappa}B p50 and p65 bind to the {kappa}B site in vivo. ChIP assay was performed as mentioned in Materials and Methods. The specific DNA fragments were immunoprecipitated with rabbit IgG or Abs specific for NF-{kappa}B p50 and p65 as indicated in IL-2-treated NK92 cells. The input contained fragmented DNA from prior immunoprecipitation reactions. Each of the PCR amplification products was identical to the expected size.

 
To further obtain direct in vivo evidence that NF-{kappa}B subunits interact with the {kappa}B binding site, ChIP assays were performed. NK92 cells were treated with IL-2 and were fixed with formaldehyde to cross-link DNA with proteins. Then, the fragmented chromatin from cell lysates was immunoprecipitated with Abs specific to NF-{kappa}B p50, p65, or control rabbit IgG under stringent conditions. As shown in Fig. 6D, in IL-2-activated NK92 cells, specific DNA fragments containing the putative {kappa}B site were detected following p50 or p65 immunoprecipitation. These data suggest that IL-2-induced NF-{kappa}B activation results in p50 and p65 heterodimer nuclear translocation followed by binding to the {kappa}B site. So, this newly identified {kappa}B binding site is also functional in vivo.

NF-{kappa}B signaling regulates human granzyme B gene transcription in primary human NK cells

To address whether the NF-{kappa}B pathway is also activated in primary NK cells, primary NK cells were isolated from the PBMC of healthy individuals. Fresh human NK cells were stimulated with 200 U/ml IL-2 for 4 h and EMSA was performed using NF-{kappa}B consensus probe. As shown in Fig. 7A, IL-2 activated NF-{kappa}B signaling and this process was suppressed by NF-{kappa}B inhibitors PDTC and SSZ. RT-PCR assays indicated that NF-{kappa}B activation was also associated with the up-regulation of human granzyme B gene transcription (Fig. 7B). These data are consistent with the results in NK92 cells.


Figure 7
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  		FIGURE 7. NF-{kappa}B signaling regulates human granzyme B gene transcription in primary human NK cells. Human primary NK cells were isolated from PBMC as mentioned in Materials and Methods. These enriched NK cells were then stimulated with 200 U/ml IL-2 for 4 h in the presence of 500 nM PDTC or 2 mM SSZ. A, Nuclear extracts were isolated from primary NK cells. The NF-{kappa}B consensus motif was labeled with [{gamma}-32P]ATP and used as a probe in EMSA. Cold competition assay was performed with a 100-fold molar excess of unlabeled NF-{kappa}B consensus WT or mutant motif. B, Total RNA was isolated from primary NK cells. RT-PCR was performed using the primers specific to human granzyme B and GAPDH genes as indicated. The ratio of granzyme B-GAPDH is shown as indicated and the ratio of starved group were assigned a value of 1.0 and the values from other groups were normalized based on this value.

 
NF-{kappa}B up-regulates granzyme B gene expression in Jurkat T cells

Granzyme B gene is mainly expressed in NK cells, LAK cells, and activated T cells. Recently, it was reported that the granzyme B is also expressed by T regulator cells (39). To address whether NF-{kappa}B signaling also plays a regulatory role for granzyme B transcription in activated T cells, Jurkat T cells were stimulated with PHA plus PMA for 2 h in the presence or absence of PDTC or SSZ and then granzyme B expression was determined. As shown in Fig. 8A, when Jurkat T cells were activated with PHA plus PMA, granzyme B transcription was up-regulated. In contrast, exposure to the NF-{kappa}B inhibitors PDTC and SSZ suppressed this process. Furthermore, p50 and p65 increased reporter activity of phgrB-{kappa}B vector in Jurkat T cells (Fig. 8B). A ChIP assay suggests that in Jurkat T cells, NF-{kappa}B p50 and p65 could also bind to the {kappa}B site in vivo in response to PHA plus PMA (Fig. 8C). These data indicate that the newly identified NF-{kappa}B binding site is functional not only in NK cells, but also in Jurkat T cells.


Figure 8
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  		FIGURE 8. NF-{kappa}B up-regulates granzyme B gene expression in Jurkat T cells. A, Jurkat T cells were activated with PHA (1 µg/ml) plus PMA (50 ng/ml) for 2 h, in the presence of 50 µM PDTC or 2 mM SSZ. Human granzyme B and GAPDH mRNA expression was determined by RT-PCR analysis. The ratio of granzyme B-GAPDH from all groups was normalized as mentioned before. B, p65 increased reporter activity of the phgrB-{kappa}B vector in Jurkat T cells. Jurkat T cells were cotransfected with the phgrB-{kappa}B vector, the phRL-SV40 vector, and expression constructs as mentioned in Fig. 3. Luciferase activities were normalized to the Renilla luciferase activity and are expressed relative to the pcDNA3 control. C, ChIP assay was performed as mentioned before.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IL-2 has been widely used in clinical cancer therapeutic research. Its mechanism of antitumor efficacy closely relates to its ability to expand and activate NK and T cells. The stimulation of NK cells with IL-2 results in an enhancement of cytotoxic function, recognizing a broader range of targets and killing with increased lytic potential. It is well known that IL-2 could induce perforin, granzyme B, Fas ligand, and TRAIL expression in NK cells (22, 23, 40, 41, 42), and these effector molecules are essential for NK cytotoxic function in cancer immunotherapy. In this report, we demonstrate that IL-2-induced stimulation of NK cells results in NF-{kappa}B activation, which up-regulates human granzyme B gene transcription in NK cells. An unrelated study indicated that perforin gene expression is also regulated by NF-{kappa}B signaling in IL-2-stimulated NK cells (24). Accordingly, NF-{kappa}B signaling may be one of the most important pathways for control of NK cell function, at least for its cytotoxic activity. This realization may give important insight into improving cancer therapeutic research by manipulating NK cell function.

To date, triggering of NF-{kappa}B activation through IL-2R-induced signaling is not well known. IL-2 ligation to its cognate receptor initiates the activation of several intracellular components including the tyrosine kinases, such as Jak1, Jak3, Syk, p56lck, and p70 S6 kinase. The IL-2R also recruits the adapter protein Shc and consequently it activates Ras/MAPK and PI3K/Akt (43, 44). In some cell types, there is serial control of NF-{kappa}B activation that depends on MAPK pathway stimulation. This concept is supported by the finding that MAPK kinase–MEKK1–stimulates the I{kappa}B-kinase complex (45). However, in our experiments, when NK92 cells were treated with MAPK pathway inhibitors and the PI3K inhibitor (LY 294002), they did not influence IL-2-triggered human granzyme B gene expression (data not shown). These results suggest that neither the MAPK nor the PI3K-signaling pathways affect IL-2R-mediated NF-{kappa}B activation in NK cells.

In this report, although we have identified a novel {kappa}B binding site (as an enhancer element) which plays an important role in inducing granzyme B gene transcription in NK cells, we could not exclude other factors’ potential regulatory effects. Hanson et al. (46) reported that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5' to the human granzyme B transcriptional start site are both required for transcriptional activation of the granzyme B promoter in 12-O-tetradecanoylphorbol-13-acetate + bt2cAMP-stimulated PEER cells. By mutation analysis of the human granzyme B promoter, Wargnier et al. (13) demonstrated that both the Ikaros binding site (–143 to –114) and the AP-1/CBF binding site (–103 to –77) are essential for the activation of human granzyme B transcription in PHA-activated peripheral blood lymphocytes. Babichuk et al. (14) observed a strong DNase1 hypersensitive site that coincides with the closely associated AP-1, CBF, Ikaros, and CRE elements which is present in activated murine CD8+ T cells, but not in resting T cells. Furthermore, both in vitro and in vivo footprints were observed at these sequence elements in activated CTLs. All the data indicate that some transcription factors such as Ikaros, AP-1, and CRE are also involved in granzyme B activation in T cells. In contrast, how these factors regulate granzyme B activation in NK cells is unknown. In this report, we demonstrated in NK92 cells that IL-2 induces granzyme B activation through the NF-{kappa}B pathway by binding to a newly identified enhancer element on the granzyme B gene. We also observed that CRE and JunB could be induced by IL-2 stimulation in NK92 cells (data not shown). Taken together, it is likely there are other factors besides NF-{kappa}B that may also modulate granzyme B activation in NK92 cells.

In summary, we demonstrate that the NF-{kappa}B-signaling pathway is involved in the transcriptional regulation of the human granzyme B gene in NK cells. A novel NF-{kappa}B binding site (GGAGATTCCC) responsible for granzyme B gene activation has been identified downstream from this gene. This NF-{kappa}B binding site is functional in vitro and in vivo, indicating that this binding site plays a pivotal role in human granzyme B gene transcriptional regulation.


   Acknowledgments
 
We thank Dr. Jonathan D. Licht for providing us with the p50 expression plasmid and Dr. Chen Wang for the p65 and pcDNA3- I{kappa}B{alpha} AA expression plasmids. We thank Dr. Peter Reinach for critical reading of the manuscript.


   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 the Technology Commission of Shanghai Municipality (Grants 04DZ14902, 04DZ19108, and 03JC14085), National Key Basic Research Program of China (Grant 2001CB510006), National Natural Science Foundations of China (Grants 30421005, 30530700, and 30170888), the Outstanding Young Scientist Fund by the National Natural Science Foundation of China (Grants 30228016 and 30325018), and a grant from E-Institutes of Shanghai Universities Immunology Division. Back

2 Address correspondence and reprint requests to Dr. Bing Sun, Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China. E-mail address: bsun{at}sibs.ac.cn Back

3 Abbreviations used in this paper: LAK, lymphokine-activated killer; CBF, core-binding factor; TPCK, N{alpha}-tosyl-Phe chloromethyl ketone; PDTC, ammonium pyrrolidinedithiocarbamate; SSZ, sulfasalazine; ChIP, chromatin immunoprecipitation; hgrB, human granzyme B; PI, propidium iodide; CRE, cAMP response element. Back

Received for publication July 25, 2005. Accepted for publication January 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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