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The Journal of Immunology, 2001, 166: 6188-6195.
Copyright © 2001 by The American Association of Immunologists

A Prominent Role for Activator Protein-1 in the Transcription of the Human 2B4 (CD244) Gene in NK Cells1

Samuel S. Chuang, Hoang-Tuan K. Pham, Pappanaicken R. Kumaresan and Porunelloor A. Mathew2

Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cell surface glycoprotein 2B4 (CD244) of the Ig superfamily is involved in the regulation of NK and T lymphocyte functions. We have recently identified CD48 as the high affinity counterreceptor for 2B4 in both mice and humans. The cytoplasmic domain of 2B4 associates with src homology 2 domain-containing protein or signaling lymphocyte activation molecule-associated protein, whose mutation is the underlying genetic defect in the X-linked lymphoproliferative syndrome. In this study, we report the molecular cloning and characterization of the human 2B4 (h2B4) promoter. Through primer extension analysis, we found that the transcription of the h2B4 gene initiates at multiple start sites. We isolated h2B4 genomic clones and PCR amplified the 5' untranslated region containing the promoter elements. We have identified a functional AP-1 site that lies between (-106 to -100) through transient transfection analysis in YT cells, a human NK cell line. EMSAs with Abs specific for various protein factors of the AP-1 family revealed that multiple members of the Jun family are involved in the regulation of the h2B4 gene. Mutation of the AP-1 site not only abolishes protein/DNA interactions but also promoter activity. These results demonstrate a significant role for AP-1 in the transcriptional regulation of the h2B4 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The surface molecule 2B4 (CD244) is implicated in the activation of NK cell-mediated cytotoxicity (1, 2, 3). 2B4 was originally identified on mouse NK cells and the subset of T cells that mediate non-MHC-restricted killing (4, 5, 6, 7). In addition to modulating non-MHC-restricted killing, 2B4 activation of NK cells induces cytokine production as well as invasiveness (4, 8). It is a member of the Ig superfamily and belongs to the CD2 subset that includes signaling lymphocyte-activation molecule (SLAM), CD48, CD58, CD84, and Ly9. Human 2B4 (h2B4)3 is found on all NK cells, a subset of CD8+ T cells, monocytes, and basophils (2). We have recently identified CD48 as the high affinity counterreceptor of 2B4 in both mice and humans (9). CD48-2B4 interactions are physiologically important because they enhance the lytic function of human NK cells (2). It has been reported that 2B4 may function as a coreceptor in human NK cell activation (10). It has been found that 2B4 associates with the signaling adapter molecule SLAM-associated protein (SAP)/Src homology 2 domain-containing protein (11). Mutations in the Src homology 2 domain of SAP/Src homology 2 domain-containing protein have been identified as the genetic defect in X-linked lymphoproliferative disease (XLPD), and a number of recent studies demonstrated that 2B4 signaling is defective in XLPD (12, 13, 14, 15, 16, 17). Another study of XLPD patients showed that not only does 2B4 fail to transduce activating signals in NK cells but that CD48-2B4 interactions inhibited activating signals transduced by other stimulatory molecules including CD16, NKp46, NKp44, and NKp30 (18). Furthermore, a recent report by Peritt et al. demonstrated that the expression of 2B4 on CD8+ T cells is a better predictor of disease progression in AIDS patients than CD4+ T cell levels (19). Although no polymorphism has been reported in humans, our studies of 2B4 in inbred mouse strains demonstrate polymorphism in the extracellular domain (20).

Over the last few years, a wealth of information has been accumulated on the identification, ligand interactions, and signaling pathways of several receptors expressed on NK cells. However, knowledge of the mechanisms underlying the regulation of expression of these receptors is fragmentary. To understand the transcriptional regulation as well as the mechanisms controlling the restricted expression of the 2B4 gene, we conducted preliminary analysis in characterizing the promoter of the mouse 2B4 (m2B4) gene. Our studies demonstrated that transcription of m2B4 did not start at a conserved initiation site and was driven by a TATA-less promoter (21). Moreover, transient transfections with fragments of the 2B4 promoter revealed cell-specific promoter activity in cells that express 2B4 only (21).

This study was performed to understand the mechanisms that regulate the expression of the h2B4 gene. To accomplish this, we isolated a genomic clone of h2B4 from a {lambda} phage library and identified >1 kb of 5' flanking sequence of the h2B4 gene. We undertook the task of characterizing the 5' flanking region of the h2B4 gene by constructing 5' nested deletion constructs and assayed their promoter activity by transient transfection of YT cells, a NK tumor cell line. We present evidence that the presence of an AP-1 site located at -106 to -100 of the h2B4 promoter strongly activates transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

YT (human NK tumor cell line) and K562 (human tumor erythroleukemia cell line) were cultured in 4 + RPMI complete medium (RPMI 1640 supplemented with 10% FBS (HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 10 mM nonessential amino acids). NK-92 (human NK tumor cell line) cells were cultured in {alpha} complete medium ({alpha} MEM supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2-ME, 0.02 mM folic acid, 200 U/ml rIL-2, 12.5% horse serum, and 12.5% FBS). Growth was at 37°C in a humidified 5% CO2/95% air incubator. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) unless otherwise noted. C1.7 Ab, which recognizes h2B4 (11), was purchased from Coulter (Orlando, FL). Affinity-purified rabbit anti-mouse Abs against c-Jun (catalog no. SC-822X), c-Fos (catalog no. SC-253X), and JunB (catalog no. SC-8051X) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All enzymes were purchased from New England Biolabs (Beverly, MA) unless otherwise stated. pGL2 and pRL vectors and dual-luciferase reporter assay system were purchased from Promega (Madison, WI). Poly(dI:dC) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All custom synthesized oligonucleotides used in this study were supplied by Integrated DNA Technologies (Coralville, IA).

Cloning of the h2B4 promoter and plasmid construction

A human genomic DNA library constructed in Fix II phagemid vector (Stratagene, La Jolla, CA) was screened with full-length [{alpha}-32P]dCTP-labeled h2B4 cDNA as probe. Full-length labeled h2B4 cDNA probe was generated using [{alpha}-32P]dCTP (DuPont-NEN, Boston, MA) and the Megaprime kit from Amersham Pharmacia Biotech. Using Escherichia coli XL1-blue as host strain, 1 x 105 plaques were plated and lifted onto nylon N(+) Hybond membrane (Amersham Pharmacia Biotech) according to standard protocols. After three successive rounds of screening, nine different positive phage clones were isolated and phage DNA was isolated by the method described by Lee et al. (22). Partial sequencing of the genomic clones was performed by automated sequencing (sequencing facility, University of Texas Southwestern Medical Center, Dallas, TX) using specific oligonucleotides based on h2B4 cDNA {lambda}19A1A (1).

Nested promoter fragments were derived by PCR using Taq polymerase (Promega) and using genomic DNA clone (F5A1) containing the 5' flanking sequence of the h2B4 gene as template (23). Various 5' 20–22 base primers were designed to incorporate a KpnI site for ease of subcloning. The 3' primer, identical for all the constructs, was designed with a XhoI site and ended at nucleotide +126 relative to the start of transcription (Table IGo). The nested promoter fragments were then cloned upstream of a promoterless and enhancerless firefly luciferase gene in the pGL2-B vector (Promega). The numbering of the constructs refers to the first nucleotide of each promoter construct relative to the start of transcription. The PCR-derived fragments were digested with KpnI and XhoI and ligated into similarly digested pGL2-B vector. All constructs were verified by nucleotide sequencing. Substitution mutant promoter constructs were generated by a two-step PCR procedure using overlapping internal primers that contain a mutant sequence, as described previously (24, 25). A Renilla luciferase reporter plasmid, pRL-CMV driven by an upstream CMV immediate-early enhancer/promoter region (Promega) was cotransfected with the pGL2 promoter constructs to adjust firefly luciferase activity for transfection efficiency. All plasmid DNA used in transient transfection assays were purified by two rounds of CsCl centrifugation.


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  		Table I. Oligonucleotides used in this study

 
Primer extension analysis

Primer extension analysis was performed using a modified procedure as described by Chen et al. (26). The primer used for primer extension analysis codes for the complementary sequence to the region (+127 to +107) from relative to the first base of the h2B4 start of transcription (see Fig. 1Go). The primer was end-labeled with [{gamma}-32P]dATP (specific activity, 6000 Ci/mmol) (DuPont-NEN) and T4 polynucleotide kinase (New England Biolabs). Six micrograms total RNA from YT cells was heated to 70°C for 10 min and then iced for 2 min. Labeled primer (200 fmol) was annealed to YT total RNA at 85°C for 5 min and then cooled to 42°C. Primer extension assays were conducted using Superscript II reverse transcriptase (Life Technologies) at 42°C for 2 h and resolved on a 6% polyacrylamide sequencing gel. A G+A sequencing reaction was conducted and run in parallel with the samples during electrophoresis as a marker. The G+A ladder was generated using a Maxam-Gilbert sequencing reaction as described previously (27).



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  		FIGURE 1. Nucleotide sequence of the 5' flanking region of the h2B4 gene. Nucleotide sequence was analyzed and screened for potential transcription factor binding sites using TESS: transcription element search site (http://agave.humgen.upenn.edu/tess). Several potential transcription factor-binding sites are underlined. *, First base of the h2B4 cDNA clone. Arrows indicate the positions of initiation start sites revealed by primer extension analysis. The numbers on the left indicate the nucleotide positions of the h2B4 promoter based on the most 3' initiation start site designated as +1.

 
Transfection and luciferase assays

YT cells were transfected with each of the 4 µg of firefly luciferase reporter constructs, and 400 ng of pRL-CMV, a Renilla luciferase reporter plasmid, for internal normalization of transfection efficiency. The cells were transfected using the DMRIE-C reagent at a 2.2:3 ratio of µg DNA/µl DMRIE-C reagent as per the manufacturer’s instructions (Life Technologies). Cell lysates were then harvested 40 h poststimulation. Firefly and Renilla luciferase assays were performed using the Dual-Luciferase reporter assay system as per manufacturer’s instructions (Promega). Each test promoter construct was cotransfected with pRL-CMV into YT cells in at least four independent trials.

Human NK cell isolation

Peripheral blood from healthy donors was diluted with two volumes of PBS and then layered over Ficoll Paque (Pharmacia, Piscataway, NJ). It was then subjected to centrifugation at 400 x g for 30 min. PBMC were then extracted from the interface. The cells were then washed with three volumes of HBSS followed by an additional wash with one volume of HBSS. The cell pellet was then resuspended in 1 ml PBS (containing 0.5% BSA). The NK cells were then purified using an NK cell isolation kit from Miltenyi Biotec (Auburn, CA) per manufacturer’s instructions.

Nuclear extraction and EMSA

Nuclear extracts were isolated from YT cells, NK-92 cells, and freshly isolated human NK cells (28), and protein-DNA binding reactions were conducted (29). A typical binding reaction mixture contained 2 µg of nuclear protein, 1 µg of poly(dI:dC), and radiolabeled oligonucleotide (20,000 cpm, ~0.2 ng) in 10 µl reaction volume. All double-stranded oligonucleotide sequences are listed in Table IGo. The mixture was incubated on ice for 30 min and then electrophoresed through a 4% polyacrylamide gel under nondenaturing condition in 0.25x Tris-borate-EDTA at 200 V for 70 min. The gel was dried and then exposed to film. The bands were visualized by autoradiography. For competition analysis, 100 and 200 molar excess amounts of unlabeled double-stranded oligonucleotides were included in the binding reactions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of 5' flanking sequences of the h2B4 gene

To obtain h2B4 genomic clones and to isolate the 5' flanking sequence, we screened a human genomic library constructed in FIX II phagemid vector (Stratagene) using [{alpha}-32P]ATP-labeled full-length h2B4 cDNA as probe. Nine genomic clones were isolated and EcoRI endonuclease digestion was conducted on each clone. Four different genomic clones with insert sizes ranging from 20 to 30 kb were chosen for further study. One of the genomic clones, F5A1, contained a 5' flanking sequence relative to the h2B4 gene. Approximately 1500 bases of the promoter region (GenBank accession no. AF297616) 5' to the h2B4 gene was amplified by using forward primer F5A1-FP and reverse primer F5A1-RP (Fig. 1Go). The characterization of the genomic structure of h2B4 has already been reported (23).

Transcription of the h2B4 gene is initiated at multiple start sites

To identify the transcription initiation site of the h2B4 gene, we performed primer extension using total RNA extracted from YT and NK92 cells. We have shown previously that YT cells express h2B4 (8). We found through FACS analysis with C1.7 mAb ({alpha}-h2B4) conjugated with FITC, that NK92 cells also express h2B4 (data not shown). Primer extension analysis using total RNA from YT cells revealed several extended extension products (Fig. 2Go). Similar to the m2B4 gene, the h2B4 gene does not have a conserved initiation start site nor does it contain a TATA box near the transcription initiation start sites (21). Primer extension analysis repeated on total RNA extracted from NK92 cells produced identical extension products (data not shown).



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  		FIGURE 2. Primer extension analysis of the h2B4 promoter identified multiple initiation start sites. An h2B4 specific primer annealing to sequences downstream of the first base of the cDNA clone of h2B4 was annealed to 6 µg of YT total RNA. The primer extension assay was conducted as described in Materials and Methods. The same primer was used with an upstream primer to generate the G+A ladder as described previously (27 ). Arrows and numbers indicate the primer extension products resolved and their positions relative to the most 3' primer extension product designated as +1.

 
Identification of 5' flanking sequences required for the induction of h2B4 promoter activity

To identify the regions of the promoter that play a role in the transcriptional regulation of the h2B4 gene, we performed transient transfection assays on YT cells. A series of promoter mutants that contain successive deletions from the 5' end were inserted upstream of a firefly reporter gene within the pGL2 vector (Fig. 3GoA). It has been reported that YT cells are poor candidates for transient transfection (30). Transient cotransfection of various constructed promoter vectors and a Renilla luciferase CMV promoter-driven reporter plasmid using DMRIE-C reagent were conducted. Using a ratio of 4.4 µg plasmid DNA to 6 µl DMRIE-C reagent, we were able to achieve 2.5–4% transfection efficiency, enough to observe measurable luciferase activity. The firefly and Renilla luciferase activity was then measured 40 h posttransfection. Transient transfections of these 5' deletion promoter constructs into YT cells revealed several important regions of the 2B4 promoter that regulate transcription. Promoter regions (-188 to -80) and (-852 to -704) were identified to have a positive effect on transcription, whereas the promoter regions (-414 to -342) and (-704 to -505) had a negative effect. Transfections conducted revealed that maximal luciferase activity was achieved with the promoter fragment (p-342) produced luciferase activity that was almost 4-fold higher than the smallest promoter fragment (p-80), which we designated a luciferase activity value of 1 (Fig. 3GoB). When the promoter fragment was reduced further from (-188 to -88) relative to the start of transcription, there was a significant reduction of activity by 72% (p < 0.001). These results reveal that the DNA sequences between -188 and +126 are sufficient for strong promoter activity in YT cells and that at least one essential DNA binding element is located within the -188 to -80 region of the h2B4 promoter.



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  		FIGURE 3. 5'-deletional analysis of the h2B4 promoter. A, YT cells were transfected with a series of 5' promoter deletion mutants in the pGL2B reporter vector, along with a pRL3-CMV control plasmid. Each promoter fragment was inserted in front of the firefly luciferase gene. The most 5' base position relative to +1 is denoted on the right. Each construct ends at the +126 nucleotide position. Transfected cells were cultured in complete medium for 40 h. B, Firefly luciferase activity, following normalization to Renilla luciferase activity, are expressed as the mean relative luciferase activity + SE (from four to six independent experiments) to the firefly luciferase activity expressed when the p-80 construct was transfected into YT cells. The normalized firefly luciferase activity expressed in YT cells transfected with the p-80 construct was assigned a value of 1.

 
Identification of an AP-1 binding site

Sequence analysis of the DNA sequence that lies between -188 and -80 relative to the start of transcription reveals a consensus binding site for AP-1. To determine the relevance of this element, we performed EMSAs with nuclear protein extracts from YT and NK-92 cells. We used double-stranded oligonucleotides coding for the promoter sequence (-111 to -89) radiolabeled with [{gamma}-32P]-dATP as probes in EMSAs. Using nuclear protein extracts from NK92 cells in the DNA binding reactions showed multiple protein-DNA complexes (Fig. 4Go). However, when unlabeled double-stranded oligonucleotides coding for the promoter sequence (-111 to -89) and for the consensus DNA binding sequence for AP-1 were used as cold competitors at 100- and 200-fold excess to the amount of labeled probe used, only one band was competed away. One hundred and two hundred molar excess amounts of unlabeled custom synthesized double-stranded oligonucleotides coding for the consensus DNA binding sequence for NF-{kappa}B and coding for the h2B4 promoter sequence spanning -111 to -89 with the mutated AP-1 binding site as (TGccgCA) were added to the DNA binding reactions. The specific protein-DNA complex failed to be competed away by these competitors respectively (Fig. 4Go). EMSAs conducted in identical fashion using nuclear protein extracts from YT cells produced very similar results (data not shown). These results indicate that the upper protein-DNA binding complex is specific.



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  		FIGURE 4. AP-1 binding activity at -106 to -100 of the h2B4 promoter detected by EMSA. An EMSA experiment was performed with a radiolabeled double-stranded oligonucleotide coding for the h2B4 promoter sequence spanning -111 to -89 relative to the start of transcription. –ve lane, Binding reaction performed in the absence of NK92 cell nuclear extract. +ve lane, Binding reaction performed in the presence of 2.7 µg NK92 cell nuclear extract. Unlabeled competitor DNAs were included in the binding reactions containing NK92 nuclear extract at 100 and 200 molar excess, respectively. The competitor DNAs used were wt (wild type cold probe), AP-1 (double-stranded oligonucleotides coding for AP-1 consensus binding site), NF-{kappa}B (NF-{kappa}B consensus binding site), and Mut (double-stranded oligonucleotides coding for the h2B4 promoter sequence spanning -111 to -89 with the mutated AP-1 binding site as TGccgCA). ], Specific AP-1 protein DNA interactions. N.S., Nonspecific bands. Free, Unbound probe.

 
To determine the identities of the protein-bound fraction involved in the upper protein-DNA complex, we conducted the DNA binding reactions with nuclear protein extracts from NK92 cells in the presence of a panel of Abs. In the presence of Abs specific for c-Jun and JunB, the presence of a supershifted protein DNA complex was observed (Fig. 5Go and data not shown). However, when an isotype control Ab (22B5) was used, no detectable additional band or any change in the presence of the other protein-DNA complexes could be detected. The c-Fos family is often associated with the Jun family of proteins to form the AP-1 protein complex (31). When we incubated anti-c-Fos Ab in the DNA binding reactions, we observed the presence of the specific upper protein-DNA complex severely diminished. Instead, the anti-c-Fos Ab has clearly shifted the band (Fig. 5Go). Thus, Ab-mediated supershift analysis in EMSAs indicate that AP-1 interacts with the h2B4 promoter and that there may be multiple forms of AP-1 involved in the transcription regulation of the h2B4 gene.



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  		FIGURE 5. The specific binding of AP-1 demonstrated with anti-c-Jun and anti-c-Fos Abs in EMSA. EMSA was performed with radiolabeled double-stranded oligonucleotides coding for the h2B4 promoter sequence spanning -111 to -89 relative to the start of transcription as outlined in Materials and Methods. The arrow indicates the supershifted protein/DNA complexes. –ve lane, Binding reaction performed in the absence of NK92 cell nuclear extract. +ve lane, Binding reaction performed in the presence of 2.7 µg NK92 cell nuclear extract. ], Where the AP-1 protein-DNA complex is absent. N.S., Nonspecific bands. Free, Unbound probe.

 
To verify the constitutive expression of AP-1 in human NK cells, we isolated untouched human primary cells from the PBMCs of healthy adult donors by cell exclusion using a human NK cell isolation kit (Miltenyi Biotec). Nuclear protein extracts were generated from the isolated cells and used in EMSAs using double-stranded oligonucleotides coding for the promoter sequence (-111 to -89) radiolabeled with [{gamma}-32P]dATP as probe (Fig. 6Go). The presence of DNA-protein complex suggests that AP-1 in the nuclear extracts of primary human NK cells interacts with h2B4 promoter. Ab-mediated supershift analysis using anti-c-Jun, anti-JunB, and anti-c-Fos further confirmed that AP-1 in nuclear extracts from primary NK cells interacts with the h2B4 promoter.



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  		FIGURE 6. AP-1 specific binding to the h2B4 promoter detected in human primary NK cells. EMSA was performed incubating nuclear protein extracts from isolated human primary NK cells from peripheral blood incubated with radiolabeled double-stranded oligonucleotides coding for the h2B4 promoter sequence spanning -111 to -89 relative to the start of transcription. Arrow indicates the supershifted protein/DNA complexes. –ve lane, Binding reaction performed in the absence of human NK cell nuclear extract. +ve lane, Binding reaction performed in the presence of 1.2 µg human NK cell nuclear extract. ], Where the AP-1 protein-DNA complex is absent. N.S., Nonspecific bands. Free, Unbound probe.

 
The AP-1 site (-106 to -100) regulates the h2B4 promoter

Upon the determination of AP-1 binding to the promoter sequence within the -111 to -89 sequence of the h2B4 promoter and the identification of the consensus DNA binding site sequence for AP-1 within this sequence, we wanted to examine the functional role of AP-1 in the transcription of the h2B4 gene. To this end, we synthesized two complementary oligonucleotides that code for the promoter sequence within the -111 to -89 sequence of the h2B4 promoter with the AP-1 binding site mutated from TGAGTCA to TGccgCA. These oligonucleotides were annealed and radiolabeled with [{gamma}-32P]dATP to form a double-stranded EMSA probe. EMSAs using this mutant probe revealed the loss of the specific protein-DNA complex (Fig. 7GoA).



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  		FIGURE 7. The mutation in the AP-1 binding site inhibits AP-1 protein binding and h2B4 promoter function. A, Using radiolabeled double-stranded oligonucleotides coding for the h2B4 promoter sequence spanning -111 to -89 with the mutated AP-1 binding site as TGccgCA in EMSAs with NK92 nuclear extracts, specific AP-1 protein binding was abolished. -ve lane, Binding reaction performed in the absence of NK92 cell nuclear extract. +ve lane, Binding reaction performed in the presence of 2.7 µg NK92 cell nuclear extract. ], Where the AP-1 protein-DNA complex is absent. N. S., Nonspecific bands. Free, Unbound probe. B, AP-1 binding site mutant constructs were created and used in the transient promoter reporter assay. Lower case letters are nucleotides used to mutate the underlined letters within the AP-1 binding site. Each promoter fragment was inserted in front of the firefly luciferase gene. Transfected cells were cultured in complete medium for 40 h. C, Firefly luciferase activity, following normalization to Renilla luciferase activity, are expressed as the mean relative luciferase activity + SE (from four to six independent experiments) to the firefly luciferase activity expressed when the p-80 construct was transfected into YT cells. The normalized firefly luciferase activity expressed in YT cells transfected with the p-80 construct was assigned a value of 1. *, p < 0.001 (from the respective wild-type counterpart).

 
To assess the functional significance of AP-1 DNA binding site in the h2B4 promoter, we synthesized mutant promoter constructs (p-M188 and p-M342) using the oligonucleotides outlined above and using the p-188 and p-342 as templates in multiple rounds of PCR (as described in Materials and Methods), respectively (Fig. 7GoB). Transient transfections with the mutant promoter constructs revealed the mutation of the AP-1 site significantly reduced luciferase activity (p < 0.001) of both constructs (Fig. 7GoC). The data suggest that AP-1 regulates the transcription of the h2B4 gene.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have cloned the h2B4 promoter region and identified a significant role for AP-1 in the transcriptional regulation of the h2B4 gene. The expression of h2B4 has been found on all human NK cells, a subset of CD8+ T cells, monocytes, and basophils (2). In an effort to identify cis- and trans-acting factors that can regulate the h2B4 promoter, we isolated a genomic clone that contained the 5' flanking region of the h2B4 gene. To identify those regions in the promoter that play a role in regulating the transcription of the h2B4 gene, 5' nested deletion constructs were created and inserted in a forward orientation in front of a firefly luciferase reporter gene. Transient transfection analysis revealed the presence of the region (-188 to -80) had a 3.5-fold positive effect on transcription over the minimal promoter (-80 to +126) (Fig. 3GoB). Several other regions may also play a role in regulating the transcription of the h2B4 gene (Fig. 8Go). Within the -188 to -80 region of the h2B4 promoter is a putative AP-1 DNA binding site that matches the consensus binding site for AP-1. Comparison to the 5' flanking sequence of the m2B4 promoter shows a striking resemblance. There is a putative AP-1 binding site in the m2B4 promoter whose location is similar relative to the multiple initiation start sites as we have found in the h2B4 promoter. The presence of the mouse AP-1 site also had a significant positive effect on transcription activity (21).



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  		FIGURE 8. Schematic diagram showing the regulating regions of the h2B4 gene. + and -, Positive and negative cis-acting sequences active in YT cells, respectively, and their relative strength. Numbers indicate nucleotide positions defining the regions relative to the start of transcription (+1).

 
In an effort to show that AP-1 interacts with the -188 to -80 site, we performed EMSAs using nuclear protein extracts from YT and NK-92 cells and labeled double-stranded oligonucleotides coding for the -111 to -89 region of the h2B4 promoter in the presence of various specific and nonspecific unlabeled double-stranded oligonucleotide competitors. As we expected, only those competitors that contained an AP-1 binding site were able to compete away the protein DNA complex as shown in Fig. 4Go. Additionally, heat treatment of nuclear protein extract from either YT or NK92 cells at 85°C for 5 min abolished the specific protein-DNA complex (data not shown). To identify whether the protein within the specific protein/DNA complex was in fact AP-1, we conducted supershift assays with Abs that were specific to various members of the Jun and Fos families. The AP-1 transcription factor consists of heterodimers between members of the Jun and Fos families of proteins. Interestingly, both Abs specific for c-Jun and JunB partially supershifted the protein/DNA complex. Ab specific for the Fos family supershifted the entire protein/DNA complex (Fig. 4Go). Similar results were observed when we used nuclear protein extracts from isolated human primary NK cells from peripheral blood (Fig. 6Go). Thus, it appears that there are multiple AP-1 complexes that may bind the h2B4 promoter. Activation of the MAP kinase pathways (c-Jun N-terminal kinase, MAP/extracellular signal-related kinase kinase, extracellular signal-related kinase) not only controls the overall levels of Jun and Fos proteins, but also leads to the phosphorylation states necessary for transcriptional activity (32). Thus, transcription of the h2B4 gene may be regulated by the various signaling pathways that can stimulate the various MAP kinase pathways.

The significance of initiation of transcription at multiple transcription start sites (Fig. 2Go) is not known. It has been shown that there are multiple splice variants of 2B4 in both mice and human (18, 23, 33). Characterization of the genomic structure of h2B4 revealed that the different transcripts are derived from a single gene (23). Therefore, it is tempting to speculate that the multiple transcription initiation sites present in the h2B4 gene promoter may have a role in the production of different splice variants. In the mouse, the splice variants give rise to different isoforms of 2B4 that display opposing signals to NK cells (33, 34). However, the functional roles of h2B4 splice variants are not known (18, 23). It is possible that the multiple transcription start sites present in the h2B4 promoter may be differentially used under different conditions of activation of NK cells.

The cytoplasmic tail of h2B4 contains four novel tyrosine-based motifs (1). These motifs are similar to those found on other CD2 subfamily members including SLAM, Ly9, CS1, and CD84 (35, 36, 37, 38) and have been shown to interact with protein tyrosine phosphatase, SHP-2 and the Src homology-2 domain containing adaptor molecule, SAP (14, 35). Mutations in SAP have been identified as the genetic defect in XLPD (14). Although h2B4 on the NK cells of these patients are normal, 2B4 could not interact with the mutant SAP (18). Immunoprecipitations of h2B4 from NK cells from these patients coprecipitated SHP-1. This is contrary to previous reports that showed SHP-2, not SHP-1 is able to associate with the h2B4 cytoplasmic tail (10). Additionally, h2B4 has been found to localize to the glycolipid-enriched microdomains and to associate with another adaptor molecule, linker for activation of T cells (39, 40). Linker for activation of T cells has been found to be constitutively associated to 2B4, and that engagement of 2B4 results in the recruitment of other signaling molecules including phospholipase C{gamma} and Grb2 (40). Recently, it has also been shown that 2B4 signaling can be regulated by NK inhibitory receptors engaged by their MHC class I ligands on resistant cells (41). This is caused by the recruitment of SHP-1, which blocks phosphorylation of the 2B4 cytoplasmic tail. Although our understanding of the signaling pathway(s) h2B4 uses to increase cytolytic activity and cytokine release is incomplete, there is evidence that transcriptional events are necessary for NK cell cytotoxicity. A recent study found NK cell treatment of inhibitors of the AP-1 pathway prevented natural cytotoxicity of susceptible target tumor cells by NK cells (42). We have found that h2B4 stimulation of NK cells triggers many events that may be controlled through transcription, including an increase in transcription of IFN-{gamma} and MMP-2 (8, 43).

We have identified and begun to characterize the h2B4, which modulates NK cell functions (1, 8). It would be very interesting to investigate the role that AP-1 may play in the signal transduction pathway of h2B4 in NK cells. AP-1 has been found to transcriptionally regulate many immune response genes including IFN-{gamma} (44, 45), IL-3 (46), granzyme B (47), IL-2 (48, 49), IL-5 (50), and now h2B4. Many studies done on various NK cell stimulation pathways have found involvement of AP-1 and the MAP kinase pathways (50, 51, 52, 53, 54, 55, 56, 57, 58, 59). Thus it appears that AP-1 may play a major role in the signaling activation of NK cells and that h2B4 gene expression, regulated through AP-1, may be controlled via many different pathways.


   Acknowledgments
 
We thank Kent S. Boles for insightful discussions and critical review of the manuscript. We also thank Dr. Vinay Kumar (University of Chicago Medical School, Chicago, IL) and Dr. Michael Bennett (University of Texas Southwestern, Dallas, TX) for the gift of mAb 22B5.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI 38938. Back

2 Address correspondence and reprint requests to Dr. Porunelloor Mathew, Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699. Back

3 Abbreviations used in this paper: h2B4, human 2B4; SLAM, signaling lymphocyte-activation molecule; SAP, SLAM-associated protein; XLPD, X-linked lymphoproliferative disease; m2B4, mouse 2B4; MAP, mitogen-activated protein. Back

Received for publication September 1, 2000. Accepted for publication March 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Boles, K. S., H. Nakajima, M. Colonna, S. S. Chuang, S. E. Stepp, M. Bennett, V. Kumar, P. A. Mathew. 1999. Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4. Tissue Antigens 54:27.[Medline]
  2. Nakajima, H., M. Cella, H. Langen, A. Friedlein, M. Colonna. 1999. Activating interactions in human NK cell recognition: the role of 2B4-CD48. Eur. J. Immunol. 29:1676.[Medline]
  3. Nakajima, H., M. Colonna. 2000. 2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism. Hum. Immunol. 61:39.[Medline]
  4. Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, V. Kumar. 1993. A novel function-associated molecule related to non-MHC-restricted cytotoxicity mediated by activated natural killer cells and T cells. J. Immunol. 151:60.[Abstract]
  5. Mathew, P. A., B. A. Garni-Wagner, K. Land, A. Takashima, E. Stoneman, M. Bennett, V. Kumar. 1993. Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells. J. Immunol. 151:5328.[Abstract]
  6. Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, A. Takashima. 1995. Activation of murine epidermal {gamma}{delta} T cells through surface 2B4. Eur. J. Immunol. 25:1117.[Medline]
  7. Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, A. Takashima. 1995. 2B4, a new member of the immunoglobulin gene superfamily, is expressed on murine dendritic epidermal T cells and plays a functional role in their killing of skin tumors. J. Invest. Dermatol. 105:592.[Medline]
  8. Chuang, S. S., M. H. Kim, L. A. Johnson, P. Albertsson, R. P. Kitson, U. Nannmark, R. H. Goldfarb, P. A. Mathew. 2000. 2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness. Immunology 100:378.[Medline]
  9. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J. Exp. Med. 188:2083.[Abstract/Free Full Text]
  10. Sivori, S., S. Parolini, M. Falco, E. Marcenaro, R. Biassoni, C. Bottino, L. Moretta, A. Moretta. 2000. 2B4 functions as a co-receptor in human NK cell activation. Eur. J. Immunol. 30:787.[Medline]
  11. Tangye, S. G., S. Lazetic, E. Woollatt, G. R. Sutherland, L. L. Lanier, J. H. Phillips. 1999. Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP. J. Immunol. 162:6981.[Abstract/Free Full Text]
  12. Nichols, K. E., D. P. Harkin, S. Levitz, M. Krainer, K. A. Kolquist, C. Genovese, A. Bernard, M. Ferguson, L. Zuo, E. Snyder, et al 1998. Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proc. Natl. Acad. Sci. USA 95:13765.[Abstract/Free Full Text]
  13. Coffey, A. J., R. A. Brooksbank, O. Brandau, T. Oohashi, G. R. Howell, J. M. Bye, A. P. Cahn, J. Durham, P. Heath, P. Wray, et al 1998. Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene. Nat. Genet. 20:129.[Medline]
  14. Sayos, J., C. Wu, M. Morra, N. Wang, X. Zhang, D. Allen, S. van Schaik, L. Notarangelo, R. Geha, M. G. Roncarolo, et al 1998. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature 395:462.[Medline]
  15. Tangye, S. G., J. H. Phillips, L. L. Lanier, K. E. Nichols. 2000. Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome. J. Immunol. 165:2932.[Abstract/Free Full Text]
  16. Benoit, L., X. Wang, H. F. Pabst, J. Dutz, R. Tan. 2000. Defective NK cell activation in X-linked lymphoproliferative disease. J. Immunol. 165:3549.[Abstract/Free Full Text]
  17. Nakajima, H., M. Cella, A. Bouchon, H. L. Grierson, J. Lewis, C. S. Duckett, J. I. Cohen, M. Colonna. 2000. Patients with X-linked lymphoproliferative disease have a defect in 2B4 receptor-mediated NK cell cytotoxicity. Eur. J. Immunol. 30:3309.[Medline]
  18. Parolini, S., C. Bottino, M. Falco, R. Augugliaro, S. Giliani, R. Franceschini, H. D. Ochs, H. Wolf, J. Y. Bonnefoy, R. Biassoni, et al 2000. X-linked lymphoproliferative disease: 2B4 molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill Epstein-Barr virus-infected cells. J. Exp. Med. 192:337.[Abstract/Free Full Text]
  19. Peritt, D., D. A. Sesok-Pizzini, R. Schretzenmair, R. R. Macgregor, N. M. Valiante, X. Tu, G. Trinchieri, M. Kamoun. 1999. C1.7 antigen expression on CD8+ T cells is activation dependent: increased proportion of C1.7+CD8+ T cells in HIV-1-infected patients with progressing disease. J. Immunol. 162:7563.[Abstract/Free Full Text]
  20. Kumaresan, P. R., V. T. Huynh, P. A. Mathew. 2000. Polymorphism in the 2B4 gene of inbred mouse strains. Immunogenetics 51:758.[Medline]
  21. Chuang, S. S., Y. Lee, S. E. Stepp, P. R. Kumaresan, P. A. Mathew. 1999. Molecular cloning and characterization of the promoter region of murine natural killer cell receptor 2B4. Biochim. Biophys. Acta 1447:244.[Medline]
  22. Lee, S. H., J. B. Clark. 1997. High-yield method for isolation of {lambda} DNA. BioTechniques 23:598.[Medline]
  23. Kumaresan, P. R., P. A. Mathew. 2000. Structure of the human natural killer cell receptor 2B4 gene and identification of a novel alternative transcript. Immunogenetics 51:987.[Medline]
  24. Chuang, S. S., H. Zhuang, S. R. Reisher, S. I. Feinstein, H. K. Das. 1995. Transcriptional regulation of the apolipoprotein B-100 gene: identification of cis-acting elements in the first nontranslated exon of the human apolipoprotein B-100 gene. Biochem. Biophys. Acta 215:394.
  25. Chuang, S. S., H. K. Das. 1999. A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells. Biochim. Biophys. Acta 1436:600.[Medline]
  26. Chen, B. P., G. Liang, J. Whelan, T. Hai. 1994. ATF3 and ATF3 {delta} zip: transcriptional repression versus activation by alternatively spliced isoforms. J. Biol. Chem. 269:15819.[Abstract/Free Full Text]
  27. Maxam, A. M., W. Gilbert. 1977. A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74:560.[Abstract/Free Full Text]
  28. Schreiber, E., P. Matthias, M. M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  29. Chuang, S. S., D. Banerjee, H. K. Das. 1999. Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I. Eur. J. Biochem. 263:773.[Medline]
  30. Nagashima, S., R. Mailliard, Y. Kashii, T. E. Reichert, R. B. Herberman, P. Robbins, T. L. Whiteside. 1998. Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood 91:3850.[Abstract/Free Full Text]
  31. Wisdom, R.. 1999. AP-1: one switch for many signals. Exp. Cell Res. 253:180.[Medline]
  32. Karin, M., Z. Liu, E. Zandi. 1997. AP-1 function and regulation. Curr. Opin. Cell Biol. 9:240.[Medline]
  33. Stepp, S. E., J. D. Schatzle, M. Bennett, V. Kumar, P. A. Mathew. 1999. Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains. Eur. J. Immunol. 29:2392.[Medline]
  34. Schatzle, J. D., S. Sheu, S. E. Stepp, P. A. Mathew, M. Bennett, V. Kumar. 1999. Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4. Proc. Natl. Acad. Sci. USA 96:3870.[Abstract/Free Full Text]
  35. Cocks, B. G., C. C. Chang, J. M. Carballido, H. Yssel, J. E. de Vries, G. Aversa. 1995. A novel receptor involved in T-cell activation. Nature 376:260.[Medline]
  36. de la Fuente, M. A., P. Pizcueta, M. Nadal, J. Bosch, P. Engel. 1997. CD84 leukocyte antigen is a new member of the Ig superfamily. Blood 90:2398.[Abstract/Free Full Text]
  37. Sandrin, M. S., M. M. Henning, M. F. Lo, E. Baker, G. R. Sutherland, I. F. McKenzie. 1996. Isolation and characterization of cDNA clones for Humly9: the human homologue of mouse Ly9. Immunogenetics 43:13.[Medline]
  38. Boles, K. S., P. A. Mathew. 2001. Molecular cloning of CS1, a novel human natural killer cell receptor belonging to the CD2 subset of the immunoglobulin superfamily. Immunogenetics 52:302.[Medline]
  39. Klem, J., M. Bennett, V. Kumar, J. Schatzle. 2000. The NK receptor, 2B4, is constitutively localized in glycolipid-enriched microdomains and associates with LAT. FASEB J. 14:A1026.
  40. Bottino, C., R. Augugliaro, R. Castriconi, M. Nanni, R. Biassoni, L. Moretta, A. Moretta. 2000. Analysis of the molecular mechanism involved in 2B4-mediated NK cell activation: evidence that human 2B4 is physically and functionally associated with the linker for activation of T cells. Eur. J. Immunol. 30:3718.[Medline]
  41. Watzl, C., C. C. Stebbins, E. O. Long. 2000. NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244). J. Immunol. 165:3545.[Abstract/Free Full Text]
  42. Bernard, K., A. Cambiaggi, S. Guia, F. Bertucci, S. Granjeaud, R. Tagett, C. N'guyen, B. R. Jordan, E. Vivier. 1999. Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line. J. Immunol. 162:4062.[Abstract/Free Full Text]
  43. Johnson, L. A., R. H. Goldfarb, P. A. Mathew. 2000. Regulation of IFN-{gamma} production following 2B4 activation in human NK cells. In Vivo 14:625.[Medline]
  44. Penix, L. A., M. T. Sweetser, W. M. Weaver, J. P. Hoeffler, T. K. Kerppola, C. B. Wilson. 1996. The proximal regulatory element of the interferon-{gamma} promoter mediates selective expression in T cells. J. Biol. Chem. 271:31964.[Abstract/Free Full Text]
  45. Barbulescu, K., C. Becker, J. F. Schlaak, E. Schmitt, K. H. Meyer zum Buschenfelde, M. F. Neurath. 1998. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-{gamma} promoter in primary CD4+ T lymphocytes. J. Immunol. 160:3642.[Abstract/Free Full Text]
  46. Nimer, S., J. Zhang, H. Avraham, Y. Miyazaki. 1996. Transcriptional regulation of interleukin-3 expression in megakaryocytes. Blood 88:66.[Abstract/Free Full Text]
  47. Wargnier, A., C. Lafaurie, S. Legros-Maida, J. F. Bourge, F. Sigaux, M. Sasportes, P. Paul. 1998. Down-regulation of human granzyme B expression by glucocorticoids: dexamethasone inhibits binding to the Ikaros and AP-1 regulatory elements of the granzyme B promoter. J. Biol. Chem. 273:35326.[Abstract/Free Full Text]
  48. Iacobelli, M., W. Wachsman, K. L. McGuire. 2000. Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein. J. Immunol. 165:860.[Abstract/Free Full Text]
  49. Yea, S. S., K. H. Yang, N. E. Kaminski. 2000. Role of nuclear factor of activated T-cells and activator protein-1 in the inhibition of interleukin-2 gene transcription by cannabinol in EL4 T-cells. J. Pharmacol. Exp. Ther. 292:597.[Abstract/Free Full Text]
  50. Mori, A., O. Kaminuma, T. Mikami, S. Inoue, Y. Okumura, K. Akiyama, H. Okudaira. 1999. Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis. J. Allergy Clin. Immunol. 103:S429.[Medline]
  51. Santana, M. A., G. Pedraza-Alva, N. Olivares-Zavaleta, V. Madrid-Marina, V. Horejsi, S. J. Burakoff, Y. Rosenstein. 2000. CD43-mediated signals induce DNA-binding activity of AP-1, NF-AT and NF{kappa}B transcription factors in human T lymphocytes. J. Biol. Chem. 275:31460.[Abstract/Free Full Text]
  52. Dong, C., D. D. Yang, C. Tournier, A. J. Whitmarsh, J. Xu, R. J. Davis, R. A. Flavell. 2000. JNK is required for effector T-cell function but not for T-cell activation. Nature 405:91.[Medline]
  53. Billadeau, D. D., S. M. Mackie, R. A. Schoon, P. J. Leibson. 2000. Specific subdomains of Vav differentially affect T cell and NK cell activation. J. Immunol. 164:3971.[Abstract/Free Full Text]
  54. Matthews, J. S., L. A. O’Neill. 1999. Distinct roles for p42/p44 and p38 mitogen-activated protein kinases in the induction of IL-2 by IL-1. Cytokine 11:643.[Medline]
  55. Mortellaro, A., S. Songia, P. Gnocchi, M. Ferrari, C. Fornasiero, R. D’Alessio, A. Isetta, F. Colotta, J. Golay. 1999. New immunosuppressive drug PNU156804 blocks IL-2-dependent proliferation and NF-{kappa}B and AP-1 activation. J. Immunol. 162:7102.[Abstract/Free Full Text]
  56. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, A. Weiss. 1998. LAT is required for TCR-mediated activation of PLC{gamma}1 and the Ras pathway. Immunity 9:617.[Medline]
  57. Torgerson, T. R., A. D. Colosia, J. P. Donahue, Y. Z. Lin, J. Hawiger. 1998. Regulation of NF-{kappa}B, AP-1, NFAT, and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-{kappa}B p50. J. Immunol. 161:6084.[Abstract/Free Full Text]
  58. Matsui, K., S. Omura, H. Cui, S. L. Schauer, G. E. Sonenshein, S. T. Ju. 1997. Proteasome regulation of Fas ligand cytotoxicity. Eur. J. Immunol. 27:2269.[Medline]
  59. Valle Blazquez, M., I. Luque, E. Collantes, E. Aranda, R. Solana, J. Pena, E. Munoz. 1997. Cellular redox status influences both cytotoxic and NF-{kappa}B activation in natural killer cells. Immunology 90:455.[Medline]



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