Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We describe the existence of multiple transcriptional start sites upstream of DAP10 exon 1 and identify the location of the basic promoter upstream of these starting sites. The promoter is active in NK and CD8+ T cells, but not in CD4+ T cells. We demonstrate TCR-mediated up-regulation of DAP10 transcription and found that a 40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription. Using pull-down and chromatin immunoprecipitation assays, we show that the DAP10 promoter interacts with Ap-1 transcription factors in primary CD8+ T and NK cells in vitro and in vivo. Overexpression of c-Jun or c-Fos in NK and T cells led to enhanced DAP10 promoter activity and DAP10 protein expression. Taken together, our data indicate that Ap-1 is an important transcription factor for regulating DAP10 gene expression in human NK and T cells, and that Ap-1 plays a key role in the transactivation of DAP10 promoter following TCR stimulation.
The human NKG2D gene is located between the CD94 and NKG2F genes within the NK gene complex on chromosome 12 and encodes a type II protein expressed by NK cells, γδ T cells, and CD8+ αβ T cells (1, 2). NKG2D itself does not possess signaling capacity. In humans, NKG2D exists on the cell surface in complex with the DAP10 adaptor protein that contains a YxxM motif that, upon tyrosine phosphorylation after NKG2D/DAP10 ligation, couples the receptor complex to the PI3K/Grb2-Vav pathway (3, 4). Murine NKG2D is encoded by two splice variants (5). The long isoform (mNKG2D-L) associates only with DAP10, whereas the short isoform (mNKG2D-S) associates with DAP10 or DAP12 (5, 6). The pairing of NKG2D with either the DAP10 or DAP12 adaptor proteins is a unique feature for murine NKG2D, as there is no evidence for the short form of human NKG2D (7). Moreover, the artificial creation of a short form of a human NKG2D showed that it failed to associate with DAP12 (8).
NKG2D/DAP10 is an activating receptor that can trigger NK cells and costimulate CD8+ T cells (9, 10). The ligands for human NKG2D/DAP10 are structurally diverse and include the MHC class I chain-related (MIC)4 proteins A and B and the UL16-binding proteins 1 through 4 (9, 11, 12). These ligands are usually absent on normal cells, but are often up-regulated on infected or cancer cells, or by cells otherwise undergoing stress (13). The expression of NKG2D/DAP10 ligands by such stressed cells makes them susceptible to killing by NK and CD8+ T cells (9, 14, 15). This killing can be inhibited by blocking the NKG2D receptor with mAb against NKG2D or its ligands (16). Recent studies have revealed that tumors can evade immune recognition by NKG2D bearing NK and CD8+ T cells by shedding soluble NKG2D ligands that mediate NKG2D/DAP10 internalization and degradation (17, 18, 19). In addition, elevated levels of TGF-β in cancer patients have been shown to inhibit the expression of NKG2D/DAP10, thereby impairing NK cell cytotoxicity (20). In contrast to the positive aspects of NKG2D/DAP10 ligand recognition for the immune response to diseased cells, NKG2D/DAP10 ligand recognition can also have adverse effects for the host, particularly in the case of certain autoimmune diseases. Inappropriate activation of NK/CD8+ T cells by NKG2D/DAP10 ligation has been implicated in the pathology of celiac disease (21), rheumatoid arthritis (22), and autoimmune diabetes in NOD mice (23). Thus, the modulation of NKG2D/DAP10 receptor expression or blockade of NKG2D/DAP10 signaling has therapeutic implications for a variety of disease states.
Because of these observations, the determination of the factors that regulate expression of NKG2D/DAP10 receptors is an area of significant interest. In this study, we focused on the transcriptional regulation of the human DAP10 gene. We identified multiple transcriptional start sites of the human DAP10 gene and localized its promoter to a segment upstream of these sites. We demonstrated that TCR stimulation up-regulates DAP10 promoter activity and show that the Ap-1 proteins c-Fos and c-Jun play a key role in this up-regulation. We also show that these transcription factors are involved in regulating DAP10 expression by NK cells.
Materials and Methods
Abs and flow cytometry
To measure NKG2D cell surface expression, we used PE-conjugated anti-NKG2D (R&D Systems). PE-conjugated isotype-matched control mAb (eBioscience) was used to monitor background staining levels. Flow cytometric analyses were performed on a FACSort cytofluorometer (BD Immunocytochemistry Systems). Purified anti-CD3 (clone UCHT1; eBioscience) was used for cell stimulations. Anti-DAP10 (clone N-17; Santa Cruz Biotechnology), anti-c-Fos (Active Motif) and anti-β-actin (Sigma-Aldrich) Ab were used for Western blot analyses.
Cell isolation and culture conditions
Polyclonal human NK cells and CD8+ and CD4+ T cells were isolated by negative selection from peripheral blood using NK cell or CD8+ or CD4+ T cell isolation kits (Miltenyi Biotec). All isolated cell populations were routinely found to be >97% in purity. Freshly purified cells were cultured in IMDM (BioWhittaker) supplemented with 10% human AB serum (BioWhittaker), and 2 mM GlutaMax (BioSource International). The human NK leukemia cell line, NKL, was cultured in RPMI 1640 (BioSource International) supplemented with 2 mM GlutaMax, 1 mM sodium pyruvate (Invitrogen Life Technologies), 100 U/ml recombinant IL-2 (Biological Resources Branch, National Cancer Institute, Frederick, MD), 5 μg/ml plasmocin (InvivoGen), and 10% FBS (BioWhittaker). Jurkat T cells were cultured in RPMI 1640 supplemented with 2 mM GlutaMax, 10 mM nonessential amino acids, 10% FBS, and 5 μg/ml plasmocin. All cells were cultured at 37°C under an atmosphere of 5% CO2. Where appropriate, cells were stimulated with plate-bound anti-CD3 or isotype-matched control mAb (eBioscience) at different concentrations (0.05–1 μg/ml) for 2–5 days before analysis.
RNA isolation, 5′-RACE, and RT-PCR
Before RNA isolation, cells were stored in RNAlater (Ambion) to prevent RNA degradation. Total RNA was isolated using the RNAqueous-4PCR kit (Ambion), including DNase treatment, according to the manufacturer’s instructions. The 5′-RACE was conducted with the First Choice RLM-RACE kit (Ambion), according to the manufacturer’s instructions. Primers were 5′-RACE inner DAP10 5′-gat ggatcatggtggactgg-3′ and 5′-RACE outer DAP10 5′-agagagagggacccacatcc-3′.
Synthesis of cDNA was performed with the iScript cDNA synthesis kit (Bio-Rad). Samples were analyzed by real-time PCR (DNA Engine Opticon 2; MJ Research) using the iQ SYBR Green PCR kit (Bio-Rad). A melting curve was performed at the end of each run to verify that there was a single amplification product and a lack of primer dimers. Standard curves, obtained using a serial 10-fold dilution of human primary CD8+ T cell cDNA, were generated to determine the level of each amplified transcript, and all samples were normalized to the amount of 18 S rRNA transcript present in each sample. Primers for 18 S rRNA were from QuantiTect Primer Assay for 18 S rRNA (Qiagen). Primers for DAP10 were (forward) 5′-tccatctgggtcacatcctcttcc-3′ and (reverse) 5′-gagtgatgatctctctcctggagtcgtctgagctg-3′. Real-time PCRs were performed in triplicate.
All DNA fragments were derived by PCR using platinum Taq polymerase (Invitrogen Life Technologies) with human genomic DNA (Promega) as the template. A DAP10 luciferase construct was subcloned into the KpnI and NheI restriction sites of the pGL3-basic reporter plasmid (Promega). The primers used to generate the DAP10 luciferase constructs were: P (reverse) NheI 5′-ctagctagcgaagaggatgtgacccagatg-3′; P1 (forward) KpnI 5′-ggggtaccccctccctctttctccatttc-3′; P2 (forward) KpnI 5′-ggggtaccttcttggccctacctcc-3′; P3 (forward) KpnI 5′-ggggtacctgagttcgttcaccaaaggc-3′; and P4 (forward) KpnI 5′-ggggtacctcaacacacacaggaagc-3′. Mutant constructs were generated with a QuikChange XL Site-directed Mutagenesis kit (Stratagene), using a pair of overlapping internal primers that contained a mutant sequence. The primers used for the promoter mutation constructs were: Ap-1mut1 (forward) 5′-tctgaccctcccGGTACCttcgttcaccaa-3′ and Ap-1mut2 (forward) 5′-cctccccctgagGGTACCcaccaaaggcag-3′. The pcDNA3/c-Jun and pcDNA3/c-Fos plasmids were gifts from Dr. N. Colburn (National Cancer Institute, Frederick, MD).
All plasmid DNA used for transfections were purified with Qiagen Plasmid Maxi kits, according to the manufacturer’s protocol. Restriction enzymes were purchased from New England Biolabs. Custom synthesized oligonucleotides were supplied by Sigma-Genosys. The correctness of the plasmid inserts was verified by sequence analysis.
Transfection and luciferase assay
All cell types (3–5 × 106) were transiently transfected with 5 μg of the reporter plasmid and 0.25 μg of the pRL-nul Renilla control vector (Promega) that acts as an internal control for the normalization of transfection efficiency. Electroporation was performed using the Amaxa nucleofection system, according to the manufacturer’s protocols. Cells were harvested at 16–24 h posttransfection and lysed. Specific luciferase activity in the cell lysates was measured with a Veritas Microplate Luminometer (Promega). All luciferase assays were performed at least three times, each in triplicate. The dual luciferase double reporter assay system and substrates were purchased from Promega.
For overexpression studies, 5 × 106 NKL or Jurkat T cells were transiently transfected with 5 μg of the pcDNA3, pcDNA3/c-Jun, or pcDNA3/c-Fos plasmids together with 3 μg of the reporter plasmid and 0.15 μg of the pRL-nul Renilla control vector using the Amaxa nucleofection system. Cells were harvested at 24 h posttransfection, lysed, and analyzed using luciferase assays.
Western blot analysis of DAP10 protein levels
Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting as previously described (24). Cells transfected with c-Jun or c-Fos expressing plasmids were harvested at 36 h posttransfection, lysed, and analyzed for protein expression by Western blot. Band intensity was quantified using UN-SCAN-IT gel software (Silk Scientific).
Primary CD8+ T cells stimulated with plate-bound anti-CD3 mAb (0.5 μg/ml) for 16 h were harvested, washed, and lysed using a Nuclear Extract kit (Active Motif). Nuclear extracts were reconstituted in binding buffer containing 10 mM Tris-HCl, 50 mM NaCl, 1 mM MgCl, 5% glycerol, 0.5 mM EDTA, 5 mM DTT, 0.5% Nonidet P-40, 1% phosphatase inhibitor mixture 1 (Sigma-Aldrich), 1% phosphatase inhibitor mixture 2 (Sigma-Aldrich), and 1% protease inhibitor mixture (Sigma-Aldrich). Cell lysates were precleared by incubation with streptavidin-agarose beads (Dynabeads M-280; Invitrogen Life Technologies) for 1 h at 4°C. Cell lysates were incubated with streptavidin-agarose beads coupled to biotinylated double-stranded oligonucleotides (Sigma-Genosys) containing a wild-type (5′-tggtctctctgaccctcccccTGAGTTCgttcaccaaagg-3′) or mutated (5′-tggtctctctgaccctcccccAAAAAAAAttcaccaaagg-3′) Ap-1 binding site (shown in capital letters) from the DAP10 promoter region. The binding reactions were performed for 1 h at 4°C. Unbound proteins were removed by six washes with lysis buffer. After washing, the oligonucleotide-bound proteins were released in Laemmli sample buffer (Sigma-Aldrich), boiled for 5 min, resolved on 10% SDS-PAGE, and detected by immunoblotting as described earlier.
Chromatin immunoprecipitation (ChIP) assay
Chromatin preparation and immunoprecipitation were performed with a EZ-ChIP kit (Upstate Biotechnology) with minor modifications. Protein-DNA cross-linking was achieved by incubating primary CD8+ T or NK cells (2 × 106 cells per condition) with 1% formaldehyde for 10 min at room temperature with gentle agitation. Cross-linking was blocked by adding glycine at a final concentration of 0.125 M and incubating at room temperature for 5 min. Cells were collected by centrifugation and washed in cold PBS containing 1% protease inhibitor mixture (Sigma-Aldrich). Pelleted cells were resuspended in 200 μl of lysis buffer (Upstate Biotechnology) and incubated on ice for 10 min. Chromatin was then sonicated to an average length of 0.2–1 kb using a Branson Sonifier 450 (Branson Ultrasonics). Sonicated chromatin was then immunoprecipitated with anti-c-Fos, anti-c-Jun Ab (Santa Cruz Biotechnology) or isotype matching control Ab attached to microbeads using a protocol provided by Upstate Biotechnology. The bound protein-DNA complexes were eluted with freshly prepared elution buffer (1% SDS and 0.1 M NaHCO3). After reversing the protein-DNA cross-linking and subsequent DNA recovery, to detect DAP10 promoter region sequences, samples were analyzed by quantitative PCR (DNA Engine Opticon 2; MJ Research) with iQ SYBR Green Supermix (Bio-Rad). The amount of DAP10 promoter related sequence present in each reaction was calculated relative to a standard curve obtained using a serial 10-fold dilution of human genomic DNA (Promega). Results were expressed as fold enrichment over control Ab. The following primers were used in the ChIP assays: human DAP10 promoter, sense 5′-cagcaaattttcttggccctacctc-3′ and antisense 5′-gttactgcctttggtgaacga-3′; and negative control primers, sense 5′-atggttgccactggggatct-3′ and antisense 5′-tgccaaagcctaggggaaga-3′. Negative control primers were from a ChIP-IT kit (Active Motif).
Human DAP10 gene has multiple transcriptional start sites in freshly isolated primary NK and CD8+ T cells
Sequence analysis of the 5′-flanking region of the human DAP10 gene with a transcriptional factor database search (TFSearch) showed that it lacked a TATA box (data not shown), a feature often associated with the existence of multiple transcriptional start sites (25). To investigate whether this was the case for the DAP10 gene and to identify the 5′ ends of the transcripts, RLM-RACE using total RNA from freshly isolated human primary NK and CD8+ T cells was performed. Sequence analyses of multiple 5′-RACE clones from three different donors for each cell type demonstrated the existence of multiple transcriptional start sites (Fig. 1 A). All transcriptional start sites in NK and CD8+ T cells occurred within a 136-bp region upstream of the DAP10 gene start site of translation (ATG, +1). Similar results were obtained using cDNA from spleen and thymus (data not shown).
Mapping of the human DAP10 promoter
To determine whether the sequence upstream of the first exon contained a promoter, we cloned a genomic fragment that began 0.7 kb upstream and extended downstream through the ATG translational start site (−698 to +27, construct P1). A series of 5′ deletion mutant constructs of this genomic fragment were created to locate the region critical for DAP10 promoter activity. These constructs were inserted into the pGL3-basic luciferase reporter vector, and transfected into DAP10-expressing NKL and Jurkat T cells. Deletion of the segment from −698 to −292 bp (construct P2) had a positive effect on DAP10 promoter activity in both cell types, but deletion from −292 to −180 bp (construct P3) resulted in a 3- to 5-fold reduction in luciferase activity (Fig. 1 B). The smallest deletion construct (−140 to +27 construct P4) began only 5 bp upstream of the first transcription starting site and possessed little promoter activity (1- to 3-fold over pGL3-basic). These results indicate that the sequence between −292 and −140 bp contains the major promoter regulatory region important for DAP10 gene transcription.
Cell type-specific activity of the DAP10 promoter in NK and T cells reflects the activity of the endogenous gene
To test whether the genomic segment containing the DAP10 promoter also contained elements responsible for controlling DAP10 cell type-specific expression in primary cells, we transfected the P2 DAP10 promoter construct, which possessed maximum promoter activity in Jurkat T cells and NKL cells (see Fig. 1,B) into primary NK cells and CD8+ or CD4+ T cells isolated from healthy donors. The promoter was active in NK and CD8+ T cells, but had little or no activity in CD4+ T cells (Fig. 2,A), which is consistent with the fact that endogenous DAP10 protein is expressed in NK and CD8+ T cells, but not usually in CD4+ T cells (Fig. 2 B). As the activity of the identified DAP10 promoter reflected the activity of the endogenous gene, we concluded that the major regulatory elements necessary for cell type-specific DAP10 expression were present in the P2 promoter construct.
TCR ligation enhances DAP10 expression in T cells
The TCR plays a major role in defining the specificity of an immune response and together with coreceptors, such as CD28 and NKG2D/DAP10, controls T cell activation (26, 27, 28). We were interested in determining the effect of TCR ligation on DAP10 expression. To make this determination, CD8+ T cells isolated from healthy donors were activated with immobilized anti-CD3 mAb for 2–5 days. NKG2D/DAP10 cell surface expression increased significantly by exposure to anti-CD3 mAb compared with cells cultured with immobilized control Ig mAb (Fig. 3,A). This TCR-mediated up-regulation of NKG2D/DAP10 was observed in T cells isolated from multiple human donors (data not shown). To check whether this increase in NKG2D/DAP10 cell surface expression correlated with an increase in DAP10 protein level, Western blot analyses were performed with cell lysates from freshly isolated and anti-CD3-stimulated CD8+ T cells. An increase in the DAP10 protein level was detected in CD3 activated CD8+ T cells (Fig. 3,B). This increase in the DAP10 protein level was observed for a range of anti-CD3 mAb concentrations from 0.05 to 1 μg/ml (data not shown). We also observed a very small but consistent appearance of DAP10 protein level in CD4+ T cells after TCR stimulation (data not shown). A significant increase in DAP10 transcripts after both 2 and 5 days of culture with immobilized anti-CD3 mAb was also evident (Fig. 3 C). After 5 days, we observed a ∼5-fold induction in DAP10 transcripts over that observed of freshly isolated CD8+ T cells. Taken together, these results demonstrate that TCR ligation positively regulates the expression of the DAP10 gene, which leads to enhanced DAP10 protein expression that, in turn, up-regulates NKG2D/DAP10 cell surface expression.
The DAP10 promoter is a target for activation by TCR ligation
We examined in more detail how TCR signals regulate DAP10 transcription using the DAP10-positive Jurkat T cell line. Stimulation of Jurkat T cells with anti-CD3 mAb markedly increased DAP10 protein levels, as well as mRNA levels (Fig. 4, A and B). To study the effect of TCR stimulation on DAP10 promoter activity, we used the P2 construct (see Fig. 1,B). As shown in Fig. 4 C, Jurkat T cells transfected with the P2 DAP10 promoter construct had a consistent 2-fold increase in the promoter driven luciferase activity in the presence of immobilized anti-CD3 mAb compared with transfected Jurkat cells treated with control mAb. Taken together, these data indicate that TCR ligation enhances DAP10 mRNA and protein levels through transactivation of the DAP10 promoter.
Role of an Ap-1-binding site in the DAP10 promoter activity
To localize the region of the DAP10 gene that mediates its induction in response to TCR ligation, each of the DAP10 deletion promoter constructs (Fig. 5,A) was transiently transfected into Jurkat T cells and the luciferase activity was measured with or without anti-CD3 mAb stimulation. DAP10 promoter constructs P1 (−698 to +27) (data not shown), P2 (−292 to +27), and P3 (−180 to +27) showed similar response to TCR ligation, but the shortest promoter construct P4 (−140 to +27) showed only marginal up-regulation after TCR ligation (Fig. 5 A). Because the P3 construct was the shortest construct showing TCR responsiveness, we concluded that the 40 bp sequence located between −180 and −140 bp contains a key regulatory segment important for the regulation of DAP10 promoter activity following TCR ligation.
The nucleotide sequence upstream of exon 1 plus a portion of the exon 1 sequence is shown in Fig. 5,B. A transcriptional factor database search (TFSearch) of this sequence revealed the presence of a putative binding site for Ap-1 transcription factors located between −180 and −140 bp (Fig. 5 B). Ap-1 transcription factors are well-characterized mediators of T cell activation that can regulate the transcription of multiple cytokine, chemokine, and cell surface receptor genes (29, 30, 31).
Because the identified Ap-1 motif is a putative Ap-1 binding site, we tested for its functionality in primary CD8+ T cells. Ap-1 is a homodimer or heterodimer containing Fos (c-Fos, FosB) and Jun (c-Jun, JunB, JunD) proteins that can be activated by TCR stimulation (29, 30, 31). Using pull-down assays, we investigated whether TCR activated primary CD8+ T cells contain c-Fos protein capable of binding to the putative Ap-1 binding site present in the DAP10 promoter region. To investigate, CD8+ T cells isolated from healthy donors were activated with immobilized anti-CD3 mAb for 16 h. Nuclear extracts were incubated with streptavidin-agarose beads coupled to biotinylated double-stranded oligonucleotides containing either a wild-type or mutated Ap-1 binding site (see Materials and Methods) designed from the DAP10 promoter. Fig. 5 C shows that the oligonucleotide containing the wild-type Ap-1 binding site was significantly more efficient at binding c-Fos protein than the oligonucleotide containing the mutated Ap-1 binding site. These results indicate that the c-Fos protein from TCR activated primary CD8+ T cells can specifically bind to the Ap-1 binding site present in the DAP10 promoter region.
To investigate the involvement of this Ap-1 binding site in the regulation of DAP10 promoter activity, we created two mutated versions (Ap-1mut1 and Ap-1mut2) of the Ap-1 binding site in the P2 DAP10 promoter construct. Each mutation significantly reduced the DAP10 promoter activity in both NKL and Jurkat T cells (Fig. 5 D), indicating that this site is involved in the transcriptional regulation of the basal activity of the DAP10 promoter.
Ap-1 binding site is involved in the TCR-mediated regulation of DAP10 promoter activity
To investigate whether the Ap-1 binding site plays a role in the induction of DAP10 gene expression following TCR ligation, the P2 DAP10 promoter constructs (wild-type, Ap-1mut1, or Ap-1mut2) were transfected into Jurkat T cells, followed by stimulation with immobilized anti-CD3 or control mAb (Fig. 5,E). Anti-CD3 stimulation resulted in a 2-fold increase in luciferase activity for the wild-type P2 construct. In contrast, this up-regulation was significantly diminished (over 50%) with the DAP10 promoter constructs that had mutated Ap-1 binding sites (Fig. 5 E). These data indicate that the Ap-1 binding site is required for optimal activation of the DAP10 promoter following TCR ligation.
Ap-1 transcription factors are able to transactivate the human DAP10 promoter and up-regulate DAP10 protein expression
To determine whether c-Fos and c-Jun transcription factors can enhance DAP10 promoter activity, the P2 DAP10 promoter construct was cotransfected with expression vectors for c-Jun or c-Fos proteins into NKL or Jurkat T cells that were subsequently stimulated with immobilized anti-CD3 mAb. Overexpression of c-Jun or c-Fos up-regulated DAP10 promoter activity ∼1.5– to 2-fold in both Jurkat T cells (Fig. 6,A) and NKL cells (Fig. 6 B). These results provide further evidence that TCR ligation activates the DAP10 promoter in T cells via Ap-1 transcription factors and support the notion that Ap-1 transcription factors are also involved in the transcriptional regulation of the DAP10 gene promoter activity in NKL cells.
To assess whether overexpression of Ap-1 caused an increase in endogenous DAP10 protein levels, Jurkat T cells and NKL cells were transfected with c-Jun or c-Fos expression vectors, and after 48 h, levels of DAP10 protein were measured by Western blot analyses (Fig. 6, C and D). Endogenous DAP10 protein levels were increased 2.5-fold upon overexpression of c-Jun or c-Fos compared with the control pcDNA3.1 vector in Jurkat T cells that had been cultured with anti-CD3 mAb (Fig. 6 C). In NKL cells, we observed 1.3- or 2-fold up-regulation of DAP10 protein levels upon overexpression of c-Jun or c-Fos transcription factors, respectively. Taken together these results provide evidence that Ap-1 transcription factors are biologically relevant regulators of DAP10 expression.
Ap-1 transcription factors interact with the DAP10 promoter in vivo
To demonstrate that the up-regulation of DAP10 expression by activated CD8+ T cells correlated with binding of Ap-1 transcription factors to the DAP10 promoter in vivo, we performed ChIP assays. For CD8+ T cells, freshly isolated cells from multiple donors were cultured for 24 h with TCR (anti-CD3) stimulation. Fig. 7 A shows that for all four donors significant amplification of DNA containing the relevant DAP10 Ap-1 site could be detected in Ab-specific immunoprecipitates derived from these cells. No significant binding was observed in immunoprecipitates derived from freshly isolated T cells (data not shown).
To investigate the in vivo interaction between Ap-1 transcription factors and the DAP-10 promoter in NK cells, primary NK cells from multiple donors were cultured for 24 h in IL-2 (required for viability) before ChIP analyses. All four donors showed significant amplification of DNA containing the relevant Ap-1 binding site in the c-Fos immunoprecipitates (Fig. 7 B). No significant binding of relevant DNA was observed in immunoprecipitates derived from freshly isolated NK cells (data not shown). Taken together, these data demonstrate that Ap-1 proteins interact with the DAP10 promoter in primary NK and CD8 T cells in vivo.
Human NKG2D/DAP10 is a well-described activation receptor expressed by NK cells that is also capable of providing costimulatory signals for CD8+ T cells (10, 28, 32). NKG2D/DAP10 has been shown to play a role in controlling the progression of certain tumors and infectious diseases, as well as exacerbating certain autoimmune diseases (21, 22, 33, 34). Several mechanisms are known to regulate the cell surface expression of the human NKG2D receptor, including the differential action of cytokines (IL-2/IL-15/IL-21, TNF-α, and TGF-β1) (22, 28, 35, 36, 37, 38, 39), interaction of the receptor with soluble and membrane bound ligands (40, 41, 42, 43, 44) and availability of the DAP10 adaptor protein (3, 8). We are interested in how the expression of this receptor is regulated. Given that DAP10 is critical for NKG2D/DAP10 signaling capacity (3) and cell surface expression (3, 8), we focused our initial study on the transcriptional regulation of the human DAP10 gene.
The human DAP10 gene is located on chromosome 19q13.1 only 130 bp downstream of the DAP12 gene in opposite transcriptional orientation (3). The human DAP10 gene is relatively small (spans <2 kb) and has four exons. DAP10 cDNA is ∼500 bp and encodes a 93 aa type I transmembrane protein (3). We show that the DAP10 gene has multiple transcriptional start sites (∼16), a common feature of genes with TATA-less promoters (25). The partner of DAP10, NKG2D, also has a TATA-less promoter and multiple transcriptional start sites (45, 46), which is a feature common for other members of the NK complex, such as NKG2A (47, 48, 49) and CD94 (50, 51). We did not see a predominance in use of particular transcriptional start sites with NK cells compared with CD8+ T cells, either in freshly isolated or IL-2 cultured cells (data not shown). We then localized the DAP10 promoter to a region between −292 and +27 bp (Fig. 1,B). This location agrees with the location of the transcriptional start sites, as most promoters occur within 150 bp upstream of the transcriptional start sites. Using deletion constructs, we localized two regions that affected DAP10 basal promoter activity. The region from −698 to −292 bp contains an element with a negative effect on DAP10 promoter activity, whereas the region from −292 to −140 bp contains an element essential for DAP10 maximum promoter activity (Fig. 1,B). Using luciferase reporter assays, we showed that the defined DAP10 promoter was functional in NK and CD8+ T cells, but not in CD4+ T cells derived from healthy donors (Fig. 2,A). This observation is directly correlated with the DAP10 protein expression by those cell types (Fig. 2 B) and with the cell surface expression of NKG2D on PBL from healthy donors (data not shown).
Under certain conditions, NKG2D/DAP10 can be expressed on the surface of subsets of CD4+ T cells. For example, in some cancer patients, a rare NKG2D+CD4+ T cell population has been reported (52). This CD4+ subset was found in patients with MIC-positive tumors and stimulation with MICA ligand was shown to contribute to the expansion of this CD4+ T cell population. In addition, NKG2D (as well as perforin) can also be induced on CD4+ T cells in vitro following infection of PBMC from healthy seropositive individuals with human CMV (53). Coligation of NKG2D with TCR was shown to potently induce proliferation and cytokine production by these cells. Of related interest, we have observed that TCR-stimulated CD4+ T cells in culture are able to express small amounts of DAP10 protein (data not shown). As Ap-1 appears to be integral to CD4+ T cell function, the failure of most CD4+ T to express DAP10 most likely relates to epigenetic modulating events, such as chromatin modification or DNA methylation status.
We show that TCR ligation up-regulates NKG2D/DAP10 cell surface expression by CD8+ T cells (Fig. 3,A). This up-regulation of NKG2D/DAP10 expression correlates with enhanced DAP10 mRNA and protein expression (Fig. 3, B and C). Taken together, these results suggest that TCR ligation positively regulates expression of the DAP10 gene, which in turn promotes enhanced cell surface expression of NKG2D/DAP10 in human T cells. Previous studies have shown that TCR-induced stimulation of CD8+ T cells increases cell surface expression of murine NKG2D within 4 days (16, 54), which agrees with a reported increase in human DAP10 protein and mRNA levels by day 10 (37). However, our data show that the up-regulation of DAP10 expression occurs after only 2–5 days of TCR stimulation.
The responsiveness of DAP10 promoter activity to TCR ligation was mapped to the 40-bp segment of the promoter that contained a Ap-1 binding site (Fig. 5,B). In line with this observation, it is known that the induction of promoter activity by coreceptors, such as CD28, 4-1BB, and 2B4, is often dependent on activation by transcription factors of the NFAT, NF-κB, and Ap-1 families (55, 56, 57). Mutations of the identified Ap-1 binding site lead to reduced reporter transcriptional activity and a reduced response by the DAP10 promoter construct to TCR stimulation in Jurkat T cells (Fig. 5, D and E). The Ap-1 transcription factor is comprised of Fos and Jun homodimers or heterodimers. Upon activation through TCR ligation, they become phosphorylated, bind to their target DNA binding sites, and act as potent transactivators of transcription (58, 59, 60). Using pull-down (Fig. 5,C) and ChIP (Fig. 7,A) assays, we show for primary CD8+ T cells that the DAP10 promoter interacts with Ap-1 transcription factors in vitro and in vivo. Moreover, overexpression of c-Jun or c-Fos in Jurkat T cells led to enhanced promoter activity, as well as increased DAP10 protein expression (Fig. 6). Although the detailed mechanism of DAP10 induction by Ap-1 transcription factors remains to be defined, these results provide evidence, for the first time, that Ap-1 transcription factors are biologically relevant regulators of DAP10 expression in human T cells. These results do not rule out the possibility that TCR ligation may regulate DAP10 gene transcription via additional mechanisms.
Ap-1 also plays an important role in NK cell functions. NK cell treatment with inhibitors of the Ap-1 pathway prevents NK cell natural cytotoxicity (61). Ap-1 regulates transcription of many genes, such as ifnγ (62, 63), il3 (64), granzyme B (65), il2 (66, 67), and il5 (68). Ap-1 binding sites are present in the NKG2A, 2B4, and proximal CD94 promoters (69). In NK cells, Ap-1 can be activated by stimulation with cytokines such as IL-2 (70). Ap-1 activity is also regulated at the posttranscriptional level by the activation of JNK (71). A decrease in human NKG2D/DAP10 cell surface expression after treatment of NK92 cells with JNK inhibitor has been recently reported (72). Consistent with these observations and the fact that NKG2D/DAP10 expression can be positively regulated by IL-2 stimulation in NK cells, we found that the basal level of DAP10 promoter activity in NKL cells was significantly decreased by mutations of the Ap-1 binding site. Moreover, using ChIP assays, we showed that in primary NK cells c-Jun protein binds to the DAP10 promoter region in vivo. Ap-1 family members were also capable of up-regulating DAP10 promoter activity, as well as the levels of endogenous DAP10 protein, in NKL cells. However, unlike in CD8+ T cells, for reasons yet to be determined, we did not observe any significant binding of c-Fos protein to DAP10 promoter in primary NK cells. Among the possible explanations for no significant binding observed is that different multiprotein complexes are formed in the DAP10 promoter region in primary NK and CD8+ T cells and NK cells may lack transcription factors necessary to form the complex containing c-Fos protein. This interpretation agrees with the fact that the Fos-Jun family members can form over 15 different homodimers and heterodimers; moreover, interactions between various Fos-Jun family members and over 50 different proteins have been reported (73). Another possibility is that other epigenetic events, such as chromatin modifications or DNA methylation status, affect the regulatory specificities of Fos-Jun family proteins in NK and CD8+ T cells.
In summary, our studies clearly demonstrate that Ap-1 transcription factors play a significant role in regulating DAP10 expression in human NK and T cells, and that in CD8+ T cells their role can be enhanced by TCR ligation.
We thank Robert Valas for technical assistance and cell isolations, and Drs. Madhan Masilamani, Xiaobin Tang, Gul’nar Fattakhova, and Sriram Narayanan for thoughtful discussion.
The authors have no financial conflict of interest.
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.
This work was supported by funds from the Intramural program of the National Institute of Allergy and Infectious Diseases.
Abbreviations used in this paper: MIC, MHC class I chain-related; ChIP, chromatin immunoprecipitation.