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J. Biol. Chem., Vol. 281, Issue 41, 30419-30430, October 13, 2006

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Transcriptional Regulation of ULBP1, a Human Ligand of the NKG2D Receptor^*

Alejandro López-Soto¹, Adolfo Quiñones-Lombraña, Rubén López-Arbesú, Carlos López-Larrea, and Segundo González²

From the Departamento de Biología Funcional, Facultad de Medicina, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo and the Unidad de Histocompatibilidad y Transplantes, Hospital Universitario Central de Asturias, Oviedo, Spain

Received for publication, May 19, 2006 , and in revised form, August 3, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tumor cells expressing ligands of the NKG2D receptor stimulate anti-tumor immunity mediated by natural killer and T cells. In humans, NKG2D ligands (NKG2DL) are encoded by MIC and ULBP proteins. NKG2DL exhibit highly restricted expression in healthy tissues but are widely expressed in tumors. However, regulation of each NKG2DL differs substantially in different cancer cells. In this study, we characterized the mechanisms that regulate the expression of ULBP1. We show that the transcription of ULBP1 strictly depends on the binding of Sp1 and Sp3 to a CRE(1) site located in the ULBP1 minimal promoter. The mutation or deletion of this Sp1/Sp3 binding site abolished the transcription of ULBP1. It also diminished the transactivation of ULBP1 promoter by Sp3 overexpression, but not by Sp1, indicating that Sp3 is the main transcription factor that regulates ULBP1 through the CRE(1) site. Experiments in SL2 cells showed that the ULBP1 promoter was inactive in the absence of the Sp proteins and indicate that Sp3 is the essential activator of ULBP1 transcription, because the overexpression of Sp3 up-regulated its promoter activity >500-fold. Additionally, we demonstrated that AP-2 repressed the expression of ULBP1 in HeLa cells by interfering with the binding of Sp3 and Sp1 to the ULBP1 promoter. These data indicate that Sp1, Sp3, and AP-2 may play an important role in the immunosurveillance against cancer. Finally, the definition of ULBP1 regulation may have implications for development of new therapeutic strategies against cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
NKG2D is an activating receptor expressed by NK³ cells, NK T cells, CD8 T cells, and T cells (1, 2). Whereas most NK receptors bind to ubiquitously expressed major histocompatibility complex class I molecules, a peculiarity of NKG2D consists in the multitude of ligands that are induced subsequently to harmful events such as genotoxic stress or infection (3–7). NKG2D ligands (NKG2DL) are also frequently expressed on cancer cells but absent in benign tissues. Tumor cells expressing NKG2DL become susceptible to NK cell killing even if the tumor cells have normal HLA class I expression (1, 3). NKG2DL expression in mice is induced by carcinogens and genotoxic stress, and tumor cells expressing these proteins were readily eliminated by NK and CD8 T cells in vivo (6, 8, 9). Similarly, mice lacking T cells are highly susceptible to epithelial tumors, and these cells could kill skin carcinoma cells by a NKG2D-dependent mechanism in vitro (10). In accordance with the role of NKG2DL in the anti-tumor immunity, cancer cells develop several means to avoid the immune system controlling the expression of NKG2D receptors on immune cells or their ligands on tumor cells (11–14). These experiments strongly support a role of the NKG2D/NKG2DL system in tumor rejection and surveillance and render this system as an interesting target for immunotherapy (15–20).

In humans, the ligands of NKG2D are MICA, MICB (21, 22), and UL16-binding proteins 1–5 (ULBP1–5) (5, 23–25). MIC and ULBP molecules exhibit highly restricted expression in healthy tissues but are widely expressed on epithelial tumors and hematological malignancies in vivo (15, 17, 19, 22, 26). These proteins are up-regulated in nontumor cell lines by genotoxic stress (6). Thereby, DNA-damage response, which arrests the cell cycle and enhances DNA repair functions, or triggers apoptosis, also alerts the immune system in response to potentially dangerous cells through the activation of NKG2D. It is still a matter of controversy as to why so many different ligands exist for the same receptor, because the stimulatory activity of the immune cells of different NKG2DLs are very similar. In the context of tumor transformation or infection, the multitude of ligands could be explained as a strategy to avoid the evasion of the immune system. Several studies indicate that the regulation of individual NKG2DL expression differs substantially in a particular tumor or infected cells (15, 17, 19), suggesting that the multitude of NKG2DLs do not simply reflect the redundant expression of molecules with the same function. Concordantly, despite the strong conservation of the coding sequence among all ULBPs, the sequence of the 5'-flanking regions of these genes has little homology, and this suggests that ULBPs could be specifically regulated in response to different stimuli or pathologies. Nevertheless, very little is known about the mechanisms of NKG2DL induction and expression.

Here, we have analyzed for the first time the mechanism that controls ULBP1 expression. We show that ULBP1 transcription strictly depends on the CRE(1) site located at –115, because the mutation or deletion of this site practically abolished the transcription of ULBP1. We also demonstrate that the Sp1 and Sp3 transcription factors are bound to the CRE(1) sequence, and the expression of ULBP1 depends on the activity of these transcription factors. Nevertheless, chromatin immunoprecipitation analysis (ChIP) demonstrated that Sp1 and Sp3 were the main transcription factors bound to this promoter in vivo. Mutation of the CRE(1) site significantly diminished the transactivation of ULBP1 promoter by Sp3 expression, but not the Sp1, indicating that Sp3 is the main transcription factor that regulates ULBP1 through the CRE(1) site. Furthermore, co-transfection experiments into Drosophila SL2 cells lacking Sp factors demonstrated that ULBP1 promoter was practically inactive in the absence of the Sp proteins and showed that the long isoform of Sp3 was a 90-fold stronger activator of ULBP1 transcription than Sp1 was. This indicates that Sp3 plays an essential role in the expression of ULBP1 suggesting that the regulation of the expression of Sp3 isoforms or its activity may play a key role in the induction of ULBP1 expression that is observed in cancer cells. We also show that AP-2 may mediate transcriptional repression of the ULBP1 gene through competition with the transcriptional activator Sp1 and Sp3 for binding to the ULBP1 minimal promoter. AP-2 is a transcription factor that acts as a tumor suppressor that favors epithelial differentiation and induces cell cycle arrest (27–29). Loss of AP-2 expression has been associated with progression of melanoma, breast, and colon cancer (30–35). Thus, our results indicate that Sp1, Sp3, and AP-2 may play an important role in the immunosurveillance against cancer cells regulating the expression of ULBP1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—HeLa (human cervix carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA), and human embryonic kidney 293 (HEK 293) were obtained from Dr. P. Sanchez Lazo (Universidad de Oviedo, Spain). Cells were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and gentamicin in a humidified 95% air, 5% CO₂ incubator. SL2 cells were a gift from Dr. P. Dominguez (Universidad de Oviedo) and were grown in Schneider's Drosophila medium (Invitrogen) with 10% fetal calf serum, 2 mM L-glutamine, and gentamicin at 28 °C without CO₂.

Mapping of the Transcription Start Sites of ULBP1 Gene—The transcription start sites of the ULBP1 gene in HeLa and HEK 293 cells were determined by 5'-rapid amplification of cDNA ends (5'-RACE) as previously described (36). Briefly, total RNA was isolated from cell cultures by TRIzol reagent (Invitrogen). A synthetic oligonucleotide (GSP1) complementary to mRNA, spanning bases +334 to +314 (5'-CAC GTC TCT TAG TGT TTC AG-3'), was used to generate 5'-end ULBP1 dA-tailed. This cDNA was subjected to two rounds of PCR. The first PCR was conducted with a specific primer (GSP2) spanning bases +266 to +242 (5'-C AAA GGC TTT GGC GTT GTG GTT AA-3') and an oligonucleotide containing deoxythymidines at the 3'-end, Sp6-(dT)₁₈ (5'-ATT TAG GTG ACA CTA TAG AAT ACG TCG ACA TCG ATG G(dT)₁₈-3'). The second one was performed with a nested specific primer (GSP3) spanning bases +28 to +10 (5'-CCG TGG ACA GGA GGC GCT-3') and the Sp6 primer lacking the dT tail. All PCR products were 5'-end phosphorylated using T4 polynucleotide kinase (Amersham Biosciences) and blunt ended with T4 DNA polymerase (Promega, Madison, WI) and then cloned in the EcoRV site of the Bluescript II KS plasmid (Stratagene, La Jolla, CA).

Generation of ULBP1 Promoter Reporter, Mutant, and Deletion Constructs—A 900-bp ULBP1 promoter fragment was amplified by PCR from human genomic DNA using the following primers: ULBP1s, 5'-TAA GCC CTC CCT TGC GGC C-3', and the reverse ULBP1as,5'-CCG TGG ACA GGA GGC GCT-3'. The fragment obtained was ligated into the SmaI site of the pGL2 Basic vector (Promega). Different deletions were produced by PCR using this construct as a DNA template with the following forward primers: –603, 5'-CAC AAC TGA TCA CAT CAT GAT TGA TCA C-3'; –288, 5'-GGG GTA ATG ATA AAT GCT GTG TTG AA-3'; –200, 5'-CGC ACG CCG CCA CCA A-3'; –137, 5'-GTC AGA TGA CGA GCC CGG G-3'; –75, 5'-CCC GCC CAG TGT ATC CCT G-3'; and –42, 5'-GGG CTG GGC AGC TTT ATA AAC AG-3'. The PCR products were subcloned into the SmaI site of pGL2 Basic vector. The integrity of constructs was confirmed by DNA sequencing. Mutant promoter constructs were generated using the QuikChange kit (Stratagene) with the following primers (only forward primers are shown; mutated oligonucleotides were underlined): mTATA, 5'-CCG GGC TGG GCA GCT TCG CGA ACA GCC GTG GTG TG-3'; mGC(1), 5'-ATC CCT GCG CGC GGC TTG CCG GGC TGG GCA GC-3'; mGC(2), 5'-GCC TAG GAT CCC GAA CAG TGT ATC CCT GCG CGC-3'; mNF-B, 5'-GGC GTG ACG GGG TGG AGC ATA AAC AAA AAA GTG CAT GCC-3'; mGC(3), 5'-GCC CGG GGC GTG ACG GTT TGG AGC ATC CCC-3'; mCRE(1), 5'-GAG CCC GGG GCG GCT GTG GGT GGA GCA TCC C-3'; mGC(4)/AP-2, 5'-GAT GAC GAG CCC GGT TCG TGA CGG GGT GGA GC-3'; and mCRE(2), 5'-GCC AGG CCG CTG TCA GGC TGT GAG CCC GGG GCG TGA CGG-3'. All these constructs were verified for their nucleotide sequence and reading frame by DNA sequence analysis.

Expression and Dominant Negative Plasmids—The constructs for mammalian expression pN3-Sp1, pN3-Sp3FL (a cytomegalovirus-driven expression vector for full-length Sp3 with both upstream AUGs) and pN3-Sp4 (37) and the Drosophila expression vectors pPac, pPacSp1, pPacSp3 (which expresses a short isoform of Sp3), pPacUSp3 (expression vector for long Sp3 isoform), and pPacSp3FL (expression vector for full-length Sp3, which is equivalent to the mammalian vector pN3-Sp3FL) (38, 39) were kindly provided by Dr. G. Suske (Philips-Universitat Marburg, Germany). Human AP-2 and murine AP-2 in pCMX-PL1 vector were generously provided by Dr. R. Buettner (40). pCDNA3.1/AP-2 was a gift from Dr. R. J. Weigel (41). The AP-2B vector pSG5/AP-2 and the expression vectors pCDNA6/AP-2-(1–165) and -(165–437) (42) were kindly provided by Dr. P. Kannan (Metro Health Medical Center, Case Western Reserve University). The reporter plasmid pGL3-waf1-LUC was described before (43).

Transient Transfections and Luciferase Reporter Assays—HeLa and HEK 293 cells were transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer's recommendations. Briefly, 5 x 10⁴ cells were transfected in 24-well plates with 300 ng of each reporter construct and 12 ng of pRL-null vector (Promega) as an internal control plasmid. Cells were harvested at 24 h and assayed for luciferase activity using the Dual Luciferase Reporter System (Promega). For co-transfection assays with dominant negative or expression vectors, pRSV -galactosidase plasmid was included and luciferase measures were normalized with -galactosidase activity. In some experiments, 200 nM mithramycin A (Sigma) was added 4 h after DNA transfections, and luciferase activity was assayed 24 h later. Drosophila SL2 cells (5 x 10⁵ cells) were co-transfected in 24-well plates using Lipofectamine 2000 with 500 ng of reporter plasmid plus 300 ng of each expression vector. Cells were harvested 48 h post-transfection, and luciferase activities were normalized against the total protein concentration as measured using the BCA Protein Assay kit (Pierce).

Real-time PCR and Flow Cytometry—Real-time PCR was performed using SYBR Green from reverse-transcribed cDNA samples for relative quantification of mRNA levels for the genes of interest. The mRNA level of the GAPDH gene was determined in each assay to normalize the expression of genes of interest. The primers used were as follows: ULBP1 sense, 5'-ATC AGC GCC TCC TGT CCA C-3'; ULBP1 antisense, 5'-AAA GAC AGT GTG TGT CGA CCC AT-3'; MICA sense, 5'-AAT GGA ACC TAC CAG ACC TGG G-3'; MICA antisense, 5'-ACA TGG AAT GTC TGC CAA TGA CT-3'; GAPDH sense, 5'-CGG AGT CAA CGG ATT TGG TC-3'; and GAPDH antisense, 5'-AAT CAT ATT GGA ACA TGT AAA CCA TGT AGT-3'. Cell surface expression of ULBP1 and MICA/B were detected by flow cytometry using a BD Biosciences FACSCalibur (Sunnyvale, CA). Detached cells were incubated with a monoclonal antibody anti-ULBP1 (4G7) or a monoclonal anti-MICA/B (6G6), which were kindly provided by Dr. Thomas Spies (Fred Hutchinson Cancer Research Centre, Seattle, WA), for 30 min on ice; followed by incubation with a phycoerythrin-labeled goat anti-mouse IgG1 secondary monoclonal antibody (BD Pharmingen, San Diego, CA) for another 30 min on ice.

To investigate the effect of Sp1, Sp3, and AP-2 on endogenous ULBP1 expression, HeLa and HEK 293 cells were grown in 60-mm dishes and co-transfected with 2 µg of pN3, pN3-Sp1, pN3-Sp3FL, pCMX, or pCMX-AP-2 expression vectors and with 0.5 µg of pCS2-GFP plasmid (44). After 48 h, ULBP1 surface expression was analyzed in GFP-positive cells using a monoclonal antibody anti-ULBP1 (4G7) as described above.

Electrophoretic Mobility Shift Assay—90% confluent HeLa and HEK 293 cells were used for preparing nuclear extracts as previously described (45). The protein concentration of the extracts was determined by using BCA assay (Pierce). A wild-type double-stranded oligonucleotide corresponding to positions –128/–99 of ULBP1 promoter (5'-GAG CCC GGG GCG TGA CGG GGT GGA GCA TC-3') was 5'-end-labeled using T4 polynucleotide kinase and [-³²P]ATP (3000Ci/mmol, Nucliber, Spain). The DNA binding reactions were performed in a volume of 20 µl containing 5 µg of nuclear protein together with the ³²P-end-labeled wild-type probe and 2 µg of poly(dI-dC) (Amersham Biosciences) in binding buffer (10 mM HEPES-KOH, pH 7.9; 5% (v/v) glycerol; 50 mM KCl; 5 mM MgCl₂; 0.5 mM EDTA; 0.1% Nonidet P-40; and 1 mM dithiothreitol) on ice for 35 min. The binding reactions were electrophoresed on 6% native acrylamide gels in Tris-Borate-EDTA buffer. For competition experiments, a 100 molar excess of unlabeled probes was incubated with the extracts at 25 °C for 15 min before adding the radioactive probe. The oligonucleotides used in competition assays were as follows: Sp1 consensus, 5'-ATT CGA TCG GGG CGG GGC GAG C-3'; CREB consensus, 5'-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3'; AP-2 consensus, 5'-GAT CGA ACT GAC CGC CCG CGG CCC GT-3'. Mutant ULBP1 promoter probes included the same punctual mutations described above. In supershift assays, nuclear extracts and 2 µg of commercial antibody were preincubated for 60 min on ice prior to the addition of the remaining components of the binding reaction. Rabbit polyclonal antibodies anti-Sp3 (sc-644X), anti-AP-2 (sc-184X), and mouse monoclonal antibody anti-Sp1 (sc-420X) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-CREB (ab503, ABCAM, United Kingdom) was a generous gift from Dr. JM Freije (Universidad de Oviedo).

Chromatin Immunoprecipitation—ChIP was performed using the ChIP assay kit (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY) following the manufacturer's recommendations. Briefly, HeLa and HEK 293 cells were treated with 1% formaldehyde, pelleted, and resuspended in SDS lysis buffer. Chromatin was sonicated to sizes between 200 and 500 bp using a Branson Sonifier 450. Diluted chromatin was precleared with 80 µl of salmon sperm DNA/protein A-agarose-50% slurry (Upstate%20Biotechnology">Upstate Biotechnology) and then incubated overnight at 4 °C with 5 µg of anti-Sp1, -Sp3, -AP-2 antibodies or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology). 20 µl of NaCl 5 M was added, the immune complexes were eluted, and cross-links were reversed by overnight incubation at 65 °C. DNA samples were purified by phenol/chloroform extraction and ethanol precipitation. A 95-bp fragment of the ULBP1 proximal promoter spanning the region from –137 to –43 was amplified by PCR using the following primers: 5'-GTC AGA TGA CGA GCC CGG G-3' (forward) and 5'-GGC CCG CCG CGC GCA-3' (reverse). The samples were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

Western Blotting—To obtain total protein extracts, HeLa and HEK 293 cells were lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 50 mM NaF, 1 mM dithiothreitol, complete inhibitor mixture (Roche Applied Science) and phosphatase inhibitor cocktails I and II (Sigma). Soluble proteins were then separated by centrifugation at 15,000 x g for 5 min at 4 °C. Protein concentration was determined using BCA assay (Pierce). Nuclear extracts from HeLa and HEK 293 cells were obtained as described above. Following heat denaturation, samples containing 15 µg of protein were loaded on 10% SDS-PAGE gels. Proteins were then transferred to nitrocellulose membranes and stained with Ponceau to verify that similar amounts of protein had been loaded. Blots were blocked with 3% nonfat dry milk and incubated overnight at 4 °C with a dilution 1/600 of the rabbit polyclonal antibodies anti-Sp3 (sc-644X) and anti-AP-2 (sc-184X) and of the mouse monoclonal antibody anti-Sp1 (sc-420X). Finally, blots were incubated with 1/7500 donkey anti-rabbit-horseradish peroxidase or goat-anti-mouse-horseradish peroxidase (Amersham Biosciences) in 1.5% nonfat dry milk washed and developed with West Pico Chemiluminescent substrate (Pierce).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Analysis of expression of ULBP1. A, expression of ULBP1 mRNA in HeLa and HEK 293 cells was analyzed by real-time PCR. The level of ULBP1 mRNA was normalized using the GAPDH level within each cell line. The results represent the mean ± S.E. of three independent experiments. B, protein expression was analyzed by immunofluorescence and FACS analysis using 4G7 (anti-ULBP1) and 6G6 (anti-MICA/B) monoclonal antibodies. Isotype controls are represented in black, ULBP1 in dark gray, and MICA in light gray. The histogram shows one representative experiment.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. RACE 5' analysis of the transcription start site of ULBP1 gene. A, RACE 5' was conducted using two adapter primers (AP1 and AP2) and three specific ULBP1 primers (GSP1, -2, and -3). The GSP1 primer was used to generate the 5'-end ULBP1 dA-tailed cDNA. This cDNA was subjected to two rounds of PCR using the GSP2 and the AP1 primers, and GSP3 and AP2 primers, respectively. B, analysis on agarose gel of the nested PCR products obtained from HeLa and HEK 293 mRNA. C, nucleotide sequence of the 5'-untranslated region and proximal promoter of ULBP1 gene. The arrows indicate the transcription start sites. The main transcription start site is numbered +1. The potential binding sites for transcription factors are underlined.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To determine the transcription initiation site(s) of the ULBP1 gene, 5'-RACE was performed using total RNA from HeLa and HEK 293 cells (Fig. 2A). Three and two ULBP1-specific transcription start sites were identified in HeLa and HEK 293, respectively (Fig. 2B). Sequencing of an additional PCR product that appeared between bands 2 and 3 indicated that it did not correspond to a ULBP1 transcription start site (data not shown). The sequence of the PCR products 2 and 3 revealed that both cell lines shared two common transcription start sites located 43 bp and 108 bp upstream of the ATG codon. HeLa presented an additional transcription start site located 132 bp relative to ATG (Fig. 2B, band 1). Fragment 3, shared by HEK 293 and HeLa, showed the highest intensity of all three bands and is located 28 bp downstream of a canonical TATA box. This site corresponds with the previously determined 5'-end of the ULBP1 mRNA (5) suggesting that this is the main transcription start site of the ULBP1 gene (Fig. 2C).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Identification of the minimal promoter region of ULBP1 using progressive deletions of the 5'-region in luciferase reporter constructs. The schematic presentation of the luciferase reporter constructs is shown. These plasmids were transiently transfected in HeLa and HEK 293 cells, and luciferase activity was assayed after 24 h. To correct the differences in transfection efficiencies, the Renilla luciferase plasmid pRL-null was included in each transfection. The activity of the luciferase reporter construct under the control of the SV40 promoter (pGL2-Control) was set as 100. The results represent the mean ± S.E. of three independent experiments made in triplicate.

The analysis of the minimal promoter (–137/+28) for consensus motifs for transcription factors revealed a canonical TATA box located at –28, three GC boxes, one overlapping sequence GC(4)/AP-2, two CRE-like sequences (CRE(1) and CRE(2)), and one site for NF-B (Fig. 2C). The functional significance of these elements was studied by mutation of their sequences in the –862/+28 construct (Fig. 4A). Mutation of the TATA box decreased the activity of the ULBP1 promoter in HeLa and HEK 293 by 19 and 80%, respectively (Fig. 4B). Remarkably, the most significant results were obtained with the mutation of the sequences located in the region –137/-75. The mutation of the CRE(1) at –116 practically abolished the ULBP1-mediated transcription activity in both cell lines (Fig. 4B). The sequences juxtaposed to this CRE(1) also showed functional activity. Thus, the mutation of the GC(3) box, located just downstream, reduced the transcriptional activity by 25 and 55% in HeLa and HEK 293, respectively; and the mutation of GC(4)/AP-2, which is located just upstream of the CRE(1) site, resulted in a 65% increase of luciferase activity in HeLa cells, but it had little effect on HEK 293 cells. This indicates that the GC(4)/AP-2 site is a negative regulator of ULBP1 transcription in HeLa. Finally, the mutation of the CRE(2) site also showed an important reduction of the ULBP1-mediated transcription in HeLa and HEK 293 cells (Fig. 4B).

Interaction of the ULBP1 Proximal Promoter with Transcription Factors Sp1, Sp3, and AP-2—The mutational analysis showed that several potential binding sites for transcription factors are functional in the minimal promoter of ULBP1. In this study, we focused on the GC(4)/AP2, CRE(1), and GC(3) sequences, which as we have shown, played a crucial role in the expression of ULBP1. EMSA was performed to determine the transcription factors that bind to these sequences. A radiolabeled double-stranded oligonucleotide encompassing the potential GC(4)/AP-2, CRE(1), and GC(3) sites (Fig. 5A) was incubated with nuclear extracts from HeLa cells, which resulted in the formation of three complexes (called C1, C2, and C3) (Fig. 5B, lane 2), whereas only two complexes (C1 and C3) were observed using nuclear extracts from HEK 293 cells (Fig. 5C, lane 2). These complexes were specific, because they competed with a 100-fold molar excess of the unlabeled probe (Fig. 5, B and C, lane 3). To characterize the cis-acting elements of ULBP1 promoter that interacted with these three complexes, competition experiments with mutated double-stranded oligonucleotides were performed. The unlabeled probe with a mutated GC(3) box competed with complexes C2 and C3, and it only partially competed with C1, indicating that C1 was partially bound to the GC(3) box (Fig. 5, B and C, lane 4). The mutant CRE(1) oligonucleotide only abolished complex C2 in HeLa, and it had no effect on HEK 293, indicating that the complexes C1 and C3 were bound to this sequence in both cell lines (Fig. 5, B and C, lane 5). The mutant GC(4)/AP-2 oligonucleotide was unable to compete with any complex, indicating that the three complexes were bound to this sequence (Fig. 5, B and C, lane 6). Together, these results indicate that complexes C1 and C3 were originated by interaction with CRE(1) and GC(4)/AP-2 sites in HeLa and HEK 293, and the binding of C1 was also extended to the GC(3) box. The complex C2 resulted from interaction with the GC(4)/AP-2 site exclusively in HeLa cells. To further characterize the transcription factors that composed these complexes, competition experiments using unlabeled double-stranded oligonucleotides encoding consensus sequences for CREB, Sp1, and AP-2 transcription factors, and supershift analysis using specific antibodies were performed. CREB consensus probe had no effect on any of these complexes, indicating that CREB was not present in them (Fig. 5, B and C, lane 7). The Sp1 consensus sequence competed with complexes C1 and C3 (Fig. 5, B and C, lane 8), suggesting that both complexes were composed by members of the Sp1 family. Complex C1 was supershifted with Sp1 antibody, and the Sp3 antibody abolished the complex C3 in both cell lines (Fig. 6, A and B). These data demonstrate that Sp1 and Sp3 transcription factors are bound in HeLa and HEK 293 to the CRE(1) site of ULBP1 promoter. Additionally, consensus AP-2 double-stranded oligonucleotide competed in HeLa with complex C2, and this complex was abolished with an AP-2 antibody (Fig. 6C), but there was no effect on HEK 293 (Fig. 6D). This indicates that AP-2 was bound in HeLa cells to the GC(4)/AP-2 site, which was a negative regulator of ULBP1 promoter activity. Next, we determined the binding of Sp1, Sp3, and AP-2 to ULBP1 proximal promoter in HeLa and HEK 293 cells in vivo using ChIP analysis. As shown in Fig. 6E, Sp1 and Sp3 were bound to the ULBP1 promoter in both cell lines (lanes 3 and 4). In accordance with the EMSA, in vivo, AP-2 was only bound to the ULBP1 promoter in HeLa cells (lane 5).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Site-directed mutagenesis of the potential binding motifs of the ULBP1 proximal promoter. A, schematic presentation of the potential binding sequences in the ULBP1 promoter that have been mutated by site-directed mutagenesis. B, the mutated reporter constructs were transiently transfected in HeLa and HEK 293 cells, and luciferase activity was assayed after 24 h. To correct the differences in transfection efficiencies, the Renilla luciferase plasmid pRL-null was included in each transfection. The activity of the wild-type promoter is set as 100%. The results represent the mean ± S.E. of three independent experiments made in triplicate.

View larger version (51K): [in this window] [in a new window]   FIGURE 5. Characterization of transcription factors interacting with ULBP1 proximal promoter. A, sequence of the probe used in EMSA assays. The probe spans the sequence –128/-99 of ULBP1 promoter and encompasses the potential GC(3), CRE(1) and GC(4)/AP-2 sites. This labeled probe was incubated with nuclear extract proteins from HeLa (B) and HEK 293 cells (C). Lane 1 shows the free probe, and lane 2 shows the binding between the probe and the nuclear extracts. As indicated in lanes 3–8, unlabeled oligonucleotide competitors were included in the binding reaction in 100-fold molar excess over probe. In lane 3, an unlabeled ULBP1 probe was included. Lanes 4–6 show the complexes observed in presence of probes containing mutations for GC(3), CRE(1), and GC(4)/AP-2 sites, in lanes 7 and 8, a consensus probe for Sp1 and CREB transcription factors were included.

View larger version (64K): [in this window] [in a new window]   FIGURE 6. Identification of proteins in complexes by supershift assays. Antibodies for CREB, Sp1, and/or Sp3 were preincubated with HeLa (A) and HEK 293 (B) nuclear extracts before the addition of the probe. In C and D, HeLa and HEK 293 nuclear extracts were incubated with unlabeled probe (lane 3), AP-2 consensus probe (lane 4), and antibodies for AP-2 (lane 5) or CREB (lane 6). Lane 1 shows the free probe, and lane 2 shows the binding between the probe and the nuclear extracts. E, in vivo binding of Sp1, Sp3, and AP-2 to ULBP1 minimal promoter was examined using ChIP analysis. Control (lane 2), Sp1 (lane 3), Sp3 (lane 4), and AP-2 (lane 5) were immunoprecipitated from the sonicated lysates of HeLa and HEK 293 cells using normal rabbit IgG or antibodies against human Sp1, Sp3, and AP-2 proteins. After reversing the cross-linking, DNA was precipitated, and PCR was performed using primers to amplify the ULBP1 core promoter DNA. The PCR products were then run on 2% agarose gels and visualized by ethidium bromide staining. Lane 1 contains input DNAs used as controls. These data are representative from two independent experiments with similar results. F, Western blotting of the expression of Sp1, Sp3, and AP-2 using cytoplasmic and nuclear extracts from HeLa and HEK 293 cells. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of protein had been loaded in each lane.

The transfection of Sp1, Sp3, and Sp4 in HEK 293 cells also transactivated the activity of the ULBP1 promoter (2-, 2.1-, and 0.97-fold, respectively) (data not shown). To confirm the role of Sp transcription factors on ULBP1 expression in HEK 293, which expresses significant amounts of ULBP1, we used mithramycin A, which is an inhibitor of Sp transcription factor binding (47). Treatment of HEK 293 cells with 200 nM mithramycin A for 24 h reduced the ULBP1 and p21-mediated promoter activities by 70 and 28%, respectively (48) (Fig. 7C).

The Long Isoform of Sp3 Is a Potent Activator of the ULBP1 Promoter in Drosophila SL2 Cells—To determine the role of Sp1 and Sp3 on the transcriptional activity of the ULBP1 promoter, transient expression experiments were performed in Drosophila SL2 cells, which lack Sp proteins (49). ULBP1 promoter was practically inactive in this cell line indicating that Sp1 and Sp3 are essential for its promoter activity. The cotransfection of the ULBP1 reporter with the pPacSp1 plasmid approximately increased 6-fold the ULBP1 transcriptional activity relative to the empty vector (pPac) (Fig. 8). The overexpression of Sp4 also led to an increase of 3-fold of ULBP1 promoter activity. The overexpression of different isoforms of Sp3 had a dual effect. The short isoform (pPacSp3) had practically no effect on ULBP1 promoter activity (1.3-fold induction); however, the long isoform of Sp3 (pPacUSp3) led to a 517-fold increase of ULBP1 promoter activity. Transfection of the full-length Sp3 (pPac-Sp3FL), which expresses the short and long isoforms of Sp3 and is equivalent to the mammalian pN3-Sp3FL expression vector previously used in HeLa and HEK 293 cells, only increased the promoter activity of ULBP1 13-fold (Fig. 8). A combination of Sp1 and Sp3 or Sp4 expression vectors showed little change in activity, suggesting that Sp1 and Sp3 do not interact synergistically on the ULBP1 promoter. Collectively, these data indicate that transcription factor Sp3 plays an essential role in the transcription of the ULBP1 gene, because it was a 90-fold stronger activator of ULBP1 promoter activity than Sp1 was.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. The transcription factors Sp1 and Sp3 and Sp4 activate ULBP1 promoter activity. A, the ULBP1 promoter construct (–862/+28) (200 ng) was cotransfected with 400 ng of the expression plasmids for Sp1, Sp3, and Sp4 in HeLa cells. The cotransfection of p21 promoter reporter and the Sp1 expression vector was used as a control in these experiments. B, the mutant CRE(1) construct was cotransfected with the expression plasmids for Sp1 and Sp3. To correct the differences in transfection efficiencies, the pRSV- galactosidase plasmid was included in each transfection. The -fold induction represents the -fold increase in luciferase activity relative to that obtained from cotransfection of backbone of the expression vector. Data are the mean ± S.E. from three independent experiments with triplicates for each experiment. C, HEK 293 cells were transfected with 300 ng of the ULBP1 promoter construct (–862/+28) or p21 promoter construct and 30 ng of the pRSV- galactosidase plasmid and treated with 200 nM of mithramycin A or Me2SO. Cells were harvested after 24 h, and luciferase and galactosidase activity was measured. Relative luciferase activity is expressed as arbitrary units that represent the mean ± S.E. of three independent experiments with triplicates for each experiment. The luciferase activity was normalized by -galactosidase activity as described under "Experimental Procedures."

The mechanism of AP-2 repression of ULBP1 transcription was further analyzed in HeLa cells. First, the role of the AP-2 binding site (GC(4)/AP-2) in response to Sp expression vectors was studied. The mutation of the AP-2 site significantly increased the transactivation capacity of the Sp expression vectors compared with the wild-type promoter. The overexpression of Sp1, Sp3, and Sp4 increased the activity of the ULBP1 promoter 6.9-, 4.8-, and 6-fold (Fig. 9B). Concordantly, the expression vector AP-2-(165–437), which only expresses the DNA binding domain of AP-2, repressed the ULBP1-mediated luciferase activity (Fig. 9C). These observations indicate that AP-2 represses ULBP1 transcription, at least in part, by preventing the binding or activity of Sp1 and Sp3 transcription factors to the CRE(1) element, which overlaps with the AP-2 binding site (Fig. 2C). To further analyze the mechanism of repression of ULBP1 transcription by AP-2, two expression vectors that lack the DNA binding domain, AP-2B and AP-2-(1–165), were cotransfected with the ULBP1-luciferase promoter construct. AP-2B is a naturally occurring splice variant of AP-2, which lacks the DNA binding domain, and AP-2-(1–165) is an artificial construct in which the DNA binding domain has been deleted. Both expression vectors also significantly repressed the ULBP1-mediated luciferase activity (Fig. 9C). These data suggest that AP-2 may repress the ULBP1 promoter activity in HeLa cells by both mechanisms, dependent and independent of DNA binding.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. The long isoform of Sp3 is a potent activator of ULBP1 transcription in SL2 cells. The ULBP1 promoter construct (–862/+28) was cotransfected with the Drosophila expression plasmids for Sp1 (pPacSp1), for the short isoform (pPacSp3), the long isoform (pPacUSp3), and the full-length of Sp3 (pPacSp3FL) and Sp4 (pPacSp4) in Drosophila SL2 cells. The amount of plasmid DNA was kept constant in all experiments using the pPac vector. Data are expressed as -fold induction of normalized luciferase activity relative to that obtained following co-transfection of the ULBP1 promoter vector with empty pPac. The data are the mean ± S.E. of luciferase activity from three independent experiments with triplicates for each experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It is well established that transformation can induce NKG2DL expression, and cancer cells expressing NKG2DL become susceptible to NK cell killing in vitro and in vivo (1–3, 8, 22). These strongly support a role of the NKG2D/NKG2DL system in tumor rejection and surveillance, rendering this system as an interesting target for immunotherapy (15–20). However, the mechanisms involved in NKG2DL induction are largely unknown. Several studies have shown that the expression of individual NKG2DL expression differs substantially in tumor cells, suggesting that individual NKG2DL expression may be induced in response to different stimuli or pathologies. To understand the mechanisms that induce the expression of NKG2DL in cancer cells, we have functionally characterized for the first time the regulation of ULBP1 transcription. Our data clearly indicate that Sp3 and Sp1 play a critical role in ULBP1 promoter activity and protein expression. These transcription factors bind to the CRE(1) site located in the minimal promoter of the ULBP1 gene in vitro and in vivo (Fig. 11), and we demonstrated that the expression of ULBP1 strictly depends on Sp1/Sp3 activity. Thus, the mutation or deletion of the Sp1/Sp3 binding site practically abolished the activity of the ULBP1 promoter in HeLa and HEK 293 cells and in several cell lines analyzed (data not shown). Furthermore, experiments in SL2 cells showed that ULBP1 promoter was practically inactive in absence of the Sp1 family proteins, and it also indicates that Sp3 is the essential activator of ULBP1. Sp1 and Sp3 are ubiquitously expressed transcription factors. Consistent with the high conservation of the DNA-binding zinc finger domain, both Sp1 and Sp3 bind to GC boxes with identical affinity (50–53). This suggested, at first, that both proteins share similar functional properties. However, the data obtained from the analysis of Sp1 and Sp3 knock-out mice and from functional analysis indicated that the physiological roles of these proteins appear to be different (52). This is due to the presence in Sp3 of an inhibitory domain, which suggests that this transcription factor may be an important target of regulatory events (39). Thus, while Sp1 usually stimulates transcription, Sp3 has a dual function as an activator or repressor whose activity is dependent upon context of DNA-binding sites in a promoter. Additionally, the transcriptional role of Sp3 is more complicated, because there are at least four isoforms derived from alternative transcription start sites. The short isoforms lack the transactivation domain, and they are inactive or weak activators (54, 55). With respect to ULBP1, transfection experiments with SL2 cells revealed that the long isoform of Sp3 was a potent regulator of ULBP1 transcription, increasing >500-fold the activity of the ULBP1 promoter, whereas the short isoform of Sp3 was practically inactive. These data together indicate that the regulation of the expression of the different isoforms of Sp3 or the relative levels of Sp1 and Sp3 may have a dramatic impact on ULBP1 expression, and, thus, in the immune response against cancer or infected cells. ULBP1 and other NKG2DLs exhibit a highly restricted pattern of expression in benign tissues, but they are frequently expressed by many epithelial tumors and some hematological malignancies (15, 17, 19, 22, 26). We observed that Sp3 plays an essential role in the expression of ULBP1 in cell lines such as HEK 293 cells, which express significant amounts of ULBP1, suggesting that regulation of the expression of Sp3 isoforms or its activity may be involved in the induction of ULBP1 expression in cancer cells. A significant shift toward the long isoforms of Sp3 is observed in Sp1^–/– cells demonstrating that the Sp3 isoform expression can change in vivo (52, 56). Unfortunately, it is not known under which physiological conditions the alteration of Sp3 isoform ratio may take place.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. The transcription factors AP-2, -, and - repressed ULBP1 promoter activity in HeLa cells. A, the ULBP1 promoter construct (–862/+28) (200 ng) was cotransfected with 400 ng of the expression vectors for AP-2, -, and - in HeLa cells. B, the ULBP1 promoter construct with the mutation of the GC(4)/AP-2 site was cotransfected with the expression plasmids for Sp1, Sp3, and Sp4 in HeLa cells. C, two AP-2 expression vectors, which lack the DNA binding domain, AP-2B and AP-2-(1–165) and the expression vector AP-2-(165–437), which only expresses the DNA binding domain of AP-2, were cotransfected with the ULBP1 promoter construct. In all the experiments, the pRSV- galactosidase plasmid was included to correct the differences in transfection efficiencies. Data are the mean ± S.E. of luciferase activity from three independent experiments with triplicates for each experiment.

View larger version (22K): [in this window] [in a new window]   FIGURE 10. Regulation of ULBP1 protein expression by Sp1, Sp3, and AP-2. HEK 293 (A) and HeLa (B) were transiently transfected with pN3, pN3-Sp1, pN3-Sp3FL, pCMX, or pCMX-AP-2 plasmids (2 µg) and with a GFP expression vector (0.5 µg), as indicated. After 48 h, surface expression of ULBP1 protein was analyzed in GFP-expressing cells by flow cytometry using a monoclonal antibody anti-ULBP1 (4G7) as described under "Experimental Procedures." Numbers in the upper right corner indicate the mean fluorescence intensity from ULBP1. These data are representative from three independent experiments with similar results.

View larger version (18K): [in this window] [in a new window]   FIGURE 11. Model of regulation of ULBP1 transcription. Schematic representation of the ULBP1 promoter and the transcription factors involved in its regulation and shown.

	FOOTNOTES

 
^* This work was supported in part by Grant 03/0067 from the Fondo de Investigaciones Sanitarias from the Spanish Ministerio de Sanidad y Consumo. 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 Recipient of a grant from the Fondo de Investigaciones Sanitarias.

² To whom correspondence should be addressed: Tel.: 34-985-106-264; Fax: 34-985-103-606; E-mail: segundog{at}uniovi.es.

³ The abbreviations used are: NK, natural killer; ULBP1, UL16-binding protein 1; NKG2DL, NKG2D ligand; MIC, major histocompatibility complex class I-related chain; HEK 293, human embryonic kidney 293; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; ChIP, chromatin immunoprecipitation; RACE, rapid amplification of cDNA ends; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CREB, cAMP-response element-binding protein.

	ACKNOWLEDGMENTS

 
We thank Dr. Carlos López Otín for the critical review of the manuscript, Alicia R. Folgueras for technical assistance, Dr. Thomas Spies and Dr. Jose María Freije for providing antibodies, and Dr. G. Suske, Dr. Perry Kannan, Dr. Reinhard Buettner, and Dr. Ronald J. Weigel for the vectors they kindly provided us.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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