Transcriptional Regulation of Mouse δ-Opioid Receptor Gene

δ-Opioid receptors (DOR) present on T cells have been shown to mediate the immunomodulatory effects of endogenous and synthetic DOR agonists on T cells. Considerable evidence indicates that there is stimulated transcription of DOR gene in activated T cells, which is correlated with augmented expression of DOR and enhanced capacity of DOR agonists to affect the T-cell's functions. However, the molecular mechanism underlying the stimulated transcription of the DOR gene in activated T cells is still unclear. In the present study, we analyzed a 1.3-kb DNA fragment immediately upstream of the translation start site (−1300 to +1 bp, with the translation start site designated as +1) of the mouse DOR gene in EL-4 cells, a mouse lymphoma T cell line that exhibits enhanced expression of DOR transcripts when activated by phytohemagglutinin. Through both in vivo and in vitro experiments, we have demonstrated that increased binding activity of Ikaros at the Ikaros-binding site (−378 to −374) in the DOR promoter is required for the stimulated transcription of DOR gene in phytohemagglutinin-activated T cells.


␦-Opioid receptors (DOR) present on T cells have been shown to mediate the immunomodulatory effects of endogenous and synthetic DOR agonists on T cells. Considerable evidence indicates that there is stimulated transcription of DOR gene in activated T cells, which is correlated with augmented expression of DOR and enhanced capacity of DOR agonists to affect the T-cell's functions. However, the molecular mechanism underlying the stimulated transcription of the DOR gene in activated T cells is still unclear.
In the present study, we analyzed a 1.3-kb DNA fragment immediately upstream of the translation start site (؊1300 to ؉1 bp, with the translation start site designated as ؉1) of the mouse DOR gene in EL- 4

cells, a mouse lymphoma T cell line that exhibits enhanced expression of DOR transcripts when activated by phytohemagglutinin. Through both in vivo and in vitro experiments, we have demonstrated that increased binding activity of Ikaros at the Ikarosbinding site (؊378 to ؊374) in the DOR promoter is required for the stimulated transcription of DOR gene in phytohemagglutinin-activated T cells.
Endogenous and synthetic ␦-opioids have been shown to modulate T-cell proliferation, cytokine production, and calcium mobilization, through the ␦-opioid receptor (DOR) 1 on T cells (1)(2)(3)(4). For example, ␤-endorphin was shown to enhance intracellular calcium mobilization in murine splenic T cells, which was inhibited by naltrindole, a selective DOR antagonist, whereas the selective -opioid receptor antagonist was ineffective (2). In addition, the enhancement of human T-cell proliferation by certain methionine-enkephalin analogs could be completely abolished by naloxone and selective DOR antagonists (3). It was also reported that DOR agonists such as deltorphin and SNC-80 could concentration dependently suppress the expression of human immunodeficiency virus-1 in DOR-transfected human T cells (4).
DOR transcripts and DOR protein have been detected at very low levels in mouse splenic and thymic T cells, as well as in some human or murine T-cell lines (5). However, the expres-sion of DOR transcripts can be significantly enhanced during T-cell activation (6,7). It was reported that the expression of DOR transcripts could be markedly increased in murine splenic T cells stimulated by concanavalin A or anti-CD3-⑀; since the DOR mRNA stability was not altered during T cell activation, the enhanced expression of DOR transcripts was apparently through a transcriptional mechanism (6). In addition, concurrent with the enhanced expression of DOR transcripts, the expression of DOR protein was significantly increased in activated T cells, correlated with greater capacity of ␦-opioids to affect the T-cell's functions (7). Thus, understanding the molecular mechanism underlying the stimulated transcription of the DOR gene in activated T cells may raise the possibility of regulating the immunomodulatory effects of ␦-opioids on T cells, by manipulation of the inducible expression of DOR.
In the present study, we analyzed a 1.3-kb DNA fragment immediately upstream of the translation start site (Ϫ1300 to ϩ1 bp, with the translation start site designated as ϩ1) of the mouse DOR gene in EL-4 cells, a mouse lymphoma T cell line that constitutively expresses low level of DOR transcripts (5) and can be activated by low concentrations of phytohemagglutinin (PHA) (8). Through both in vivo and in vitro experiments, we have demonstrated that increased binding activity of Ikaros at the Ikaros-binding site (Ϫ378 to Ϫ374) in the DOR promoter is required for the stimulated transcription of the DOR gene in PHA-activated EL-4 cells.

MATERIALS AND METHODS
Cell Culture-Mouse lymphoma EL-4 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 4 mM L-glutamine, and 4.5 g/liter glucose. The cells were incubated at 37°C in an atmosphere of 10% CO 2 and 90% air.
RT-PCR Analysis-Total RNA was isolated from an equal amount of unstimulated or PHA-activated EL-4 cells using the TRI Reagent kit (Molecular Research Center) according to the instructions of the manufacturer. 1.5 g of total RNA was reverse transcribed using Superscript TM First-strand Synthesis System (Invitrogen) according to the instructions of the manufacturer. The resultant reverse transcription products were amplified by PCR. The sense primer was 5Ј-ATCTTCAC-CCTCACCATGATG-3Ј and the antisense primer was 5Ј-CGGTCCT-TCTCCTTGGAACC-3Ј. The expected PCR product is 355 bp (9). The PCR conditions were 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 40 s followed by a final extension at 72°C for 10 min. Reverse transcribed products of ribosomal RNA (18 S) was measured for normalization, using 25 cycles of PCR amplification with QuantumRNA 18 S primers (Ambion). The PCR products were resolved on 1% agarose gels containing ethidium bromide and photographed on a UVP Transluminator. The density of the DOR transcripts in each lane of the gels was calculated using ImageQuant software (Molecular Dynamics) and normalized to the density of the RT-PCR-amplified 18 S rRNA fragment. The resultant relative DOR transcripts levels were analyzed using Student's t test.
Cloning and Sequencing of PCR Product-PCR product was purified after 1% agarose gel electrophoresis and inserted into EcoRV site of the cloning vector pBluescript (Stratagene). Sequencing reactions were per-formed using Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (U. S. Biochemical Corp.).
Plasmid Construction-Luciferase fusion plasmids pD1300, pD890, pD400, pD360, and pD262 were constructed as described previously (10). The mutant constructs pD400-MIK1, pD400-MIK2, and pD400-MSP were constructed using Altered Sites II in vitro Mutagenesis System (Promega) according to the instructions of the manufacturer. The dominant-negative-Ikaros expression vector was prepared by polymerase chain reaction using the reverse transcription products from EL-4 cells total RNA as templates. The upper primer bears the essential Kozak sequence and the lower primer bears the XbaI site. The PCR product encodes partial amino acid sequence (from 282 to 517) of Ikaros-1 and was subcloned into EcoRV and XbaI sites of pcDNA3 vector (Invitrogen). All the correct clones were confirmed by sequencing.
Transient Transfection and Reporter Gene Activity Assay-EL-4 cells were transfected using the DOTAP (Roche Molecular Biochemicals) Lipofection method as described previously (11). Briefly, cells were transfected with an equimolar amount of each plasmid. After a 24-h culture with or without PHA (1.5 g/ml), cells were harvested and lysed with lysis buffer (Promega). A one-fifth molar ratio of PCH110 plasmid (Amersham Bioscience) containing the ␤-galactosidase gene driven by the SV40 promoter was included in each transfection for normalization.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts were prepared from an equal amount of unstimulated or PHA-activated EL-4 cells using the method described by Johnson et al. (12). EMSA was performed with 32 P-labeled double-stranded oligonucleotides that were incubated with nuclear extract in EMSA buffer (10 mM Tris, pH 7.5, 5% glycerol, 1 mM EDTA, pH 7.1, 50 mM NaCl, 1 mM dithiothreitol, and 0.1 mg/ml poly(dI-dC)). For oligonucleotide competition analysis, a 75-fold molar excess of competitor oligonucleotide was also added to the mixture. After incubation at room temperature for 30 min, the mixture was analyzed on 5% nondenaturing polyacrylamide gels. For the supershift assays, 2 g of goat polyclonal anti-Ikaros antibody (Santa Cruz Biotechnology) was added to the mixture. The reaction was then incubated on ice for 1 h. Protein-DNA complexes and free DNA were fractionated on 5% polyacrylamide gels in 1 ϫ Tris borate-EDTA electrophoresis buffer at 4°C and visualized by autoradiography.
In Vivo Formaldehyde Cross-linking and Immunoprecipitation of Chromatin-EL-4 cells (ϳ2 ϫ 10 7 cells per sample) were fixed by formaldehyde (Fisher Scientific) at a final concentration of 1%. Fixation proceeded at room temperature for 10 min and was stopped by the addition of glycine to a final concentration of 0.125 M. Nuclei were collected as described by Ausubel et al. (13). The following chromatin immunoprecipitation was performed according to the protocols described by Farnham and co-workers (14). Briefly, the nuclei were sonicated on ice to shear the chromatin to an average length of 400 bp. The chromatin solutions were precleared with the addition of Staph A cells for 15 min at 4°C. Precleared chromatin was incubated with 2 g of goat polyclonal antibody (Santa Cruz: anti-Ikaros M-20), 1 l of goat preimmune control serum, or no antibody at 4°C overnight. After immunoprecipitation, washing, and elution (15), cross-links were reversed by addition of NaCl to a final concentration of 200 mM; RNA was removed by addition of 10 g of RNase A per sample followed by incubation at 65°C for 5 h. Samples were ethanol-precipitated and resuspended for incubation with proteinase K. Then DNA was extracted and precipitated by standard protocols. DNA pellets were collected and analyzed through PCR. Approximately 2 ng of DNA was used as a template in a 50-l PCR reaction mixture using 1 unit of pfu polymerase. For DOR amplification, a sense primer corresponding to

FIG. 1. RT-PCR analysis of the expression of DOR transcripts in unstimulated and PHA-activated EL-4 cells.
A, total RNA from an equal amount of EL-4 cells cultured for 24 h with or without PHA (1.5 g/ml) was amplified by RT-PCR (30 cycles) and separated on agarose gels. RT-PCR-amplified 18 S rRNA fragment was measured for normalization (22 cycles). B, the relative DOR transcripts level was calculated as described under "Materials and Methods." The histograms represent the means of relative DOR transcripts level from three independent experiments using different preparations of total RNA. The error bars indicate the range of standard deviations. *, p Ͻ 0.05 compared with the "unstimulated" group.

FIG. 2. Deletional analysis of the mouse DOR promoter activity in unstimulated and PHA-activated EL-4 cells.
A series of DOR promoter/luciferase constructs were prepared and introduced into EL-4 cells. After a 24-h culture with or without PHA (1.5 g/ml), cells were harvested for luciferase activity assay. The line graph on the left is a schematic representation of DOR gene promoter regions that were included in each construct. Each construct was named by the relative position of the 5Ј-end nucleotide of the inserted DOR promoter region. The luciferase activity of the transfectants was normalized to ␤-galactosidase activity and then expressed as luciferase/␤-galactosidase activity ratio. The histograms represent mean values of four independent transfection experiments with two different plasmid preparations. Error bars indicate the range of standard errors.
the DOR promoter sequence from Ϫ458 to Ϫ439 and an antisense primer corresponding to the DOR promoter sequence from Ϫ339 to Ϫ320 were used. A set of primers specific to the GAPDH exon 8 was used for GAPDH amplification. The amplification was performed using one cycle at 95°C for 2 min, 35 cycles at 95°C for 40 s, 68°C (for DOR) or 64°C (for GAPDH) for 30 s, and 72°C for 30 s.
Western Blot Analysis-Nuclear extracts were prepared from an equal amount of unstimulated or PHA-activated EL-4 cells using the method described by Johnson et al. (12). 16 g of nuclear extracts were loaded onto 10% SDS-polyacrylamide gels. Proteins were blotted onto a polyvinylidene difluoride microporous membrane (Millipore). Membranes were incubated for 1 h with a 1/1000 dilution of anti-Ikaros serum (Santa Cruz Biotechnology), and then washed and revealed using donkey anti-goat IgG horseradish peroxidase conjugate (1/5000, 1 h). Peroxidase was revealed with an Amersham Bioscience ECF kit. Proteins were quantified before being loaded onto the gel, and equal loading of extracts was verified by Ponceau coloration.

Enhanced Expression of DOR Transcripts in PHA-activated EL-4 Cells-EL-4 is a mouse lymphoma T cell line that consti-
tutively expresses low levels of DOR transcripts and can be activated by low concentrations of PHA. To determine whether the expression of DOR transcripts could be enhanced in activated EL-4 cells, the cells were treated with PHA (1.5 g/ml) for 24 h. Then total RNA was extracted from an equal amount of unstimulated or PHA-activated EL-4 cells and subjected to RT-PCR analysis. As shown in Fig. 1A, DOR transcripts is expressed at a relatively low level in unstimulated EL-4 cells. However, the expression of DOR transcripts is apparently enhanced in PHA-activated EL-4 cells. The density of the DOR fragment in each lane was calculated and normalized to the density of RT-PCR-amplified 18 S rRNA fragment. Student's t test revealed significant difference between groups in the relative DOR transcript level (Fig. 1B). The PCR-amplified fragments were confirmed to be the desired DOR cDNA fragment by cloning and sequencing of the PCR products.
Deletional Analysis of the Mouse DOR Promoter Activity in Unstimulated and PHA-activated EL-4 Cells-To determine the regulatory elements that contribute to the stimulated transcription of DOR gene in activated T cells, serial deletional analyses were performed using the 1.3-kb DNA fragment im-mediately upstream of the translation start site (Ϫ1300 to ϩ1 bp, with the translation start site designated as ϩ1) of the mouse DOR gene. A primary luciferase reporter construct and its serial deletion constructs were prepared form this 1.3-kb DNA fragment as described under "Materials and Methods." This primary construct, designated as pD1300, and its serial 5Ј-deletion constructs, designated as pD890, pD400, pD360, and pD262, are illustrated in Fig. 2. The pGL3-basic plasmid (designated as basic) containing neither promoter nor enhancer was included as a negative control. The promoter activity of each construct was tested by transient transfection assays in EL-4 cells. As shown in Fig. 2, all the luciferase reporter constructs showed similar luciferase activities in unstimulated EL-4 cells, with insignificant variations. In contrast, in PHAactivated EL-4 cells, pD1300, pD890, and pD400 showed a significant increase (over 2-fold) in the luciferase activity compared with that in unstimulated EL-4 cells; further 5Ј-deletion down to Ϫ360 abolished the increase of promoter activity. These data suggest that the sequence between Ϫ400 and Ϫ360 in the DOR promoter contains a regulatory element(s) that is essential for the stimulated promoter activity of DOR gene in activated T cells.
Functional Identification of the Ikaros-binding Site in the DOR Promoter-The 40-bp DNA sequence between Ϫ400 and Ϫ360 in the DOR promoter was analyzed by sequence comparison using the Transcription Factors Data base (TRANSFAC 4.0), 2 which revealed two consensus Ikaros-binding sites (Ϫ385/Ϫ381 and Ϫ378/Ϫ374, respectively) and a consensus Sp1-binding site (Ϫ380/Ϫ375) that partially overlaps the Ikaros-binding sites (Fig. 3A). To determine which site(s) was functional, mutational analyses were performed. As shown in Fig. 3A, the mutant constructs pD400-MIK1 and pD400-MIK2 have the same sequence of pD400 except for a point mutation in the consensus Ikaros-binding sites at Ϫ385/Ϫ381 and Ϫ378/ Ϫ374, respectively, without altering the Sp binding motif at Ϫ380/Ϫ375; the mutant construct pD400-MSP has the same sequence of pD400 except for the Ikaros-binding site at Ϫ385/ 2 The site for TRANSFAC 4.0 is transfac.gbf.de/TRANSFAC/. Ϫ381 and the Sp1-binding site being disrupted by "AAGCTT," while leaving the Ikaros-binding motif at Ϫ378/Ϫ374 intact. As shown in Fig. 3B, the mutant constructs displayed similar luciferase activity to that of the wild type construct (pD400) in unstimulated EL-4 cells. In contrast, while the mutation in the Ikaros-binding site at Ϫ385/Ϫ381 (pD400-MIK1) or in the Sp1binding site (pD400-MSP) did not show apparent effects on the stimulated DOR promoter activity in PHA-activated EL-4 cells, the mutation in the Ikaros-binding site at Ϫ378/Ϫ374 (pD400-MIK2) abolished the increase in the promoter activity. These results indicate that the Ikaros-binding site at Ϫ378/Ϫ374 is essential for the stimulated DOR promoter activity in activated EL-4 cells.
In Vitro Protein-DNA Binding Activity at the Putative Ikarosbinding Site in the DOR Promoter-EMSAs were performed to determine the protein binding activity at the putative Ikarosbinding site. As shown in Fig. 4A, four oligonucleotides, D397/ 364, MIK1, MIK2, and MSP were synthesized, corresponding to the DOR promoter sequence Ϫ397 to Ϫ364 in constructs pD400, pD400-MIK1, pD400-MIK2, and pD400-MSP, respectively. First, EMSAs were performed in the presence of nuclear extracts from an equal amount of unstimulated or PHA-activated EL-4 cells, using D397/364 as the probe. As shown in Fig.  4B, nuclear extracts of unstimulated (lanes 2-7) or PHA-activated EL-4 cells (lanes 8 -11) formed two major protein-DNA complexes with D397/364, which was specific because the formation of the complexes could be abolished by molar excess of unlabeled probe (lane 3). As molar excess of unlabeled consensus Ikaros-binding sequence (lanes 4 and 9) and Sp1-binding sequence (lane 5) could, respectively, abolish the formation of one of the two complexes, the complexes were designated as Ikaros-complex and Sp-complex, respectively. Anti-Ikaros antibody supershifted the Ikaros-complex to a higher position while the control serum did not (lanes 6 and 7 and lane 11), indicating that Ikaros proteins (16) could bind to the Ϫ397/ Ϫ364 region of the DOR promoter. Interestingly, the binding activity of Ikaros in the Ϫ397/Ϫ364 region of the DOR promoter was very low in unstimulated EL-4 cells (lane 2); however, there was a marked increase in the Ikaros binding activity in PHA-activated EL-4 cells (lane 8), which was correlated with the increased DOR promoter activity in PHA-activated EL-4 cells. To correlate the in vitro protein-DNA binding activities with the in vivo promoter activities of pD400, pD400-MIK1, pD400-MIK2, and pD400-MSP, EMSAs were performed with oligonucleotides D397/364, MIK1, MIK2, and MSP in the presence of nuclear extracts from PHA-activated EL-4 cells. As shown in Fig. 4C, the binding of Ikaros to D397/364 could be abolished by 75-fold excess of unlabeled MIK1 (lane 4), but not MIK2 (lane 5). Moreover, when MIK2 was used as the probe, there was no Ikaros-complex formed while the Sp-complex formation was not affected (lane 8). In contrast, when MIK1 or MSP was used as the probe, the Ikaros-complex formation was not affected (lanes 7 and 9). Combined with the data from the mutational analyses (Fig. 3), the EMSAs results indicate that increased binding activity of Ikaros to the Ikaros-binding site at Ϫ378/Ϫ374 is required for the stimulated DOR promoter activity in PHA-activated EL-4 cells.
In Vivo Binding Activity of Ikaros in the DOR Promoter in EL-4 Cells-To further determine whether Ikaros could bind to the DOR promoter in vivo, we performed in vivo formaldehyde cross-linking assays. First, an equal amount of unstimulated or PHA-activated EL-4 cells were treated with 1% formaldehyde. Then the cross-linked chromatin was immunoprecipitated by using antibody against Ikaros (anti-Ikaros M-20, Santa Cruz Biotechnology). As negative controls, we included a sample without the addition of antibody, a sample with the addition of preimmune serum, and a non-cross-linked sample. After immunoprecipitation and reversal of the cross-links, enrichment of the endogenous DOR promoter fragment in each sample was monitored by PCR amplification using primers specific for the DOR promoter (as described under "Materials and Methods"). As shown in Fig. 5, in PHA-activated EL-4 cells, the PCRamplified DOR promoter fragment was readily detectable in samples with the addition of anti-Ikaros antibody, while no detectable PCR products were observed in the negative controls. The binding detected in the DOR promoter was specific, because the antibody against Ikaros did not enrich the GAPDH gene fragments to the same level as they enriched the DOR promoter fragments (Fig. 5, lower). The in vivo Ikaros binding activity was not detectable when the same set of experiments was carried out in unstimulated EL-4 cells (data not shown), which may be explained by the very low binding activity of  1 and 2) or presence of different unlabeled competitors as indicated (lanes 3-6). Lane 7, MIK1 was the probe. Lane 8, MIK2 was the probe. Lane 9, MSP was the probe.
Ikaros in the DOR promoter in unstimulated EL-4 cells (Fig.  4B, lane 2). Together these results demonstrated that Ikaros could bind to the DOR promoter in vivo and suggest that the in vivo Ikaros binding activity in the DOR promoter is significantly increased in PHA-activated EL-4 cells.
Dominant-negative Ikaros Inhibits the Stimulated DOR Promoter Activity in PHA-activated EL-4 Cells-To confirm the functional role of Ikaros in the stimulated expression of DOR transcripts in PHA-activated EL-4 cells, we constructed a dominant-negative-Ikaros (DNIK) expression vector that encodes a mutant form of Ikaros containing no N-terminal zinc fingers. As at least three N-terminal zinc fingers are required for the sequence-specific and high-affinity binding of Ikaros to DNA, the DNIK is unable to bind DNA, but still able to dimerize with wild-type Ikaros (16). Thus, it functions by dimerizing with the wild-type Ikaros and preventing it from binding DNA. First, pD400 was co-transfected with the DNIK expression vector into unstimulated or PHA-activated EL-4 cells. As shown in Fig. 6, in unstimulated EL-4 cells, the DNIK did not show apparent effect on the promoter activity of pD400, in agreement with the low Ikaros binding activity in the DOR promoter in unstimulated EL-4 cells. However, the DNIK exerted an over 2-fold inhibition on the pD400 promoter activity in PHAactivated EL-4 cells, confirming that Ikaros contributes to the stimulated DOR promoter activity in PHA-activated EL-4 cells.
To determine the DNIK's effect on the expression of DOR transcripts in EL-4 cells, the DNIK expression vector was transfected into unstimulated and PHA-activated EL-4 cells, followed by RT-PCR analyses to measure the expression of DOR transcripts. As shown in Fig. 7A, the DNIK did not affect the DOR transcripts expression in unstimulated EL-4 cells; however, in PHA-activated EL-4 cells, it apparently reduced the expression of DOR transcripts by more than 2-fold, almost to the level in the unstimulated cells. This observation was confirmed by quantitation (Fig. 7B). Together these results demonstrate that the DNA binding of Ikaros is required for the stimulated DOR promoter activity and thus the enhanced expression of DOR transcripts in PHA-activated EL-4 cells.
Western Blot Analysis of the Nuclear Expression of Ikaros in Unstimulated and PHA-activated EL-4 Cells-To determine the mechanism underlying the increased binding activity of Ikaros in the Ϫ378/Ϫ374 region of the DOR promoter in PHAactivated EL-4 cells, the nuclear expression of Ikaros was measured by Western blot analysis using nuclear extracts from an equal amount of unstimulated or PHA-activated EL-4 cells. As shown in Fig. 8, while the expression of Ikaros 1 was at similar levels in both unstimulated and PHA-activated EL-4 cells, the expression of Ikaros 2/3 was apparently increased in PHA-activated EL-4 cells, which provides an explanation for the augmented binding activity of Ikaros in the DOR promoter in PHA-activated EL-4 cells. DISCUSSION The DOR present on T cells have been shown to mediate the immunomodulatory effects of ␦-opioids on T cells. Although the expression of DOR transcripts is at very low levels in resting T cells, it is significantly increased in activated T cells through a transcriptional mechanism, correlated with augmented expression of DOR protein. To understand the mechanism underlying the stimulated transcription of the DOR gene in activated T cells, we analyzed the DNA sequences immediately upstream of the translation start site of the mouse DOR gene in the mouse EL-4 T cell line and have demonstrated that increased binding activity of Ikaros at the Ikaros-binding site (Ϫ378 to Ϫ374) in the DOR promoter is required for the stimulated transcription of DOR gene in PHA-activated EL-4 cells.
The EMSAs data (Fig. 4) indicated that the Ϫ378/Ϫ374 Ikaros-binding site was required for Ikaros binding to the DOR promoter. Interestingly, in EMSAs where the radiolabeled probe was in much excess of the probe-binding factors, it was observed that Ikaros and Sp family proteins could, respectively, form complexes with the D397/364 probe and there was no Ikaros-Sp-complex formed (Fig. 4B), which suggests that Ikaros and the Sp family proteins could not bind to the probe simultaneously but compete for the composite Ikaros/Sp1-binding site. However, combined analysis of the data from both mutational analyses (Fig. 3) and EMSAs (Fig. 4) revealed that the Sp family proteins had no effect on the DOR promoter even though they may bind to the Ikaros/Sp1-binding site. We noted that in unstimulated EL-4 cells, there was little Ikaros-complex formed in EMSAs, in agreement with the low level of the DOR promoter activity as shown in Fig. 1A. In contrast, Ikaros showed a markedly increased binding activity at the Ϫ378/ Ϫ374 Ikaros-binding site in PHA-activated EL-4 cells, correlated with the augmented DOR promoter activity. This observation was corroborated by the in vivo formaldehyde crosslinking assays. While the in vivo binding activity of Ikaros was not detectable in the DOR promoter in unstimulated EL-4 cells, it was detected in the PHA-activated cells (Fig. 5), indicating that the Ikaros binding activity was significantly increased in PHA-activated EL-4 cells so that it could be detected by the in vivo cross-linking assay that has a relatively lower sensitivity than the EMSA. In combination with the data from mutational analyses, all these results indicate that increased binding of Ikaros to the Ϫ378/Ϫ374 Ikaros-binding site is indispensable for the stimulated DOR promoter activity in PHA-activated EL-4 cells. Moreover, the function role of the binding of Ikaros to the DOR promoter was confirmed by using a dominantnegative Ikaros that prevented the wild-type Ikaros from binding DNA (Fig. 7).
The Ikaros gene encodes a family of hemopoietic-specific zinc finger transcription factors capable of high affinity DNA binding to sites that contain the GGGA core motif (17,18). Homoand heterodimers formed between the DNA-binding Ikaros isoforms (Ikaros 1, 2, and 3) or heterodimers formed between Ikaros and its dimerization partners (such as Helios and Aiolos) can greatly increase Ikaros affinity for DNA and consequently its ability to activate transcription (16). Since the antiserum against Ikaros dimerization partners did not supershift the Ikaros-complex in EMSAs (data not shown), the Ikaros-complex supershifted by the anti-Ikaros antibody in the EMSAs (Fig. 4C) was predominantly comprised of Ikaros proteins. Western blot analysis revealed that the expression of nuclear Ikaros 2/3 was apparently increased in PHA-activated EL-4 cells compared with that in the unstimulated cells (Fig.  8), thereby providing a mechanistic basis for the increased binding activity of Ikaros in the DOR promoter in PHA-activated EL-4 cells. In addition, interactions between the three DNA-binding Ikaros isoforms may generate six homo-and heterodimeric complexes with distinct combinations of two DNAbinding domains, which may confer different DNA binding potential to the complexes. Thus, we suggest that the increased expression of Ikaros 2/3 may also change the ratio of the homoand heterodimers of Ikaros, and thus may result in an apparent increase in the binding affinity of Ikaros to the Ϫ378/Ϫ374 Ikaros-binding site in the DOR promoter context. Further studies will be needed to uncover the underlying mechanism(s).
T lymphocytes are exposed to endogenous opioid peptides in vivo. For example, circulating ␤-endorphin originating from the pituitary and enkephalin peptides originating from the adrenal medulla continuously bathe T lymphocytes. In addition, T lymphocytes may also produce and release their own opioids (19,  20). As Ikaros has been reported to set threshold for T-cell activation (21) and plays an important role in the T-cell homeostasis (17), the link between Ikaros and the stimulated transcription of the DOR gene in activated T cells implicate an active role for endogenous opioids in modulating the functions and homeostasis of activated T cells.