Transcriptional Regulation of Mouse delta -Opioid Receptor Gene

IKAROS-2 AND UPSTREAM STIMULATORY FACTOR SYNERGIZE IN TRANS-ACTIVATING MOUSE delta -OPIOID RECEPTOR GENE IN T CELLS*

Ping SunDagger and Horace H. Loh

From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, August 9, 2002, and in revised form, October 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Considerable evidence indicates that transcription of the delta -opioid receptor (dor) gene is correlated with both the expression of DOR on T cells and the capacity of DOR agonists to modulate the immunological functions of the T cell. We previously reported that increased Ikaros (Ik) binding activity over an Ik-binding site at -378 to -374 (with the translation start site designated as +1) in the mouse dor promoter was required for the enhanced transcription of dor gene in phytohemagglutinin-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR. In the present study, we have analyzed further the mouse dor promoter in EL-4 cells and have demonstrated that Ik-2 homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. Moreover, an E box (-185 to -180) that binds upstream stimulatory factor is essential for the dor promoter activity in both resting and phytohemagglutinin-activated T cells. Furthermore, we have demonstrated that Ik-2 and upstream stimulatory factor synergize in trans-activating the dor promoter via the putative Ik-binding site and the E box, respectively.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endogenous and synthetic delta -opioids have been shown to modulate T-cell proliferation, cytokine production, and calcium mobilization through the delta -opioid receptor (DOR)1 on T cells (1-4). For example, beta -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 µ-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 in mouse splenic and thymic T cells, as well as in some human or murine T-cell lines (5). Considerable evidence indicates that the transcription of the dor gene is correlated with both the expression of DOR on T cells and the capacity of DOR agonists to affect the functions of the T cell (6-10). Thus, understanding the molecular mechanism underlying the transcriptional regulation of the dor gene in T cells may raise the possibility of regulating the immunomodulatory effects of delta -opioids on T cells by manipulation of the expression of DOR.

Previously, we analyzed a 1.3-kilobase pair DNA fragment immediately upstream of the translation start site (-1300 to +1 base pair, with the translation start site designated as +1) of the mouse dor gene in a mouse neuronal cell line and identified a minimum promoter region (-262 to -141); a GC box (-226 to -221) and a composite Ets-1-binding site/E box (-192 to -180) were found crucial for the promoter activity (11, 12). Subsequent studies revealed that the minimum dor promoter was also sufficient to confer constitutive dor promoter activity in EL-4 cells, a mouse T cell line that constitutively expresses DOR. In addition, increased binding activity of Ikaros (Ik) at an Ik-binding site (-378 to -374) was demonstrated to account for the significantly enhanced dor promoter activity in phytohemagglutinin (PHA)-activated EL-4 cells (13). In the present study, further analyses were carried out in EL-4 cells. Through both in vivo and in vitro experiments, we have demonstrated that Ik-2 homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. Moreover, the E box (-185 to -180), which binds upstream stimulatory factor (USF), is essential for the dor promoter activity in both resting and PHA-activated T cells. Furthermore, we have demonstrated that Ik-2 and USF synergize in trans-activating the dor promoter via the putative Ik-binding site and the E box, respectively.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- The luciferase fusion plasmids pD1300 and pD400 were constructed as described previously (13). The mutant constructs pD400MIK2 and pD400ME were constructed using the Altered Sites II in vivo mutagenesis system (Promega) according to the instructions of the manufacturer. The Ik-2 expression vector was created by polymerase chain reaction (PCR) using the reverse transcription products from the total RNA of the EL-4 cell. The upper primer bears the essential Kozak sequence and the lower primer bears the XbaI site. The PCR product was inserted into the EcoRV and XbaI sites of pcDNA3 vector (Invitrogen). The pD1300IK and pD400AS constructs were generated by PCR. All of the correct clones were confirmed by sequencing.

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% CO2 and 90% air.

Transient Transfection and Reporter Gene Activity Assay-- EL-4 cells were transfected using SuperFect transfection reagent (Qiagen) according to the instructions of the manufacturer. Briefly, cells were transfected with equimolar amount of each plasmid. After a 24-hour culture with or without PHA (1.5 µg/ml), cells were harvested and lysed with lysis buffer (Promega). A one-fifth molar ratio of PCH110 plasmid (Amersham Pharmacia Biotech) containing the beta -galactosidase gene driven by an SV40 promoter was included in each transfection for normalization.

Recombinant Protein Purification-- Bacterially expressed recombinant human USF-1 and mouse Ik-2 were prepared as described by Pognonec et al. (14). Briefly, competent JM109 bacteria cells were transformed with human USF-1 or mouse Ik-2 expression vectors. The cultures were grown and induced at 28 °C overnight. The bacteria pellet was lysed by sonication in ice-cold lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1% (v/v) aprotinin (Sigma), 0.1% Nonidet P-40). The sonicated sample was centrifuged at 4 °C for 10 min at 10,000 × g. Then, saturated ammonium sulfate was added dropwise to the supernatant to a final concentration of 33% (v/v). After 15 min on ice, the sample was centrifuged at 4 °C for 10 min at 10,000 × g. The pellet was resuspended in lysis buffer and centrifuged again. Then the supernatant was diluted with lysis buffer devoid of NaCl. The partially purified recombinant protein products, proved to comprise predominantly the desired recombinant proteins by 10% SDS-polyacrylamide gel electrophoresis plus Coomassie Blue staining, were used in subsequent gel retardation assays in which the purification product from mock-transformed bacteria cells was employed to incubate with the radiolabeled probe as a control.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared from resting or PHA-activated EL-4 cells using the method described by Johnson et al. (15). The plasmids pD400, pD400MIK2, and pD400ME were digested with KpnI, dephosphorylated with calf intestinal alkaline phosphatase, end-labeled with [gamma -32P]ATP, and then digested with NcoI to generate the 5'-labeled 400-base pair probes, which were purified by polyacrylamide gel electrophoresis. The double-stranded oligonucleotide D198/169 was 5'-end labeled with [gamma -32P]ATP. The probes were incubated with EL-4 nuclear extracts or the indicated amounts of recombinant protein(s) 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 competition analysis, a 75-fold molar excess of cold probe was added to the mixture and incubated at room temperature for 30 min. For supershift assays, 2 µg of anti-Ik, anti-USF-1, or anti-USF-2 antibody (Ab) (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× Tris borate-EDTA electrophoresis buffer at 4 °C and visualized by autoradiography.

Western Blot Analysis-- 16 µg of nuclear extracts prepared from an equal amount of unstimulated or PHA-activated EL-4 cells was 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-USF-1 or anti-USF-2 Ab and then washed and revealed using anti-rabbit IgG horseradish peroxidase conjugate (1/5000, 1 h). Peroxidase was revealed with an Amersham Pharmacia Biotech ECL kit. Proteins were quantified before being loaded onto the gel, and equal loading of extracts was verified by Ponceau coloration.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ik-2 Homodimers Bind to the -378/-374 Ik-binding Site and Trans-activates the dor Promoter-- Previously we reported that in parallel with the augmented expression of nuclear Ik proteins, the increased binding of Ik family members at an Ik-binding site (-378 to -374) in the mouse dor promoter enhanced the dor promoter activity in PHA-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR (13). As only Ik-1 and Ik-2 are the predominant Ik isoforms capable of DNA binding in the nucleus (16, 17), we employed the mouse Ik-1 and Ik-2 expression vectors in the present study to determine the individual roles of these proteins in trans-activating the dor promoter. The Ik-1 or Ik-2 expression vector was transfected into EL-4 cells with pD400, a mouse dor promoter/luciferase fusion plasmid encompassing the dor promoter sequence from -400 to +1, or pD400MIK2, a mutant of pD400 with a point mutation in the core binding motif of the -378/-374 Ik-binding site (Fig. 1A). As shown in Fig. 1B, pD400 and pD400MIK2 displayed similar promoter activities in resting EL-4 cells. Overexpressed Ik-2 enhanced the promoter activity of pD400 by ~2-fold, almost to the level of that activated by PHA, while showing no effect on pD400MIK2. In addition, overexpressed Ik-1 exerted no detectable effect on the promoter activity of pD400. These results indicate that Ik-2 but not Ik-1 can trans-activate the dor promoter via the -378/-374 Ik-binding site.


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Fig. 1.   Ik-2 trans-activates the dor promoter via the -378/-374 Ik-binding site. A, the luciferase reporter construct pD400 contains the dor promoter sequence from -400 to +1. The mutant construct pD400MIK2 was made by introducing a point mutation into the core binding motif of the -378/-374 Ik-binding site in pD400. The putative Ik-binding site in pD400 is underscored, and the mutant nucleotide in pD400MIK2 is shown in boldface. B, EL-4 cells were transfected with 2.0 µg of pD400 or pD400MIK2 and 0.5 µg of the Ik-1 or Ik-2 expression vector. In addition, pD400 was transfected into EL-4 cultures with or without PHA activation (1.5 µg/ml). Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as fold activation to the luciferase activity of pD400, which is defined as 1. Results are means of three independent experiments. Error bars indicate the range of standard errors. The empty expression vector (pcDNA3, Invitrogen) was added to make an equal amount (2.5 µg) of DNA for each transfection.

To determine the binding activity of individual Ik proteins at the putative Ik-binding site, EMSAs were performed with recombinant Ik-1 and Ik-2. Two DNA fragments, D400 and MIK2, corresponding to the dor promoter sequences from -400 to +1 in constructs pD400 and pD400MIK2, respectively, were employed as probes (Fig. 2A). As shown in Fig. 2B, 50 ng of Ik-2 readily formed a complex with D400 (lane 4), which was completely abolished by molar excess of unlabeled D400 (lane 6). In contrast, the cold competitor MIK2 with a mutation in the -378/-374 Ik-binding site was not able to reduce the Ik-2·D400 complex (lane 7). In addition, anti-Ik Ab shifted the complex to a higher position (lane 9). Together these results demonstrate that Ik-2 can specifically bind to the -378/-374 Ik-binding site in the dor promoter. Interestingly, it was observed that no detectable DNA-protein complex was formed using 50 ng of Ik-1 (lane 2), and a higher concentration of Ik-1 (250 ng) formed only a faint band with D400 (lane 3). Moreover, the Ik-2·D400 complex apparently was reduced in the presence of 50 ng of Ik-1, without the formation of any other detectable DNA-protein complex (lane 5). As the Ik isoforms bind to DNA through dimerization (16, 17), these results demonstrate that both Ik-1 homodimers and Ik-1/Ik-2 heterodimers have a very low affinity for the -378/-374 Ik-binding site, whereas Ik-2 homodimers can exert efficient binding to the putative Ik-binding site. This is consistent with the results in the functional assays that overexpression of Ik-2 but not Ik-1 significantly increased the dor promoter activity via the -378/-374 Ik-binding site (Fig. 1). Collectively, these results indicate that Ik-2 binds to the -378/-374 Ik-binding site and trans-activate the dor promoter mainly in the form of Ik-2 homodimers.


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Fig. 2.   DNA binding activities of Ik proteins at the -378/-374 Ik-binding site in the dor promoter. A, two DNA fragments were used in EMSAs. D400 contains the dor promoter sequence from -400 to +1, encompassing the -378/-374 Ik-binding site (underscored). MIK2 contains the same sequence as D400 except for a point mutation (indicated in boldface) in the putative Ik-binding site. B, EMSAs were performed by using D400 as the probe in the presence of recombinant Ik-1 and/or Ik-2 as indicated. Lane 1, control reaction (as described under "Materials and Methods"); lane 2, 50 ng of recombinant Ik-1; lane 3, 250 ng of recombinant Ik-1; lane 4, 50 ng of recombinant Ik-2; lane 5, 50 ng of recombinant Ik-1 plus 50 ng of recombinant Ik-2. Subsequently, 50 ng of recombinant Ik-2 was used in the presence of different unlabeled competitors (lanes 6 and 7), control serum (lane 8), or anti-Ik Ab (lane 9).

A USF-binding E box Is Essential for dor Promoter Activity-- We previously reported the identification of a minimum mouse dor promoter (-262 to -141) in mouse neuronal cell lines; this region contains a GC box (-226 to -221) and a composite Ets-1-binding site/E box (-192 to -180) that contribute to the constitutive dor promoter activity (11, 12). The minimum dor promoter was found sufficient to confer the constitutive promoter activity in resting T cells as well (13). Mutation in the GC box or the Ets-1-binding site did not result in a significant decrease in the dor promoter activity in either resting or PHA-activated EL-4 T cells (data not shown). However, mutation in the E box as present in pD400ME (Fig. 3A) almost completely abolished the promoter activity of pD400 in both resting and PHA-activated EL-4 cells (Fig. 3B), indicating that the E box is required for the basal dor promoter activity in T cells.


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Fig. 3.   Functional analysis of the E box in the dor promoter. A, the luciferase reporter construct pD400 contains the dor promoter sequence from -400 to +1. The mutant construct pD400ME was made by replacing the core binding motif of the E box in pD400 with a HindIII site. The E box in pD400 is underscored, and the mutant sequence in pD400ME is shown in boldface. B, luciferase activities of 2.0 µg of pD400 or pD400ME in both unstimulated and PHA-activated (1.5 µg/ml) EL-4 cells are expressed as luciferase/beta -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.

EMSAs were performed to determine the protein binding activity at the E box. Oligonucleotides D198/169 and Dm185, synthesized as probes, correspond to the dor promoter sequence from -198 to -169 in constructs pD400 and pD400ME, respectively (Fig. 4A). As shown in Fig. 4B, nuclear extracts from resting EL-4 cells formed a major complex with D198/169 (lane 2). Molar excess of unlabeled D198/169 abolished the complex formation (lane 3). In contrast, the cold competitor Dm185 with a mutation in the E box was not able to reduce the complex formation (lane 4). In addition, both anti-USF-1 Ab and anti-USF-2 Ab shifted the complex to a higher position (lanes 6 and 7). These results demonstrate that USF family members specifically bind to the E box in the dor promoter in EL-4 cells. Moreover, it was noted that the USF-binding activity at the E box basically did not change in PHA-activated EL-4 cells (lanes 8 and 9), consistent with the results from Western blot analysis that the expression of USF-1 or USF-2 is not changed in PHA-activated EL-4 cells compared with that in the unstimulated cells (Fig. 4C). Combined with the data from the mutational analysis (Fig. 3), these results indicate that USF family members specifically binds to the E box and confer basal dor promoter activity in either resting or activated T cells. However, USF binding is not the rate-limiting step for the dor promoter activity increment in activated T cells. This is in agreement with our previous conclusion that the increased Ik binding activity at the -378/-374 Ik-binding site triggers the augmentation of dor promoter activity in activated T cells (13).


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Fig. 4.   Protein binding activity at the E box in the dor promoter. A, two oligonucleotides were used in EMSAs. D198/169 contains the dor promoter sequence from -198 to -169 including the E box. Dm185 contains the same sequence as D198/169 except that the core binding motif of the E box is replaced by a HindIII site. B, EMSAs were performed by using D198/169 as the probe in the presence of nuclear extracts from an equal amount of unstimulated (lanes 2-7) or PHA-activated (lanes 8-9) EL-4 cells. Lane 1, probe only; lane 2 and lane 8, normal reaction; lanes 3 and 4, different unlabeled competitors as indicated; lane 5, control serum; lanes 6 and 9, 2 µg of anti-USF-1 Ab; lane 7, 2 µg of anti-USF-2 Ab. C, nuclear extracts from an equal amount of unstimulated or PHA-activated EL-4 cells were analyzed by Western blot using anti-USF-1 and anti-USF-2 Abs, respectively. The positions of USF-1 and USF-2 are indicated.

Ik-2 and USF Synergistically Trans-activate the dor Promoter-- To determine whether Ik-2 could functionally interact with USF, plasmid pD400, pD400MIK2 (Ik binding site mutated), or pD400ME (E box mutated) was co-transfected into EL-4 cells with expression vectors for mouse Ik-2 and/or human USF-1, which shows over 95% amino acid identity to murine USF-1. As shown in Fig. 5, overexpressed Ik-2 or USF-1 alone elevated the promoter activity of pD400 slightly more than 2-fold or 1-fold, whereas the combined overexpression of Ik-2 and USF-1 resulted in a more than 4-fold activation. The latter activation was abolished either by mutation of the putative Ik-binding site (pD400MIK2) or the E box (pD400ME). In addition, overexpressed Ik-2 could not rescue the promoter activity that was abolished by the E box mutation, indicating that Ik-2 needs to function through the E box-bound USF, which confers the basal dor promoter activity. Similar results were observed in co-transfection assays using Ik-2 and USF-2 (data not shown). Taken together, these data demonstrate that Ik-2 synergizes with USF in trans-activating the dor promoter via the -378/-374 Ik-binding site and the E box, respectively.


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Fig. 5.   Ik-2 and USF synergistically trans-activate the dor promoter. EL-4 cells were transfected with 2.0 µg of pD400, pD400MIK2, or pD400ME and 0.5 µg of the Ik-1 and/or USF-1 expression vector. Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as fold activation to the luciferase activity of pD400, which is arbitrarily defined as 1. Results are means of three independent experiments. Error bars indicate the range of standard errors. The empty expression vector (pcDNA3, Invitrogen) was added to make an equal amount (2.5 µg) of DNA for each transfection.

The -378/-374 Ik-2-responsive Element Is Position- but Not Orientation-dependent-- To further understand the functional properties of the -378/-374 Ik-binding site, several constructs were generated. Plasmid pD1300IK was created by inserting a duplicate of the Ik-2-responsive element (-400 to -360) upstream of -1300 while silencing the original Ik-2-responsive element by mutating the Ik-binding site at -378 to -374. pD400AS was generated by inserting the Ik-2-responsive element upstream of -360 in the antisense orientation (Fig. 6A). As shown in Fig. 6B, although overexpressed Ik-2 enhanced the promoter activity of pD400, pD400AS and pD1300 by about 2-fold in EL-4 cells, the promoter activity of pD1300IK was not affected. These results demonstrate that the -378/-374 Ik-binding site functions in a position-dependent, orientation-independent manner, indicating that the Ik-2-responsive element acts as an upstream promoter element rather than an enhancer for the dor promoter (18, 19). Interestingly, similar results were observed when the Ik-2 treatment was replaced by PHA activation in EL-4 cells (Fig. 6C), suggesting that the enhanced dor promoter activity in PHA-activated EL-4 cells results from increased binding activity of Ik-2 at the -378/-374 Ik-binding site.


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Fig. 6.   The -378/-374 Ik-2-responsive element is position- but not orientation-dependent. A, luciferase reporter constructs pD1300 and pD400 contain the dor promoter sequences from -1300 to +1 and from -400 to +1, respectively. The mutant construct pD1300IK was made by inserting a duplicate of the Ik-2-responsive element (-400 to -360) upstream of -1300 while silencing the original Ik-2 responsive element by mutating the Ik-binding site at -378 to -374. Mutant construct pD400AS was generated by inserting the Ik-2-responsive element upstream of -360 in the antisense orientation. B, EL-4 cells were transfected with 2.0 µg of pD1300, pD400, pD1300IK, or pD400AS and 0.5 µg of the Ik-2 expression vector. The empty expression vector (pcDNA3, Invitrogen) was added to make an equal amount (2.5 µg) DNA for each transfection. C, 2.0 µg of pD1300, pD400, pD1300IK, or pD400AS was transfected into EL-4 cultures with or without PHA activation (1.5 µg/ml). Luciferase activities were normalized to beta -galactosidase activity from a co-transfected LacZ vector (pCH110) and expressed as fold activation to the luciferase activity of pD400, which is arbitrarily defined as 1. Results are means of three independent experiments. Error bars indicate the range of standard errors.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we reported that increased Ik binding activity at an Ik-binding site (-378 to -374) in the mouse dor promoter is required for the enhanced dor promoter activity in PHA-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR. In the present study, we further analyzed the mouse dor promoter in EL-4 cells and have demonstrated that Ik-2 homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. Moreover, an E box (-185 to -180) that binds upstream stimulatory factor is essential for the dor promoter activity in both resting and PHA-activated T cells. Furthermore, we have demonstrated that Ik-2 and USF synergize in trans-activating the dor promoter via the putative Ik-binding site and the E box, respectively.

The Ikaros gene encodes a family of hemopoiesis-specific zinc finger transcription factors by means of alternative splicing. Three Ik isoforms, Ik-1, Ik-2, and Ik-3, are capable of binding DNA. Ik-1 and Ik-2 are detected predominantly in the nucleus, whereas Ik-3 is present mainly in the cytoplasm. Interactions between the three DNA-binding Ikaros isoforms generate six homo- and heterodimeric complexes with distinct combinations of two DNA-binding domains that can interact with a range of regulatory sequences with different affinities (16, 17, 20). This is in agreement with our observation that Ik-2 homodimers can bind efficiently to the -378/-374 Ik-binding site in the dor promoter, whereas Ik-1 homodimers or Ik-1/Ik-2 heterodimers show very low affinity for the same site (Fig. 2). In addition, although overexpressed Ik-1 exerted no effect on the dor promoter activity in EL-4 cells, the overexpression of Ik-2 elevated the dor promoter activity almost to the level of that activated by PHA (Fig. 1). These results, together with our previous observation that the expression of the nuclear Ik proteins was increased in PHA-activated EL-4 cells (13), indicate that the increased Ik binding activity at the -378/-374 Ik-binding site in PHA-activated EL-4 cells is mainly due to the augmented formation of Ik-2 homodimers, which readily bind to the putative Ik-binding site and enhance the dor promoter activity.

On the other hand, a USF-binding E box (-185 to -180) is essential for dor promoter activity. The E box mutant exhibited almost total loss of dor promoter activity in both resting and PHA-activated EL-4 cells (Fig. 3), suggesting that the USF-bound E box functions as the basal dor promoter. This proposition is supported by several lines of evidence from other reports. First, USF has been shown to be able to interact with TFIID (21, 22), increases the rate or stability of TFIID binding (23), and stabilize formation of the preinitiation complex (24). Second, USF is able to interact with other basal transcription factors, including TAFII55 (25), TFII-I (26, 27), and transcriptional co-factor PC5 (28). Third, the USF-bound E box has been found to function in a manner similar to the initiator element (27) and to direct transcription in a TATA- and initiator-less promoter (29). In addition, multiple transcription initiation sites are present within the 40-base pair region immediately downstream of the USF-binding E box in the TATA- and initiator-less dor promoter (30). Thus, we propose that the E box-bound USF can recruit TFIID and/or other components of the basal transcription machinery to the dor promoter and facilitate the assembly of the preinitiation complex.

USF consists of two ubiquitous polypeptides (31), USF-1 (43 kDa) and USF-2 (44 kDa), both of which bind the E box (Fig. 4) and interact functionally with Ik-2 in trans-activating the dor promoter (Fig. 5). Interestingly, overexpressed Ik-2 could not rescue the promoter activity that was abolished by the E box mutation, in agreement with the notion that the USF-bound E box may functions as the basal promoter. It was observed that USF expression as well as USF binding activity at the E box basically did not change in PHA-activated EL-4 cells compared with the unstimulated cells (Fig. 4). This indicates that USF binding at the E box is not rate-limiting for the dor promoter activity increment in PHA-activated T cells, consistent with our conclusion that increased binding activity of Ik-2 homodimers triggers the augmentation of the dor promoter activity in PHA-activated EL-4 cells.

The -378/-374 Ik-binding site functions in a position-dependent manner (Fig. 6), indicating that the Ik-2-responsive element does not act as an enhancer (18, 19) but an upstream promoter element. Although the functional synergy between Ik-2 and USF in trans-activating the dor promoter was obvious in EL-4 cells (Fig. 5) and some other mouse T cell lines (data not shown), no direct interaction between Ik-2 and USF was detected in this study (data not shown). Thus, Ik-2 probably exerts its trans-activation effect on the dor promoter by promoting the assembly of the basal transcription machinery initiated via the E box-bound USF through interaction with components of the preinitiation complex. Collectively, our data support such a model; the USF-bound E box confers the constitutive dor promoter activity in resting T cells, wherein the binding activity at the -378/-374 Ik-binding site is weak (13) because of inadequate Ik-2 homodimers. However, in activated T cells, the augmented expression of nuclear Ik proteins results in increased formation of Ik-2 homodimers, which in turn leads to the increased binding activity at the putative Ik-binding site and enhances the dor promoter activity via functional synergy with the E box-bound USF.

T lymphocytes are exposed to endogenous opioid peptides in vivo (32, 33). Because Ik has been reported to set threshold for T-cell activation (34) and to play an important role in T-cell homeostasis (35), the link between Ik-2 and the transcriptional regulation of the dor gene in T cells implies an active role for endogenous opioids in modulating the functions and homeostasis of T cells in different physiological settings. In addition, as Ik proteins are specific to the hemopoietic system, particularly T cells (16, 17, 36), this study also provides insight into the tissue-specific transcriptional regulation of the dor gene.

    ACKNOWLEDGEMENTS

We thank Dr. Michéle Sawadogo (University of Texas Cancer Center) for the kind gifts of human USF-1 and mouse USF-2 expression vectors. We thank Dr. Li-Na Wei (University of Minnesota) for the kind gift of the mouse Ik-1 expression vector.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DA-00546, DA-01583, DA-11806, and KO5-DA-70554 and by the A. and F. Stark Fund of the Minnesota Medical Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St., S. E., Minneapolis, MN 55455. Tel.: 612-626-6539; Fax: 612-625-8408; E-mail: sunx0078@tc.umn.edu.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M208162200

    ABBREVIATIONS

The abbreviations used are: DOR, delta -opioid receptor; Ik, Ikaros; PHA, phytohemagglutinin; USF, upstream stimulatory factor; TF, transcription factor; EMSA, electrophoretic mobility shift assay; Ab, antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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