©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Analysis of the Human Interleukin 2 Receptor Chain Gene Promoter (*)

(Received for publication, August 1, 1994; and in revised form, January 3, 1995)

Kazuyuki Ohbo Naruhiko Takasawa Naoto Ishii Nobuyuki Tanaka Masataka Nakamura (§) Kazuo Sugamura

From the Department of Microbiology, Tohoku University School of Medicine, Sendai 980-77, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The third component of the interleukin (IL) 2 receptor, chain, is essential not only for IL-2- but also for IL-4-, IL-7-, IL-9-, and IL-15-induced proliferation of lymphocytes. To elucidate the mechanisms by which the chain is expressed, we have analyzed the promoter region of the chain gene. The 633-base pair fragment upstream of the initiation codon showed the promoter activity in human hematopoietic cell lines, Jurkat and THP-1, when linked to the luciferase gene. With a series of 5`-deletion mutants, the basal promoter activity was found in a fragment from nucleotide 80 to 58 upstream from the RNA start site, including an Ets binding sequence. Treatment of cells with either 12-O-tetradecanoylphorbol-13-acetate or phytohemagglutinin but not forskolin induced transcription from the chain gene promoter. A viral trans-acting transcriptional activator, Tax, of human T-cell leukemia virus type I elevated expression of the chain gene. In contrast, IL-2 decreased transcription from the IL-2 receptor chain promoter. These results suggest that expression of the chain is regulated at the transcription level by extracellular stimuli and may be implicated in immune response.


INTRODUCTION

Interleukin (IL) (^1)2 functions as a main physiological regulator in T cell growth through binding to a specific receptor, IL-2 receptor, on cell surface(1, 2, 3) . We have recently demonstrated that the high affinity IL-2 receptor complex (K, 10 pM) includes, in addition to the previously identified alpha and beta chains, the chain and that the beta and chains are essential for the intracellular growth signal transduction(4, 5) .

Mutations of the chain gene have been demonstrated to cause X-linked severe combined immunodeficiency in human, characterized by a complete lack of mature T cells in the peripheral (6, 7) . Thus, the function of the chain is required for T cell development. In this context, it is interesting that we and others (8, 9, 10, 11, 12, 13) have recently shown that the chain participates in the receptor complexes for interleukin 4, interleukin 7, interleukin 9, and interleukin 15.

The regulation of the expression of the alpha chain has been well characterized. T cell activators such as phytohemagglutinin (PHA)(14) , phorbol 12-myristate 13-acetate(15, 16) , human T cell leukemia virus type I (HTLV-I) Tax(17, 18, 19, 20, 21, 22, 23) , and IL-2 itself (24, 25) stimulate expression of the alpha chain, probably, at least in part, through a kappaB site in the regulatory region of the alpha chain gene(20, 21, 22, 26) . An exclusive case is adult T cell leukemia-derived cell lines, which constitutively express the alpha chain(27) . On the other hand, little is known about the regulation of the expression of the beta chain. It has been indicated that putative binding sites for the octamer binding factor, Ap-1, Ap-2, and Sp1 are present in the region upstream of the beta chain coding region(28, 29) . Recently, c-Ets1 and GA binding proteins were reported to play a role in regulating basal and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced beta chain promoter activity(30) .

Previous studies indicate that the chain is expressed in almost all of the hematopoietic but not non-hematopoietic lineage cells(4, 31, 32) . Since the chain is indispensable for T cell growth and development, its expression could be one of important steps critical for regulation of both differentiation in the T cell development and activation in the T cell response. It is thus intriguing to see the mechanism by which the expression of the chain is regulated.

In this study, to elucidate the regulatory mechanism of the chain gene expression, we examined function of the transcriptional regulatory region of the chain gene in response to T cell stimulants. Our results illustrate that the regulatory region contains elements positively responsive to TPA, Tax, and PHA and negatively responsive to IL-2.


MATERIALS AND METHODS

Cell Line

MOLT4, TL-OmI, Jurkat, MT-1, MT-2, ILT-Mat, TCL-Kan, and HUT 102 are human T cell lines. Jurkat alphabeta-17 is a subline of Jurkat, which constitutively expresses IL-2 receptor from the exogenously introduced cDNA for the alpha and beta chains with the endogenous chain. The IL-2 receptor on Jurkat alphabeta-17 binds IL-2 with a high affinity, which can mediate internalization of IL-2 and tyrosine phosphorylation of the IL-2 receptor beta and chains. JPX-9 is a transformant of Jurkat expressing inducibly Tax from pMAXRHneo-1(33, 34) . Peripheral blood lymphocyte (PBL) was prepared by Ficoll-Conray gradient centrifugation. PBL was cultured with 1 µg/ml PHA (Difco) and recombinant IL-2 (500 pM, Ajinomoto, Tokyo) in RPMI 1640 containing 10% fetal calf serum. Raji, BALL-1, RAMOS, and LCL-Kan are human B cell lines. YT-C3 is a human natural killer cell line. M-TAT is a human megakaryoblastic cell line. K562, EoL-3, HL-60, and THP-1 are human myelomonocytic cell lines. These cells were maintained in RPMI 1640 medium containing 10% fetal calf serum. HeLa, a human epithelial cell line, was grown in Dulbecco's modified minimum essential medium supplemented with 5% calf serum.

Plasmids

A reporter plasmid, PGV-B (Toyo Ink, Tokyo) was used to measure the promoter activity of the human IL-2 receptor chain gene. Another luciferase vector, pSV(2)-Luc, served as a positive control(35) . A beta-galactosidase expression vector, pRSV-beta gal, was used for the internal reference except for the experiment with TPA(35) . pMAXRHneo-1 is a metallothionein promoter-directed HTLV-I Tax expression vector, and pMAXRHneo/M expresses an inactive mutant Tax(33, 34) . Tax and mutant Tax were induced in Jurkat cells with CdCl(2) for 16 h.

Isolation of Human Genomic Clones for the Chain Gene

A human genomic library in EMBL 3 was obtained from Japanese Cancer Research Resources Bank. The library was screened with a [alpha-P]dCTP-labeled probe encompassing human IL-2 receptor chain (whole) cDNA(4) . Prehybridization and hybridization were performed as previously described(36) . Five positive clones were rescreened with a double strand 25-mer oligonucleotide probe, which was labeled at the 5` termini with [-P]ATP (6000 Ci/mmol; Amersham, United Kingdom) and T4 polynucleotide kinase. One out of five, -5, was positive. The nucleotide sequence has been given the DDBJ, EMBL, and GenBank accession number D16358.

DNA Sequence Analysis

The nucleotide sequence was determined by the dideoxy termination method (Sequenase, U. S. Biochemical Corp.) using synthetic primers as previously described (36) .

Polymerase Chain Reaction (PCR)

The PCR mixture contained 1 ng of template DNA, 100 pM oligonucleotide primers, 200 µM dNTPs, and 2.5 units of Taq DNA polymerase in reaction buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl(2), and 0.01% (w/v) gelatin). The reaction was carried out with a DNA thermal cycler (Perkin-Elmer Corp.) for 25 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 2 min, and polymerization at 72 °C for 3 min.

Plasmid Construction

The 633-bp HindIII-HaeII fragment of -5 was inserted into the SmaI site of PGV-B. The resultant plasmid carrying the fragment in the sense orientation was named pPB600 and in the antisense orientation pPBA600. A series of 5`-deletion mutants of the IL-2 receptor chain promoter was constructed by PCR (Fig. 4). Amplification of the promoter sequences was performed between a synthesized oligonucleotide primer for the 3`-end of the promoter, 3P (TTGCTCTTCATTCCCTGGGTGTAGT), and one of following synthesized oligonucleotide primers for the truncated 5`-ends of the promoter: 5P-398 (CTTTGACAGAGATTTAAGGGTGACC), 5P-161 (AGCACCTAATCTCCTAGAGACTTA), 5P-116 (ATTTGCCACACCCTCTGTAAAGCCC), 5P-91 (TGGTTATAAGGTTCTTTCCACCGG), 5P-80 (GTTCT TCCACCGGAAGCTAGACAGAGGA), and 5P-58 (AAGAGGAAACGTGTGGGTGGGGA). A substitutional mutation in pPB80cc was introduced by PCR amplification between primer 5P-80M (GTTCTTCCACCCCAAGCTAGACAGAGGA) and 3P. The amplified fragment between 5P-80M and 3P was used to generate the other substitutional mutants, pPB398cc and pPB161cc, along with 5P-398 and 5P-161, respectively. The amplified PCR products were subsequently cloned into the SmaI site of PGV-B. All newly constructed plasmids were verified by sequencing.


Figure 4: Promoter activity of deletion mutants. 5`-Deletion mutants of the chain promoter linked to the luciferase gene are shown. Numbers indicate 5`-far end nucleotides derived from the chain promoter. Reporter plasmids were introduced into Jurkat cells. Normalized results are shown. The luciferase activity of pPBA600 was assigned a value of 1.0.



RNA Isolation and Northern (RNA) Blot Analysis

Total RNA was prepared by the guanidinium thiocyanate method as previously described(36) . 20 µg of total RNA were electrophoresed through a 1.0% agarose gel containing 6% formaldehyde and blotted onto Hybond-N nylon membranes. After UV cross-linking with Stratalinker (model 1800, Stratagene), the membranes were hybridized with the full-length cDNA probe, washed twice in 2 times SSC containing 0.1% SDS at 65 °C for 30 min, and exposed using an imaging plate. The imaging plate was subjected to a bio-image analyzer (BAS2000, Fuji Film, Tokyo).

Primer Extension

The synthetic oligonucleotides, GPE1 and GPE2 (Fig. 2C), were end labeled with T4 polynucleotide kinase and [-P]ATP for 1 h at 37 °C. Poly(A) RNA (1 µg) was purified from MOLT4 and HeLa cells by two rounds of chromatography on oligo(dT)-cellulose (Collaborative Research, Bedford, MA) as previously described (36) and incubated overnight at 37 °C with 10^5 cpm of the primers in hybridization buffer (0.167 M Hepes (pH 7.5), 1 M NaCl, and 0.3 mM EDTA (pH 8.0)). After ethanol precipitation, the precipitate was resuspended in 25 µl of reverse transcriptase mix (3.5 µl of 4 mM dNTPs, 1 µl of RNAsin (50 units/µl, Takara, Kyoto, Japan), 2.5 µl of 10 times reverse transcriptase buffer (0.5 M Tris-HCl (pH 8.0), 50 mM MgCl(2), 50 mM dithiothreitol, 0.5 M KCl, and 0.5 mg/ml bovine serum albumin), and 18 µl of H(2)O), and then 40 units of murine mammary tumor virus reverse transcriptase were added. The samples were incubated for 90 min at 42 °C, treated with 20 µg/ml pancreatic ribonuclease-A for 30 min at 37 °C, and precipitated and resuspended in TE buffer. The samples were subsequently electrophoresed on a 6% polyacrylamide sequencing gel with 7 M urea with sequencing ladders generated with the same primers.


Figure 2: Structure of the chain regulatory region. Panel A, physical map. The upperhorizontalboldline indicates an isolated genomic fragment, which hybridized with the 5`-oligoprobe. The restriction sites in the DNA fragment are denoted by verticallines. The bottomhorizontalline shows a fragment subcloned into PGV-B. The initiation codon is shown. Panel B, primer extension analysis. The -P-labeled 35-mer oligonucleotide primer, GPE-1, complementary to the 5`-coding region of chain mRNA, was annealed to 1 µg of mRNA from MOLT4 (lane1) and HeLa (lane2), and polymerase reaction was conducted. Sequence ladder (lanesC, T, A, and G) was obtained with GPE-1. Arrow heads on the left indicate products of primer extension, and arrows on the right show position of potential transcription initiation sites, which are denoted in the nucleotide sequence shown in Fig. 2C. Panel C, nucleotide sequence. The potential binding sequences for the transcription factors are shown; PEA-3 site, GT-boxes, Ets binding site, and TATA-like sequence are underlined, P.U.1 sites are boxed, and the CACCC box is boldlyunderlined. The nucleotides corresponding to the transcription initiation sites are indicated by asterisks. Among them, a strong initiation site is numbered as +1. Two oligonucleotides of 35-mer (GPE1 and GPE2, underlined) in the first exon were synthesized as primers. Triangles and a square on the sequence represent deletions and substitution compared with sequence previously reported (30) .



Luciferase Assay

Supercoiled plasmid DNA was transfected into 5 times 10^6 cells of Jurkat, Jurkat alphabeta-17, and THP-1 cells by the DEAE-dextran method as previously described(33, 34) . In brief, cells were washed twice and resuspended in 1 ml of RPMI 1640. Plasmid DNA in 1 ml of RPMI 1640 containing 1 mg/ml DEAE-dextran (DEAE-dextran stock solution, 5 mg/ml DEAE-dextran in 1 M Tris-HCl (pH 7.5)) (Pharmacia, Uppsala, Sweden) was mixed with the cell suspension, and the cells were incubated for 30 min at 37 °C. Na-heparin (1 unit/ml in RPMI 1640) (Wako, Osaka, Japan) was then added. The cells were cultured for 40 h at 37 °C. PHA (1 µg/ml), TPA (5 ng/ml or 1 ng/ml), or a combination of these reagents were added to the culture 12 h after transfection. Treatment with Cd ion was performed for 16 h by adding indicated concentrations of CdCl(2). The cells were lysed in 100 µl of 25 mM Tris-phosphate buffer (pH 7.5) containing 1% Triton X-100. Soluble extracts were prepared by centrifugation (800 times g for 5 min) and assayed for the luciferase and beta-galactosidase activities(35) . The luciferase assay was performed with a PicaGene assay kit (PGK-L100; Toyo Ink). Light intensity was measured by Lumat (LB9501, Berthold, Wildbad, Germany). Luciferase activity was expressed as light intensity after correction of transfection efficiency by the beta-galactosidase activity(35) . Relative luciferase activity was expressed as values of ratio compared with the light intensity of pPBA600. The protein concentration was determined with a protein assay kit (Bio-Rad) and used for normalization of the luciferase assays. The luciferase assay results shown in figures are representative of experiments independently repeated at least 3 times.


RESULTS

Expression of IL-2 Receptor Chain in Human Cell Lines

Various cells including normal and PHA-activated peripheral blood leukocytes were examined for the chain expression by the Northern blot analysis. In addition to lymphoid lineage cell lines (MOLT4, Jurkat, MT-1, MT-2, ILT-Mat, and Raji) previously tested (4) , the other hematopoietic cell lines (HUT102, BALL-1, RAMOS, LCL-Kan, TCL-Kan, YT-C3, M-TAT, K562, EoL3, and HL-60) expressed at least the 1.8-kilobase transcript from the chain gene (Fig. 1). The 3.6-kilobase message hybridized with the chain probe was seen with PHA-PBL, MOLT4, Jurkat, LCL-Kan, YT-C3, and M-TAT. Among hematopoietic lineage cell lines tested so far, THP-1 was exceptional; it did not show any appreciable expression of mRNA for the chain (Fig. 1). No expression of the chain mRNA was seen with glioma (Onda11, U-251MG, and Hs683), neuroblastoma (SK-N-SH), epithelial (HeLa), hepatocytic (HepG2), and fibroblastic (WI-38 and Detroit 532) cell lines (data not shown).


Figure 1: Expression of human IL-2 receptor chain mRNA in various cell lines. Total RNA (20 µg) was probed with the entire IL-2 receptor chain cDNA: PBL, PHA-PBL, T lymphocyte-derived cell lines (MOLT4, Jurkat, MT-1, MT-2, ILT-Mat, TCL-Kan, and HUT102), B lymphocyte-derived cell lines (Raji, BALL-1, RAMOS, and LCL-Kan), natural killer cell line (YT-C3), megakaryoblastic cell line (M-TAT), and myelomonocytic cell lines (K562, EoL-3, HL-60, and THP-1).



Isolation of the 5`-Flanking Region and Primer Extension

A human PBL genomic library in EMBL 3 was screened with the full-length cDNA probe. Finally, one clone obtained, -5, was analyzed in terms of physical mapping (Fig. 2A). The clone encompasses the exon containing the ATG codon and approximately the 5.0-kilobase non-coding upstream region. To identify the transcription initiation site of the chain gene, we carried out primer extension by using mRNA from MOLT4 cells with a P-labeled primer, GPE1. Electrophoresis of synthesized cDNA revealed three major fragments of 91, 95, and 97 bp, indicating the transcription initiation sites at A nucleotides 32, 36, and 38 bp upstream from the ATG codon (Fig. 2B). The same result was obtained with a different primer, GPE2 (data not shown). These transcription initiation sites were identical to those previously observed(37) . No primer-extended product was seen with mRNA from HeLa and primer GPE1 (Fig. 2B).

The 633-bp HindIII-HaeII fragment of the -5 clone had seven nucleotides different from those in the previous report (37) (Fig. 2C). A region upstream of the transcriptional initiation sites contains several sequences similar to previously identified transcription regulatory elements: four P.U.1 recognition sites (two GAGGAA at -468 and -55 and two inverted complementary sequences, TTCCTC at -581 and -541)(38) , one PEA-3 site (ACACTTCCT at -545)(39, 40) , one CACCC box (GCCACACCC at -112)(41) , three GT boxes (GGGTGGG at -497, -334, and -43)(42, 43) , one TATA-like sequence (TTTATAA at -88), and one Ets binding site (CCGGAAGC at -70) (44) .

Promoter Activity

To determine the promoter ability, the HindIII-HaeII fragment was ligated to the luciferase gene in PGV-B, generating pPB600. The luciferase activity in Jurkat introduced with pPB600 was profoundly higher than that in the same cells transfected with pPBA600 carrying the HindIII-HaeII fragment in an antisense orientation, which gave a background level of the luciferase activity as low as that with the promoterless, enhancerless PGV-B plasmid (Fig. 3). These results indicate that the 633-bp HindIII-HaeII fragment contains the basic promoter and probably other regulatory elements. To clarify those elements, a series of mutants of the fragment was examined for their transcriptional activities. A fragment deleted up to nucleotide position -398 (pPB 398) exhibited 60% of the luciferase activity with the HindIII-HaeII fragment, the -161 (pPB161), -116 (pPB116), and -91 (pPB 91) mutants exhibited approximately 25, 12, and 30%, respectively, and the -80 mutant (pPB 80) exhibited 28% (Fig. 4). pPB 80 and pPB 91 reproducibly exhibited higher luciferase activity than pPB116. The other mutant fragment carrying up to nucleotide position -58 (pPB 58) showed the activity indistinguishable from the background activity with PGV-B. These data suggest that there are two positively regulatory regions (-600 to -161 and -80 to +35) and probably a negatively regulatory region (-161 to -116).


Figure 3: Promoter activity of the cloned HindIII-HaeII fragment. Each cell line was transfected with 10 µg of pPB600 or pPBA600. To normalize transfection efficiency, 10 µg of pRSV-beta-gal was cotransfected. The luciferase activity corrected by protein concentration was adjusted to the level of the beta-galactosidase expression and is shown as a ratio to the value obtained with pPBA600.



Although the endogenous chain expression was not detected on THP-1, pPB600 exhibited significant luciferase activity in THP-1 (Fig. 3).

The chain promoter contains a TATA-like sequence, TTTATAA, at -88. To assess the activity of the TATA-like sequence, we introduced a substitutional mutation in the sequence, in which TTTATAA was converted to TTGCTAA named pPB91 gc. Jurkat transfected with the construct gave the same luciferase activity as the parental reporter plasmid, pPB91 (Fig. 4). The result suggests that the TATA-like sequence, TTTATAA, does not have the conventional activity as a TATA-box in this gene.

Based on the above studies, the basic promoter activity is obviously present in the region downstream from -80, which contains one GT box and two consensus sequences for the c-ets family, the Ets binding site at -70, and the P.U.1 site at -55. The transcription factors belonging to the c-ets family regulate transcriptional initiation from a variety of cellular and viral gene promoter/enhancer elements(45) . To determine the functional significance of the Ets binding site at -70, we constructed substitutional mutants of the Ets binding site, pPB398cc, pPB161cc, and pPB80cc. The mutant plasmids have two nucleotide substitutions in the core motif of GGAA yielding CCAA. All of the substitutional mutants showed the background luciferase activities (Fig. 4). Taking into account the lack of the basal promoter activity with pPB 58, which contains putative one P.U.1 box and GT box, the region from -80 to -58, carrying the putative Ets binding sequence, is essential for the basal promoter activity of the human IL-2 receptor chain gene.

Activation of the Chain Promoter

TPA, an activator of protein kinase C, leading to the activation of the AP-1 and kappaB sites, is reported to enhance expression of the IL-2 gene and IL-2 receptor alpha chain gene(20, 22, 46) . Jurkat and THP-1 introduced with pPB600 were treated with TPA. Interestingly, despite the absence of typical TPA-responsive element in the cloned HindIII-HaeII fragment, TPA strikingly enhanced transcriptional activity in both cell lines (Fig. 5A). To confirm the enhancing effect of TPA, we further examined expression of the endogenous chain mRNA in Jurkat and THP-1. The level of chain mRNA in Jurkat increased within 3 h after exposure to TPA, reached a maximum at 12 h, and decreased (Jurkat, Fig. 5B). The increases were 2.3-fold by the densitometric analysis. Little, if any, effect of TPA on endogenous expression of the beta chain was seen with Jurkat (data not shown). Even though THP-1 did not endogenously express mRNA for the chain, TPA treatment induced the chain expression (THP-1, Fig. 5B). Expression of the chain was induced within 3 h, increased in a time-dependent manner, and reached a maximum level at 48 h after TPA exposure.


Figure 5: TPA-dependent up-regulation of the cloned promoter. Panel A, TPA-dependent up-regulation of the cloned promoter. After introduction with reporter plasmid, Jurkat cells were treated with 5 ng/ml TPA for 28 h. The luciferase activity was normalized. The luciferase activity of pPBA600 was assigned a value of 1.0. Panel B, TPA effects on endogenous mRNA expression. Jurkat and THP-1 cells were treated with 10 ng/ml TPA for indicated periods. Total RNA (20 µg) was examined by Northern blot analysis with the whole chain probe. Filters were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).



To identify a TPA-responsive region in the chain promoter, luciferase assays were performed in Jurkat transiently transfected with different constructs, pPB600, and a series of the 5`-deletion mutants, pPB398, pPB161, pPB116, pPB91, pPB80, and pPB58. The five constructs, pPB600, pPB398, pPB161, pPB116, and pPB91 showed 7.3-8.2-fold increase in the luciferase activities in response to TPA (Fig. 6A). pPB80 showed 5.4-fold induction of the luciferase activity in response to TPA, whereas 4.0-fold induction was seen with pPB58. PGV-B showed little or no increase. These results suggest that complete TPA-responsive elements are present downstream from nucleotide position -91.


Figure 6: TPA-responsive region of the chain promoter. Panel A, effects of TPA on 5`-deletion mutants. Jurkat cells transfected with reporter plasmids were treated with 5 ng/ml TPA for 28 h. The luciferase activity was normalized. The results are shown as a ratio to the value obtained with the same plasmid without TPA treatment. Panel B, synergistic activation of transcription by TPA and PHA. Jurkat cells transfected with 10 µg of pPB600, pPB398, and pPB161 were treated with 1 ng/ml TPA and/or 1 µg/ml PHA for 28 h. TPA (1 ng/ml) and PHA (1 µg/ml) used gave 50 and 100%, respectively, of the maximum responses when each alone was used. The luciferase activity was normalized. All values are relative ratio compared with those of non-stimulated (NS) cells with pPBA600.



Similar experiments were carried out to examine effect of PHA on the promoter activity in Jurkat cells. Cells transfected with pPB600 were cultured in the presence of 1 µg/ml PHA. The transcriptional activity of the HindIII-HaeII fragment was slightly enhanced by treatment with PHA (1.5-fold, Fig. 6B). Concurrent activation of the chain promoter by suboptimal TPA (1 ng/ml) and optimal PHA (1 µg/ml) stimulation showed additive effect (Fig. 6B). pPB398 and pPB161 also showed similar results with pPB600, suggesting the presence of a PHA-responsive element downstream from nucleotide position -161.

Forskolin, an activator of cyclic AMP-dependent protein kinase, did not appreciably affect the promoter activity of the same fragment in Jurkat cells (data not shown).

Down-regulation by IL-2

To examine effects of IL-2 on the chain promoter activity, the luciferase assay was performed with a subclone of Jurkat, Jurkat alphabeta-17, which expresses the IL-2 receptor alpha and beta chains from exogenously introduced cDNAs besides the endogenous chain, resulting in the high affinity IL-2 receptor (data not shown). Treatment with 100 pM IL-2 of Jurkat alphabeta-17 transfected with pPB600 caused a significant decrease in the luciferase activity. Level of the activity was 60% of the control (Fig. 7A). A similar result was seen with another human T cell line, TL-OmI (data not shown). TL-OmI is HTLV-I genome-positive but negative for viral expression and expresses the high affinity IL-2 receptor constitutively. We further investigated by Northern blot analysis if IL-2 affected expression of the endogenous chain gene in Jurkat alphabeta-17. Repression of endogenous mRNA for the chain was obvious within 12 h and continued at least up to 48 h after exposure to IL-2 (Fig. 7B). A similar repression of the endogenous chain was also seen with TL-OmI (data not shown). Results from the two different assays clearly show that IL-2 down-regulates expression of the chain in contrast to its effect on the IL-2, IL-2 receptor alpha and beta chains(24, 25, 26, 47) .


Figure 7: Negative regulation of the chain promoter activity by IL-2. Panel A, IL-2 suppressive effect in transient assay. A total of 10 µg of plasmid DNA of each designated construct was transfected into Jurkat alphabeta-17 cells. Cells were treated with 100 pM IL-2 immediately after transfection. After 40 h, the luciferase activity was determined and normalized. All values are relative ratios compared with those of non-stimulated (NS) cells with pPBA600. Panel B, IL-2 suppressive effect on endogenous mRNA expression. Jurkat alphabeta-17 cells were treated with 100 pM IL-2 for indicated periods. Total RNA (20 µg) was subjected to Northern blot analysis with the whole chain probe. Filters were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Intensity of the bands was quantitated with a bio-image analyzer and expressed as a percentage of the non-treated control.



In Jurkat alphabeta-17 cells, the IL-2-induced inhibition of the chain promoter activity was clearly observed with pPB600 but not with pPB398 and pPB161, suggesting that a region responsible for the IL-2-dependent inhibitory effect is between nucleotide positions -600 and -398 (Fig. 7A).

Transactivation by HTLV-I Tax

The regulatory regions of genes for IL-2 and the IL-2 receptor alpha chain contain Tax-responsive elements(17, 18, 19, 20, 21, 22, 23) , and HTLV-I-transformed cells constitutively express the high affinity IL-2 receptor. These facts encouraged us to investigate whether Tax activates transcription from the chain promoter. The reporter plasmid, pPB600, was cotransfected into Jurkat along with pMAXRHneo-1. The luciferase activity from pPB600 was significantly enhanced by HTLV-I Tax, whereas an inactive Tax mutant from pMAXRHneo/M did not show any appreciable effect (Fig. 8, A and B). HTLV-I Tax-induced enhancement of the chain promoter activity was seen with a series of the 5`-deletion mutants, even with pPB58 carrying only a fragment -58 to +35 (Fig. 8B). These results suggest that a Tax-responsive element may exist between nucleotide positions -58 and +35. Additionally, we studied expression of the endogenous chain gene in response to Tax with JPX-9, which is stably introduced with the Tax expression plasmid in Jurkat capable of inducing Tax expression in a CdCl(2)-dependent manner (Fig. 9A). Kinetics of induction of the chain by Tax in JPX-9 was also examined (Fig. 9B). Induction of the chain gene appeared within 12 h and reached a plateau from 24 to 72 h after exposure to 10 µM CdCl(2). JPX-9, when treated with CdCl(2), increased the level of mRNA for the chain in parallel with induction of Tax (data not shown), implying that Tax transactivates the chain promoter despite the lack of elements previously identified as targets for HTLV-I Tax.


Figure 8: Effects of HTLV-I Tax on the chain promoter. Panel A, enhancement of the promoter activity by HTLV-I Tax. pPB600 was introduced into Jurkat cells along with indicated amounts of pBR322, pMAXRHneo-1, and pMAXRHneo/M. The luciferase activity was normalized. The luciferase activity with pPBA600 and pBR322 was assigned a value of 1.0. Panel B, identification of Tax-responsible region. 5`-Deletion mutant vectors (10 µg) were introduced into Jurkat cells along with 10 µg of pBR322, pMAXRHneo-1, and pMAXRHneo/M. Normalized results are shown. The luciferase activity of pPBA600 with pBR322 was assigned a value of 1.0.




Figure 9: Induction of endogenous chain mRNA by Tax. Panel A, expression of the chain mRNA induced by Tax. JPX-9 cell were treated with indicated concentrations of CdCl(2) for 16 h. Total RNA (20 µg) was subjected to Northern blot analysis with the whole probe. Northern blot bands were quantitated with a bio-image analyzer. Panel B, kinetics of induction of the chain mRNA by Tax in JPX-9 cells. JPX-9 cells were treated with 10 µM CdCl(2) for indicated periods. Total RNA (20 µg) was subjected to Northern blot analysis with the whole probe. Intensity of the chain mRNA was quantitated with a bio-image analyzer and is expressed as a percentage of that of non-treated control.




DISCUSSION

The common usage of the chain for multiple cytokine receptors may account for the present data depicting a wide distribution of the chain among various hematopoietic cell lines (Fig. 1). This stems from the fact that, although IL-2 is known to exert its function on B cells, natural killer cells, and macrophage/monocytes besides T cells, IL-4, IL-7, IL-9, and IL-15 also affect, in addition to T and B cells, natural killer cells, macrophages/monocytes, mast cells, thymocytes, and hematopoietic stem cells. Thus, expression of the chain may determine action of cytokines and the fate of these cells. Moreover, X-linked severe combined immunodeficiency is associated with mutation of the chain gene(7) . In this context, it is important to elucidate how transcription of the chain is regulated.

Our results indicate that the 633-bp fragment upstream of the exon including the ATG codon encompasses the transcriptional regulatory region of the chain, which contains the putative P.U.1 sites, PEA-3 site, CACCC box, GT boxes, and Ets binding site. The analysis with the 5`-deletion mutants revealed that a critical region required for minimal expression of the IL-2 receptor chain gene is between nucleotide positions -80 and -58. The potential Ets binding site present in this region is located at nucleotide position -70. Analysis with three substitutional mutants of the core sequence of the Ets binding site at -70, GGAA to CCAA, revealed that the Ets binding site is essential for the basic promoter activity (Fig. 4). The Ets binding site may interact with the putative P.U.1 binding site at -55 where a factor, P.U.1, belonging to the c-ets proto-oncogene family through binding to the factor bound to the putative P.U.1 sites, presumably resulting in activation of transcription. On the other hand, a TATA-like AT-rich sequence, TTTATAA, at nucleotide position -88, does not seem to be involved in transcription.

TPA significantly enhanced transcription from the chain promoter, although known TPA-responsive elements such as the AP-1 site, AP-2 site, serum-responsive element, and kappaB site are not present in the cloned 633-bp fragment. The chain gene may be regulated by a different mechanism from cases of the IL-2 and alpha chain genes, which are induced by TPA through the kappaB site(20, 21, 22, 26) . Some genes encoding ets-related transcription factors, such as PEA-3, c-Ets 1, c-Ets 2, and GA binding protein, were reported to contribute to TPA response(30, 39, 40) . Although we did not detect endogenous expression of the beta chain induced by TPA in Jurkat and THP-1 (data not shown), Lin et al.(30) recently showed that the isolated regulatory region of the beta chain promoter contains a PMA-responsive element and a GGAA Ets binding site. Therefore, it is interesting to see whether the Ets binding site (at -70) and P.U.1 site (at -55) contribute to TPA response in chain expression.

THP-1 is exceptional among hematopoietic lineage cell lines tested so far in that it shows little or no expression of the chain mRNA. Expression of the chain mRNA in THP-1 was profoundly induced by TPA treatment. Interestingly, THP-1 was reported to be converted to a macrophage-like cell by TPA treatment(48) . Induction of the chain by TPA preceded a morphological change of the phenotype; however, at present, we do not know whether the TPA-induced chain expression is implicated in the differentiation. Because the beta chain was not induced by TPA exposure in THP-1 (data not shown), the chain may function as a subunit of the other receptors rather than for the IL-2 receptor. THP-1 showed significant expression of the reporter gene directed by the isolated regulatory region. Thus, there are likely to be sequences further upstream from the isolated regions, which may function as a suppressor element in THP-1.

Upon exposure to antigens, T cells initiate proliferation through interaction of IL-2 and the functional IL-2 receptor, both of which are transiently expressed by T cells activated with antigens. Our finding that transcription of the chain is down-regulated by treatment with IL-2 indicates that the chain, as well as the alpha and beta chains, and IL-2 are involved in transient proliferation of antigen-activated T cells. The down-regulation started 6 h after exposure to IL-2 and continued at least up to 48 h. On the contrary, HTLV-I Tax enhanced expression of the chain. This may be important for development of adult T cell leukemia. IL-2-IL-2 receptor interaction-induced proliferation of HTLV-I-infected T cells is thought to be a possible process at the early stage of adult T cell leukemia development; that is, down-regulation of the chain by IL-2 seen in the normal immune response may be compensated for by enhancement by Tax in HTLV-I-infected cells.

A region negatively regulated by IL-2 was mapped from -600 to -398 of the isolated chain promoter. There are several elements in this region similar to identified ones, such as P.U.1, PEA-3, and GT box; however, identification of an element responsible for IL-2-induced suppression will need further investigation. Our luciferase assay with the 5`-deletion mutants of the chain promoter region indicated that a sequence downstream from -58 is responsive to Tax, which includes potential binding sites for Sp1 (at -43) and P.U.1 (at -55). The IL-2 receptor chain promoter does not, however, contain the sequences previously identified as Tax-responsive sequences, such as the ATF/CREB site, kappaB site, and CArG box(49, 50, 51) . Very recently, Tax was shown to interact with a TATA binding protein(52) . Furthermore, Armstrong et al.(53) reported that Tax increases the activity of Sp1, ATF-1, and GAL-4 to bind to DNA. Taken together, our result that Tax-responsive element is downstream from -58, the chain basal promoter activity may be transactivated by Tax.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan, grants from special coordination funds of the Science and Technology Agency, a grant-in-aid from the Ministry of Health and Welfare of the Japanese Government for the comprehensive 10-year strategy for cancer control, a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Naito Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-22-273-9073; Fax: 81-22-273-2787.

(^1)
The abbreviations used are: IL, interleukin; PBL, peripheral blood lymphocyte; PCR, polymerase chain reaction; bp, base pair(s); TPA, 12-O-tetradecanoylphorbol-13-acetate; HTLV-1, human T cell leukemia virus type I; PHA, phytohemagglutinin.


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