(Received for publication, August 1, 1994; and in revised form, January 3, 1995)
From the
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.
Interleukin (IL) ()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
and
chains, the
chain and that the
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 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
chain, probably, at least in part, through a
B site in the
regulatory region of the
chain
gene(20, 21, 22, 26) . An exclusive
case is adult T cell leukemia-derived cell lines, which constitutively
express the
chain(27) . On the other hand, little is
known about the regulation of the expression of the
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
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
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.
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 pPBA
600 was assigned a value of
1.0.
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) .
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).
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) .
Figure 3:
Promoter activity of the cloned HindIII-HaeII fragment. Each cell line was
transfected with 10 µg of pPB600 or pPBA
600. To normalize
transfection efficiency, 10 µg of pRSV-
-gal was cotransfected.
The luciferase activity corrected by protein concentration was adjusted
to the level of the
-galactosidase expression and is shown as a
ratio to the value obtained with
pPBA
600.
Although
the endogenous chain expression was not detected on THP-1,
pPB
600 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 pPB
91
gc. Jurkat transfected with the construct gave the same luciferase
activity as the parental reporter plasmid, pPB
91 (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, pPB
161cc, and pPB
80cc.
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.
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, pPB
600,
and a series of the 5`-deletion mutants, pPB
398, pPB
161,
pPB
116, pPB
91, pPB
80, and pPB
58. The five
constructs, pPB
600, pPB
398, pPB
161, pPB
116, and
pPB
91 showed 7.3-8.2-fold increase in the luciferase
activities in response to TPA (Fig. 6A). pPB
80
showed 5.4-fold induction of the luciferase activity in response to
TPA, whereas 4.0-fold induction was seen with pPB
58. 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 pPB
600, pPB
398, and pPB
161 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
pPBA
600.
Similar experiments were carried out to examine effect of
PHA on the promoter activity in Jurkat cells. Cells transfected
with pPB
600 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).
pPB
398 and pPB
161 also showed similar results with
pPB
600, 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).
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
-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 pPBA
600. Panel B, IL-2 suppressive effect on
endogenous mRNA expression. Jurkat
-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 -17 cells, the IL-2-induced inhibition of the
chain promoter activity was clearly observed with pPB
600 but not
with pPB
398 and pPB
161, suggesting that a region responsible
for the IL-2-dependent inhibitory effect is between nucleotide
positions -600 and -398 (Fig. 7A).
Figure 8:
Effects of HTLV-I Tax on the chain
promoter. Panel A, enhancement of the promoter activity by
HTLV-I Tax. pPB
600 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
pPBA
600 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 pPBA
600 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
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
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.
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
B 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
chain genes, which are induced by TPA through the
B
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
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
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
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
and
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,
B 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.