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*
Laboratoire d’Immunologie Moléculaire, Université de Bordeaux II, Bordeaux, France;
Unité d’Immunogénétique Cellulaire, Institut Pasteur, Paris, France; and
Shanghai Institute of Biochemistry, Shanghai, China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-tubulin), oncogene-regulating proteins (CCCTC-binding factor, Jun
inhibitor factor-1), and transcription factors (E2F-4, cyclic
AMP-responsive element-binding protein, zhx-1). IL-2 also regulated the
expression of genes coding for multifunctional proteins, e.g.,
ß-catenin and nucleolin. These results were verified using Con
A-induced T cell blasts stimulated or not by IL-2. The in vivo
expression of four of these genes was also analyzed in spleen and lymph
node cells of IL-2-deficient and MRL/lpr mice, which
both have high numbers of activated cells, but the latter have intact
IL-2 expression. The expression of ß-catenin, CCCTC-binding factor,
Jun inhibitor factor-1, and nucleolin was significantly higher in
MRL/lpr animals. A similar analysis of thymocytes from
IL-2-/- and IL-2+/- mice demonstrated the
same expression patterns of the 4 sequences in these strains. The
expression of the IL-2-induced genes described herein is similar to the
regulatory pattern of IL-2R. Taken together, our data provide
additional evidence for the pleiotropic action of IL-2 in the periphery
and IL-2 independence of molecular processes involved in thymocyte
differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2Rß, and IL-2R
.
IL-2R enhances ligand affinity, and the heterodimer IL-2Rß
triggers downstream signaling events. IL-2 activates three major
signaling pathways: the Ras and mitogen-activated protein kinase
cascade, involved in mitogenesis; the phosphatidylinositol 3-kinase
pathway, involved in cytoskeleton reorganization; and the Janus
kinase-STAT pathway 2 .
We and others have demonstrated that in vitro IL-2 induces the
expression of its own -chain receptor gene 3, 4, 5, 6, 7 . Using
IL-2-/- animals 8 , we have verified that in
vivo IL-2R expression is IL-2 dependent in secondary lymphoid
organs. However, in IL-2-/- animals, IL-2R is normally
expressed in thymus and bone marrow 9 . IL-2-responsive elements have
been localized at 1.7 and 3.7 kb upstream from the transcription start
site of the mouse and human IL-2R genes, respectively
10, 11, 12 . These responsive elements bind multiple factors, including
STAT5, Elf-1, high mobility group family-(Y), and GATA family proteins;
STAT5 homodimer might be the major IL-2-induced factor involved in the
up-regulation of the transcription rate of the IL-2R gene
12, 13 . More recently, we showed that the TNF-ß gene is
induced by IL-2, and we demonstrated that there is a STAT5-binding site
with GAS sequence upstream from the transcription initiation
point of this gene 14 . Except for IL-2R and TNF-ß,
the target genes of the IL-2-induced signaling pathways are not well
characterized. The oncogenes c-myc, c-jun,
c-fos, c-myb, bcl-2, pim-1,
and c-raf-1 and the tyrosine kinase gene ITK have
been reported to be IL-2-inducible immediate-early genes 15 . However,
these genes are commonly induced by a variety of other growth factors.
In addition to genes implicated in growth and activation, IL-2 may induce the expression of genes negatively controlling the immune response, as suggested by the phenotype of IL-2-deficient mice, which develop a lymphoproliferative disorder involving uncontrolled T cell activation and autoimmune disease 8 . Thus, immune cell activation and differentiation are induced in the absence of IL-2, but the negative control of the immune response cannot be engaged in the absence of this cytokine. The molecular mechanisms implicated in this negative regulation are still under investigation 16, 17, 18 .
To identify new IL-2-inducible genes involved in these different
activities, we used a cDNA subtraction hybridization method derived
from genomic RDA5, 19 and a
mouse T cell line grown in either IL-2- or IL-4-containing medium.
Sixty-six nonredundant sequences were characterized including 16
previously identified genes. Herein, we report the influence of IL-2 on
the in vitro expression of eight of these genes, which include
sequences encoding cytoskeleton proteins, oncogene regulators, and
transcription factors. Their in vivo expression was analyzed in both
the absence and the presence of IL-2, by comparing mRNA levels in LN
and spleen cells from IL-2-deficient and MRL/lpr mice. Both
strains develop a lymphoproliferative disorder with accumulation of
activated lymphocytes in LN and spleen, but MRL/lpr mice
have intact IL-2 gene expression 8, 20 . Furthermore, the
expressions of the IL-2-induced genes in the thymuses of
IL-2-/- and of IL-2+/- mice were compared.
Taken together, the data suggest that the regulation of the identified
sequences differs for thymocytes and mature lymphocytes and follows the
pattern of IL-2R expression previously described 9 .
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2-deficient mice were previously described 8 . MRL/lpr mice were obtained from Harlan (Gannat, France). All mice were 6 to 9 wk old at the time of the experiments.
Cell lines, cell culture, and proliferation assay
Two cell lines were used in the course of the present work. T
cell line 18.111 was used for the preparation of the cDNA subtraction
library. It has been obtained after transfection by human IL-2R of a
murine cell line (8.2) expressing constitutively IL-2Rß and IL-2R.
When grown in IL-2- or in IL-4 containing medium, cell line 18.111 is
continuously in an activated state. It has been shown to express murine
IL-2R only when cultured in IL-2 21 . The cytotoxic T cell line
C30.1 (initially provided by Dr. F. Denizot) was used for analysis of
the expression of IL-2-induced genes. In the absence of cytokines,
C30.1 cells become resting and can be subsequently activated either by
IL-2 or by IL-4 21 .
For standard culture in IL-2, complete medium (RPMI 1640 medium containing 5% heat-inactivated FCS, 2 mM glutamine, 10 mM HEPES, 2 µM 2-ME, penicillin, streptomycin, and Fungizone) was supplemented with 20 ng/ml human rIL-2 (kindly donated by Roussel-Uclaf, Romainville, France). For standard culture in IL-4, mouse rIL-4 was added at a final concentration of 20 U/ml. HeLa subline (H28) transfected with the plasmid pKCRIL-4 Neo was used as a source of murine IL-4. This line was provided by Pr. T. Honjo (Kyoto University, Kyoto, Japan).
Proliferation assays were performed as previously described 22 .
Preparation and stimulation of Con A blasts
Spleen cells from IL-2+/- or IL-2-/-
animals on the same 129/Ola x C57BL/6 background were used. T
cells were purified from spleen cell suspensions after incubation with
anti-CD14 rat mAb followed by immunodepletion with magnetic beads
coated with polyclonal anti-mouse Ig (Dynabeads, Dynal, Oslo,
Norway). Two rounds of negative selection were performed to remove
monocytes and B lymphocytes efficiently. The remaining cell population
was mainly composed of T cells (
95%). The purified T cells from
IL-2+/- animals were cultured at low density
(105 cells in 200 µl of RPMI supplemented medium/well) in
96-well microtiter plates (Costar, France). Cells were cultured during
18 h either alone or in the presence of Con A (5 µg/ml) or Con A
+ IL-2 (10-9 M). Cells from IL-2-/- animals
were cultured at higher density (3 x 105 cells/well),
and when indicated they were stimulated during 18 h either by Con
A or by Con A + IL-2, or by Con A + IL-4 (100 U/ml).
cDNA subtractive hybridization and amplification
To identify IL-2-induced genes in mouse T cells, a cDNA subtraction library was characterized mainly from the mouse CD8+ T cell line 18.111 grown in IL-2-containing medium (18.111.2). The same cells grown in IL-4 (18.111.4), were used as a negative cell population. We used a subtractive cDNA hybridization and amplification method based on RDA, which combines hybridization in solution of cDNA populations and PCR selection of target sequences. This methodology has been proved to be able to adequately demonstrate qualitative and quantitative differences between two cDNA samples 19 .
Briefly, 2 µg of poly(A)+ RNA from 18.111.4 cells were used to synthesize the driver cDNA using a random-PCR method 23 . The tester cDNA were synthesized from 2 µg of poly(A)+ RNA purified from 18.111.2 cells. Double-stranded tester cDNA were primed with an oligo(dT) (CGGGAATTCGCTCGACATGTTTTTTTTTTTTTTTV) and digested by the restriction enzyme NlaIII. Then, the AD1 adapter (CGGGAATTCGCTCGACATG) was ligated, generating molecules with defined ends. The subtractive cDNA hybridizations were performed between cDNA from 18.111.4 and 18.111.2 cells 19 . Driver cDNA (50 µg) and tester cDNA (250 ng) were mixed in 5 µl of hybridization buffer (30 mM HEPES (pH 8), 1 mM EDTA, 1 M NaCl) and allowed to hybridize for 24 h at 68°C. Then, digestion with S1 nuclease prevented the amplification of the common sequences. After S1 nuclease digestion, the cDNA mixture was precipitated in the presence of 25 µg of driver cDNA. After three rounds of hybridization-S1 digestion, the target sequences were selected by PCR. The amplification reactions were set up by combining 1 µl of the subtracted product, 1 µM AD1 primer, 0.4 U of Taq polymerase (Goldstar, Eurogentec, Serainq, Belgium), and then 30 cycles (94°C, 1 min; 57°C, 1 min; 72°C, 3 min) were performed. Then, the products were cloned in pBluescript vector (Stratagene, La Jolla, CA). The enrichment in target sequences was controlled by hybridization of subtracted and unsubtracted cDNA probes on cDNA dot blots corresponding to the two cell lines. Densitometry of the hybridization signals showed a fivefold enrichment of 18.111.2 cDNA target sequences (data not shown).
Sequence analysis and characterization of the subtractive cDNA libraries
The single-stranded cDNA inserts were purified with magnetic beads 24 , and sequence reactions were performed on this solid phase as previously described 25 . The sequences were read on an automated sequencer (ALF, Pharmacia, Uppsala, Sweden). The database comparisons were performed with BLAST software.
From the cDNA library, 122 clones were randomly selected and sequenced;
they represent 66 nonredundant sequences. Three major groups have been
distinguished. The first group, representing 16 of our sequences,
corresponds to sequences sharing homologies with previously
characterized molecules (Table I
). Among
them, different subgroups could be defined. Sequences encoding for
cytoskeleton proteins (-tubulin), transcription factors (CREB,
E2F-4, and zhx-1), oncogene regulators (CTCF and JIF-1), nuclear
proteins (mMIS-5, heterogeneous nuclear ribonucleoprotein, and U2-small
nuclear ribonucleoprotein), ribosomal-binding proteins (ribosomal
protein S12, S15, L35, and L37a), membrane transport protein
(Na+/K+ ATPase), and multifunctional proteins
(ß-catenin and nucleolin) were found. The second group, representing
16 of our sequences, matches sequences registered in the EST databases;
they were isolated from various human and mouse cDNA libraries, but
their corresponding proteins are unknown. The third group comprising 34
sequences has not been found in the public databases. All our sequence
data have been registered in the EMBL database.
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Total RNA were prepared using RNA-B solution (Bioprobe System, Montreuil, France), and mRNA were purified using Oligotex mRNA kits (Qiagen, Hilden, Germany). Northern blot analyses were performed with specific probes as previously described 22 . Loaded RNA was measured with 18S or 28S ribosomal probes prepared from total RNA by random PCR.
When indicated, poly(A)+ mRNA were prepared before performing the Northern blot analysis. The specific hybridization signals were evaluated with specific probes, and the total amount of loaded RNA was measured using Genogold staining.
Hybridization signals were quantified by phosphorus-stimulated luminescence (PhosphorImager Molecular Dynamics, Evry, France). The specific hybridization signals were compared with the 18S or 28S signals. In the results, the ratios of specific hybridization signals to 18S, 28S, or Genogold signals are reported. When indicated, the mean ratios ± SD are reported. Student’s t test was used to evaluate the significance of the results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
corresponding molecules have not been previously reported to be induced
by IL-2. For our study, we retained the sequences encoding: 1)
ß-catenin and -tubulin because of the cytoskeleton involvement in
signal transduction and cell regulation; 2) the CTCF transcription
factor and the JIF-1, because of their previously reported roles in
c-myc and c-jun oncogene regulation, respectively
26, 27 ; 3) the E2F-4, zhx-1, and CREB transcription factors, because
they could be implicated in IL-2-induced gene expression. Nucleolin was
also examined because it is a multifunctional protein with
transcriptional activity. mRNA levels of selected sequences in response to in vitro IL-2 stimulation
To analyze the kinetics of induction of our selected sequences, we
use C30.1 T cells, which can be easily obtained in a resting or in an
activated state. The kinetics of cell size increase, as measured by
flow cytometry, were similar after IL-2 or IL-4 stimulation, while IL-2
generated an amplitude threefold higher than the increment recorded
with IL-4 (Fig. 1A). The
kinetics of [3H]thymidine incorporation were comparable
until 48 h of stimulation (Fig. 1B). As additional
controls, the mRNA levels of the three IL-2R chains were examined
during IL-2 and IL-4 stimulation of C30.1 cells; IL-2Rß mRNA was
transiently induced by IL-2 but not IL-4 and returned to the basal
level by 18 h (data not shown). Induction of IL-2R mRNA by IL-2
but not IL-4 confirmed our previously reported results (Fig. 1, C and D) 21 . The IL-2R is used by IL-2 and
IL-4 receptors, but its mRNA was induced only by IL-4 after 18 h
of stimulation, reaching a maximum at 36 h. This finding is
consistent with our observation that the IL-2R mRNA level is higher
in IL-4-dependent T cell lines than in IL-2-dependent T cell lines
(data not shown).
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The mRNA levels corresponding to the cytoskeleton proteins ß-catenin,
-tubulin, and ß-actin were investigated first (Fig. 2A). Rapid increases of
ß-catenin and ß-actin mRNA were observed in response to IL-2, with
maxima being reached at 6 h. The -tubulin mRNA level also
increased rapidly after IL-2 stimulation but remained elevated for
36 h. These increases of mRNA encoding cytoskeletal proteins might
reflect the increased cell size observed after IL-2-induced activation
(Fig. 1A).
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B).
c-myc mRNA peaked 6 h after IL-2 stimulation and then
declined regularly until 48 h. During this second period, the CTCF
mRNA level continued to rise, reaching its maximum at 36 h. These
kinetics suggest a relationship between the expression of the oncogene
with respect to the expression of its regulator. In contrast, under our
experimental conditions, the kinetics of induction of c-jun
and JIF-1 mRNA were similar and do not allow any conclusion to be drawn
concerning the involvement of JIF-1 in c-jun expression.
mRNA levels of the nuclear factors CREB, E2F-4, zhx-1, and nucleolin
were also investigated, (Fig. 2C). E2F-4 mRNA increased
after IL-2 stimulation with two peaks at 6 and 36 h. Because E2F-4
is involved in cell cycle regulation, these two peaks might correspond
to two cell division cycles during the 48 h of the experiment.
CREB and zhx-1 were induced by IL-2, but not IL-4, attaining their
maximum levels after 6 h of cytokine stimulation. The basal
nucleolin mRNA level at t = 0 was high and then
declined sharply over 3 h in the presence of IL-2 before rising
again to peak at 24 h. A more precipitous and prolonged decrease
of the nucleolin mRNA level was observed during IL-4 stimulation, and
this level, despite a slight rise, never returned to baseline.
Taken together, these different induction kinetics for the selected sequences after cytokine stimulation of murine T cells suggest that the responses might be attributable to specific functions acquired after IL-2 stimulation.
In vitro expression of selected sequences by T cell blasts
Purified T cells from IL-2+/- animals were analyzed
for in vitro expression of IL-2R, ß-catenin, nucleolin, and JIF-1
sequences. We found that culturing the cells during 18 h at low
density (105/well) limits their proliferation. Under those
experimental conditions, nonstimulated cells and Con A blasts produce
low amounts of mRNA for the four sequences analyzed (Fig. 3A). On the contrary, Con A blasts stimulated
by IL-2 produce significant quantities of mRNA for IL-2R, ß
catenin, nucleolin, and JIF-1, therefore demonstrating that these
sequences are IL-2 dependent for their expression (Fig. 3A).
To further confirm these data, we performed a similar experiment with
cells from IL-2-/- animals (Fig. 3B). These
cells do not produce IL-2, and nonstimulated or Con A-stimulated
cultures produce very low amounts of mRNA for IL-2R, ß-catenin,
nucleolin, and JIF-1. In the presence of IL-2, the studied sequences
are induced (Fig. 3B shows the results obtained with
IL-2R and ß-catenin). As control, we see that IL-4 does not lead
to a similar induction.
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). It is striking
to see that cells from IL-2+/- animals produce more mRNA
before culture than after 18 h in vitro without stimulation. With
cells from IL-2-/- animals, this phenomenon is less
striking (as shown with ß-catenin, Fig. 3B), therefore
suggesting that IL-2 is involved in this in vivo stimulation. IL-2-dependent expression of the selected sequences in spleens and LN of IL-2-/- and MRL/lpr mice
IL-2-deficient mice generated by gene targeting 8 provide an
additional experimental model to investigate the influence of IL-2 on
the expression of the selected genes. In these animals, spleen and LN
cells are activated, as assessed by the expression of the CD69 and CD71
markers on the CD4+ and CD8+ subpopulations
9 . Because activation was observed in the absence of IL-2, expression
of the genes under consideration might be negatively modulated. In
agreement with this hypothesis, IL-2R was not expressed at the
surface of mature CD4+ T lymphocytes from
IL-2-/- animals. To compare cell populations with similar
activation status, we chose MRL/lpr mice, the lymphocytes of
which are spontaneously activated 20 . The presence of IL-2, mRNA
levels in MRL/lpr spleen, and LN were evaluated by RT-PCR
(data not shown).
The IL-2R mRNA was barely detectable in spleen cells from
IL-2-/- mice, in contrast to their strong expression in
MRL/lpr spleen cells (Fig. 4).
These results confirm our previous observations made by FACS analysis
9 . Similarly, the expression of ß-catenin, CTCF, JIF-1, and
nucleolin mRNA was higher in MRL/lpr than
IL-2-/- spleen cells. Although variations among different
animals were found, the mean hibridization ratios were 4.5- to
8-fold higher in MRL/lpr than in IL-2-/-
spleen (0.001 < p < 0.01).
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). As expected, IL-2R was not
expressed in IL-2-/- animals, and ß-catenin, JIF-1,
CTCF, and nucleolin levels were low in these mice. The differences
between expression levels of the five genes analyzed, assessed by their
hybridization ratios in MRL/lpr and IL-2-/-
LN, were statistically significant (0.001 < p <
0.008).
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Because FACS analysis showed that 30% of the
CD4-CD8- thymocytes from
IL-2-/- mice, like the IL-2+/- strain used
as controls, express IL-2R 9 , expression of this chain at the
surface of IL-2-/- thymocytes seems normal. These data
were confirmed by Northern blotting. Fig. 6A shows that IL-2R mRNA is
normally expressed in the thymuses of IL-2-/- animals;
when compared with the IL-2R mRNA levels in the thymuses of
IL-2+/- animals, the ratios obtained were not
statistically different (p = 0.29).
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and 5, we found significant differences between the
mRNA levels of IL-2R in IL-2+/- and
IL-2-/- animals; IL-2+/-mice express
significantly more IL-2R than IL-2-/- animals
(0.0006 < p < 0.01) (Fig. 6B).
Under the same experimental conditions, we investigated the expression
of some of our selected sequences: ß-catenin, CTCF, JIF-1, and
nucleolin. As for IL-2R, we found that these sequences were
comparably well expressed in the thymuses from IL-2-/-
and IL-2+/- animals (0.1 < p < 0.4)
(Fig. 6A). We also compared the expression of these
sequences in spleen and LN of IL-2-/- and
IL-2+/- mice. Spleen and LN cells from
IL-2+/- animals express more ß-catenin, JIF-1, and
nucleolin mRNA than the corresponding cells from IL-2-/-
animals (p < 0.05). As expected from the
frequency of activated cells, the ratios calculated for the expression
of theses genes in IL-2+/- animals were lower than those
determined for MLR/lpr (Figs. 4 and 5). Unlike IL-2R,
ß-catenin, JIF-1, and nucleolin, the amounts of CTCF mRNA were
comparable in IL-2+/- and IL-2-/- animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression has been the
subject of intense research efforts. IL-2 is the main biological signal
influencing IL-2R expression. In this study, we characterized
families of genes with regulation patterns paralleling that of
IL-2R. They correspond to different types of molecules (cytoskeleton
structural proteins, oncogene regulators, and transcription factors)
and depend on IL-2 for their expression in secondary lymphoid organs.
These data highlight the pleiotropic molecular activity of IL-2. In
contrast, the expression of these genes in the thymus is IL-2
independent, a result that emphasizes the absence of any major
influence of IL-2 on thymic differentiation.
Using a cDNA subtraction procedure with mRNA from a T cell line grown
in medium containing either IL-2 or IL-4, we characterized 66
nonredundant sequences preferentially induced by IL-2 (Table I and data
not shown). Our simplified cDNA RDA method gives rise to few false
positives, unlike many other subtractive procedures or differential
display PCR. Our methodology, based on cDNA amplification, can be
applied to very small amounts of starting material and can be used in a
variety of systems to isolate mRNA specifically modulated throughout
cell activation or cell differentiation processes. Analysis of the mRNA
levels of eight selected sequences, which encode cytoskeleton proteins,
oncogene regulators, and transcription factors, was conducted both in
vitro and in vivo. In vitro, T cells were stimulated by either IL-2 or
IL-4, as a control. Fig. 1 shows that both cytokines stimulate cell
proliferation, although the increased cell size induced by IL-2
suggests a major contribution of the cytoskeleton after IL-2
stimulation (see below). All sequences studied were induced to various
degrees by IL-2 (Fig. 2). In most cases, maximum induction occurred at
6 h, although -tubulin and nucleolin exhibited different
kinetic profiles. In vitro experiments performed with T cells blasts
from IL-2+/- or IL-2-/- animals confirm that
IL-2 is required for the induction of IL-2R, ß-catenin, nucleolin,
and JIF-1. In vivo studies were conducted to compare the expression of
four sequences (ß-catenin, JIF-1, CTCF, and nucleolin) in spleen and
LN cells from IL-2-/- and MRL/lpr mice. In
both strains, lymphocytes were activated 9 , but only
MRL/lpr animals expressed intact IL-2. ß-Catenin, JIF-1,
CTCF, and nucleolin mRNA levels were significantly higher in
MRL/lpr than IL-2-/- spleen and LN cells
(Figs. 4 and 5). Since MRL/lpr and IL-2-/-
spleen and LN cells are activated by different mechanisms, they may
contain subsets of lymphocytes of distinct phenotypes and express
different sets of genes independently of IL-2 expression. However, the
role of IL-2 in the control of the selected sequences was verified by
comparing the results obtained with spleen and LN cells from
IL-2+/- and IL-2-/- animals (Fig. 6). We
also show that thymuses from IL-2-/- animals express
normally ß-catenin, JIF-1, CTCF, and nucleolin mRNA as they express
IL-2R (Fig. 6).
In this report, we have shown that IL-2 induced the expression of
ß-catenin, -tubulin, and ß-actin mRNAs, corresponding to
molecules comprising part of the cytoskeleton network. The enhanced
synthesis of these structural proteins may reflect the increased cell
size and intense membrane movements observed after IL-2 stimulation but
not after exposure to IL-4 (Trautmann et al., unpublished
observations). - and ß-catenin provide a link between cell
surface-expressed cadherins and the actin cytoskeleton filaments and
represent key molecules connecting cellular adhesion to signal
transduction pathways. Cadherin molecules expressed on T lymphocytes
28 form a complex with ß-catenin 29 and might be involved in the
interactions between activated T cells and their cellular targets or
the extracellular matrix. In addition, ß-catenin could participate in
IL-2-induced activation, acting as a cofactor for members of the
high mobility group family of transcription factors such as lymphocyte
enhancer factor-1 or T cell factor-1 30, 31 .
The precise regulation of c-myc and c-jun gene expression in response to IL-2 is essential for controlling cell growth. Although the downstream IL-2R-signaling pathways leading to c-myc and c-jun oncogene induction have been extensively studied 32, 33, 34 , little is known about the molecules involved in the regulation of these oncogenes. We have identified two oncogene regulators with mRNA levels that are enhanced after IL-2 stimulation of mouse T cells. First, the CTCF transcription factor, initially described as a c-myc transcriptional repressor, is characterized by a DNA-binding domain consisting of 11 zinc fingers and 2 repression domains 26 . Its singular property of combining different sets of zinc fingers to bind different nucleotide sequences suggests that it could have several target genes. CTCF is therefore a multivalent transcription factor the activity of which might depend on the activation status of the cell. We described herein a relationship between the decrease of c-myc mRNA and the increase of CTCF mRNA in IL-2-stimulated C30.1 cells, suggesting the possible CTCF repression of c-myc. The second regulator, JIF-1, was initially described as a jun inhibitory factor and is a highly conserved protein with tumor suppressor properties 27 . This factor binds c-Jun in a Zn2+-dependent manner and inhibits the AP1 complex formation. The role of IL-2 in the control of AP1 formation is therefore supported by our observation.
Four sequences encoding nuclear factors were isolated in our
cDNA-subtraction library. 1) E2F-4 transcription factor belongs to the
E2F family, which comprises five E2F-related proteins and three DP
proteins crucial for cell cycle progression and DNA replication 35, 36 . It has been reported that in quiescent primary T cells and
CD34+ hemopoietic progenitors, the major E2F transcription
factor is composed of E2F-4 complexed with DP-1 and p130 37 . 2) CREB
plays a crucial role in T cell activation and proliferation, as
indicated by the markedly impaired thymocyte proliferation and IL-2
production in transgenic mice expressing a dominant-negative CREB 38 .
CREB triggers the expression of growth-related genes, including the
peripheral cell nuclear Ag, cyclin A, as well as c-fos,
c-jun, fra-2, and fosB involved in AP1
complex formation, which are essential for IL-2-induced proliferation
38, 39 . 3) Zhx-1 is a newly described transcription factor
characterized by two zinc finger motifs and five tandem homeodomains
isolated by screening a mouse cDNA library of bone marrow stroma cell
lines 40 . 4) Nucleolin is a ubiquitously expressed multifunctional
protein involved in ribosomal biogenesis, DNA replication, and
transcription. Herein we showed that IL-2 induced E2F-4, CREB, zhx-1,
and nucleolin mRNA, but their different kinetics of induction (Fig. 2)
suggest the sequential involvement of these transcription factors after
IL-2 stimulation.
The similarities between the expression patterns of the genes isolated
with our subtractive cDNA hybridization procedure and the IL-2R
gene, both in vitro and in vivo, suggest that some common factors could
regulate their transcription. Three proximal regulatory regions have
been characterized in the IL-2R gene promoter 11, 12 .
Transcription factor STAT5 may account for most of the IL-2-induced
expression of the IL-2R gene. Among the
previously described promoters of ß-catenin, CREB, and nucleolin gene
sequences 41, 42, 43 , we have not found either GAS or regulatory
sequences analogous to the 5'-region of the IL-2R gene.
The three 5'-flanking regions are characterized by the presence of CpG
islands and numerous Sp1 transcription factor sites, both of which are
features of regulated housekeeping genes. Therefore, these genes could
be constitutively expressed at low levels and up-regulated in secondary
lymphoid organs by transcription factors modulated directly or
indirectly by the IL-2-IL-2R system. The phosphatidylinositol 3-kinase
pathway or the Ras/mitogen-activated protein kinase cascades could be
implicated. The expression of these genes in the thymus would be
enhanced by yet unknown signals. More genomic sequences corresponding
to the genes characterized as inducible by IL-2 are needed to draw more
definitive conclusions concerning the mechanism(s) involved in the
activation of this cytokine.
The selective regulation by IL-2 of cytoskeleton proteins, oncogene regulators, and transcription factors provides new elements contributing to a better understanding of the role of IL-2 at the level of mature lymphocytes and thymocytes. The observation that the corresponding mRNAs were expressed normally in the thymuses of IL-2-/- mice is in agreement with the normal thymic differentiation observed in these animal and suggests that at the thymic level, a cell surface molecule or another cytokine might be involved in the up-regulation of the genes under consideration. The possible involvement of IL-15 was explored, but no up-regulation in the IL-2-/- thymus was detected 9 . The underexpression of these genes in the peripheral lymphocytes of IL-2-/- animals might contribute to the phenotype of these animals. Abnormal proliferation may be facilitated by the uncontrolled expression of oncogenes and transcription factors. Similarly faulty adhesion due to defective cadherin-ß-catenin complexes may result in abnormal lymphocyte circulation and homing and may also allow lymphocytes to escape normal regulatory signals. Taken together, underexpression of these sets of genes may contribute, at least in part, to the lymphoproliferation and autoimmune disorders observed in IL-2-/- animals.
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Acknowledgments |
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Footnotes |
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2 Current address: Laboratoire d’Hematopoïèse et Leucémie, IRCM, 110, avenue des Pins Ouest, Montreal, H2W 1R7, Canada.
3 Current address: Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K.
4 Address correspondence and reprint requests to Dr. Jacques Thèze, Unité d’Immunogénétique Cellulaire, Institut Pasteur, 25, rue du Docteur-Roux, 75724 Paris Cedex 15, France. E-mail address:
5 Abbreviations used in this paper: RDA, representational difference analysis; CREB, cAMP-responsive element binding protein; CTCF, CCCTC-binding factor; JIF-1, Jun inhibitor factor-1; LN, lymph node.
Received for publication June 29, 1998. Accepted for publication December 15, 1998.
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