From the Laboratory of Molecular Immunology, NHLBI,
National Institutes of Health, the
Metabolism Branch, Center for
Cancer Research, and the
Biometric Research
Branch, National Cancer Institute, Bethesda, Maryland 20892
Received for publication, September 4, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and
IL-21 form a family of cytokines based on their sharing the common
cytokine receptor The common cytokine receptor Only limited information is available regarding the genes that are
activated by Cell Cultures--
Peripheral blood mononuclear cells (PBMCs)
were isolated using Ficoll (Amersham Biosciences) density
gradient centrifugation, stimulated for 18 h with
phytohemagglutinin (PHA, Roche Molecular Biochemicals, 2 µg/ml), and
expanded for 2 weeks in RPMI 1640 medium containing 10% fetal bovine
serum, 100 units/ml penicillin and streptomycin, and 2 mM glutamine (complete RPMI medium) supplemented with PHA
(500 ng/ml) and IL-2 (50 units/ml). The cells were washed twice and
resuspended in complete medium without PHA or IL-2 for 3 days. The
purity of T cells was evaluated by flow cytometry, and only cultures
with >95% CD3+ T cells were used for further studies.
Cells were then not stimulated or stimulated with IL-2 (100 units/ml),
IL-4 (100 units/ml), IL-7 (200 units/ml), or IL-15 (200 units/ml) for
4 h. We also separately performed more detailed time course
experiments (0.5, 2, 4, 6, or 8 h) with IL-2 and IL-4. In some
instances, PBMCs were stimulated for 1, 3, 6, or 24 h with phorbol
2-myristate 3-acetate (PMA) (50 ng/ml) and ionomycin (1.5 µM) (PI).
B lymphocytes (>98% pure by flow cytometry) were isolated from PBMCs
by negative magnetic selection using StemCell Technologies human B-cell
enrichment mixture and cultured for 0, 1, 3, 6, or 24 h at 5 × 106 cells/ml in complete RPMI medium containing 50 µg/ml anti-IgM (Jackson Laboratories).
Cell Lines and Chronic Lymphocytic Leukemia (CLL)
Samples--
Cell lines (Jurkat, Ramos, SUDHL10, L428, MCF-7, and
PC-3) were cultured in complete RPMI medium. OCI-Ly8 cells were grown in Iscove's modified Dulbecco's modified Eagle's medium in the presence of 20% human plasma and 1% penicillin/streptomycin. CLL cells were obtained from untreated patients, and CD19+
cells were purified by magnetic selection.
RNA Isolation--
For cDNA microarray analyses, mRNA
was prepared using the Fast-Track kit (Invitrogen). Total RNA was
isolated using the Trizol reagent (Invitrogen).
cDNA Microarray Analyses--
Microarray analysis was
performed as described in the "Microarray Procedures" section under
"Methods" in Ref. 16, using Lymphochips that contained either 7,296 or 12,672 array elements. RNA samples from cytokine-stimulated T cell
cultures were reverse transcribed, labeled with Cy5, and these probes
were hybridized to microarrays together with Cy3-labeled probes
generated from nonstimulated control cultures. Cy5-labeled probes were
also generated from T cells stimulated with IL-2 or IL-4 for various
time periods, PBMCs activated with PI, B-cells stimulated with anti-IgM
beads, and cell lines and CLL cells. These were hybridized together
with Cy3-labeled cDNA generated from a common reference mRNA
pool (16). This allowed us to compare the relative expression level of
a given gene across all of our experiments. Microarrays were analyzed on a GenePix scanner (Axon Instruments), and data files were entered into a custom data base maintained at the National Institutes of Health
(nciarray.nci.nih.gov). We used a standard global normalization approach for our expression data analogous to that previously used (16,
17). We extracted data for clustering analysis (programs "Cluster"
and "TreeView") (19) that fulfilled the following requirements:
spot size of at least 25 µm, minimum intensities of 100 relative
fluorescent units (RFU) in the Cy3 and Cy5 channels or minimum
intensity of at least 1000 RFU in one of the two channels. The 100 RFU
criteria are as previously used for the Lymphochip and Axon 4000A
scanner (17). Unless stated otherwise, the expression of a gene was
considered induced or repressed if the median induction or repression
was at least 2-fold with any one cytokine in at least two of three
experiments (five experiments were done with IL-2, three with IL-4,
four with IL-7, and 3 three with IL-15).
Northern Blotting and Real Time PCR--
Total RNA (15 µg/lane) was northern blotted using PCR-amplified inserts of the
following IMAGE clones: 176940, 203132, 1336478, 714453, 342378, 1304437 for Mal, TRAIL, MAPKAPK3, IL-4R Transient and Stable Transfections--
293T cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin. For IL-2 receptor
reconstitution experiments, cells were transfected in 10-cm plates with
plasmids containing cDNAs for IL-2R Western Blotting, Immunoprecipitation, and Kinase
Assays--
For Western blotting, cells were harvested, washed with
phosphate-buffered saline, and lysed in lysis buffer (50 mM
Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 10 µg/ml each of leupeptin and aprotinin, and 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride and centrifuged at 14,000 × g at 4 °C for 15 min. 15 µg of protein per lane was
Western blotted. For immunoprecipitations, 150 µg of protein lysate
was used. ERK-1/2 kinase activity was determined using Elk-1 as a
substrate and the p44/42 MAP kinase assay kit (Cell Signaling).
We used antibodies to phosphorylated ERK-1/2,
phosphorylated-MEK1, ERK-1/2, phosphorylated Stat5 (all from Cell
Signaling), MEK1 (Transduction Laboratories), Myc-epitope (9E10),
IL-2R IL-2, IL-7, and IL-15 Induce a Highly Similar Set of Genes, whereas
IL-4 Induces Both Overlapping and Distinct Genes--
Using cDNA
microarrays, we identified 137 genes that were induced at least 2-fold
by IL-2, IL-4, IL-7, or IL-15 in preactivated T lymphocytes (genes
induced or repressed at least 2-fold are in Figs.
1-3 and will be made available at
llmpp.nih.gov/cytokines). A one-sample t test was
performed on the log ratios to evaluate the significance of the
observed fold changes. We observed that 99% of IL-2-regulated (five
experiments), 100% of IL-7-regulated (four experiments), 74% of
IL-4-regulated (three experiments), and 54% of IL-15-regulated (three
experiments) genes were differentially expressed to a degree that
achieved statistical significance (two-sided p < 0.05). We hypothesize that the lower significance for the IL-4 and
IL-15 groups results in large part from the lower power associated with
having only three experiments for each of these cytokines. The induced
genes (Figs. 1 and 2)
included many genes previously known to be regulated by IL-2, such as
those encoding the IL-2R
The gene expression data were analyzed using average linkage
hierarchical clustering (the Pearson correlation coefficient was used
as the distance metric) (18), yielding dendrograms that group
experiments and genes based on the degree of similarity of their
expression patterns (see Fig. 1). We separately analyzed genes that
were induced by IL-2, IL-4, IL-7, or IL-15 (Fig. 1A), and
the expression profiles seen with IL-2, IL-7, and IL-15 were very
similar (see for example Fig. 1, B-D). Some
genes were induced in a similar fashion by all four cytokines (Fig.
1B). A number of genes were strongly induced by IL-2, IL-7,
and IL-15 but not induced or only weakly induced by IL-4 (Fig.
1C). A third set of genes was preferentially induced by IL-4
(Fig. 1D).
To determine whether the differences between the gene expression
profiles for IL-2 and IL-4 represented differences in the kinetics of
gene expression between the cytokines rather than induction of
different genes, we studied gene expression 0.5, 2, 4, 6, and 8 h
after cytokine treatment (Fig. 1, E and F). In the four panels on the left, multiple experiments
are shown at the 4-h time point for IL-2, IL-7, IL-15, and IL-4,
whereas in the three panels on the right data are shown for
two time courses for IL-2 and one for IL-4. Most genes showed increased
expression in a cytokine-restricted manner between 0.5 and 2 h,
and this increase was sustained for at least 8 h. For example,
leukemia inhibitory factor (LIF) and biliary glycoprotein were induced by IL-2 at 30 min but were not induced by IL-4 at any time point (second and third rows from the
top in Fig. 1E).
Most Genes Induced by IL-2, IL-4, IL-7, or IL-15 Are Induced by
Multiple Stimuli--
We sought to identify a set of genes that could
distinguish a Confirmation of Microarray Results by Northern Blotting--
We
confirmed the induction or repression of select genes (those encoding
IL-2R DUSP5 Regulates IL-2-Dependent Phosphorylation and Catalytic
Activity of ERK-1/2--
One of the genes induced by IL-2, IL-7, and
IL-15 but not by IL-4 was that encoding DUSP5, a dual-specificity
phosphatase originally cloned from mammary epithelial (13) and liver
(14) cell lines. DUSP5 is also known as hVH-3 (14) and is a
dual-specificity phosphatase induced by serum stimulation and heat
shock (13). DUSP5 can hydrolyze proteins at both phosphotyrosine and
phosphoserine/threonine residues, and recombinant DUSP5 can decrease
the catalytic activity of purified ERK-1 protein in vitro
(13, 14); but its physiological role has not been evaluated, and it has
not previously been shown to be expressed in T cells.
Because IL-2 can activate ERK-1/2 as well as induce DUSP5 expression,
we hypothesized that DUSP5 induction might be part of a negative
feedback loop controlling IL-2-induced MAPK activity. We first
confirmed that IL-2 could induce ERK-1/2 phosphorylation in the T cells
(Fig. 5A, top
panel, lanes 1-3, see arrow). IL-15 also induced phosphorylation of ERK-1/2, but neither IL-4 nor IL-7
shared this property (Fig. 5A, top panel,
lanes 10-12 versus 4-6
and 7-9). In the same experiments IL-2, IL-7,
and IL-15 induced phosphorylation of Stat5 (Fig. 5A,
middle panel, lanes 1-3,
7-9, and 10-12), and IL-4
induced phosphorylation of Stat6 (Fig. 5A, bottom
panel, lanes 4-6), demonstrating the
responsiveness of the cells to all of these cytokines. Using real time
PCR, we confirmed the induction of DUSP5 mRNA in preactivated PBMCs
within 30 min of stimulation with IL-2, with a subsequent decline (Fig.
5B). Similarly, an antiserum raised against a DUSP5·GST
fusion protein Western blotted an IL-2-inducible band of a molecular
weight appropriate for DUSP5 (Fig. 5C, upper
panel), whereas a control anti-serum could not (data not
shown), suggesting that IL-2 stimulation also increases DUSP5 protein
levels.
We next investigated whether DUSP5 could regulate IL-2-induced ERK-1/2
phosphorylation using an IL-2 receptor reconstitution system (24) in
293T cells. In this setting, ERK-1/2 phosphorylation was induced by
IL-2 (Fig. 5D, lane 2 versus
lane 1), and this phosphorylation was inhibited
when the cells were also transfected with wild type DUSP5 (Fig.
5D, lanes 3 and 4). Transfection of an
inactive (C263S) mutant of DUSP5 did not affect ERK-1/2 phosphorylation (Fig. 5D, compare lane 6 to lane 2),
which is consistent with the fact that 293T cells express very low
levels of endogenous DUSP5 (data not shown). As expected, 293T cells
transfected with constitutively active MEK1 showed increased ERK-1/2
activity that was independent of IL-2 (Fig. 5D, lanes
7 and 8).
To further evaluate the possible role of DUSP5 in the regulation of
IL-2-induced MAPK activity, we stably transfected
IL-2-dependent CTLL-2 cells with wild type and inactive
DUSP5. CTLL-2 is a murine T cell line that has been widely used to
study IL-2 biology and signaling (see, for example, Refs. 25 and 26).
We studied two clones that constitutively express wild type DUSP5 (WT1,
WT2) and three clones that constitutively express an inactive mutant of
DUSP5 (M1, M2, M3), as demonstrated by Western blotting with anti-Myc
epitope antibody (Fig. 6A,
top panel, lanes 1-20). The clones
expressing the inactive form of DUSP5 showed markedly increased phosphorylation of ERK-1/2 (doublet band, Fig. 6A,
second panel from the top, lanes 9-20) in
response to IL-2 when compared with parental CTLL-2 cells (lanes
21-24) or clones expressing wild type DUSP5 (lanes
1-8). Similarly, as evaluated by an in vitro kinase
assay, clones expressing inactive DUSP5 exhibited higher IL-2-induced
ERK-1/2 kinase activity than did the parental cells (Fig.
6B, lanes 7-15 versus
16-19), whereas clones expressing wild type
DUSP5 showed decreased activity (Fig. 6B, lanes
1-6). As ERK-1/2 protein levels were not affected by DUSP5
expression (Fig. 6A, third panel from
the top, lanes 1-24), these results indicate that DUSP5 negatively regulates the activity of ERK-1 and
ERK-2. We also studied the effect of DUSP5 on IL-2-induced phosphorylation of MEK1 and Stat5 (Fig. 6A,
fourth and fifth panels from the top) and
IL-2R In this study, we investigated genes regulated by IL-2, IL-4,
IL-7, and IL-15. Although some data regarding genes regulated by these
cytokines have been previously generated, comparative data on the
effects of these cytokines on large numbers of genes have not been
available. Genes induced by IL-2 have been most extensively studied,
and IL-2 is known to induce a number of genes including, for example,
those encoding c-Myc (15), c-Fos (15), c-Jun (15),
IL-2R Hierarchical clustering of these genes revealed that the induced genes
fell into two major groups, i.e. those regulated
preferentially by IL-2, IL-7, and IL-15, and those regulated by IL-4.
It was noteworthy that IL-2, IL-7, and IL-15 induced almost identical gene expression patterns in T lymphocytes, at least at the doses we
used. This supports the concept that multiple cytokines can similarly
provide survival/growth signals and that the specificity of cytokine
action is largely determined by cell type or developmental stage
specific expression of cytokine receptor(s) and the availability of
ligand(s) (30-32). However, it is possible that cell
type-dependent differences also exist. For example, it has
been suggested that IL-15 uses different receptor or signaling pathways
depending on cell type (33, 34), which could also explain why IL-2 and IL-15 induce similar gene responses and proliferation in T cells, whereas only IL-15 is essential for NK cell differentiation.
Although IL-4 induced a set of genes overlapping those induced by IL-2,
IL-7, and IL-15, there were also many differences. Most genes that were
induced by IL-2, IL-7, or IL-15 were not induced by IL-4 or were
induced at only a very low level. However, some genes (e.g.
those endocing SOCS-1, CIS1, and Bcl-2) were induced in
a similar fashion by all of the cytokines, whereas others
(e.g. those encoding IL-4R Most of the genes that we found to be induced by
Only a few of the genes whose expression was regulated by
Studies in the 32D myeloid cell line revealed that IL-2R It this study, we have shown that IL-2 potently activates ERK-1 and
ERK-2 as well as DUSP5, which negatively regulates ERK-1 and ERK-2. In
contrast, IL-4 does not potently activate either ERK-1 or ERK-2 nor
does it induce DUSP5, thus providing an example of how differential
gene regulation by chain,
c, which is mutated
in X-linked severe combined immunodeficiency (SCID). As a step toward
further elucidating the mechanism of action of these cytokines in
T-cell biology, we compared the gene expression profiles of IL-2, IL-4,
IL-7, and IL-15 in T cells using cDNA microarrays. IL-2, IL-7, and
IL-15 each induced a highly similar set of genes, whereas IL-4 induced distinct genes correlating with differential STAT protein activation by
this cytokine. One gene induced by IL-2, IL-7, and IL-15 but not IL-4
was dual-specificity phosphatase 5 (DUSP5). In
IL-2-dependent CTLL-2 cells, we show that IL-2-induced
ERK-1/2 activity was inhibited by wild type DUSP5 but markedly
increased by an inactive form of DUSP5, suggesting a negative feedback
role for DUSP5 in IL-2 signaling. Our findings provide insights into
the shared versus distinctive actions by different members
of the
c family of cytokines. Moreover, we have
identified a DUSP5-dependent negative regulatory pathway
for MAPK activity in T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
c chain
(
c)1 is
essential for normal immune development and function. Mutations in
c result in X-linked severe combined immunodeficiency
(XSCID) (1), a disease in which affected individuals are highly
susceptible to infections resulting from the defective development of T
and NK cells and nonfunctional B cells.
c is a component
of the receptors for multiple cytokines, including IL-2, IL-4, IL-7,
IL-9, IL-15, and IL-21 (2). These cytokines have both redundant and
distinctive actions on lymphocytes. IL-2 is a growth and survival
factor for activated T lymphocytes and is also essential for
activation-induced cell death (AICD) and the prevention of T cell
anergy, processes involved in the maintenance of peripheral tolerance
(3). IL-4 is also a T-cell growth factor and is necessary for the
development of Th2 cells (4), which regulate humoral immune responses, whereas IL-7 is required for the development and growth of T
lymphocytes but can also support the growth of peripheral T lymphocytes
(5-7). IL-15 has overlapping functions with IL-2, but, unlike IL-2,
IL-15 is essential for NK-cell differentiation and is more important than IL-2 in the generation of CD8+ memory T cells (8).
Although transgenic expression of IL-9 causes thymomas, suggesting that
IL-9 can regulate lymphocyte growth (9), IL-9 knock-out mice do not
manifest abnormalities in T-cell development or function but exhibit
defective mast cell proliferation and mucus production (10). Based on
in vitro studies, IL-21 has potential roles for T-cell,
B-cell, and NK-cell biology (11).
c-dependent cytokines. To
investigate the basis for overlapping and distinctive actions of
c-dependent cytokines, we used the
"Lymphochip" microarray, a specialized cDNA array enriched for
genes expressed in lymphocytes (12), to identify genes regulated by
IL-2, IL-4, IL-7, and IL-15 in peripheral blood T lymphocytes. IL-2,
IL-7, and IL-15 regulated most genes in a similar manner, whereas the
pattern seen with IL-4 was more distinctive. Although most
c-dependent genes are redundantly regulated
by various stimuli, certain cytokine-specific patterns of gene
expression were noted. One gene induced by IL-2, IL-7, and IL-15 but
not IL-4 was dual-specificity phosphatase 5 (DUSP5) (13,
14). IL-2 is known to activate several signaling pathways, including
Ras-MAP kinase, Jak-STAT, and phosphoinositol 3-kinase/Akt/p70 S6
kinase pathways (15), and we now provide evidence for a role for DUSP5
as part of a negative feedback loop that controls IL-2-induced MAPK
activity in T cells. This is the first demonstration of a role for
DUSP5 in T-cell biology and identifies a mechanism by which
IL-2-mediated MAPK activation can be controlled.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, DUSP5, and TSC-22R,
respectively, as well as probes for IL-2R
(X01075), and pHe7
(which corresponds to a housekeeping gene). Real time PCR was performed
using a Light Cycler. 10 ng of total RNA was amplified for 40 cycles
with either DUSP5 (sense 5'-AAAGGGGGATA TGAGACTTTC-3', antisense
5'-TTGGATGCATGGTAGGC ACTT-3') or
-actin (Clontech)-specific primers. A standard curve was
prepared by amplifying different amounts of total RNA prepared from
IL-2-stimulated PBMCs. Results obtained with DUSP5-specific primers
were normalized against
-actin.
(2 µg),
c
(0.5 µg), Jak3 (0.25 µg), and with control vector pRV (3 µg), wild type DUSP5 (3 µg), or inactive mutant of DUSP5 (C263S mutant) (3 µg) or the constitutively active MEK1 (3 µg) using
EffecteneTM transfection reagent (Qiagen). For stable
transfections, CTLL-2 cells were grown in complete RPMI medium
supplemented with 50 units/ml of IL-2 and electroporated (5 × 106 cells, 170 V, 960 µF) with Myc-epitope tagged DUSP5
wild type or mutant cDNAs (36 µg) as well as with pBabe-puro (4 µg) to confer puromycin resistance. Transfected cells were plated on
6-well plates, and puromycin (2.5 µg/ml) was added the next day. The cDNAs for Myc-epitope tagged wild type and mutant (C263S) DUSP5, human Jak3, and constitutively active MEK1 were kindly provided by Jack
E. Dixon, John O'Shea, and Silvio Gutkind, respectively.
phosphotyrosine antibody (PY99) (all from Santa Cruz
Biotechnology), and anti-
-actin (AC-15, Sigma). DUSP5 anti-serum was
a gift from Dr. Jack E. Dixon.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
chain, cyclin D2 (15), SOCS1 (19), CIS1
(20), and Pim-1 (21), indicating the validity of our analysis. IL-4R
was also induced by IL-4, as reported previously (22). We also identified 34 repressed genes (Fig. 3).
Although the use of 2-fold induction criteria is common, smaller
changes in gene expression levels can also be biologically significant,
and certain known IL-2-induced genes were not identified by the 2-fold
criteria. We therefore searched the microarray data for genes whose
expression was induced or repressed by more than 40% (based on
microarray analysis) in at least any 8 of the 15 experiments. 95 additional genes fulfilled these less stringent criteria (data not
shown but will be made available at llmpp.nih.gov/cytokines). This
group included c-myc (15) and Bcl-XL
(23), genes whose expression levels are known to be regulated by IL-2.
Thus, it is important to evaluate genes whose mRNA levels are
changed less than 2-fold as well.
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Fig. 1.
Hierarchical clustering of genes induced by
IL-2, IL-4, IL-7, and IL-15. The color intensity reflects
the magnitude of induction (red squares) or repression
(green squares). Gray
squares indicate missing or excluded data.
Asterisks indicate genes whose identity was verified by
re-sequencing. A, array dendrogram obtained by hierarchical
clustering of expression data from 137 induced genes in a total of 15 array experiments comprising four different cytokines. Samples
stimulated for 4 h by IL-2, IL-7, and IL-15 cluster together
(black branches), indicating a similar gene
expression response, whereas the samples induced by IL-4 form a
separate group (blue branches). B,
hierarchical clustering of genes similarly induced at least 2-fold by
IL-2, IL-4, IL-7, and IL-15. Each column represents data from one
experiment, and each row represents the measurements for a given gene
across all experiments. C, hierarchical clustering of genes
induced more potently by IL-2, IL-7, and IL-15 than by IL-4. Genes in
this cluster were induced more than 2-fold by IL-2, IL-7, and IL-15,
and their expression was at least two times higher in IL-2-, IL-7-, and
IL-15-stimulated cultures than in IL-4-stimulated cultures.
D, hierarchical clustering of genes preferentially induced
by IL-4. Genes in this cluster were induced at least 2-fold by IL-4,
and their expression was at least two times higher in IL-4 stimulated
cultures than in cultures stimulated by IL-2, IL-7, or IL-15.
E, kinetics of IL-2- and IL-4-induced gene expression. Gene
expression data for highly induced genes after 4 h of stimulation
with IL-2 (five experiments), IL-7 (four experiments), IL-15 (three
experiments), and IL-4 (three experiments) and corresponding
measurements in three time course experiments (two IL-2 experiments and
one IL-4 experiment) at 0, 0.5, 2, 4, 6, and 8 h. The genes were
ranked by their average up-regulation in response to IL-2, IL-7, and
IL-15. Data from the 20 most induced genes are shown. F,
genes induced more than 2-fold by IL-4 that, in addition, were at least
2-fold more highly induced by IL-4 than by IL-2, IL-7, or IL-15. In
panels B-D the "gene tree" on the
left indicates the degree of similarity in gene expression
across all the experiments. The blue circles
correspond to genes whose expression was also studied by Northern
blotting in Fig. 3.
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Fig. 2.
Analysis of expression patterns of
cytokine-induced genes in PI-stimulated PBMCs (PBMC
Act.), B cells after anti-IgM crosslinking
(B-cell Act.), proliferating cell lines, and CLL
cells. A, a set of genes induced by IL-2, IL-4, IL-7,
or IL-15 that were 3-fold more expressed in proliferating cell lines
than in CLL cells. B, cytokine-inducible genes that are
induced >2-fold by PI stimulation of PBMCs and BCR stimulation of B cells but showed variable expression in the cell
lines and CLL samples. C, cytokine-inducible genes that are
induced >2-fold by PI treatment of PBMCs but not by BCR cross-linking
of B cells. D, genes induced >2-fold with BCR cross-linking
of B cells but not induced by PI. E, genes specifically
induced by IL-2, IL-4, IL-7, or IL-15. Cytokine-inducible genes were
assigned to this cluster based on lack of induction by PI stimulation
of PBMCs or BCR cross-linking of B cells and variable expression cell
lines and CLL samples. In panels A and
B the blue circles correspond to genes
whose expression was also studied by Northern blotting (Fig. 4). The
time-course for PI stimulation and BCR cross-linking was 0, 1, 3, 6, and 24 h. The letters below the cell lines and CLL cells
correspond to following: A, Jurkat; B, Ramos;
C, OCI-Ly8; D, SUDHL10; E, L428;
F, MCF-7; G, PC-3; H-L,
CD19+ cells from five different CLL patients. Data are shown for one of
three representative experiments. The data for IL-2, IL-7, and IL-15
were averaged.
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Fig. 3.
Genes repressed by IL-2, IL-4, IL-7, and/or
IL-15. Data are shown for 34 genes after 4 h stimulation with
cytokines and with corresponding measurements in time-course
experiments, PI stimulated PBMCs, B-cells after BCR cross-linking,
proliferating cell lines, and CLL cells. The blue
circles correspond to genes whose expression was also
studied by Northern blotting in Fig. 4.
c-Dependent Cytokines Induce a Group of Genes That
Are Highly Expressed in Proliferating Cell Lines but Are Expressed at a
Low Level in CLL Cells--
Most of the genes we identified have not
been previously linked to cytokine responses and many are functionally
uncharacterized. One strategy for finding clues to the functions
mediated by these genes is to define expression "signatures"
characterizing cellular processes. One such expression signature is
defined by a set of genes whose expression correlates with cell
proliferation in that they are highly expressed in proliferating cell
lines (Fig. 2A, third panel from
the left, lanes
A-G) but are expressed at a low level in CLL
cells (lanes H-L) which are relatively quiescent in their growth properties (16). ~20% of the genes induced by IL-2,
IL-4, IL-7, and/or IL-15 fulfilled these criteria.
c-dependent cytokine response
from other activation events, and we compared genes induced or
repressed by IL-2, IL-4, IL-7, and IL-15 with those regulated in PBMCs
(>70% T cells) by PI and in B cells after antigen receptor
cross-linking (Fig. 2). We found that 73% of the genes induced by the
cytokines were also induced in PI-stimulated PBMCs (Fig. 2,
A-C, first column
of panels from the
left). The induction typically occurred within 1 h,
minimizing the possibility that PI-dependent cytokine
production was responsible for the PI effect. BCR stimulation of B
cells induced less overlapping (41%) gene expression profiles (Fig. 2,
A and B versus C and
D, compare first and second
panels from the left), consistent with the use of nonshared
signaling pathways or lineage-specific differences in expression. 23%
of the
c-dependent genes showed a more
restricted expression pattern, as they were not up-regulated by either
PI treatment of PBMCs or BCR cross-linking of B cells.
c-Dependent Cytokines Repress Certain Genes, Some of
Which Are Highly Expressed in CLL Cells--
We also identified 34 genes that were consistently repressed by IL-2, IL-4, IL-7, and/or
IL-15 (Fig. 3), most of which were also repressed in PBMCs treated with
PI or B cells treated with anti-IgM. Analogous to many of the activated
genes being poorly expressed in CLL cells, many of the repressed genes
were more highly expressed in CLL cells than in highly proliferating
cell lines (Fig. 3, third panel from the left,
lanes H-L versus
A-G), suggesting a correlation between the
expression of these genes and establishing or maintaining a more
quiescent state.
, TRAIL, MAPKAPK3, DUSP5, Mal, IL-4R
and TSC-22R; blue circles in Figs. 1-3) by Northern blot
analysis (Fig. 4). IL-2R
, TRAIL, and
DUSP5 were more potently induced by IL-2, IL-7, and IL-15 than by IL-4.
Mal and IL-4R
were most strongly induced by IL-4. MAPKAPK3 was
induced by all four cytokines, and TSC-22R was repressed by all of the
cytokines.
View larger version (65K):
[in a new window]
Fig. 4.
Northern blot analysis of selected
genes. PBMCs were either not stimulated ( ) or stimulated (+) for
3 h with PI (lanes 1 and 2), and cultured T
cells were either not stimulated (
) (lane 3) or stimulated
with the indicated cytokines for 4 h (lanes 4-7). Data
are shown for one of three representative experiments.
View larger version (41K):
[in a new window]
Fig. 5.
IL-2-induced ERK-1/2 phosphorylation is
inhibited by DUSP5. A, T cells were cultured as
described under "Materials and Methods," rested for 3 days, and
stimulated with IL-2, IL-4, IL-7, or IL-15 for 0, 10, or 30 min. 15 µg of protein lysate per lane was Western blotted using antibodies
for phosphorylated-ERK-1/2 (top panel), phosphorylated Stat5
(middle panel), or phosphorylated Stat6 (bottom
panel). B, PBMCs were activated with PHA for 48 h,
rested for 2 days, and stimulated with IL-2 for the indicated times.
DUSP5 mRNA expression was analyzed by real-time PCR, and the
expression levels were normalized against -actin expression. Shown
is the average of three independent experiments (± S.D.).
C, 30 µg of total cellular protein lysates
(TCL) from the experiment portrayed in panel
B (lanes 1-6) were Western blotted using
antibodies for DUSP5 (top panel) or
-actin
(bottom panel). Lanes 7 and
8 are lysates from parental CTLL-2 cells (
) or
DUSP5-transfected CTLL cells (+), as controls. Shown is one of three
representative experiments. D, 293T cells were transfected
with IL-2 receptor components (see "Materials and Methods") and
either the pRV control vector, wild type DUSP5, inactive mutant of
DUSP5 (DUSP5mut), or constitutively active MEK1
(MEK1act). After 2 days, cells were not
stimulated or stimulated with IL-2 (1000 units/ml), lysed, and 15 µg
of protein lysates were Western blotted using an antibody specific for
phosphorylated ERK-1/2. Data shown are from one of five experiments
with similar results.
(Fig. 6C), but no changes were observed. Thus,
DUSP5 appears to selectively regulate ERK-1 and ERK-2 activity in
IL-2 signaling.
View larger version (68K):
[in a new window]
Fig. 6.
DUSP5 regulates ERK-1/2 activity in CTLL-2
cells. IL-2 signaling events in clones of CTLL-2 cells expressing
wild type DUSP5 (WT1 and WT2), three clones
expressing an inactive DUSP5 mutant (M1-M3), or parental
CTLL-2 cells. The cells were rested for 10 h in 5% fetal bovine
serum in the absence of IL-2 and then either not stimulated or
stimulated for 5, 15 or 30 min with 100 units/ml of IL-2. A,
15 µg of total cellular lysates were Western blotted using antibodies
for Myc-epitope (to validate the expression levels of transfected DUSP5
proteins, top panel), phosphorylated ERK-1/2
(second panel from top), total ERK-1/2
(third panel from top), phosphorylated MEK1
(fourth panel from top), phosphorylated Stat5
(fifth panel from top), and -actin (bottom
panel). B, ERK-1/2 kinase assay. Total cellular lysates
(250 µg) were immunoprecipitated with antibodies to phosphorylated
ERK-1/2 followed by kinase reaction in the presence of Elk1 substrate
protein. The proteins were Western blotted using an antibody to
phosphorylated Elk-1. C, total cellular lysates (250 µg)
were immunoprecipitated with antibodies to IL-2R
and Western blotted
with anti-phosphotyrosine. Data are shown for one of four
representative experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
chain (15), Pim-1 (21), Bcl-2 (15), and the
SOCS family proteins SOCS1 (19) and CIS1 (20). Previous studies have
sought to identify IL-2-regulated genes in a more systematic way
(27-29) but have revealed largely nonoverlapping sets of IL-2-induced
transcripts, suggesting that many other IL-2-regulated genes remained
to be identified. In our microarray analysis137 genes appeared to be
induced, and 34 genes appeared to repressed by IL-2, IL-4, IL-7, or
IL-15. A significant number of these genes (20%) are related to cell
proliferation based on their high level expression in proliferating
cell lines but low expression in relatively quiescent CLL cells (16),
in accord with the known mitogenic function of these cytokines on
activated T cells.
and Mal) were more
strongly induced by IL-4. The basis for this more distinctive pattern
for IL-4 may be explained by the fact that IL-4 activates primarily Stat6, whereas the other cytokines preferentially activate Stat3, Stat5a, and Stat5b (35). For example, the promoter for IL-4R
, which
is regulated by IL-4, contains Stat6 binding sites (36), whereas the
IL-2R
promoter, which is regulated by IL-2, contains binding sites
for Stat5a and Stat5b (37-39). It will be important to determine
whether genes induced preferentially by IL-4, such as Mal,
contribute to functions unique to IL-4 such as the induction of Th2
differentiation among T lymphocytes.
c-dependent cytokines were also induced by
the combination of PMA plus ionomycin (74%), and many were induced
after BCR stimulation of B lymphocytes (57%). One of these genes, the
IL-2R
gene is regulated by at least five positive
regulatory regions (PRRs) (15, 37-41). PRRI is presumably a T cell
receptor response element as it contains an NF-
B binding site
that is required for IL-2R
promotor activity in response to PHA or
PMA (15), whereas PRRIII and PRRIV (37-40) are both required for
IL-2-induced IL-2R
induction. A fifth element is a CD28 response
element (41). Thus, in the IL-2R
gene, different enhancer-like
elements differentially respond to different stimuli. The coexistence
of antigen and cytokine response elements in other genes as well might
account for the highly overlapping gene expression profiles between
c-dependent cytokines and PI-stimulation. In this regard, IL-15 and T cell receptor stimulation were recently shown
to induce many of the same genes in CD8+ memory T cells
(42).
c-dependent cytokines were previously
identified as functionally relevant target genes, such as those
encoding IL-2R
(43-45) and Bcl-2 (46), and most of the
genes we identified have not been characterized as part of a cytokine
response. One such gene is DUSP5, which we now show is
induced by IL-2, IL-7, and IL-15, but not by IL-4. DUSP5 was previously
shown in vitro to be capable of dephosphorylating ERK-1, but
the physiological context was not investigated (13, 14). IL-2 signaling
has been extensively studied, and along with the Jak-STAT and PI
3-kinase/Akt pathways, the MAP kinase pathway has been described as
important (15). The activation of Stat5 proteins is mediated by
phospho-tyrosine docking sites (Tyr-392 and Tyr-510) on the IL-2
receptor
chain (47, 48) and has been functionally linked to the
regulation of proliferation and activation-induced cell death (48, 49). Activation of the PI 3-K/Akt pathway regulates
IL-2-dependent cell survival (50) and may contribute to
cell proliferation (50). Regarding the MAPK pathway, Tyr-338 on
IL-2R
directly binds the phospho-tyrosine binding (PTB) domain of
Shc (48) and mediates the activation of ERK-1/2 by IL-2 (49, 51, 52). We provide evidence that DUSP5 induction by IL-2 may negatively regulate IL-2-dependent activation of ERK-1/2.
Tyr-338 is
required for IL-2-dependent proliferation, suggesting that
Shc-coupled MAPK activation is vital for proliferation (48). However,
in primary T cells, simultaneous inactivation of Tyr-338 and Stat5
binding sites was required to reveal a decrease in proliferation, suggesting that in these cells either MAPK or Stat5-coupled pathways by
themselves are sufficient for proliferation (49). This is consistent
with our finding in CTLL-2 cells that DUSP5 alone did not decrease
IL-2-mediated proliferation (data not shown). However, it is possible
that MAPK and DUSP5 may have other effects in IL-2 biology.
c-dependent cytokines can
modulate cytokine-specific actions. As noted above in our microarray
analysis, DUSP5 was induced in T cells either following treatment with
cytokines or PMA plus ionomycin. This suggests that
DUSP5-dependent negative feedback regulation of MAPK is not restricted to cytokine signaling. ERK-1/2 activation has been indicated
in a range of important functions in T-cells and NK cells (53).
Remarkably, in ERK-1-deficient mice the only observed defect is in
thymic T-cell development at the double positive stage resulting in
~50% reduction in the numbers of single positive lymphocytes (54).
Accordingly, we recently generated mice expressing DUSP5 transgene,
which in preliminary experiments show an even more complete block in T
lymphocyte development with ~70% reduction in numbers of single
positive lymphocytes (data not shown). This is consistent with our
observation that DUSP5 regulates both ERK-1 and ERK-2 in T cells and
suggest that DUSP5 regulation of MAPK is a general theme in T
lymphocyte activation and signaling.
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ACKNOWLEDGEMENTS |
---|
We thank Jack E. Dixon for kindly providing the DUSP5 cDNA constructs and antiserum and Drs. Keiji Zhao, Joost Oppenheim, Jian-Xin Lin, and John Kelly for critical comments.
![]() |
FOOTNOTES |
---|
* 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.
§ These authors should be considered as equal first authors.
¶ Supported in part by grants from the Maud Kuistila Foundation, the Finnish Cultural Foundation, the Emil Aaltonen Foundation, and the Academy of Finland.
** Supported in part by a grant from the Deutsche Krebshilfe, Bonn, Germany.
§§ These authors should be considered as equal last authors.
¶¶ To whom correspondence should be addressed. Fax: 301-402-0971; E-mail: wjl@helix.nih.gov.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209015200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
c, cytokine receptor
chain;
IL, interleukin;
DUSP5, dual-specificity
phosphatase 5;
MAP, mitogen-activated protein: MAPK, MAP kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
STAT, signal transducers and activators of transcription;
PBMC, peripheral
blood mononuclear cell;
PHA, phytohemagglutinin;
PMA, phorbol
2-myristate 3-acetate;
PI, PMA plus ionomycin;
PRR, positive regulatory
region;
CLL, chronic lymphocytic leukemia;
BCR, B cell antigen
receptor.
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