From the Molecular Oncology Program, H. Lee Moffitt
Cancer Center and Research Institute, § Department of
Oncology, and Department of Biochemistry and Molecular Biology,
University of South Florida College of Medicine, Tampa, Florida
33612
Received for publication, January 2, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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We have shown previously that serum promotes T
cell proliferation by acting with T cell receptor (TCR) agonists to
efficiently down-regulate p27Kip1 and activate
cdk2-containing complexes. In the studies described here, the effect of
serum on the expression of the Engagement of T cell receptors
(TCRs)1 initiates a complex
program of events that leads to the proliferation and differentiation of mature resting T cells (1). A key player in this program is
interleukin-2 (IL-2), a lymphokine first identified in supernatants of
antigen-primed T cells (2-4). The receptor for IL-2 (IL-2R) is
noncatalytic and consists of three subunits termed IL-2R Of the three IL-2R subunits, IL-2R As in all cell types, T cell proliferation is governed by the ordered
activation of cyclin-dependent kinases (CDKs) (20). CDK
activity is controlled by cyclins, which are positive regulators, and
CDK inhibitors, which repress activity. Cyclins, which are expressed
periodically, combine with CDKs to form active complexes at distinct
points in the cell cycle. During G0/G1, for
example, complexes containing the D cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2 are sequentially assembled and activated. Activation of these complexes is required for
G0/G1 traverse and S phase entry, and key
substrates include the Rb family of transcriptional repressors. The CDK
inhibitor family includes the Cip/Kip proteins, which inactivate
complexes containing cdk2 and, according to some reports, cdk4 and cdk6
(21-23). Of these inhibitors, p27Kip1 is thought to play a
particularly important role in T cell proliferation. p27Kip1 is present at high levels in resting T cells and is
down-regulated in response to mitogenic stimulation (24, 25). Failure
to reduce p27Kip1 levels below a critical threshold
precludes cdk2 activation and arrests T cells in
G0/G1. Moreover, T cells lacking
p27Kip1 exhibit dysregulated cdk2 activation and
proliferate in conditions that do not support the growth of wild-type
cells (Ref. 26 and accompanying article (43)). Previous studies have
shown that IL-2 elicits and is required for efficient
p27Kip1 down-regulation in primary T cells and T
lymphoblasts (24, 25). In addition, we have found that serum acts with
TCR agonists to maximally and persistently reduce p27Kip1
levels and, consequently, to activate cdk2 in naive T cells (43).
Given the pivotal role of IL-2 in T cell mitogenesis, a full
understanding of the mechanisms regulating the expression of IL-2 and
IL-2R is imperative. IL-2R density is a critical determinant of T cell
proliferation (27), and previous studies have shown that IL-2R density
is dictated by serum concentration (18). As monitored by IL-2 binding
assays, serum (at 10%) significantly increased the expression of
surface-localized IL-2Rs in antigen-treated human T cells (18).
Building on this observation, data presented here demonstrate that
serum selectively facilitates the post-transcriptional expression of
IL-2R Isolation of Splenocytes and Cell Culture--
A single cell
suspension of splenocytes was prepared by passage through nylon mesh,
and red cells were depleted using a whole blood erythrocyte lysing kit
(R&D Systems). For purification, spleen cell suspensions were loaded
onto T cell-enrichment columns (R&D Systems), and T cells were isolated
by high affinity negative selection as specified by the manufacturer.
Splenic and purified T cells were plated at 107 cells/ml
and 5 × 106 cells/ml, respectively, in RPMI 1640 supplemented with 50 units/ml penicillin, 2 mM
L-glutamine, and 10% fetal calf serum. p27-47 fibroblasts
were prepared and maintained as described previously (28). The
percentage of S phase cells in a population was determined by FACS
analysis of propidium iodide-stained cells as detailed previously
(43).
Protein Analysis--
Western blots were performed as described
previously (43). For ELISA assays, flat bottom 96-well microtiter
plates were coated overnight at 4 °C with 6 µg/ml IL-2R mRNA Analysis--
Total mRNA was isolated using TRIzol,
and Northern blots were performed as described previously (30). For
RNase protection assays, mRNA (20 µg) was hybridized overnight at
56 °C with 32P-labeled probes (105 cpm)
corresponding to the mCR-1 probe set (PharMingen). Samples were then
digested with RNase T1 and RNase A for 45 min at 30° C and proteinase
K for 15 min at 37 °C. Samples were extracted with
phenol/chloroform, collected by sodium acetate/ethanol precipitation, denatured at 90 °C for 3 min, and electrophoresed on a 5%
polyacrylamide gel. Gels were dried and exposed to x-ray film.
In Vitro Kinase Assay--
Cell extracts were incubated with
antibody to cyclin E or cyclin A for 4-12 h at 4 °C and
subsequently with protein A-agarose beads. Immune complexes were washed
twice with lysis buffer (43) and once with histone kinase buffer (50 mM Tris (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol). Washed complexes were incubated in 15 µl of kinase buffer containing 20 µM ATP, 0.1 µCi/ml
[ Materials--
ConA was purchased from Sigma Chemical Co., and
anti-CD3, recombinant IL-2, and IL-2R Serum Modulates IL-2R
To ensure that T cells remained viable when stimulated in
serum-deficient medium,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assays were performed. When examined 40 h after stimulation, T
cells treated with anti-CD3 and 0.1% serum were only slightly less
viable than those receiving anti-CD3 and 10% serum (Fig. 1A, bottom panel). This change in viability is
not sufficient to account for the low levels of IL-2R
To determine if serum concentration affected total (as well as
surface-localized) levels of IL-2R
To assess the functional consequences of serum-dependent
IL-2R
To determine if serum regulated IL-2R Serum-dependent IL-2R
In wild-type splenocytes, cdk2 activation requires a pronounced and
persistent decrease in p27Kip1 levels, and both ConA and
10% serum are needed to achieve this effect (43). In contrast, in
p27Kip1-null splenocytes, cyclin E-cdk2 activation
is constitutive and cyclin A-cdk2 activation is serum- (although not
ConA-) independent (26, 31, 43). Thus, conditions that promote cdk2
activation are the same as those that promote IL-2R
The necessity of cdk2 activity for IL-2R cdk2 Activity and IL-2 Signaling Comprise a Regulatory
Loop--
Previous studies have shown that IL-2 stimulates cyclin
E-cdk2 and cyclin A-cdk2 activity in activated T cells (33-35).
Because these investigations place cdk2 activation downstream of IL-2R activation, the need for cdk2 activity for IL-2R Engagement of the TCR results in the transcriptional up-regulation
of IL-2R The requirement for cdk2 activity for IL-2R Because IL-2 signaling is thought to be a cause rather than
an effect of cdk2 activation in T cells (24, 25), we considered the
possibility that these processes were interdependent. In support of
this hypothesis, we found that abrogation of IL-2 signaling repressed
both cdk2 expression and cdk2 activity in splenocytes stimulated with
ConA and 10% serum. Two methods were used to inhibit IL-2 signaling:
an IL-2R subunit of the interleukin-2
receptor (IL-2R
) was examined. We found that serum was required for
maximal and sustained IL-2R
protein expression and consequent IL-2
signaling in TCR-activated splenocytes. Serum had no effect on IL-2R
mRNA levels and thus modulates IL-2R
expression
post-transcriptionally. Unlike wild-type splenocytes, splenocytes
exhibiting serum-independent cdk2 activation due to loss of
p27Kip1 efficiently expressed IL-2R
in serum-deficient
medium. Conversely, serum did not promote IL-2R
accumulation in
conditions in which cdk2 activity was blocked. These findings
demonstrate that cdk2 activation is necessary and sufficient for
IL-2R
accumulation in TCR-stimulated splenocytes. On the other hand,
IL-2 signaling was required (at least in part) for cdk2 activation in
these cells. Thus, cdk2 activation, IL-2R
expression, and IL-2
signaling are interdependent events, and we suggest that this
feed-forward regulatory loop plays a key role in T cell mitogenesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-2R
,
and IL-R2
(also known as
c) (5). IL-2R
and IL-2R
are also
components of other lymphokine receptors, whereas IL-2R
is unique to
the IL-2R and thus is responsible for substrate specificity. IL-2R
is also required for high affinity (and presumably biologically relevant) IL-2 binding. In vitro, IL-2Rs lacking IL-2R
are functional in human T cells (albeit at a lower affinity than
trimeric receptors) but not in mouse T cells (6-10). On the other
hand, IL-2R
has an extremely short cytoplasmic domain and does not
participate in intracellular signaling per se. Instead,
signaling is mediated by IL-2R
and IL-2R
, which interact with a
variety of cytoplasmic effector proteins (11). These include the Janus
kinases (JAKs), Jak1 and Jak3, which associate constitutively with
IL-2R
and IL-2R
, respectively. When activated by ligand-induced
receptor oligomerization, JAKs phosphorylate IL-2R
and IL-2R
on
specific tyrosine residues to create docking sites for other effectors. Additional JAK substrates include the JAKs themselves and members of
the STAT (signal transducer and activator of transcription) family of
transcriptional regulators.
exhibits the most variable
expression. IL-2R
is not present in resting T cells and is transcriptionally up-regulated by TCR agonists (12). Such agonists include cognate antigen, anti-CD3, and concanavalin A (ConA). IL-2R
and IL-2R
, on the other hand, are expressed constitutively, and
TCR-induced changes in their expression are less dramatic (5). TCR
activation increases IL-2 production (13), and IL-2 signaling further
up-regulates IL-2R
expression in a STAT-dependent manner
(14-16). As a further prelude to IL-2 actions, TCR stimulation induces
a variety of metabolic responses that allow quiescent T cells to exit
G0 (11). IL-2 then promotes continued
G0/G1 traverse and the initiation of DNA
synthesis. T cell proliferation is also influenced by co-stimulatory
signals delivered by CD28 (17) and by mitogens contained in serum,
which is indispensable for T cell propagation in vitro (18).
The capacity of co-stimulatory signals to up-regulate IL-2R
transcription has been reported (19).
in primary splenic T cells exposed to TCR agonists.
Serum-mediated IL-2R
accumulation was accompanied by the induction
of IL-2 signaling pathways and was dependent on cdk2 activation.
Because cdk2 activity both contributed to and resulted from IL-2
signaling, we suggest that these events are interdependent. We propose
a model of T cell proliferation in which serum-dependent
p27Kip1 down-regulation initiates a feed-forward loop
consisting of cdk2 activation, IL-2R
accumulation, and IL-2R signaling.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monoclonal antibody (PC61, PharMingen) and blocked with 3% bovine
serum albumin for 1 h at 37 °C. Coated plates were treated
consecutively as follows: serial dilutions of cell lysates, 1 h at
37 °C; 1 µg/ml biotin-conjugated IL-2R
monoclonal antibody
(7D4, PharMingen), 1 h at 4 °C; streptavidin-biotinylated peroxidase complex (PharMingen), 1 h at 37 °C. Reactions were developed for 30 min with 100 µl/well 3·5'-5·5'
tetramethylbenzidine substrate (Dako) and stopped with 0.5 N H2SO4. Absorbance was measured at
450 nm on a Titertek ELISA reader. This protocol is a modified version
of Osawa et al. (29). For analysis of cell surface markers,
T cells were incubated in phosphate-buffered saline containing 2%
mouse IgG (Dako) and fluorescein isothiocyanate- or
phycoerythrin-conjugated antibodies (PharMingen) for 30 min in
the dark at 4 °C. Corresponding isotype-specific conjugated antibody
was used for detection of nonspecific binding. Analysis was performed
on a FACScan flow cytometer with Cell Quest software (Becton Dickinson).
-32P]ATP and 100 µg/ml histone H1 (Roche Molecular
Biochemicals) for 10 min at 37 °C. Reactions were stopped by boiling
in Laemmli buffer, and proteins were separated on SDS gels.
Radiolabeled proteins were visualized by autoradiography.
blocking antibody were
obtained from PharMingen. Antibodies to IL-2R
, IL-2R
, IL-2R
,
Jak1, and phosphotyrosine were purchased from Santa Cruz
Biotechnologies. p27Kip1 and cdk2 antibodies were from
Transduction Laboratories, and Jak3 antibody and cyclin A antibody were
from Upstate Biotechnology and Neomarker, respectively.
p27Kip1-deficient mice were provided by A. Koff.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Expression at a
Post-transcriptional Level--
Although the serum dependence of IL-2R
expression has been described previously (18), the receptor
component(s) targeted by serum has yet to be identified. To address
this issue, purified splenic T cells derived from Balb/c mice
were stimulated for 30 h with mitogenic concentrations of anti-CD3
and either 10% or 0.1% serum, and the cell surface expression of
IL-2R
and IL-2R
was determined by FACS analysis. These serum
concentrations were chosen because they allow maximal (10% serum) and
minimal (0.1% serum) amounts of DNA synthesis in anti-CD3-treated T
cell cultures (Fig. 1A,
top panel, and accompanying article (43)). As shown in Fig.
1B, resting T cells contained little if any
surface-localized IL-2R
, and a higher percentage of cells expressed
this protein when stimulated with anti-CD3 and 10% as compared with
0.1% serum (76% versus 33%, respectively). Moreover,
there were significantly more receptors per cell in the population
receiving 10% serum as compared with 0.1% serum (Fig. 1C).
On the other hand, one-third of the cells in the quiescent population
were IL-2R
-positive, and anti-CD3 increased this percentage
~2-fold irrespective of serum concentration (Fig. 1B).
These data show that serum regulates the overall expression and/or the
cell surface localization of IL-2R
, but not of IL-2R
, in
TCR-stimulated T cells. Levels of IL-2R
detected by this assay were
too low to be accurately quantitated.
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Fig. 1.
Proliferation, survival, and cell surface
marker expression of purified T cells as a function of serum
concentration. A, resting T cells received the
indicated combinations of anti-CD3 (5 µg/ml), anti-CD28 (2 µg/ml),
IL-2 (1000 units/ml), and either 10% or 0.1% serum. Top
panel, triplicate cultures were pulsed with 1 µCi/ml
[3H]thymidine for 16 h prior to harvest at 48 h. Incorporation was determined by scintillation counting, and results
are expressed as counts per minute ± the standard deviation.
Bottom panel, MTT assays were done on triplicate
cultures at 40 h after stimulation as specified by the
manufacturer (R&D Systems). Results are expressed as absorbance at 550 nm ± the standard deviation. B and C,
resting T cells were stimulated with 5 µg/ml anti-CD3 and either 10%
or 0.1% serum for 30 h. The cell surface expression of CD69,
CD28, IL-2R (CD25), and IL-2R
(CD132) was
determined by FACS analysis using the appropriate antibodies.
B, data are expressed as the percentage of positive cells.
C, the histogram for IL-2R
is shown. Background
fluorescence (left peak) was determined using an isotype
control antibody, and specific fluorescence (right peak) was
determined using an IL-2R
antibody.
expression and
S phase entry in T cell populations exposed to anti-CD3 and 0.1%
serum. We also found that the serum requirement for DNA synthesis and IL-2R
expression could not be overridden by high levels of IL-2 or
by antibody to CD28 (Fig. 1A, top panel, and data
not shown). These results suggest that these signals are not
rate-limiting in cells in serum-deficient medium. Although cells
receiving anti-CD3 and 0.1% serum did not initiate DNA synthesis, they
did exhibit increased expression of the early T cell activation
markers, CD69 and CD28 (Fig. 1B). This finding is consistent
with our earlier report showing that TCR-stimulated T cells partially
traverse G0/G1 in serum-deficient medium
(43).
, ELISAs and Western blots were
performed on whole cell extracts. To expedite these studies, unfractionated splenocyte populations were used. In our previous study,
which measured the effects of serum concentration on several cell
cycle-related parameters, splenocytes and purified T cells exhibited
identical responses (43). As shown in Fig.
2, A and B
(left panel), IL-2R
was barely detectable in resting
splenocytes and was weakly induced by ConA at 10 h regardless of
serum concentration. After this time, IL-2R
continued to accumulate
in cells co-stimulated with ConA and 10% serum, with peak expression
occurring at 30-40 h. In contrast, IL-2R
essentially disappeared
from cells receiving ConA and 0.1% serum. The capacity of serum to
enhance IL-2R
expression was also evident in splenocytes
stimulated with anti-CD3 (Fig. 2B, right panel).
These findings indicate that serum increases the cell surface
expression of IL-2R
, at least in part, by increasing total cellular
levels of IL-2R
. On the other hand, serum did not affect the
expression of IL-2R
or IL-2R
(Fig. 2, B and
C).
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Fig. 2.
Effect of serum on the overall expression of
IL-2R , IL-2R
, and
IL-2R
. A, quiescent
splenocytes received 2.5 µg/ml ConA and either 10% serum
(squares) or 0.1% serum (circles) for the
indicated times. IL-2R
protein levels were determined by ELISA.
B, resting splenocytes were treated with either 2.5 µg/ml
ConA (left panel) or 5 µg/ml anti-CD3 (right
panel) and the indicated amounts of serum for the indicated times.
IL-2R
and IL-2R
protein levels were determined by Western
blotting. C, resting splenocytes were stimulated with 2.5 µg/ml ConA and either 10% or 0.1% serum for 24 h. IL-2R
,
IL-2R
, and IL-2R
levels were determined by Western
blotting.
up-regulation, we assayed the activity of the IL-2 signaling intermediate, Jak3, in splenocytes treated with ConA and either 10% or
0.1% serum. To detect active Jak3, Jak3 immunoprecipitates were
immunoblotted with antibody to phosphotyrosine. As presented in Fig.
3A, increases in IL-2R
expression were paralleled by increases in Jak3 phosphorylation.
Quiescent cells contained little if any IL-2R
or phosphorylated
Jak3. Cells receiving ConA and 0.1% serum exhibited minor increases in
IL-2R
expression and Jak3 phosphorylation, whereas both responses
were substantially elevated in cells exposed to ConA and 10% serum.
Serum (at 10%) also increased IL-2R
levels and activated Jak3 when
added to cells 20 h after addition of ConA and 0.1% serum. This
result shows that cells incubated with ConA in serum-deficient medium remain viable and retain the capacity to initiate
IL-2-dependent events when subsequently exposed to 10%
serum.
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Fig. 3.
Effect of serum on
IL2R , IL-2R
, and
IL-2R
mRNA levels and JAK activity in
splenocytes. A, splenocytes were treated continuously
with 2.5 µg/ml ConA and either 10% or 0.1% serum or were exposed to
ConA and 0.1% serum for 20 h before receiving 10% serum for an
additional 20 h (indicated by +). Cell extracts were immunoblotted
with antibody to IL-2R
or immunoprecipitated with antibody to Jak3
and immunoblotted with antibody to Jak3 or phosphotyrosine
(Jak3-P). B, quiescent splenocytes received 2.5 µg/ml ConA and either 0.1% or 10% serum for the indicated times.
mRNA levels of the IL-2R components and of L3T4 and GAPDH (loading
controls) were determined by RNase protection assay.
mRNA expression, RNase
protection assays were done on splenocytes stimulated with ConA and
10% versus 0.1% serum. As shown in Fig. 3B,
IL-2R
mRNA levels rose within 5 h of addition of ConA to
cells and remained elevated for up to 25 h regardless of serum
concentration. This finding demonstrates that maximal increases in
IL-2R
mRNA levels are not sufficient for maximal expression of
IL-2R
protein and, consequently, that serum controls IL-2R
expression at a post-transcriptional level. Similar to protein levels,
mRNA levels of IL-2R
and IL-2R
were unaffected by serum concentration.
Expression Requires cdk2
Activity--
The data presented above show that T cells do not
appreciably express IL-2R
, activate JAKs, or enter S phase when
stimulated with medium containing ConA or anti-CD3 and 0.1% serum. On
the other hand, we have found that splenocytes derived from
p27Kip1-null C57b1/6 mice are capable of initiating DNA
synthesis when exposed to a TCR agonist and either 10% or 0.1% serum
(Fig. 4 and accompanying article (43)).
This finding implies that cells lacking p27Kip1 either
efficiently express IL-2R
in serum-deficient medium or no longer
require IL-2 signaling for proliferation. To distinguish between these
alternatives, we examined IL-2R
expression and Jak1 and Jak3
activity in p27
/
splenocytes exposed to ConA and either
10% or 0.1% serum. IL-2R
was not detectable in quiescent
p27
/
splenocytes but was present at high levels in
ConA-treated p27
/
splenocytes regardless of serum
concentration (Fig. 4). Moreover, both Jak1 and Jak3 were active in
p27
/
splenocytes receiving ConA and either 10% or
0.1% serum. Similar to Balb/c splenocytes, wild-type C57b1/6
splenocytes required 10% serum for both IL-2R
accumulation and JAK
activation. These data indicate that the capacity of
p27
/
splenocytes to proliferate in serum-deficient
medium results (at least in part) from the capacity of these cells to
optimally express IL-2R
in a serum-independent manner.
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Fig. 4.
Serum-independent IL-2R
expression and JAK activation in p27Kip1-deficient
splenocytes. Quiescent p27+/+ and p27
/
splenocytes were treated with 2.5 µg/ml ConA and either 0.1% or 10%
serum for 36 h. Cell extracts were immunoblotted with antibody to
IL-2R
or immunoprecipitated with antibody to Jak1 or Jak3 and
immunoblotted with antibody to phosphotyrosine (Jak1-P,
Jak3-P). To ensure that all samples contained equal amounts
of Jak1 or Jak3, immunoprecipitates were also immunoblotted with
antibodies to these proteins. The percentage of S phase cells was
determined by FACS analysis of propidium iodide-stained cells.
expression, and
it is possible, therefore, that cdk2 activity contributes to IL-2R
expression. In support of this hypothesis, we found that IL-2R
did
not accumulate in Balb/c splenocytes treated with ConA and 10% serum
in the presence of roscovitine, a potent and selective inhibitor of
cdk2 activity (Fig. 5A and
Ref. 32). Roscovitine did not inhibit the expression of cyclin E and
thus does not nonspecifically block protein synthesis. Similar to naive
splenocytes, roscovitine also precluded IL-2R
expression, as well as
cdk2 activity, in quiescent T lymphoblasts restimulated with ConA and
10% serum (Fig. 5B).
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Fig. 5.
Inhibition of IL-2R
accumulation by roscovitine and p27Kip1
overexpression. A, resting Balb/c splenocytes were
treated for 12 h with 2.5 µg/ml ConA and 10% serum prior to
addition of either DMSO (vehicle control) or roscovitine (final
concentration, 25 µM). Cells were harvested at the
indicated times. IL-2R
and cyclin E levels were determined by
Western blotting. B, to prepare T lymphoblasts, splenocytes
were treated with 5 µg/ml ConA and 10% serum for 48 h and 10%
serum alone for an additional 48 h. Quiescent lymphoblasts then
received 2.5 µg/ml Con A and 10% serum and either Me2SO
(DMSO) or 25 µM roscovitine for the indicated
times. IL-2R
expression was determined by Western blotting, and cdk2
activity (A (H1)) was determined in cyclin A immune
complexes by in vitro kinase assay. C,
exponentially growing p27-47 fibroblasts treated with or without 1 mM IPTG for 20 h were transfected with a vector alone
(pIRES2-EGFP) or vector containing IL-2R
cDNA by LipofectAMINE
(Life Technologies). Both sets of cells were cotransfected with
-galactosidase (
-gal) under control of the
cytomegalovirus promoter. After transfection, cells were incubated with
or without IPTG for an additional 20 h. Transfection efficiency
was monitored by green fluorescence protein expression using flow
cytometry. IL-2R
protein and mRNA levels were determined by
Western blotting and Northern blotting, respectively. Equal RNA loading
was ascertained by hybridization of the membrane with
-actin
cDNA probe. cdk2 activity ((A) H1) was assessed as in
B. Data showing
-galactosidase protein levels in
mock-transfected cells and
-galactosidase-transfected cells are also
presented.
accumulation was further
demonstrable in experiments in which IL-2R
was transiently expressed
in a fibroblast cell line (termed p27-47) that inducibly expresses
p27Kip1 in response to
isopropyl-
-D-thiogalactopyranoside (IPTG) (Fig. 5C and Ref. 28). As we reported previously, induction of
p27Kip1 in sparse p27-47 cells represses cdk4 and cdk2
activity but does not result in growth inhibition (28). As shown in
Fig. 5C, IL-2R
mRNA and protein were apparent in
p27-47 cells transfected with a plasmid containing IL-2R
cDNA
but not with vector alone. Although IL-2R
mRNA levels were
approximately equal in cells treated with or without IPTG, IL-2R
protein levels were substantially lower in IPTG-treated as compared
with untreated cultures. On the other hand, levels of ectopically
expressed
-galactosidase were similar in both IPTG-treated and
untreated cultures, thus indicating that p27Kip1
overexpression does not globally inhibit protein expression. Collectively, the data in Fig. 5 show that cdk2 activity is required for IL-2R
expression. Our studies, therefore, establish a series of
events in which serum facilitates cdk2 activation, which in turn
modulates IL-2R
expression at a post-transcriptional level.
accumulation seems paradoxical. It is possible, however, that these processes are
interdependent; i.e. cdk2 activity enhances IL-2R
expression and consequent IL-2 signaling promotes cdk2 activation. To
assess the dependence of cdk2 activation on IL-2 signaling in our
system, we assayed cdk2 activity in splenocytes treated with ConA and 10% serum in the presence or absence of an IL-2R
blocking antibody. As shown in Fig. 6A, cdk2
activity was substantially lower in antibody-treated cultures, as was
IL-2R
expression. AG490, a selective inhibitor of Jak activity (36),
also blocked cdk2 activation and IL-2R
expression when presented to
cells 12 h after stimulation with ConA and 10% serum (Fig.
6B). AG490 also repressed the expression of cyclin A and
cdk2; we have shown previously that the expression of these proteins in
T cells requires cdk2 activity (43). On the other hand, AG490 had no
effect on cyclin E levels and thus does not inhibit protein expression
in general. Together, the above findings show that cdk2 activation both
results from and contributes to IL-2R
expression and that cdk2
activity, IL-2R
expression, and IL-2 signaling comprise a regulatory
loop.
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Fig. 6.
Inhibition of cdk2 activity by an
IL-2R blocking antibody and AG490.
A, resting Balb/c splenocytes were treated with 2.5 µg/ml
ConA and 10% serum in the presence or absence of an IL-2R
blocking
antibody (7.5 µg/ml) for the indicated times. Cell extracts were
immunoblotted with antibody to IL-2R
or immunoprecipitated with
antibody to cyclin E for determination of cdk2 activity (H1
(E)) by in vitro kinase assay. B, quiescent
splenocytes received 2.5 µg/ml ConA and 10% serum for 12 h,
followed by Me2SO (DMSO) (vehicle control) or 50 µM AG490 for 24 h. Levels of the indicated proteins
were determined by Western blotting. cdk2 activity (H1 (A))
was measured in cyclin A immunoprecipitates by in vitro
kinase assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and numerous studies have focused on the pathways and
promoter elements involved in this response (5). Data presented here
show that IL-2R
expression is also regulated post-transcriptionally and that serum and cdk2 play prominent roles in this process. In
splenocytes exposed to ConA and 0.1% serum, IL-2R
was only weakly
and transiently expressed. In contrast, in splenocytes receiving ConA
and 10% serum, IL-2R
expression was robust and sustained. Serum
also elevated the cell surface expression of IL-2R
in purified T
cells, and IL-2R
accumulation was accompanied by activation of the
IL-2 signaling intermediates, Jak1 and Jak3. On the other hand, serum
had no effect on IL-2R
mRNA levels or on the expression of
IL-2R
or IL-2R
at either the protein or message level. These
findings clearly show that serum selectively modulates the expression
of the
subunit of the IL-2R at a post-transcriptional level and
that serum-induced increases in IL-2R
levels are biologically relevant.
accumulation was
established using two different experimental approaches. First, endogenous IL-2R
levels were determined in T cells stimulated in the
presence of the pharmacological cdk2 inhibitor, roscovitine. We found
that roscovitine abolished the capacity of serum to increase IL-2R
levels in both ConA-treated splenocytes and ConA-treated T
lymphoblasts. Second, levels of ectopically expressed IL-2R
were
measured in p27-47 fibroblasts treated with or without IPTG. As
reported previously, these cells are stably transfected with a plasmid
that expresses p27Kip1 under the control of the lac
repressor (28). When exposed to IPTG, which alleviates lac repression,
these cells produce high amounts of p27Kip1, and
consequently, cdk2 activity is inhibited. As shown here, IPTG-induced
cdk2 inactivation also markedly repressed the expression of IL-2R
protein. In contrast, IL-2R
mRNA levels were similar in
IPTG-treated and untreated cells. These findings complement those
obtained in the roscovitine experiments and show that cdk2 activity,
like serum, modulates IL-2R
expression at a post-transcriptional level.
blocking antibody and the JAK inhibitor, AG490. On the
basis of these data and those discussed above, we propose the following
regulatory loop (Fig. 7). We suggest that this loop begins with cyclin E-cdk2 activation, which results from
persistent serum-dependent p27Kip1
down-regulation. As described by others (37-39), cyclin
E-cdk2 complexes, together with cyclin D-containing complexes,
phosphorylate Rb, and thus allow the E2F-mediated transcription of
cyclin A and the consequent formation of cyclin A-cdk2 complexes. As
shown here, activation of cyclin E-cdk2 also leads to the
post-transcriptional accumulation of IL-2R
and the subsequent
induction of IL-2-mediated events, as exemplified by JAK activation.
IL-2 signaling pathways, in turn, further optimize and sustain cyclin
E-cdk2 and cyclin A-cdk2 activity. At this point in the cycle, it is
likely that both of these activities promote the continued expression
of IL-2R
.
View larger version (8K):
[in a new window]
Fig. 7.
Interdependence of cdk2 activation and IL-2
signaling. TCR agonists and serum induce a persistent loss of
p27Kip1, which leads sequentially to cyclin E-cdk2
activation, post-transcriptional IL-2R expression, IL-2 signaling,
and expression and activation of cyclin A-cdk2 complexes. At this
point, both cyclin E-cdk2 and cyclin A-cdk2 activities promote
continued IL-2R
expression and subsequent events. Treatment of cells
with roscovitine, anti-IL-2R, or AG490 stops the cycle at the point
indicated and results in the inhibition of all events in the
cycle.
The capacity of serum to facilitate splenocyte proliferation by
initiating the regulatory loop outlined in Fig. 7 is clearly demonstrated by our studies on p27Kip1-deficient
splenocytes. In these cells, cdk2 activation, IL-2R accumulation,
and JAK activation were serum-independent, as was S phase entry (Fig. 4
and accompanying article (43)). In p27
/
cells, ConA was
still required for transcriptional up-regulation of IL-2R
and for
activation of D cyclin complexes and consequent expression of cyclin A
(43). It is possible that the more proximal target of serum is IL-2R
expression (and consequent IL-2 signaling) rather than
p27Kip1 down-regulation (and consequent cdk2 activation).
However, in support of our model, it is noted that submaximal increases
in cdk2 activity have been observed in T cells treated with TCR
agonists (and 10% serum) in conditions in which IL-2 is not produced
(40, 41). Thus, we propose that ConA activates cyclin E-cdk2 to a small
extent in a serum-dependent but IL-2-independent manner and
that this limited increase in activity is sufficient to set the cycle
in motion.
Although translational regulation of IL-2R has been reported
previously (42), our studies at present do not distinguish between an
effect of cdk2 activity on the synthesis versus the stability of IL-2R
. Our transfection data indicate that the
information required for maximal IL-2R
expression is contained
within the IL-2R
coding region and a 123-bp upstream sequence. As
described previously (42), this upstream sequence contains a
translational start codon and an in-frame stop codon that are thought
to reduce translational efficiency by causing leaky scanning. Whether
this process is influenced by cdk2 activity remains to be determined. As a final point, we note that many of the effects described here have
also been observed in splenocytes receiving ConA and (rather than
serum) a serum substitute consisting of insulin, selenium, and
transferrin.2
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ACKNOWLEDGEMENTS |
---|
We thank Baoky Chu for technical assistance, Nancy Olashaw for manuscript preparation, Mary Zhang for preparation of the p27-47 cells and for assistance in the experiments involving these cells, and Andy Koff for p27Kip1-deficient mice. We also acknowledge the helpful service of the Flow Cytometry and Molecular Imaging Core Laboratories at the Moffitt Cancer Center.
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FOOTNOTES |
---|
* This work was supported by the Cortner-Couch Endowed Chair for Cancer Research and National Institutes of Health Grants CA72694 and CA67360.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.
¶ To whom correspondence should be addressed: H. Lee Moffitt Cancer Center, 12902 Magnolia Dr., Tampa, FL 33612. Tel.: 813-979-3887; Fax: 813-979-3893; E-mail: pledgerw@moffitt.usf.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100037200
2 S. Mohapatra and W. J. Pledger, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T cell
receptor;
IL-2, interleukin-2;
IL-2R, interleukin-2 receptor;
JAK, Janus kinase;
STAT, signal transducer and activator of transcription;
CDK, cyclin-dependent kinase;
FACS, fluorescence-activated
cell sorting;
ELISA, enzyme-linked immunosorbent assay;
bp, base pair(s);
IPTG, isopropyl--D-thiogalactopyranoside;
ConA, concanavalin A.
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