 |
INTRODUCTION |
T cell proliferation, and the consequent expansion of T cell
populations, are fundamental events in the generation of an immune response. In vivo, T cell mitogenesis is initiated by the
interaction of T cell receptors
(TCRs)1 with peptides coupled
to major histocompatibility antigen complexes located on the surface of
antigen-presenting cells (1). In vitro, the actions of
natural ligands are mimicked by a variety of agents including anti-CD3,
calcium ionophore, and lectins such as concanavalin A (ConA) and
phytohemagglutinin. TCR stimulation allows resting T cells to enter
G1 and induces the expression of interleukin-2 (IL-2) and
the
subunit of the IL-2 receptor (IL-2R
) (2-4). In the presence
of IL-2, IL-2R
combines with IL-2R
and IL-2R
to form the high
affinity IL-2R complex, which elicits the secondary signals required
for continued G1 traverse and S phase entry. T cell
proliferation also requires additional factors, many of which are
routinely provided to cultured T cells in the form of serum (5).
Although previous studies have shown that serum enhances the production
of IL-2 and IL-2R
(5), the potential contribution of serum to other
cell cycle-regulatory processes has yet to be explored.
In T cells, as in other cell types, cell cycle traverse is governed by
the ordered activation of cyclin-dependent kinases (CDKs)
(6). Activation of the CDKs requires their interaction with cyclins,
whose levels fluctuate during the cell cycle, and their phosphorylation
at specific threonine residues by constitutively expressed
cyclin-activating kinases. CDKs also interact with a group of proteins
collectively termed CDK inhibitors (CKIs). CKI levels, like cyclin
levels, vary during the cell cycle and thus contribute to the
periodicity of CDK activation (7). Two classes of CKIs have been
defined: the INK proteins, which target cdk4 and cdk6, and the Cip/Kip
proteins, which inactivate cdk2-containing complexes (8, 9).
Traverse of G0/G1 and entry into S phase is
controlled by the sequential activation of complexes containing the D
cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2.
Addition of mitogens to G0-arrested cultures induces the
expression of the D cyclins (D1, D2, and D3) and, in some cell types
(e.g. T cells), of cdk4 and cdk6 (10-13). Mitogenic
stimulation also down-regulates p27Kip1, a Cip/Kip protein
that accumulates in quiescent cells (14-17). D cyclin complexes become
active in mid G1 and phosphorylate the anti-oncogene Rb
(18). Pre-existing cyclin E-cdk2 complexes become active after
p27Kip1 levels decrease and further phosphorylate Rb in
late G1. When phosphorylated by these kinases, Rb no longer
represses the activity of the E2F transcription factors, and a variety
of genes, including those encoding cyclins E and A, are expressed
(19-21). At this point, cells pass through the restriction point in
late G1 and in a manner dependent on cyclin E-cdk2 and
cyclin A-cdk2 activity enter and traverse S phase (22).
The drop in p27Kip1 levels and the consequent activation of
pre-existing and newly formed cyclin E-cdk2 and cyclin A-cdk2 complexes are critical aspects of G0/G1 traverse. For
example, prevention of p27Kip1 down-regulation by agents
such as rapamycin and cyclic AMP blocks cdk2 activation and cell cycle
traverse, as does ectopic expression of p27Kip1 (15,
23-25). Conversely, ablation of p27Kip1 function in both
whole animals and cultured cells has been shown to promote
proliferation by impeding G0 arrest (26, 27). The mitogen-induced decrease in p27Kip1 levels is a complex and
not fully understood process that involves translational inhibition,
accelerated degradation, and perhaps also transcriptional repression
(12, 16, 28-31). Additional studies have shown that
p27Kip1 down-regulation is not sufficient for cdk2
activation, which also requires sequestration of residual
p27Kip1 molecules by complexes containing the D cyclins and
their CDK partners (7). Whether cyclin D/CDK activity is also repressed by p27Kip1 is unclear, because past reports differ in
this regard (24, 32).
Previous studies have shown that TCR agonists down-regulate
p27Kip1 and activate cdk6 and cdk2 when added to resting T
cells (11-13, 33). However, these studies were done on cells
stimulated in the presence of 10% serum. Thus, they leave open the
possibility that at least some of these responses are induced by TCR
agonists in a serum-dependent manner. This is an important
issue, because it addresses a potential mechanism by which serum might
promote T cell proliferation. In the studies described here, we
examined the contribution of serum to cyclin/CDK activation in primary splenocytes and purified T cells exposed to TCR agonists such as ConA
or anti-CD3. We show that serum acts in conjunction with mitogenic
amounts of ConA or anti-CD3 to induce the sustained down-regulation of
p27Kip1 and the activation of cdk2-containing complexes.
Consistent with the premise that serum facilitates T cell proliferation
by targeting p27Kip1, we also find that splenocytes lacking
p27Kip1 no longer require serum for cdk2 activation or S
phase entry.
 |
MATERIALS AND METHODS |
Isolation of Splenocytes and T Cells--
A single cell
suspension of mouse spleen cells was prepared by passage through nylon
mesh. Red cells were depleted using a whole blood erythrocyte lysing
kit (R&D Systems). For isolation of purified T cells, spleen cell
suspensions were loaded onto T cell enrichment columns, and T cells
were purified via high affinity negative selection as specified by the
manufacturer (R&D Systems). Splenocytes and purified T cells were
plated at 107 cells/ml and 5 × 106
cells/ml, respectively, in RPMI 1640 supplemented with 2 mM
L-glutamine, 50 units/ml penicillin, and 10% fetal bovine serum.
Cell Cycle Analysis--
For assessment of DNA synthesis,
triplicate cultures in microtiter plates were pulsed with 1 µCi/ml
[3H]thymidine (PerkinElmer Life Sciences) for the
indicated times. Incorporation was determined by scintillation
counting, and each experiment was repeated at least twice. To determine
cell cycle position, cells were washed with cold phosphate-buffered
saline (PBS) and fixed with 70% ethanol overnight at 4 °C. Fixed
cells were resuspended in PBS containing 1% bovine serum albumin,
0.5% Tween 20, 1 µg/ml propidium iodide, and 1 µg/ml RNase A and
incubated at room temperature for 2 h. Total DNA content was
analyzed on an EPICS 753 flow cytometer (Coulter Electronics,
Inc.).
Western Analysis--
Cells were rinsed with cold PBS and lysed
in buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin,
0.5 mM NaF, and 0.1 mM sodium vanadate (lysis
buffer). After a 30-min incubation, insoluble material was removed by
centrifugation. Proteins (30 µg) were resolved by SDS-polyacrylamide
gel electrophoresis and transferred to nitrocellulose membrane or
polyvinylidene difluoride membrane for cdk2. Blots were blocked in PBST
(PBS plus 0.05% Tween) containing 5% instant milk and incubated with
primary antibody in PBST. Proteins recognized by the antibody were
detected by enhanced chemiluminescence using a horseradish
peroxidase-coupled secondary antibody as specified by the manufacturer (Pierce).
Immunoprecipitation and Kinase Assay--
Cell extracts (80 µg
of protein in lysis buffer) were incubated with antibody to the
indicated cyclin or CDK for 4-12 h at 4 °C and subsequently with
protein A-agarose beads. Immune complexes were washed twice with lysis
buffer and once with either histone kinase buffer (50 mM
Tris (pH 7.4), 10 mM MgCl2, 1 mM
dithiothreitol) or Rb kinase buffer (50 mM Tris (pH 7.4),
10 mM MgCl2, 5 mM
MnCl2, 1 mM dithiothreitol). Washed complexes
were incubated in 15 µl of kinase buffer containing 20 µM ATP, 0.1 µCi/ml [
-32P]ATP, and
either 100 µg/ml histone H1 (Roche Molecular Biochemicals) for 10 min
at 37 °C or 2.5 µg/ml GST-Rb for 30 min at 30 °C. Reactions were stopped by boiling in Laemmli buffer and proteins were separated by SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were
visualized by autoradiography. For p27Kip1 immunodepletion,
cell extracts (150 µg of protein) were incubated with antibody to
p27Kip1 (or for mock depletion, with preimmune serum) for
4 h at 4 °C, and immune complexes were removed by
centrifugation with protein A-agarose beads. Removal of
p27Kip1 was confirmed by Western blotting.
Materials--
ConA and anti-CD3 were obtained from Sigma
Chemical Co. and PharMingen, respectively. Antibodies to cyclin D3,
cyclin E, and cdk6 were purchased from Santa Cruz Biotechnology.
p27Kip1 and cdk2 antibodies were from Transduction
Laboratories. Cyclin A antibody was provided by E. Leof and GST-Rb by
D. Cress. p27kip-deficient mice were obtained from by A. Koff (34), and p21Cip1-deficient mice were obtained from T. Jacks (35).
 |
RESULTS |
T Cells Require Both TCR Agonists and Serum for
G0/G1 Traverse--
Initial experiments
confirmed that TCR agonists and serum act in a concerted manner to
stimulate the proliferation of primary splenocytes and purified T
cells. The cells used in these experiments were derived from Balb/c
mice. As assessed by incorporation of [3H]thymidine into DNA, resting splenocyte populations
did not initiate DNA synthesis when treated for 40 h with serum
(0.1-10%) alone or with 0.1% serum and various concentrations of
ConA (Fig. 1A) or anti-CD3
(Fig. 1B). However, co-stimulation of splenocytes with
optimal amounts of serum (10%) and either ConA (2.5 µg/ml) or
anti-CD3 (10 µg/ml) produced a significant increase in
[3H]thymidine incorporation. Anti-CD3 and serum also
synergistically stimulated the cell cycle traverse of T cell-enriched
populations containing greater than 95% T cells (Fig. 1C).
Maximal [3H]thymidine incorporation occurred in T cell
cultures receiving 5 µg/ml anti-CD3 and 10% serum. The lower amount
of [3H]thymidine incorporation seen in cultures receiving
10 µg/ml as compared with 5 µg/ml anti-CD3 presumably reflects the
fact that many of the cells in the former condition have already exited S phase at the time of the pulse (36).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Cell cycle progression of Balb/c splenocytes
and T cells stimulated with TCR agonists and serum. A,
quiescent splenocytes were exposed to the indicated concentrations of serum
and ConA for 36 h and pulsed with 1 µCi/ml
[3H]thymidine for an additional 4 h. B,
as in A with the exception that splenocytes received
anti-CD3 instead of ConA. The concentrations of plate-bound anti-CD3
are indicated. C, resting T cells were stimulated with the
indicated amounts of plate-bound anti-CD3 and either 0.1% or 10%
serum for 50 h and pulsed with [3H]thymidine for an
additional 10 h. A-C, data are plotted as counts per
minute ± the standard deviation. D, quiescent
splenocytes were treated with 2.5 µg/ml ConA and either 0.1%
or 10% serum. E, quiescent splenocytes were
exposed to 10 µg/ml anti-CD3 and either 0.1% or 10% serum.
F, quiescent T cells were incubated with 5 µg/ml anti-CD3
and either 0.1% or 10% serum. D-F, cells were harvested
at the indicated times and cell cycle position was determined by
fluorescence-activated cell sorting analysis. The percentage of cells
in S+G2/M is shown.
|
|
Serum did not simply substitute for IL-2, because ConA (2.5 µg/ml)
and IL-2 (500 units/ml) did not induce DNA synthesis in medium
containing 0.1% serum (data not shown). When exposed to 10% serum and
either ConA or anti-CD3, quiescent splenocytes and T cells entered S
phase after a lag of 20-24 h (Fig. 1, D, E, and
F). As determined by fluorescence-activated cell sorting
analysis, 30-40% of the cells were in S phase or G2/M at
40 h after stimulation. In contrast, cultures stimulated in medium
containing 0.1% serum did not appreciably initiate DNA synthesis
during the experimental period. These findings show that mixed
splenocyte populations respond similarly to ConA and anti-CD3 and
behave similarly to purified T cells.
Serum Is Required for Sustained p27Kip1 Down-regulation
and cdk2 Activation--
The next set of experiments addressed the
possibility that serum promoted T cell proliferation by facilitating
cyclin/CDK activation. In these experiments, quiescent splenocytes and
T cells were stimulated with ConA or anti-CD3 in the presence of 10%
or 0.1% serum. These serum concentrations were chosen because they
allow maximal and minimal S phase entry, respectively (see Fig. 1).
Serum at 0.1% (rather than no serum) was used as the negative control,
because serum at this concentration prevents adsorption of IL-2 to
culture dishes (5). However, events induced by ConA or anti-CD3 in
medium containing 0.1% serum were also induced by these agents in
serum-free medium (data not shown) and thus are considered
serum-independent. Cyclin, CDK, and p27Kip1 levels were
determined by Western blotting, and cyclin/CDK activity was assessed by
in vitro kinase assay. It is noted that treatment of resting
cells with 10% serum in the absence of TCR agonist had no effect on
the responses examined below (data not shown).
p27Kip1 was present in quiescent splenocytes, and its
levels decreased to a similar extent in cells stimulated with ConA and
either 10% or 0.1% serum for 10 h (Fig.
2A; 68% versus
51% decrease, respectively). After this time, p27Kip1
levels fell much more precipitously in cultures receiving ConA and 10%
as compared with 0.1% serum. For example, at 25 h after stimulation, p27Kip1 levels were 10-fold lower in the
serum-supplemented culture than in the serum-deficient culture.
Although the kinetics differed somewhat, the pattern of
p27Kip1 down-regulation was similar in ConA-treated
splenocytes (Fig. 2B), anti-CD3-treated splenocytes (Fig.
2C), and anti-CD3-treated T cells (Fig. 2D). Note
that p27Kip1 levels re-accumulated at later times in cells
stimulated in serum-deficient medium (Fig. 2, A-D). These
findings show that p27Kip1 down-regulation in T cells is a
two-stage process, consisting of serum-independent and
serum-dependent segments.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 2.
Requirement for serum for persistent
p27Kip1 down-regulation and cdk2 activation.
A, resting splenocytes were incubated with 2.5 µg/ml ConA
and 0.1% or 10% serum for the indicated times. p27Kip1
levels were determined by Western blotting, and relative amounts of
p27Kip1 were quantitated by densitometric scanning of the
p27Kip1 bands on the Western blot. B,
splenocytes were treated as in A. C, quiescent splenocytes
were exposed to 10 µg/ml anti-CD3 and either 10% or 0.1% serum.
D, quiescent T cells received 5 µg/ml anti-CD3 and either
10% or 0.1% serum. B-D, levels of the indicated proteins
were determined by Western blotting. cdk6 activity ((Cdk6)
Rb) was measured in cdk6 immune complexes by in vitro
kinase assay using GST-Rb as substrate. cdk2 activity was measured in
cyclin E ((E) H1) and cyclin A ((A) H1) immune
complexes using histone H1 as substrate. Panels depicting
the results of kinase assays are indicated by an
asterisk.
|
|
Cyclin D3, cdk6, and cyclin E were expressed at low levels in resting
cells and were substantially up-regulated by ConA or anti-CD3
regardless of serum concentration (Fig. 2, B-D). These agents also increased cyclin D3-cdk6 activity in cells receiving either
10% or 0.1% serum. Cyclin A, on the other hand, was barely detectable
in quiescent cells and was up-regulated by ConA or anti-CD3 to a
greater extent in medium containing 10% as compared with 0.1% serum
(Fig. 2, B-D). cdk2 expression was even more dependent on
serum; elevations in the basal levels of cdk2 were seen in cells
treated with ConA and 10% but not 0.1% serum (see Fig.
3B). In cells stimulated in
medium containing 10% serum, increases in the expression of cyclin E,
cyclin A, and cdk2 were paralleled by increases in the activities of
both cyclin E-cdk2 and cyclin A-cdk2. In contrast, these complexes were
not active in cells receiving ConA or anti-CD3 in serum-deficient
medium (Fig. 2, B-D). Collectively, these findings
demonstrate that TCR agonists induce an initial but transient loss of
p27Kip1, the expression of cyclin D3, cyclin E, and cdk6,
and the activation of cyclin D3-cdk6 complexes in a serum-independent
manner. In contrast, serum is clearly required for maximal and
sustained p27Kip1 down-regulation, maximal expression of
cyclin A and cdk2, and activation of cyclin E-cdk2 and cyclin A-cdk2
complexes. Because cdk2 activity is obligatory for cell cycle traverse,
it is apparent from these studies that serum promotes T cell
proliferation by enabling this response.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Restoration of cdk2 activation and S phase
entry by serum replenishment. A, quiescent splenocytes
received 2.5 µg/ml ConA and either 0.1% ( ) or 10% ( ) serum
for the indicated times. Parallel cultures were treated with ConA and
0.1% serum for 20 h, at which point (denoted by arrow)
serum was added to a final concentration of 10% ( ). Cells were
pulsed with 1 µCi/ml [3H]thymidine for 4 h prior
to harvest. Data are plotted as counts per minute ± the standard
deviation. B, quiescent splenocytes were treated with 2.5 µg/ml ConA and either 10% or 0.1% serum for 24 or 40 h.
Parallel cultures were exposed to ConA and 0.1% serum for 20 h
prior to addition of serum to a final concentration of 10% (denoted by
"+"). Levels of p27Kip1, cyclin A, and cdk2 were
determined by Western blotting. Kinase activity was determined in
cyclin A (A (H1)) or cyclin E (E (H))
immunoprecipitates using histone H1 as substrate. Panels
depicting the results of kinase assays are indicated by an
asterisk.
|
|
Serum Replenishment Restores cdk2 Activation and S Phase Entry in
ConA-treated Splenocytes--
The data presented above demonstrate
that serum is required for events that occur in late
G0/G1 (e.g. cdk2 activation) but not
for events that begin earlier in G0/G1
(e.g. cdk6 activation). Consistent with these results, we
found that quiescent splenocytes initiated but did not complete
G0/G1 traverse in serum-deficient medium. In
these experiments, resting splenocytes were treated continuously with
ConA and either 10% or 0.1% serum, or were pretreated with ConA and
0.1% serum for 20 h prior to addition of 10% serum. DNA
synthesis was assessed by [3H]thymidine incorporation.
Similar to the data in Fig. 1D, splenocytes co-treated with
ConA and 10% serum entered S phase after an approximate 20 h lag
(Fig. 3A). In contrast, cultures receiving ConA and 0.1% serum remained (for the most part) in G0/G1.
However, when exposed to 10% serum, serum-deprived cultures initiated
DNA synthesis within 8-12 h. This observation indicates that
ConA-treated splenocytes partially traverse
G0/G1 in medium containing 0.1% serum and that the serum-dependent "checkpoint" is temporally located
in mid to late G1. In accord with its capacity to induce S
phase entry, serum replenishment also elevated the expression of cyclin
A and cdk2, maximized the loss of p27Kip1, and restored
both cyclin A-cdk2 and cyclin E-cdk2 activities (Fig. 3B).
The capacity of splenocytes pretreated with ConA and 0.1% serum to
elicit these responses when exposed to 10% serum indicates that a
substantial portion of the population remains viable in serum-deficient medium.
Cyclin-cdk2 Complexes in Splenocytes Stimulated with ConA and 0.1%
Serum Are Associated with p27Kip1--
Our data suggest
that serum elicits cdk2 activation by persistently down-regulating
p27Kip1 and by elevating the expression of cdk2 and cyclin
A. Such conditions would favor the formation of cyclin-cdk2 complexes
that do not contain p27Kip1 (designated
"p27Kip1-free") and thus are catalytically active. On
the other hand, due to higher p27Kip1 levels and lower cdk2
and cyclin A levels, inactive p27Kip1-associated complexes
would predominate in cells receiving ConA and 0.1% serum. These
predictions are confirmed by the data presented in Fig.
4. In these experiments, the amounts of
total and p27Kip1-free cyclin A-cdk2 complexes were
determined in unfractionated and p27Kip1-immunodepleted
cell lysates, respectively. As shown in Fig. 4, most if not all of the
cyclin A-cdk2 complexes in cells stimulated with ConA and 10% serum
for 24 or 40 h were present in p27Kip1-depleted
extracts and thus are not bound to p27Kip1 (compare
lanes 3 and 9, and 6 and
12). Cyclin A-cdk2 complexes were not detected in cells
receiving ConA and 0.1% serum for 24 h (lane 1) but
were present at low levels in cells treated in this manner for 40 h (lane 4). However, the majority of these complexes were
associated with p27Kip1 (i.e. were removed by
p27Kip1 antibody; compare lanes 4 and
10). Addition of serum (to 10%) to cells treated with ConA
and 0.1% serum for 20 h increased both the total amount of cyclin
A-cdk2 and the percentage of cyclin A-cdk2 complexes that do not
contain p27Kip1 (lanes 5 and 11).
Similar results were obtained in experiments examining the interaction
of p27Kip1 with cyclin E-cdk2 complexes (data not shown).
These findings demonstrate that the lack of cdk2 activity in
splenocytes stimulated in serum-deficient medium results from the
presence of p27Kip1 in cdk2-containing complexes.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Association of p27Kip1 with
cyclin A-cdk2 complexes in splenocytes stimulated in serum-deficient
medium. Quiescent splenocytes were stimulated with 2.5 µg/ml
ConA and either 0.1% or 10% serum, or received 10% serum 20 h
after addition of ConA and 0.1% serum (denoted by "+"). Cells were
harvested at 24 or 40 h after ConA addition. Cell lysates were
cleared with either preimmune serum (lanes 1-6) or antibody
to p27Kip1 (lanes 7-12), immunoprecipitated
with antibody to cyclin A, and immunoblotted with antibody to cyclin A
or cdk2. Lanes 1-6 represent total cyclin A-cdk2 complexes,
whereas lanes 7-12 represent cyclin A-cdk2 complexes that
are not bound to p27Kip1 (designated
"free").
|
|
Serum-independent cdk2 Activation and Cell Cycle Traverse in
p27kip1-deficient Splenocytes--
Previous studies have
shown that T cells derived from p27Kip1-null mice exhibit
constitutive cyclin E-cdk2 activity (37, 38). Because our data indicate
that serum promotes T cell proliferation by allowing cdk2 activation,
it was of interest to determine if splenocytes lacking
p27Kip1 initiated DNA synthesis in a serum-independent
manner. For these experiments, we used splenic cells derived from
C57b1/6 mice that express an N-terminally truncated form of
p27Kip1 that does not interact with or inhibit the activity
of cyclin-cdk2 complexes (34). Initial experiments characterized the
"cyclin/CDK profiles" of p27+/+, p27+/
,
and p27
/
splenocytes. In all three populations, and
similar to results obtained with Balb/c splenocytes (Fig.
2B), levels of cyclin D3 and cdk6 and of cyclin D3-cdk6
activity increased in response to ConA in a serum-independent manner
(data not shown). In unstimulated p27
/
cells, levels of
cdk2 and of cyclin E-cdk2 activity were comparable to those of
p27+/+ cells treated with ConA and 10% serum (Fig.
5A; compare lanes 3 and 8). Thus, in the absence of p27Kip1, cdk2 is
constitutively expressed and, as described previously, cyclin E-cdk2 is
constitutively active (37, 38). In contrast, levels of cyclin A and of
cyclin A-cdk2 activity were not substantially elevated in unstimulated
p27
/
cells. However, addition of ConA and 0.1% serum
to p27
/
cells resulted in increases in both cyclin A
expression and associated activity that were comparable to those seen
in p27+/+ cells receiving ConA and 10% (but not 0.1%)
serum (compare lanes 3 and 11). Thus, although
TCR stimulation is still required, abrogation of p27Kip1
function renders cyclin A expression and cyclin A-cdk2 activation serum-independent. The need for TCR signaling for these events presumably reflects the dependence of cyclin A expression (and hence
cyclin A-cdk2 activity) on cyclin D3-cdk6 activity and consequent Rb
phosphorylation and E2F activation (18, 20).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 5.
Serum-independent cdk2 activation and S phase
entry in p27Kip1-deficient splenocytes. A,
p27+/+, p27+/ , and p27 /
splenocytes received 2.5 µg/ml ConA and either 0.1% or 10% serum
for 20-40 h. Levels of p27Kip1, cyclin A, and cdk2 were
determined by Western blotting. cdk2 activity was determined in cyclin
A ((A) H1) and cyclin E ((E) H1) immune complexes
by in vitro kinase assay. Panels depicting the
results of kinase assays are indicated by an asterisk.
B, quiescent p27+/+, p27+/ , and
p27 / splenocytes were stimulated with 2.5 µg/ml ConA
and 0.1% or 10% serum for 36 h, and pulsed with
[3H]thymidine for an additional 4 h. Data are
plotted as the percent of maximal incorporation (counts per minute ± standard deviations).
|
|
p27+/
cells behaved similarly to p27
/
cells in terms of cyclin A expression and cyclin A-cdk2 activity (Fig.
5A, lanes 4-7). On the other hand,
p27+/
cells, unlike p27
/
cells, required
ConA (but not 10% serum) for cdk2 up-regulation and cyclin E-cdk2
activation. p27Kip1, however, was present in quiescent
p27+/
cells at levels comparable to those seen in
quiescent p27+/+ cells (compare lanes 1 and
4), and its down-regulation was mediated by ConA
(lanes 5-7). Interestingly, ConA and 0.1% serum decreased p27Kip1 levels in p27+/
cells to an extent
greater than that observed in p27+/+ cells stimulated with
ConA and 10% serum (compare lanes 3 and 7).
Together, the findings in Fig. 5A indicate that ablation of p27Kip1 function in splenocytes results in a number of
complex and presumably interdependent changes in cyclin/CDK activity.
However, it is clear from these studies that loss of
p27Kip1 (either one or both copies) eliminates the serum
dependence of splenocytes for cdk2 activation.
When stimulated with ConA and 10% serum, splenocytes isolated from
p27+/+, p27+/
, and p27
/
mice
all exhibited a 9-fold increase in [3H]thymidine
incorporation as compared with p27+/+ splenocytes receiving
ConA and 0.1% serum (Fig. 5B). The magnitude of this
increase was similar to that obtained using splenocytes from Balb/c
mice (Fig. 1A). p27+/
and p27
/
cells did not proliferate in response to 10% serum alone and thus
still required TCR signaling (data not shown). However, in contrast to
Balb/c and p27+/+ splenocytes, both p27+/
and
p27
/
populations displayed a 4- to 5-fold increase in
[3H]thymidine incorporation in response to ConA and 0.1%
serum. Thus, the loss of one or both copies of p27Kip1
allows substantial splenocyte proliferation in conditions in which
serum is limiting. Although additional actions of serum cannot be
excluded, these results demonstrate that p27Kip1 and cdk2
are key components of the serum-initiated pathway that functions in
concert with TCR signaling to promote the cell cycle traverse of
resting splenocytes.
Similar experiments were done on splenocytes lacking the
p27Kip1-related CKI, p21Cip1. Like
p27Kip1, p21Cip1 is a potent inhibitor of cdk2
activity and, consequently, of cell cycle traverse (9). However, in
splenocytes, loss of p21Cip1 did not mimic loss of
p27Kip1. As shown in Fig.
6A, p21Cip1
ablation did not result in the constitutive expression of cdk2 or the
constitutive activation of cyclin E-cdk2 complexes. Moreover, cyclin A
was not up-regulated and cyclin A-cdk2 complexes were not active in
p21
/
splenocytes receiving ConA and 0.1% serum.
p21
/
splenocytes also failed to initiate DNA synthesis
when exposed to ConA in serum-deficient medium (Fig. 6B).
All of the above responses were, however, efficiently induced in
p21
/
cells by ConA and 10% serum. These results
demonstrate that p21Cip1 does not contribute significantly
to the re-entry of quiescent T cells into the proliferative cycle.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 6.
Lack of effect of p21Cip1 loss on
cdk2 activation and S phase entry. A, primary
splenocytes were prepared from p21+/+ and
p21 / B6129 mice and treated with 2.5 µg/ml ConA and
either 0.1% or 10% serum for the indicated times. Levels of
p27Kip1, cyclin A, and cdk2 were determined by Western
blotting. cdk2 activity was determined in cyclin A ((A) H1)
immune complexes by in vitro kinase assay. Panels
depicting the results of kinase assays are indicated by an
asterisk. B, quiescent p21+/+ and
p21 / splenocytes were stimulated with 2.5 µg/ml ConA
and either 0.1% or 10% serum for 36 h and pulsed with
[3H]thymidine for an additional 4 h. Data are
plotted as the percent of maximal incorporation (counts per minute ± standard deviations).
|
|
cdk2 Activity Is Required for Enhanced Expression of
cdk2--
Data presented above show that conditions that promote cdk2
activation also enhance cdk2 expression. For example, treatment of
wild-type splenocytes with TCR agonists and 10% (but not 0.1%) serum
resulted in both cdk2 activation and maximal cdk2 expression. Conversely, in splenocytes lacking p27Kip1, cyclin E-cdk2
complexes were constitutively active, and cdk2 was expressed at high
levels in the absence of stimulus. These observations suggest that
increases in cdk2 expression might result from as well as contribute to
cdk2 activation. In accord with this premise, we found that ConA and
10% serum did not increase cdk2 protein levels when presented to
Balb/c splenocytes in combination with roscovitine, a pharmacological
agent that selectively inhibits cdk2 activity (Fig.
7) (39). Roscovitine also prevented the up-regulation of cyclin A in splenocytes exposed to ConA and 10% serum. This observation is not surprising given the dependence of
cyclin A expression on cdk2 activity, Rb phosphorylation, and E2F
transactivation (18, 20). On the other hand, cyclin E levels increased
to a comparable extent in cells stimulated in the presence and absence
of roscovitine. This finding is consistent with the capacity of ConA to
increase cyclin E expression in serum-deficient medium (i.e.
in conditions in which cdk2 is not active). Moreover, this finding
indicates that cyclin E expression in this system is
E2F-independent.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of cdk2 expression by
roscovitine. Resting Balb/c splenocytes were cultured for 12 h in the presence of 2.5 µg/ml ConA and 10% serum prior to addition
of either ME2SO (vehicle control) or roscovitine (final
concentration, 25 µM). Cells were harvested at the
indicated times. Levels of cyclin E, cyclin A, and cdk2 were determined
by Western blotting.
|
|
 |
DISCUSSION |
Previous studies have shown that the proliferation of cultured T
cells in the presence of TCR agonists requires either the addition of
serum or the use of a complex medium specifically formulated for this
purpose (5). As shown here, the percentage of resting T cells that
initiated DNA synthesis in response to ConA or anti-CD3 was clearly
dictated by the amount of serum in the culture medium. Maximal S phase
entry was achieved with 10% serum, whereas 0.1% serum was
ineffective. The mechanism by which serum promotes T cell proliferation
is, however, incompletely understood. Our studies examined the effects
of serum on the expression and/or activity of several key cell cycle
regulators (e.g. cyclins, CDKs, CKIs) and provide a
biological basis for this phenomenon. Using both splenocyte and
purified T cell populations, we show that serum facilitates cdk2
activation by acting with TCR agonists to maximally and persistently
down-regulate p27Kip1. We suggest that the capacity of
serum to modulate p27Kip1 levels accounts, in large part,
for the serum dependence of T cell proliferation. p27Kip1
has also been implicated in the growth regulation of anergic T cells
(40) and T cells treated with co-stimulatory factors (31).
In splenocytes and purified T cells, p27Kip1
down-regulation was biphasic. The first phase was induced by TCR
agonists such as ConA and anti-CD3 in a serum-independent manner. This
initial loss of p27Kip1 was not sustained and did not
result in cdk2 activation despite the presence (albeit limited) of cdk2
and its cyclin partners in cells stimulated in serum-deficient medium.
The second phase of p27Kip1 down-regulation required both
TCR agonists and 10% serum. This phase was greater in extent and more
prolonged than the first phase and was accompanied by the activation of
both cyclin E-cdk2 and cyclin A-cdk2 complexes. These findings
demonstrate that the capacity of serum to maintain p27Kip1
levels below a critical threshold correlates with its capacity to
activate cdk2. Whether similar or distinct mechanisms account for the
first and second phases of p27Kip1 down-regulation is not
known at present. It is possible that serum maintains
p27Kip1 at low levels, in part, by a positive feedback loop
in which active cdk2 complexes phosphorylate p27Kip1 and
thus accelerate its degradation (29, 30).
Additional studies demonstrated that p27Kip1 was directly
responsible for the lack of cdk2 activity in T cells stimulated with ConA or anti-CD3 in serum-deficient medium. Results of these studies showed that most if not all of the cdk2 complexes in these cells contained p27Kip1. In contrast, in cells co-treated with
ConA and 10% serum, few if any cdk2 complexes were associated with
p27Kip1. Thus, removal of p27Kip1 from cdk2
complexes apparently requires decreases in p27Kip1 levels
greater than those induced by TCR agonists alone. In addition to
reductions in overall p27Kip1 levels, cdk2 activation also
requires the sequestration of residual p27Kip1 molecules by
D cyclin complexes. However, we found that cyclin D3 levels were
comparable in cells stimulated with TCR agonists and either 10% or
0.1% serum, as were cdk6 levels. Thus, it is likely that the D
cyclin/CDK reservoir is of similar size in both conditions and that
comparable amounts of p27Kip1 are sequestered. It is noted
that cdk6 is the predominant D cyclin partner in T cells (33) and that
primary mouse splenocytes express cyclin D3 and, to a lesser extent,
cyclin D2 but not cyclin D1 (data not shown).
Our conclusion that serum promotes T cell proliferation by targeting
p27Kip1 is based on data obtained using splenocytes derived
from p27Kip1-null mice. In agreement with previous studies
(38), we found that cyclin E-cdk2 complexes were constitutively active
in splenocytes lacking p27Kip1. As novel findings, we
report that these cells also exhibit constitutive cdk2 expression and
serum-independent (although not ConA-independent) cyclin A expression,
cyclin A-cdk2 activation, and S phase entry. Thus, in the absence of
p27Kip1, cdk2 activation no longer requires serum, and T
cells proliferate in serum-deficient medium. Previous studies have
shown that p27Kip1-null mice contain many enlarged organs
(including the spleen) and consequently are bigger than their control
litter mates (34, 37, 41). Increased organ size apparently results from
inappropriate cell proliferation during development due to an impaired
ability of cells to exit the cell cycle in the absence of
p27Kip1. Given the capacity of p27
/
splenocytes to proliferate in serum-deficient medium in
vitro, it is possible that abrogated growth factor requirements
account, at least in part, for the dysregulated proliferation of
p27
/
splenocytes in vivo.
Splenocytes heterozygous for p27Kip1 expression also
initiated DNA synthesis when exposed to ConA in medium containing 0.1%
serum. Unlike p27
/
splenocytes, p27+/
splenocytes required ConA (but not 10% serum) for cyclin E-cdk2 activation and cdk2 up-regulation. The need for ConA presumably reflects the need to down-regulate p27Kip1, which is
present in quiescent p27+/
splenocytes. As shown above,
addition of ConA to these cells nearly completely eliminated
p27Kip1. Thus, deletion of one copy of p27Kip1
abrogates the serum-dependent component of
p27Kip1 down-regulation. Comparison of p27+/+,
p27+/
, and p27
/
splenocytes reveals a
progressive loss of growth factor requirements for cyclin E-cdk2
activation in vitro. If mimicked in vivo, this phenomenon may contribute to the increasing sizes of the spleens of
p27+/+, p27+/
, and p27
/
mice
in vivo (34, 37, 41).
In contrast to quiescent p27
/
splenocytes, quiescent
p21
/
splenocytes still required serum (as well as ConA)
for cdk2 activation and S phase entry. Thus, loss of
p21Cip1 does not mimic loss of p27Kip1 or, in
respect to the parameters examined, produce a phenotype that differs
from that of wild-type splenocytes. Consistent with this finding,
p21Cip1-null mice are of normal size and exhibit no obvious
abnormalities other than an increased sensitivity to radiation-induced
cell cycle arrest (35, 42). Although previous studies argue against a
role of p21Cip1 in the control of the
G0/G1 transition in resting T cells, as do
ours, they do not negate the involvement of this CKI in other aspects
of lymphocyte mitogenesis (23, 43). For example, the finding that
p21Cip1 levels are substantially higher in actively cycling
T cells as compared with resting T cells suggests that
p21Cip1 contributes to the long-term expansion of T cell
populations (23). This supposition is substantiated by studies showing
that p21Cip1 loss had no effect on the initial
TCR-dependent proliferation of naive splenocytes, whereas
the subsequent and sustained IL-2-mediated phase of splenocyte growth
was enhanced (43).
In addition to decreasing p27Kip1 levels, serum also
contributes to cdk2 activation by facilitating the expression of cyclin
A and cdk2 in TCR-stimulated splenocytes. Increases in the amounts of
these proteins, however, are dependent on and thus are secondary to
cdk2 activation. The requirement for cdk2 activity for cyclin A
expression is well documented (20, 44). Our studies show that cdk2
expression does not increase in splenocytes treated with ConA and 10%
serum in the presence of the cdk2 inhibitor, roscovitine. Moreover, in
p27
/
splenocytes, constitutive cyclin E-cdk2 activation
was accompanied by constitutive expression of cdk2. Therefore, cdk2
activity is both necessary and sufficient for cdk2 expression. The
mechanism by which serum enhances cdk2 expression remains to be
determined. Our preliminary data show that ConA and 10% serum increase
the levels of cdk2 protein but not of cdk2 mRNA in wild-type
splenocytes and thus suggest that these agents modulate cdk2 expression
post-transcriptionally. Regardless of mechanism, we suggest that
elevations in cdk2 expression play an important part in prolonging cdk2
activation in stimulated splenocytes.
Previous studies have shown that serum enhances the TCR-initiated
expression of IL-2 and IL-2R
, and that IL-2 induces
p27Kip1 down-regulation and cdk2 activation when added to
TCR-activated T cells (5, 12, 14, 31, 40). We have found that serum is
required for the maximal accumulation of IL-2R
in primary splenocytes and that the capacity of serum to elicit this response is
abrogated by roscovitine (45). This finding places cdk2 activation upstream of the serum-regulated component of IL-2R
expression. Thus,
while IL-2 signaling may contribute to continued cdk2 activation, we
suggest that serum activates cdk2, at least initially, in an IL-2-independent manner. In support of this contention, we note 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 (13, 46).
At present, the factors in serum responsible for splenocyte growth are
not known. However, we have found that a "serum substitute" consisting of insulin, transferrin, and selenium also induces p27Kip1 down-regulation, cdk2 activation, and S phase entry
in ConA-treated splenocytes (data not shown). Regardless of the factors
involved, our study provides significant insights into the mechanism of serum-dependent splenocyte proliferation. Most importantly,
we identify p27Kip1 as a key regulator of this process and
show that this CKI plays a decisive role in determining the
proliferative status of TCR-stimulated T cells.