p27Kip1 Regulates T Cell Proliferation*

Subhra MohapatraDagger §, Deepak AgrawalDagger §, and W. J. PledgerDagger §||

From the Dagger  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, October 26, 2000, and in revised form, April 6, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies addressed the mechanism by which serum acts in conjunction with T cell receptor (TCR) agonists to promote the proliferation of primary splenic T cells. When added to resting splenocytes, TCR agonists initiated G0/G1 traverse and activated cyclin D3-cdk6 complexes in a serum-independent manner. On the other hand, both TCR agonists and 10% serum were required for the activation of cyclin E-cdk2 and cyclin A-cdk2 complexes and the entry of cells into S phase. Serum facilitated cdk2 activation by maximizing the extent and extending the duration of the TCR-initiated down-regulation of the cdk2 inhibitor, p27Kip1. Although p27Kip1 levels were reduced (albeit submaximally) in cells stimulated in serum-deficient medium, nearly all of the cdk2 complexes in these cells contained p27Kip1. In contrast, in cells receiving TCR agonist and 10% serum, little if any p27Kip1 was present in cyclin-cdk2 complexes. Unlike wild-type splenocytes, p27Kip1-null splenocytes did not require serum for cdk2 activation or S phase entry whereas loss of the related cdk2 inhibitor, p21Cip1, did not override the serum dependence of these responses. We also found that cdk2 activation was both necessary and sufficient for maximal expression of cdk2 protein. These studies provide a mechanistic basis for the serum dependence of T cell mitogenesis.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  subunit of the IL-2 receptor (IL-2Ralpha ) (2-4). In the presence of IL-2, IL-2Ralpha combines with IL-2Rbeta and IL-2Rgamma 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-2Ralpha (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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.


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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.


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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% (black-square) 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% (black-triangle). 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.


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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).


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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.


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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.


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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-2Ralpha , 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-2Ralpha 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-2Ralpha 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.

    ACKNOWLEDGEMENTS

We thank Nancy Olashaw for manuscript preparation and Drs. Ed Leof for cyclin A antibody, Andy Koff for p27Kip1-deficient mice, Tyler Jacks for p21Cip1-deficient mice, and Doug Cress for GST-Rb. We also acknowledge the helpful service of the Flow Cytometry and Molecular Imaging Core Laboratories at the Moffitt Cancer Center.

    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 and Research Institute, 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, April 10, 2001, DOI 10.1074/jbc.M009788200

    ABBREVIATIONS

The abbreviations used are: TCR, T cell receptor; IL-2, interleukin-2; IL-2R, interleukin-2 receptor; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; ConA, concanavalin A.

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DISCUSSION
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