Cytokine-driven cell cycling is mediated through Cdc25A - PubMed (original) (raw)
Cytokine-driven cell cycling is mediated through Cdc25A
Annette R Khaled et al. J Cell Biol. 2005.
Abstract
Lymphocytes are the central mediators of the immune response, requiring cytokines for survival and proliferation. Survival signaling targets the Bcl-2 family of apoptotic mediators, however, the pathway for the cytokine-driven proliferation of lymphocytes is poorly understood. Here we show that cytokine-induced cell cycle progression is not solely dependent on the synthesis of cyclin-dependent kinases (Cdks) or cyclins. Rather, we observe that in lymphocyte cell lines dependent on interleukin-3 or interleukin-7, or primary lymphocytes dependent on interleukin 7, the phosphatase Cdc25A is the critical mediator of proliferation. Withdrawal of IL-7 or IL-3 from dependent lymphocytes activates the stress kinase, p38 MAPK, which phosphorylates Cdc25A, inducing its degradation. As a result, Cdk/cyclin complexes remain phosphorylated and inactive and cells arrest before the induction of apoptosis. Inhibiting p38 MAPK or expressing a mutant Cdc25A, in which the two p38 MAPK target sites, S75 and S123, are altered, renders cells resistant to cytokine withdrawal, restoring the activity of Cdk/cyclin complexes and driving the cell cycle independent of a growth stimulus.
Figures
Figure 1.
Inhibition of p38 MAPK prevents G1/S arrest after withdrawal of IL-3 or IL-7 from cytokine-dependent lymphoid cells. Cell cycle was analyzed by incorporation of propidium iodide (PI) as described in Materials and methods. Percent of cells in G1 and S phases was calculated using ModFit LT software. (A) IL-3 was withdrawn from FL5.12A, pro-B cells for 8, 12, 17, or 24 h. The p38 MAPK inhibitor, PD169316 (20 μM), was added at the time of IL-3 withdrawal. Shown is a representative example of four experiments. (B) IL-7 was withdrawn from D1, a thymic cell line, for 24 h. The p38 MAPK inhibitor, PD169316 (20 μM) was added at the time of IL-7 withdrawal. Shown is a representative experiment. (C) Mouse lymph node cells were isolated and placed in culture with IL-7 (100 ng/ml) and a submitogenic concentration of Con A (0.25 μg/ml) for 48 h. Cells were then washed and cultured for 24 h without IL-7 and with PD169316 (20 μM). Shown are the results of two combined experiments, with error bars representing ± SEM.
Figure 2.
Inhibition of p38 MAPK, but not ERK or JNK, restores S-progression and Rb phosphorylation in the absence of IL-3. Cell cycle was analyzed by incorporation of PI as described in Materials and methods. Percent of cells in S phase was calculated using ModFit LT software. (A) FL5.12A cells were cultured with or without IL-3 for 17 h. The p38 MAPK inhibitors, PD169316 (20 μM) or SB202190 (20 μM), the MEK1 inhibitor (activator of ERK), PD98059 (20 μM), and the Jun kinase inhibitor, JNK inhibitor II (20 μM), were added at the time IL-3 was withdrawn. DMSO (0.1%) was included as a negative control. Two different FL5.12A clones B3 and A2, stably transfected with a dominant negative (DN) p38 MAPK, were placed in culture without IL-3 for 17 h. Results shown are representative of two experiments. (B) FL5.12A-A2 cells, expressing DN-p38 MAPK, incorporated Brdu during 17 h IL-3 withdrawal, while untransfected cells arrested. (C) Levels of phosphorylated Rb protein were assayed in FL5.12A cells after 48 h of IL-3 withdrawal. Nuclear lysates were resolved by SDS-PAGE and immunoblotting as described in Materials and methods using an antibody specific for Rb protein phosphorylated at serine 780.
Figure 3.
Inhibition of p38 MAPK after IL-3 withdrawal induces minor increases in G1-S Cdks and cyclins in B lymphocytes. (A) FL5.12A cells were incubated for 12 h in the absence of IL-3 and then further incubated for 5 h with either PD169316 (20 μM) or IL-3 (not depicted) for a total of 17 h. Cells were then harvested and total RNA isolated. RPAs were performed as described in Materials and methods. Transcription of L32 was measured as a control for total mRNA expressed. Results were quantified using a Bio-Rad Gel Documentation system, and mRNA transcripts for Cdks and cyclins normalized to the levels of L32 expression. (B) Whole cell lysates were prepared from FL5.12A cells deprived of IL-3 for 17 h and immunoblotted as described in Materials and methods for detection of Cdk4 and cyclin D3 with specific antibodies.
Figure 4.
Inhibition of Cdk2 or Cdc25 reverses the effects of p38 MAPK inhibition on the promotion of cell cycling during IL-3 withdrawal. Cell cycling was analyzed by incorporation of PI and percent of cells in S phase was calculated using ModFit LT software as described in Materials and methods. FL5.12A cells were cultured without IL-3 and PD169316 (20 μM) in the presence of 20 μM of a pharmacological inhibitor of Cdk2 (Cdk2 inhibitor II) or 20 μM of a pharmacological inhibitor of Cdc25 (NSC 95397) for 17 h and effects of cell cycling assessed.
Figure 5.
Cdc25A protein is degraded upon cytokine withdrawal and stabilized, in the absence of cytokines, by inhibition of p38 MAPK. (A) Levels of endogenous mouse Cdc25A protein were measured in FL5.12A cells. Whole cell lysates were made from cells deprived of IL-3 for 2, 4, 8, and 16 h, then resolved by SDS-PAGE and immunoblotted as described in Materials and methods using an antibody specific for Cdc25A. Total p38 MAPK was measured as a loading control. (B) Endogenous mouse Cdc25A was measured in FL5.12A cells stably overexpressing wild-type (WT) human Cdc25A. Whole cell lysates were made from cells cultured with or without IL-3 and 20 μM of PD169316 for 16 h, and then resolved by SDS-PAGE and immunoblotted as described in Materials and methods using antibodies specific for mouse Cdc25A and for human Cdc25A. Levels of p38 were detected as a loading control. Shown are representative experiments of three such performed.
Figure 6.
Expression of Cdc25A mutated at S75 and S123 prevents G1 arrest and sustains S phase progression after withdrawal of IL-3 or IL-7. DNA synthesis was assayed by incorporation of BrdU and cell cycle was analyzed by uptake of 7AAD and assayed by flow cytometry as described in Materials and methods. Percent of cells in G1 and S phase was calculated using ModFit LT software, excluding apoptotic cells and aggregates. (A) FL5.12A cells stably expressing the dominant-positive (DP) Cdc25A (S75,123A) mutein or Cdc25A-WT were cultured with or without IL-3 for 24 h. (B) D1 cells stably expressing the Cdc25A-DP (S75, 123A) mutein or Cdc25-WT were cultured with or without IL-7 for 24 h. (C) Viability and cell size of D1 cells stably expressing Cdc25A-DP or CDc25A-WT are shown in dot blots displaying forward (FSC) and side scatter (SSC). Results shown are representative of three or more experiments performed. Apoptotic cells were not excluded (D). Lymph node cells were isolated and grown with IL-7 (100 ng/ml) and Con A (0.25 μg/ml) as previously described. Primary cells were transfected with Cdc25A-DP or Cdc25A-WT and GFP, as a selection marker. Transfected cells, were selected by gating on GFP expression for analysis of DNA synthesis and cycling by BrdU/7AAD staining after 24 h of IL-7 withdrawal as described in Materials and methods. Shown is a representative experiment of three performed.
Figure 7.
Cdc25A-DP (S75,123A) remains stable and sustains phosphorylation of Rb and Cdks after IL-3 withdrawal. (A) Protein levels of Cdc25A-DP (S75,123A) were measured in FL5.12A cells. Cells were deprived of IL-3 for 17 h, and cytosolic extracts were resolved by SDS-PAGE and immunoblotted as described in Materials and methods using an antibody specific for mouse (to detect endogenous protein) or human Cdc25A (to detect the overexpressed HA-tagged protein). Total p38 MAPK was measured as a loading control. Shown is a representative experiment of two. (B) Stability of Cdc25A was determined in FL5.12A cells overexpressing the Cdc25A-DP mutant (DP) and the Cdc25A single mutant (S75A) (SP) after IL-3 withdrawal. The immunoblot was quantified using a Bio-Rad Gel Documentation system and results shown in the adjacent panel. The graph indicates fold differences in protein levels measured with or without IL-3 for 16 h. (C) Levels of phosphorylated Rb protein and Cdk2 were measured in FL5.12A cells and FL5.12A cells stably overexpressing the Cdc25A-DP mutein. Nuclear extracts were made from cells deprived of IL-3 for 17 h, then resolved by SDS-PAGE and immunoblotted, as described in Materials and methods, using antibodies specific for phospho-Rb protein (phosphorylated at serine 780), phospho-Cdk (tyrosine 15) (detects phospho-Cdk2 and phospho-Cdk1) and phospho-Cdk (threonine 160; detects phospho-Cdk2 and phospho-Cdk1). Total Cdk2 levels were also measured with a specific antibody. Note, the same cell lysates (equally loaded) were used in the phospho-Rb, phospho-Cdks, and Cdk-2 blots.
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