Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the α isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.

Interleukin 2 induces T cell growth through binding to its high affinity receptor expressed on the surface of activated T lymphocytes (1, 2). The IL-2R is composed of three different subunits: α (55 kDa), β (75 kDa), and the common γ-chain (γc; 64 kDa), which is shared by several hematopoietic receptors (3). Signaling by the IL-2R is mediated by ligand-induced heterodimerization of the cytoplasmic domains of the IL-2Rβ and γc. The apparent function of the α-chain is to affect the affinity of the receptor, but not the mechanism of IL-2 signaling. Heterodimerization of the IL-2R β and γ subunits allows activation of the src-family tyrosine kinases (4) and the Janus kinases 1 and 3 (5) that, in turn, create docking sites for the association/activation of other signaling molecules, such as STAT3 and -5, Shc, and phosphoinositide 3-kinase (PI3K)3 (6). Several studies have addressed the signaling mechanisms that regulate cell cycle entry following IL-2 binding, but, to this date, the molecular events underlying IL-2-induced signal transduction, particularly those involved in the IL-2-induced proliferative response are not fully understood. For instance, it has been shown in Ba/F3 cells transfected with the IL-2R that mitogenesis can be fully supported by IL–2 receptors that lack the ability to activate the Ras pathway (7), suggesting the existence of other mechanisms by which IL-2R, and perhaps other cytokine receptors, control cell proliferation.

In the search for novel mechanisms transducing IL-2 signals, we have previously shown that IL-2 binding induces phosphatidic acid (PA) formation by the rapid activation of the α isoform of diacylglycerol kinase (DGK) (8). Addition of IL-2 causes the rapid translocation of this enzyme from its cytosolic location to the perinuclear region of the cell. IL-2-induced activation of αDGK can be prevented by preincubation of the cells with the DGK inhibitor R59949. Treatment of the cells with this inhibitor prevents IL-2-induced proliferation, an effect similar to that seen with other immunosuppressors, such as rapamycin or wortmannin. In the present paper, we have extended our previous work by performing a detailed study of the molecular events caused by DGK inhibition. The analysis of IL-2-induced protooncogene expression demonstrates that inhibition of IL-2-induced DGKα activation prevents the induction of c-fos, c-myc, and c-raf by this cytokine. In addition, inhibition of IL-2-induced DGK activation prevents cyclin D3 expression and, consequently, retinoblastoma tumor suppressor protein (Rb) hyperphosphorylation. Inhibition of IL-2-induced DGK activation does not affect other IL-2-induced signaling pathways known to be related to cell cycle control, such as PI3K-dependent Akt activation. Our results confirm the existence of a signal transduction pathway that relies on the generation of PA and establishes a link between IL-2R-mediated PA production and the cell cycle machinery. The relevance of this pathway in IL-2-dependent responses and its relation with other IL-2-initiated signaling pathways is further discussed.

Recombinant human IL-2 was generously provided by Hoffman-LaRoche (Nutley, NJ). Recombinant murine IL-4 was from Peprotech (Rocky Hill, NJ). DGK inhibitor II (R59949) was from Calbiochem (San Diego, CA). Nonidet P-40, protease inhibitors (leupeptin, aprotinin, and PMSF), and sn-1,2-dioleoylglycerol were obtained from Sigma (Poole, U.K.). Phosphatidylinositol and dioleoyl PA were from Avanti Polar Lipids (Alabaster, AL). Histone H2B was from Boehringer Mannheim (Mannheim, Germany). Anti-c-myc, anti-c-fos, anti-cyclin D3, and anti-cRaf 1 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-bcl-2, anti-bcl-xL, and anti-Rb Abs were from PharMingen (San Diego, CA). Anti-Akt/protein kinase B Ab was from Upstate Biotechnology (Lake Placid, NY). Anti-rabbit and anti-mouse Ig HRP-linked whole Abs and the enhanced chemiluminescence Western blotting detection system were from Amersham (Aylesbury, U.K.). Silica Gel G-60 TLC plates were obtained from Whatman (Clifton, NJ). [γ-32P]ATP were from Amersham.

CTLL-2 cells were maintained in basal medium (RPMI 1640, 2 mM l-glutamine, 50 μM 2-ME, buffered to pH 7.2 with 10 mM HEPES) supplemented with 10% (v/v) FCS and 20 U/ml recombinant human IL-2. To obtain maximal synchronization, cells were washed extensively and incubated for 8 h in basal medium. After this period of incubation, the majority of the cells were found in the G1 phase, and no apoptosis was observed.

CTLL-2 cells growing exponentially in IL-2-supplemented medium were washed free of IL-2 and FCS before resuspending in basal medium. After 8 h, the DGK inhibitor (R59949, 1 μM final) or vehicle (DMSO, 0.01% v/v final) was added 15 min before IL-2 (50 U/ml final) or IL-4 (10 ng/ml). Cells were harvested by centrifugation and washed in PBS. For cell cycle analysis, cells were resuspended in permeabilization solution (0.1% sodium citrate, 0.05% Nonidet P-40) before treatment with 50 μg/ml RNase A for 30 min at room temperature. Propidium iodide was then added yielding a final concentration of 20 mg/ml. After 20 min, the fluorescence of the propidium iodide-stained DNA was quantitated on a per cell basis with an EPICS-XL flow cytofluorometer (Coulter, Miami, FL). For PI/annexin V analysis, cells were incubated with FITC-labeled annexin V, a protein that binds to phosphatidylserine and propidium iodide (Annexin V FITC Kit; Immunotech, Marseille, France). Following 10 min of incubation, positive cells were analyzed by flow cytometry.

Exponentially growing CTLL-2 cells were washed twice and resuspended at a concentration of 2 × 105 cell/ml in IL-2- and serum-free medium. After 8 h of starvation, cells were restimulated with recombinant IL-2 (50 U/ml). When indicated, the DGK inhibitor R59949 (1 μM) was added 15 min before IL-2 addition. At the appropriate times, cells were collected by centrifugation at 4°C, washed twice with ice-cold PBS, followed by lysis of the cell pellet in radioimmunoprecipitation (RIPA) buffer. Protein concentrations were determined using the Bio-Rad (Richmond, CA) protein assay. Equal amounts of protein were resolved using SDS-PAGE, before transfer to nitrocellulose membranes (Bio-Rad) in 25 mM Tris (pH 8.3), 190 mM glycine, and 20% v/v methanol for 1 h at 200 mA. The membranes were then blocked by incubation with TBS (150 mM NaCl, 20 mM Tris-HCl (pH 7.4)) containing 0.5% w/v BSA, 0.05% v/v Tween, and 5% w/v non-fat dried milk. Proteins were detected with specific Abs and HRP-conjugated-anti-mouse or anti-rabbit Abs and visualized by enhanced chemiluminescence, according to the manufacturer’s recommendations.

CTLL-2 cells were washed free of serum and IL-2 before being placed in phosphate-free RPMI medium for 6 h. During the last 2 h, carrier-free orthophosphate (Amersham) was added at a radiochemical concentration of 400 μCi/ml. To determine the effect of the inhibitors, cells were preincubated with either DMSO (0.01% v/v (control)), R59949 (1 μM final concentration), or wortmannin (50 nM final concentration) for 15 min before IL-2 addition. Cells were stimulated with IL-2 for 10 min, after which the cells were pelleted, washed twice with ice-cold PBS, and immediately frozen on dry ice. Cellular lipids were extracted using CHCl3/MeOH/HCl followed by their deacylation as described previously (9). 3-phosphorylated phosphoinositides were separated by strong anion exchange HPLC as described (9).

Anti-Akt immunoprecipitates were resuspended in 25 μl of kinase buffer (50 mM Tris (pH 7.5), 10 mM MgCl2, 1 mM DTT) containing 2.5 μg of histone 2B (H2B). Reactions were initiated by adding [γ-32P]ATP 50 μM (3 μCi) and incubated for 20 min at room temperature. H2B phosphorylation was assessed by autoradiography of the SDS-PAGE. The amount of Akt protein in each sample was determined by Western blot analysis. For transfection experiments, CTLL-2 cells were electroporated with 30 μg of the corresponding cDNAs.

DGK activity in total cell lysates was measured as previously described (10). Briefly, 15 × 106 quiescent cells were stimulated with either buffer, 500 U/ml of recombinant IL-2, or 100 ng/ml of recombinant IL-4. When indicated, the inhibitor was added 15 min before IL-2. After the periods of time indicated in the figure legends, the cells were harvested, washed twice with ice-cold PBS, and frozen on dry ice. The cells were thawed and lysed by nitrogen cavitation (10 min at 500 p.s.i., 4°C) in a buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 50 mM NaF, 2 mM Na3VO5, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. The lysates were then centrifuged at 800 × g, and the supernatant was used to determinate DGK activity. At the end of the phosphorylation assay, lipids were extracted by the subsequent addition of 200 μl of CHCl3/MeOH (2:1 v/v), 50 μl of CHCl3, and 50 μl of 0.1 M HCl. After centrifugation at 500 × g, the organic layer was recovered, dried under a stream of nitrogen, dissolved in 20 μl of CHCl3/MeOH (2:1 v/v), and applied to silica gel G-60 plates along with dioleoyl-PA as a standard. Plates were developed with a solvent system consisting of CHCl3/MeOH/4 M NH4OH (9:7:2, v/v/v). Dried plates were subjected to autoradiography, and the bands corresponding to PA were quantified by scanning of the autoradiograms.

We have previously shown, in the human T cell line YT, that the DGK inhibitor R59949 prevents IL-2-induced activation of the DGK α isoform (8). Unfortunately, there are no specific Abs that recognize the α isoform of murine origin, and we cannot demonstrate direct inhibition of this specific isoform following treatment with the DGK inhibitor in CTLL-2 cells. However, since most of the DGK activity stimulated by IL-2 in human T lymphocytes is that of the α isoform, we decided to evaluate the effect of this inhibitor on IL-2-stimulated DGK activation in CTLL-2 cells. As can be seen in Fig. 1, when R59949 was added before IL-2, the DGK activation induced by the cytokine was impaired indicating that, as previously shown for human T cells, inhibitor treatment prevented IL-2-induced DGK activation.

FIGURE 1.

The DGKα inhibitor R59949 prevents IL-2-induced DGK activation in CTLL-2 cells. Exponentially growing CTLL-2 cells were washed twice and resuspended in IL-2- and serum-free medium. Resting cells (after 8 h of IL-2 starvation) were either preincubated for 15 min with or without 1 μM R59949 before stimulation with recombinant IL-2 (500 U/ml). DGK activity was assayed as described in Materials and Methods. The quantitation of PA was determined by ascending TLC and autoradiography. The figure illustrates the band comigrating with the PA standard on the TLC plate. Similar results were obtained in three independent experiments.

FIGURE 1.

The DGKα inhibitor R59949 prevents IL-2-induced DGK activation in CTLL-2 cells. Exponentially growing CTLL-2 cells were washed twice and resuspended in IL-2- and serum-free medium. Resting cells (after 8 h of IL-2 starvation) were either preincubated for 15 min with or without 1 μM R59949 before stimulation with recombinant IL-2 (500 U/ml). DGK activity was assayed as described in Materials and Methods. The quantitation of PA was determined by ascending TLC and autoradiography. The figure illustrates the band comigrating with the PA standard on the TLC plate. Similar results were obtained in three independent experiments.

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CTLL-2 cells can be arrested in G1 by incubation for 8 h in medium free of both IL-2 and serum. Following this period, the effect of IL-2 as both a survival and proliferative factor can be assessed without any interference from serum constituents. As can be seen in Fig. 2,A, IL-2, in the absence of serum, induced cell cycle entry of CTLL-2 cells in a synchronic fashion (59% of the cells were found in S + G2/M 48 h after IL-2 addition). Due to the absence of serum in the culture medium, there was a significant percentage of cells undergoing apoptosis following IL-2 addition (15% at 36 h). However, when IL-2 was not present in the culture medium, the percentage of apoptotic cells was much higher and, by 48 h, 55% of the cells were found in a sub-G1 population. When additional experiments were performed under these established conditions, addition of the DGK inhibitor R59949, 15 min before IL-2, prevented cell cycle entry (26% of cells in S + G2/M at 48 h) without a significant increase in cell death by apoptosis. To further assess that DGK inhibition differentially affected proliferation rather than cellular survival, the level of apoptotic cells in the various conditions was examined by FACS analysis of annexin V-positive cells. As shown in Fig. 2 B, addition of IL-2 significantly reduced the percentage of annexin V/PI-positive cells compared with that of cells cultured in the absence of the cytokine. Again, addition of the DGK inhibitor did not significantly increase the percentage of annexin V-positive cells. These results demonstrate that, in CTLL-2 lymphocytes, inhibition of DGK activity prevents IL-2-induced cell cycle entry without affecting the capacity of this cytokine as a survival factor.

FIGURE 2.

DGK inhibition prevents IL-2-induced cell cycle entry and does not increase the rate of apoptosis in synchronized CTLL-2 cells in the absence of serum. Exponentially growing CTLL-2 cells were washed twice and resuspended at a concentration of 2 × 105 cells/ml in IL-2- and serum-free medium. After 8 h of starvation, cells were restimulated with recombinant IL-2 (50 U/ml). When indicated, the DGK inhibitor R59949 (1 μM) was added 15 min before IL-2 addition. At the indicated times, cells were collected, and the cell cycle distribution, normalized to 10,000 cells, was analyzed by propidium iodide staining (A). Histograms show DNA content (x-axis) plotted vs relative cell number (y-axis). The inset number indicates the percentage of cells in sub G1, G1, and S + G2/M phases. The percentage of annexin V-positive cells was analyzed at the same times (B). Histograms show annexin V (x-axis) plotted vs PI content (y-axis). The inset number indicates the percentage of cells in the different subsets. Similar results were obtained in three independent experiments.

FIGURE 2.

DGK inhibition prevents IL-2-induced cell cycle entry and does not increase the rate of apoptosis in synchronized CTLL-2 cells in the absence of serum. Exponentially growing CTLL-2 cells were washed twice and resuspended at a concentration of 2 × 105 cells/ml in IL-2- and serum-free medium. After 8 h of starvation, cells were restimulated with recombinant IL-2 (50 U/ml). When indicated, the DGK inhibitor R59949 (1 μM) was added 15 min before IL-2 addition. At the indicated times, cells were collected, and the cell cycle distribution, normalized to 10,000 cells, was analyzed by propidium iodide staining (A). Histograms show DNA content (x-axis) plotted vs relative cell number (y-axis). The inset number indicates the percentage of cells in sub G1, G1, and S + G2/M phases. The percentage of annexin V-positive cells was analyzed at the same times (B). Histograms show annexin V (x-axis) plotted vs PI content (y-axis). The inset number indicates the percentage of cells in the different subsets. Similar results were obtained in three independent experiments.

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The binding of IL-2 to its high-affinity receptor induces expression of the protooncogenes c-myc, c-fos, bcl-xL, and bcl-2 (11). While expression of bcl-2 and bcl-xL have been implicated in the prevention of apoptosis, IL-2-induced expression of c-myc has been shown to be essential to induce proliferation (12). To determine the effect of DGK inhibition on protooncogene induction by IL-2, the expression of these proteins was analyzed by Western blotting. Fig. 3 indicates that inhibition of IL-2-induced DGK activation by R59949 had a dramatic effect on the induction of c-fos and c-myc by IL-2. Furthermore, inhibition of IL-2-induced DGK also reduced induction of c-raf-1 by this cytokine.

FIGURE 3.

The effect of DGK inhibition on IL-2-induced protooncogene expression. Quiescent CTLL-2 cells were stimulated with IL-2, with or without R59949 (1 μM) as detailed in Fig. 2. Then, 48 h after IL-2 addition, cells were collected and lysed in detergent-containing buffer. Protein expression was analyzed by Western blotting using specific Abs. Similar results were seen in four independent experiments.

FIGURE 3.

The effect of DGK inhibition on IL-2-induced protooncogene expression. Quiescent CTLL-2 cells were stimulated with IL-2, with or without R59949 (1 μM) as detailed in Fig. 2. Then, 48 h after IL-2 addition, cells were collected and lysed in detergent-containing buffer. Protein expression was analyzed by Western blotting using specific Abs. Similar results were seen in four independent experiments.

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In CTLL-2 cells, and as previously demonstrated by Broome et al. (13), deprivation of IL-2 had no apparent effect on the level of bcl-2, whereas the bcl-xL protein level was severely reduced. IL-2-induced expression of both bcl-2 and bcl-xL was not affected by preincubation with R59949, which correlates with the lack of effect on IL-2-induced survival.

The retinoblastoma tumor suppressor protein, Rb, plays a central role in the control of the G1 to S transition. The association of Rb with transcription factors, such as E2F, is thought to be responsible for Rb’s ability to block the passage of cells through the G1 checkpoint. The hyperphosphorylation of Rb renders this protein incapable of associating with these factors, causing their release and, as a consequence, allows progression of cells into S phase (14). We have previously demonstrated, using CTLL-2 cells, that IL-2 deprivation induces the rapid dephosphorylation of Rb (15). To investigate the effect of DGK inhibition on Rb hyperphosphorylation, CTLL-2 cells were pretreated with R59949 before IL-2 addition. Rb phosphorylation was then assessed by Western blotting. As shown in Fig. 4 A, inhibition of IL-2-induced DGK activation prevented Rb phosphorylation, which correlated with the inhibition of S phase entry.

FIGURE 4.

The effect of DGK inhibition on Rb hyperphosphorylation, induction of cyclin D3, and the decrease in p27 in CTLL-2 cells. Quiescent CTLL-2 cells were stimulated with IL-2, plus or minus R59949 (1 μM) as detailed in Fig. 2. Then, 48 h after IL-2 addition, the cells were collected and lysed in detergent-containing buffer. Total cell lysates were resolved by SDS-PAGE and subjected to Western blotting. Proteins were detected using specific Abs. Similar results were seen in three independent experiments.

FIGURE 4.

The effect of DGK inhibition on Rb hyperphosphorylation, induction of cyclin D3, and the decrease in p27 in CTLL-2 cells. Quiescent CTLL-2 cells were stimulated with IL-2, plus or minus R59949 (1 μM) as detailed in Fig. 2. Then, 48 h after IL-2 addition, the cells were collected and lysed in detergent-containing buffer. Total cell lysates were resolved by SDS-PAGE and subjected to Western blotting. Proteins were detected using specific Abs. Similar results were seen in three independent experiments.

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A network of different regulatory processes controls the activity of the G1/S cyclin/cyclin-dependent kinase (cdk) complexes responsible for Rb phosphorylation. Formation of an active cyclin D/cdk complex appears to rely on de novo synthesis of the cyclin protein (16). In addition, the KIP/CIP family of cdk inhibitors (cdki) can also inhibit both cyclin D- and cyclin E-activated kinase complexes (17). Finally, the cdks activated by cyclin D and cyclin E are regulated by site-specific phosphorylation and dephosphorylation (18). In T lymphocytes, the cdki p27 is elevated in resting cells, and IL-2 induces the rapid down-regulation of this protein, allowing activation of cyclin D/cdk complexes (19). Additionally, IL-2 also induces de novo synthesis of cyclins D2, D3, and E (20). As depicted in Fig. 4,B, treatment with the DGK inhibitor did not affect the down-regulation of p27 levels induced by IL-2 treatment of the cells. However, when the same membrane was analyzed for cyclin D3 expression, following DGK inhibition, expression of cyclin D3 induced by IL-2 was dramatically reduced (Fig. 4 C).

One of the earliest signaling events following IL-2 binding to its high-affinity receptor is the activation of PI3K (9, 21, 22). It has been recently shown that the activation of this enzyme has a very important effect on some of the events regulating S phase entry (23). At least one isoform of DGK (DGK ε) is known to control the level of araquidonoyl-stearoyl-PA, an intermediate in the PI cycle. Therefore, we reasoned that inhibition of DGK activity could be related with the inhibition of the generation of 3-phosphorylated lipids. We measured the generation of such lipids in vivo following the addition of IL-2 with or without DGK inhibitor. As shown in Fig. 5, treatment of the cells with the DGK inhibitor did not affect the in vivo generation of either PI3,4P2 or PI3,4,5P3, the two principal products of PI3K activity. As a positive control for PI3K inhibition, we used wortmannin, a well-recognized PI3K inhibitor, to demonstrate inhibition of stimulated PI3,4P2 and PI3,4,5P3 generation.

FIGURE 5.

The effect of pharmacological inhibition of DGK and PI3K activities on the in vivo IL-2-stimulated increases in PI3,4P2 and PI3,4,5P3. Radiolabeled CTLL-2 cells were preincubated with 0.01% DMSO (control), R59949 (15 min, 1 μM), or wortmannin (15 min, 50 nM) before treatment with or without IL-2 (500 U/ml) for 10 min. 3-phosphorylated phosphoinositides were separated using strong anion exchange HPLC, as described in Materials and Methods. Results indicate mean ± SD (n = 3). The figure is representative of two independent experiments.

FIGURE 5.

The effect of pharmacological inhibition of DGK and PI3K activities on the in vivo IL-2-stimulated increases in PI3,4P2 and PI3,4,5P3. Radiolabeled CTLL-2 cells were preincubated with 0.01% DMSO (control), R59949 (15 min, 1 μM), or wortmannin (15 min, 50 nM) before treatment with or without IL-2 (500 U/ml) for 10 min. 3-phosphorylated phosphoinositides were separated using strong anion exchange HPLC, as described in Materials and Methods. Results indicate mean ± SD (n = 3). The figure is representative of two independent experiments.

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To further confirm that cell cycle arrest following DGK inhibition was not related to PI3K-derived signaling pathways, we measured the effect of DGK inhibition on IL-2-stimulated activation of Akt, the kinase that is directly activated by 3-phosphorylated phosphoinositides (24). As can be seen in Fig. 6, Akt was activated following IL-2 treatment of CTLL-2 cells. This activation was prevented following treatment of the cells with wortmannin or by cell transfection with p85, the regulatory subunit of PI3K, which acts as a dominant negative form of this enzyme. However, treatment of CTLL-2 cells with R59949 had no effect on IL-2-stimulated Akt activation, indicating that the formation of PA in response to IL-2 was not related to the generation of 3-phosphorylated lipids.

FIGURE 6.

The effect of inhibition of DGK and PI3K on Akt activity. CTLL-2 cells were left untreated, stimulated as indicated, or transfected with a plasmid containing the cDNA for p85 or p110CAAX. Endogenous Akt was immunoprecipitated and the activity measured in a kinase assay using H2B as a substrate, as described in Materials and Methods. Proteins were then separated by SDS-PAGE. The upper part of the gel was transferred to nitrocellulose, and the amount of Akt in each sample was determined by Western blotting with anti-Akt Abs (upper panel). H2B phosphorylation was determined by autoradiography of the lower half of the dried gels (lower panel). Similar results were seen in three independent experiments.

FIGURE 6.

The effect of inhibition of DGK and PI3K on Akt activity. CTLL-2 cells were left untreated, stimulated as indicated, or transfected with a plasmid containing the cDNA for p85 or p110CAAX. Endogenous Akt was immunoprecipitated and the activity measured in a kinase assay using H2B as a substrate, as described in Materials and Methods. Proteins were then separated by SDS-PAGE. The upper part of the gel was transferred to nitrocellulose, and the amount of Akt in each sample was determined by Western blotting with anti-Akt Abs (upper panel). H2B phosphorylation was determined by autoradiography of the lower half of the dried gels (lower panel). Similar results were seen in three independent experiments.

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CTLL-2 cells are IL-2-dependent, but they can also grow in response to IL-4. We decided to test the effect of the DGK inhibitor on IL-4-induced CTLL-2 cell proliferation. As shown in Fig. 7, treatment of CTLL-2 cells with IL-4 increased DGK activity, and this activation was prevented by addition of R59949. Cell cycle analysis of IL-4-stimulated CTLL-2 cells demonstrated that, as is the case for IL-2, inhibition of DGK activation prevented IL-4-induced proliferation. Following 36 h of IL-4 addition, 54% of the cells were found in the S + G2/M phases of the cell cycle. Addition of the DGK inhibitor significantly reduced the percentage of cycling cells (36% in S + G2/M). As previously shown for IL-2, DGK inhibition did not significantly increase the percentage of apoptotic cells determined either by propidium iodide analysis (14% vs 20%) or by FACS analysis of annexin V-positive cells (data not shown).

FIGURE 7.

The DGKα inhibitor R59949 prevents IL-4-induced DGK activation and cell cycle entry in CTLL-2 cells. Exponentially growing CTLL-2 cells were washed twice and resuspended in IL-2- and serum-free medium. Resting cells (after 8 h of IL-2 starvation) were either preincubated for 15 min with 0.01% DMSO or with 1 μM R59949 before stimulation with recombinant IL-4 (10 ng/ml). A, DGK activity was assayed as described for Fig. 1. The quantitation of PA was determined by ascending TLC and autoradiography. The band comigrating with the PA standard on the TLC plate is shown. B, Cell cycle distribution was analyzed by propidium iodide staining as in Fig. 2. Similar results were obtained in three independent experiments.

FIGURE 7.

The DGKα inhibitor R59949 prevents IL-4-induced DGK activation and cell cycle entry in CTLL-2 cells. Exponentially growing CTLL-2 cells were washed twice and resuspended in IL-2- and serum-free medium. Resting cells (after 8 h of IL-2 starvation) were either preincubated for 15 min with 0.01% DMSO or with 1 μM R59949 before stimulation with recombinant IL-4 (10 ng/ml). A, DGK activity was assayed as described for Fig. 1. The quantitation of PA was determined by ascending TLC and autoradiography. The band comigrating with the PA standard on the TLC plate is shown. B, Cell cycle distribution was analyzed by propidium iodide staining as in Fig. 2. Similar results were obtained in three independent experiments.

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IL-2 regulation of T lymphocyte proliferation and survival is essential for correct homeostasis in the immune system. Dissecting the mechanisms responsible for IL-2-controlled cell proliferation is of fundamental relevance to allow the identification of new targets for therapeutic intervention. In the present work, we have extended previous observations by our laboratory where we identified PA production through DGK activation as an essential component of the proliferative response of this cytokine. Generation of PA has been shown to be an important action of several growth factors. However, and distinct from other well-characterized systems, IL-2-induced proliferation of T cells requires PA, which is not generated through the activation of a phospholipase D (25), but rather through activation of a DGK. In the last two years, several laboratories have reported the cloning and characterization of different DGKs with a very broad pattern of expression and structural features (26). We have focused our interest on the α isoform of DGK, since this isoform is primarily expressed in oligodendrocytes and lymphocytes (27). By using specific Abs against the isoform of human origin, we have previously demonstrated that IL-2 binding to its high-affinity receptor induces DGKα activation, and that this activation could be prevented by using the potent DGK inhibitor R59949 (8). However, our previous observations were performed in YT cells, which do not require IL-2 for growth. We have now examined the effect of this same inhibitor in the IL-2-dependent murine T cell line CTLL-2 to further investigate the molecular mechanisms underlying the inhibition of cell proliferation caused by prevention of DGK activation. The reason that we chose this cell line as our experimental model was that, in CTLL-2 cells, the effect of IL-2 as both an inducer of survival and as a mitogen could be demonstrated in the absence of any added serum. This is very important to properly ascertain that the signal is due only to the effect of the cytokine and not to additional growth factors present in the serum. As we have demonstrated, addition of IL-2 in the absence of the survival factors contained in the serum promoted cell cycle entry, although, as expected, a significantly higher proportion of cells underwent apoptosis. However, the population of apoptotic cells, measured as annexin V-positive cells, did not significantly increase in the presence of the DGK inhibitor. Together, these experiments demonstrate that PA production following IL-2 addition is essential for cytokine-induced cell proliferation.

Our results, presented here, demonstrate that DGK inhibition prevents IL-2 induction of c-fos, c-myc, and c-raf-1, three of the protooncogenes up-regulated by this cytokine during T cell expansion. Reciprocally, prevention of DGK activation does not affect the IL-2-regulated expression of bcl-x protein, indicating again that PA-derived signals are directly related to the proliferation machinery but not to the IL-2-regulated survival mechanisms. The involvement of both c-raf-1 and c-myc in mitogenic control is well established, although the exact mechanism underlying the regulated expression of these proteins by IL-2 has not been fully defined. Studies in freshly isolated T cells have demonstrated that the promotion of G1 progression by IL-2 was associated with an increase in c-raf protein expression (28). It has been previously suggested that c-raf-1 is constitutively expressed in CTLL-2 cells, but our experiments demonstrate that this is mostly due to the presence of serum. If CTLL-2 cells are deprived of both serum and IL-2, c-raf-1 protein expression is reduced. Readdition of IL-2 restores the levels of this protooncogene, an effect similar to that described in activated lymphocytes. Under these circumstances, inhibition of DGK activation prevents c-raf-1 induction, demonstrating for the first time that generation of PA is implicated in the control of c-raf-1 expression. Previous experiments by ourselves and others have demonstrated that addition of exogenous PA induces c-myc expression in T cells (10, 29), indicating the existence of a pathway governed by PA that is responsible for the induction of this gene. The present demonstration that DGK inhibition reduces c-myc expression further confirms that PA generation through DGK activation is essential for the induction of this protooncogene.

The binding of IL-2 to its high-affinity receptor regulates the transit of T cells through the G1 phase to reach a restriction point, after which cell proliferation is no longer controlled by growth factor receptor ligation. The transition through S phase is largely regulated by Rb hyperphosphorylation, which is in turn controlled by a complex network of events. It has been previously demonstrated that IL-2 regulates cyclin expression as well as p27 degradation. Our results here indicate that DGK inhibition prevents IL-2-induced up-regulation of cyclin D3 and does not affect the down-regulation of p27 that follows IL-2 addition. In this regard, the cell arrest induced by DGK inhibition differs from the effect caused by other immunosuppressors, such as rapamycin, since rapamycin treatment of T cells prevents the IL-2-induced decrease in the protein levels of p27 (19). Additionally, it has recently been demonstrated, using the PI3K inhibitor LY29002, that PI3K/Akt signals were required for IL-2-induced hyperphosphorylation of Rb in peripheral blood-derived T lymphoblasts and Kit225 cells (23). The same authors demonstrated that PI3K-derived signals were required for IL-2 up-regulation of cyclin D3, as well as down-regulation of p27. Our results indicate that inhibition of DGK does not interfere with either the generation of 3-phosphorylated lipids or the activation of Akt by IL-2. Moreover, G1 progression is prevented without effects on p27 down-regulation, indicating a different level of control by PA than PI3K/Akt-derived signals in IL-2-induced cell cycle progression. Previous studies with IL-2R mutants have indicated that IL-2-induced DNA synthesis required the integration of a network of signals generated by the IL-2Rβ and γ subunits (11). As previously indicated, activation of the PI3K/Akt pathway has been shown to be essential for E2F transactivation (23). In addition, there is considerable evidence indicating that the activation of STATs is required for the proliferative response of lymphoid cells (30). We demonstrate that DGK inhibition has no effect on IL-2-stimulated PI3K/Akt activation, nor does it reduce IL-2Rα surface expression (data not shown), indicating that STAT5 activation is not affected. The elucidation of the integration of the signals derived from DGK activation with other IL-2R-induced signals should clarify the mechanisms regulating cytokine-induced cell proliferation.

PA generation has been shown to have an important role in mitogenesis in response to different mitogens in a variety of cell systems. Our results now indicate that not only IL-2, but also IL-4, activates DGK in CTLL-2 cells and that inhibition of IL-4-induced DGK activation also prevents IL-4-induced cell proliferation. Previous studies have shown that IL-4 activates several proliferative pathways on its own and also primes cells for a response to IL-2-triggered proliferative signals (31). The molecular mechanism underlying these effects is the effective activation by IL-2 in IL-4-cultured cells of several molecules that play an important role in promoting proliferation, including the induction of expression of c-myc, c-fos, and cyclin D3. Interestingly, the same genes the authors described in the above-mentioned study, up-regulated by both IL-4 and IL-2, were impaired in our studies following DGK inhibition, confirming a role for this enzyme in mitogenic control. Although the importance of PA generation in proliferation is broadly accepted, to this date the exact role of this lipid is not well established. The profound effect that DGK inhibition exerts on IL-2- and IL-4-regulated proliferation indicates that the cellular levels of the mitogenic lipid PA must regulate the activity, intracellular localization, or protein-protein interactions of certain key cell components. Therefore, the intracellular concentration of PA in the cell could constitute one of the factors that the cell checks before making the decision to enter mitosis.

We thank Julian Downward and Stephen Wennstrom for the gift of the p85 and p110-CAAX plasmids, M.C. Moreno and I. Lopez-Vidriero for help with the flow cytofluorometer, Carlos Martinez-A. for helpful advice and critical discussion of the manuscript, and Hoffmann-La Roche for the kind gift of recombinant IL-2.

1

This work was supported by Grant PM97-0132 from the Dirección General de Enseñanza Superior e Investigación Científica , Grant 97-15 from the Association for International Cancer Research, and Grant 08.1/0036/98 from Comunidad de Madrid (to I.M.). The Department of Immunology and Oncology was founded and is supported by the Consejo Superior de Investigaciones Científicas and Pharmacia & Upjohn.

3

Abbreviations used in this paper: PI3K, phosphoinositide 3-kinase; DGK, diacylglycerol kinase; PA, phosphatidic acid; Rb, retinoblastoma tumor suppressor protein; cdk, cyclin-dependent kinase; cdki, inhibitor of cdk; H2B, histone 2B.

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