Research ArticleCELLULAR AND MOLECULAR

Ca2+/Calmodulin-Dependent Protein Kinase II and Cytosolic Phospholipase A2 Contribute to Mitogenic Signaling in Myeloblastic Leukemia U-937 Cells

Mubarack M. Muthalif, Farid Ljuca, J. Brent Roaten, Neelu Pentapaty, Mohammed R. Uddin and Kafait U. Malik
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 272-278;

Abstract

The signaling mechanisms downstream of growth factor-stimulated proliferation in myeloid leukemia cells have not yet been fully elucidated. Recent evidence suggests that alternate pathways to the mitogen-activated protein kinase cascade are required. We have previously shown that Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activates cytosolic phospholipase A2 (cPLA2), which is involved in the proliferation of vascular smooth muscle cells. In the present study, the contribution of this pathway was investigated in the proliferation of U-937 myeloid leukemia cells. In U-937 cells, fetal bovine serum (FBS)-induced proliferation was attenuated by CaM kinase II inhibitor KN-93 but not by its inactive analog KN-92. Inhibitors of cPLA2 (methyl arachidonyl fluorophosphonate and arachidonyl trifluoromethyl ketone) also reduced proliferation of U-937 cells. FBS-induced proliferation was also attenuated by cotransfection with cPLA2 antisense oligonucleotides. These results suggest a role for CaM kinase II and cPLA2 in the proliferation of U-937 cells. FBS stimulated CaM kinase II and cPLA2activities in a time-dependent manner. Moreover, FBS-stimulated phosphorylation and activation of cPLA2 activation was inhibited by KN-93. FBS-stimulated phosphorylation of CaM kinase II was blocked by KN-93 but not by cPLA2 inhibitors, suggesting that CaM kinase II activates cPLA2. The products of phospholipid hydrolysis produced by cPLA2, lysophosphatidylcholine but not arachidonic acid, increased [3H]thymidine incorporation in U-937 cells. These data suggest that exposure of U-937 cells to FBS promotes phosphorylation and activation of CaM kinase II, leading to stimulation of cPLA2 and generation of lysophosphatidylcholine and resultant proliferation of these cells.

The human myeloid leukemia cell line U-937 has been used as a model to study the signal transduction mechanisms involved in processes governing proliferation, differentiation, and oncogenic transformation (Harris and Ralph, 1985). Mitogen-activated protein (MAP) kinases, termed extracellular signal-regulated kinases (ERKs), have been shown to be primarily involved in the control of cell growth and differentiation in a variety of cell types (Marshall, 1994; Robinson and Cobb, 1995). Even though ERKs are considered a potential target for antineoplastic therapy, evidence for involvement of ERKs in leukemia is still lacking. Recently, Ajenjo et al. (2000) demonstrated that myeloid leukemia cells respond to mitogenic stimuli without ERK activation and that blockade of ERK activity, pharmacologically and genetically, does not affect cell proliferation and differentiation; they concluded that the ERK pathway would not be a suitable target for antileukemic therapy.

The human myeloid leukemia cell line U-937 expresses a high level of cytosolic phospholipase A2(cPLA2), which selectively hydrolyzes the sn-2 ester of arachidonyl-containing phospholipids to produce arachidonic acid (Clark et al., 1990; Dennis, 1997; Kramer and Sharp, 1997; Leslie, 1997). Arachidonic acid and products of its metabolism exert growth-regulatory functions in a wide variety of cell types (Dennis, 1997; Muthalif et al., 1998). cPLA2 is activated by micromolar concentrations of Ca2+ and is regulated post-translationally by phosphorylation and by Ca2+-dependent translocation to the nuclear envelope (Glover et al., 1995; Muthalif et al., 1996; Dennis, 1997;Kramer and Sharp, 1997; Leslie, 1997; Borsch-Haubold et al., 1998). cPLA2 is phosphorylated at serine-505 and activated by MAP kinase (Lin et al., 1993). Some studies have provided evidence for the phosphorylation and activation of cPLA2 in a MAP kinase-independent manner (Qiu and Leslie, 1994; Kramer et al., 1995). These results and the existence of multiple phosphorylation sites on cPLA2(serine-431, -454, -505, and -727; de Carvalho et al., 1996) suggest that cPLA2 may be a substrate for other kinases. cPLA2 is an attractive target for novel therapies because of its profound importance in inflammatory processes, allergic responses, reproductive physiology, postischemic brain injury, cell proliferation, and cancer (Anderson et al., 1997; Heasley et al., 1997;Uozumi et al., 1997).

We previously reported that proliferation of rabbit vascular smooth muscle in response to norepinephrine is mediated via activation of cPLA2 (Uddin et al., 1998). Our studies indicated that Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) mediates the activation of cPLA2and arachidonic acid release in response to norepinephrine (Muthalif et al., 1996, 1998). CaM kinase II is a multifunctional enzyme involved in the regulation of gene expression, cell cycle control, and differentiation in multiple cell types (Braun and Schulman, 1995). These results raise the possibility that CaM kinase II might mediate cPLA2 activation and play a role in cellular proliferation in other cell types. To test this hypothesis, we studied the phosphorylation and activation of cPLA2 and CaM kinase II and their involvement in the proliferation of U-937 cells in response to fetal bovine serum (FBS).

In this article, we provide evidence that the activation of both CaM kinase II and cPLA2 regulates the proliferation of U-937 cells by FBS. Moreover, we report that CaM kinase II mediates the activation of cPLA2 by a mechanism involving phosphorylation.

Experimental Procedures

Materials.

The polyclonal antibody of cPLA2 was a gift from Genetics Institute (Cambridge, MA). The following drugs were purchased: ATP, aprotinin, leupeptin, penicillin/streptomycin, indomethacin, and amphotericin from Sigma (St. Louis, MO); the cPLA2 inhibitors arachidonyl trifluoromethyl ketone (ATK) and methyl arachidonyl fluorophosphonate (MAFP) from Calbiochem (La Jolla, CA); 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methybenzylamine (KN-93, a selective inhibitor of CaM kinase II) and 2-[N-(4-methoxybenzenefulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-92, an analog of KN-93 that exhibits no inhibitory effect on CaM kinase II) from Seikagaku (Falmouth, MA); baicalein and 17-ODYA from BIOMOL Research Laboratories (Plymouth Meeting, PA); arachidonic acid from Nuchek (Elysian, MN); lysophosphatidylcholine from Avanti Polar Lipids (Alabaster, AL); CaM kinase II antibody and monoclonal phospho-CaM kinase II antibody from Oncogene Research Products (Cambridge, MA); RPMI medium was from Celgro (Mediatech, Herndon, VA); phosphate-free DMEM from Life Technologies (Grand Island, NY); [32P]orthophosphate from Amersham Pharmacia Biotech (Arlington Heights, IL); [γ-32P]ATP (6000 Ci/mmol) and [3H]thymidine (20 Ci/mmol) from Dupont NEN (Boston, MA); andl-[1-14C]phosphatidyl choline (57 mCi/mM) from American Radiolabeled Chemicals, Inc. (St. Louis, MO).

MTT Assay.

Proliferation of U-937 cells after various treatments was measured with the MTT reagent (Sigma) method as per the manufacturer's protocols. Briefly, after experimental interventions, MTT was added to the cells and incubated for 4 h. Cells were lysed and solubilized, and absorbance was measured spectrophotometrically at a wavelength of 550 nm. Proliferation is expressed as the ratio of absorbance of the experimental over the control in percentage.

Culture and Maintenance of U-937 Cells.

U-937 myeloid leukemia cells were grown in RPMI 1640 medium (with glutamine), 10% FBS, and supplemented with penicillin/streptomycin (100 U/ml).

Transfection of U-937 Cells with cPLA2 Antisense Oligonucleotides.

U-937 cells were exposed to 1 μM phosphorothioate antisense or control cPLA2oligonucleotides in the presence of lipofectamine. Eighteen hours later, the cells were stimulated with FBS (5%).

Measurement of [3H]Thymidine Incorporation in U-937 Cells.

U-937 cells (20 ml, 1 × 105cells/ml) were cultured for 0, 2, 4, and 6 h in the presence of KN-93 (0.1–25 μM) or its vehicle. KN-92, an analog of KN-93 that exhibits no inhibitory effect on CaM kinase II, was used as a control. Aliquots (5 ml) were removed at each time point and incubated with 20 μl of [3H]thymidine for 30 min. Cells were pelleted and washed twice with ice-cold phosphate-buffered saline (PBS). The pellet was dissolved in 0.5 ml NaOH, and 200-μl aliquots were removed for scintillation counting.

Cell Cycle Analysis.

Cells were arrested for 48 h in RPMI medium (0.05% serum) and then treated with FBS (5%) in the presence of KN-93, MAFP, ATK (1 μM), or their vehicle for 16 h. Cell suspensions were centrifuged, and the pellets were fixed in ice-cold ethanol (70%) for 30 min. The fixed cells were washed three times by centrifugation at 1000 rpm for 10 min and resuspended in 3 ml of bovine serum albumin (BSA) buffer. The washed pellet was then resuspended in BSA buffer containing 100 μg/ml propidium iodide and incubated at 37°C for 15 min in the dark. The cells were analyzed for DNA content using an Epics Profiler II (Coulter Electronics, Miami Lake, FL) with an argon laser emitting at 488 nm. The emission maximum for analysis of PI fluorescence is 610 to 630 nm. Percentage of cells in various stages of the cell cycle was determined using the “multi-cycle” program (P. Rabinovitch; Phoenix Flow Systems, San Diego, CA).

cPLA2 Activity.

PLA2activity was measured usingl-[1-14C]phosphatidyl choline as substrate as previously described (Muthalif et al., 1996). Lipase reactions were performed at 37°C for 30 min. Briefly, 11 μg of radiolabeled phospholipid was added to 0.5 ml of reaction mixture (9 μM dioleoylglycerol, 25 mM HEPES, pH 7.4); 150 mM NaCl, 5 mM CaCl2, 1 mM dithiothreitol, 1 mg/ml BSA) and sonicated on ice. The reaction mixture (50 μl) containing 25 μg of protein was incubated at 37°C for 1 h. The reaction was stopped by adding 2.5 ml of Dole's reagent (2-propanol/heptane/0.5 M H2SO4, 20:5:1 ratio), 1.5 ml of heptane, and 1 ml of water containing 20 μg of unlabeled arachidonic acid. The heptane phase containing radiolabeled fatty acid was passed through a silicic column, and radioactivity was quantified from the eluates.

CaM Kinase II Enzyme Assay.

CaM kinase II was measured in cell lysates with the CaM kinase II assay system (Upstate Biotechnology, Lake Placid, NY), which measures the CaM kinase II-catalyzed transfer of the γ-phosphate group of [γ-32P]ATP to a synthetic peptide (KKALRRQETVDAL) that is selective for CaM kinase II. The procedures were carried out as described by the manufacturer.

Measurement of Phospho CaM Kinase II by Western Blotting.

The levels of phospho CaM kinase II were determined by lysing cells that were exposed to various inhibitors in a buffer (1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 100 μg/ml aprotinin, and 200 μM Na3VO4). Equal amounts of proteins (100 μg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (12%). After transfer to nitrocellulose membranes, the blots were blocked with 5% milk powder in Tris-buffered saline buffer (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h and then incubated for 2 h with phospho-specific CaM kinase II antibodies. Phospho-specific CaM kinase II antibody is a mouse IgG1 monoclonal antibody (clone 22B1) generated from HL-1 mouse myeloma cells fused with spleen cells from BALB/c mice immunized with a thiophosphated (at threonine-286) peptide corresponding to amino acids 281 to 294 (MHRQETVDCLKKFN) of the α subunit of mouse and rat CaM kinase II (Patton et al., 1993). To evaluate total CaM kinase II levels, blots were stripped and reprobed with CaM kinase II antibody. The blots were developed with the use of biotinylated secondary antibodies and horseradish peroxidase and visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech).

Phosphorylation of cPLA2 in Intact U-937 Cells.

U-937 cells were arrested for 48 h in medium containing 0.05% FBS. The cells were preincubated with phosphate-free DMEM medium for 30 min. The cells were labeled for 4 h in phosphate-free DMEM medium containing [32P]orthophosphate (300 μCi/ml) along with inhibitors or vehicle and then stimulated with 5% FBS for 5, 15, 30, 60, and 120 min. The cells were washed with ice-cold PBS and lysed in 1 ml of buffer A (50 mM Tris, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin). The protein concentration was adjusted to 1 mg/ml, and the lysates were centrifuged at 10,000g for 10 min. The cPLA2 was immunoprecipitated from the supernatants (1 mg of protein) by incubating the cell lysates with 10 μg of cPLA2 polyclonal antibody for 4 h at 4°C, followed by protein A-agarose beads for 1 h. The immunoprecipitate was washed three times with ice-cold PBS containing phosphatase inhibitors. The samples were boiled for 5 min in 50 μl of 2× Laemmli's sample buffer, and the supernatants were subjected to SDS-PAGE and visualized by autoradiography.

Analysis of Data.

The basal incorporation of [3H]thymidine in U-937 ranged between 15,024 and 28,196 cpm (14,103 ± 2,738, mean ± S.E.) per 25-cm2 flask in different passages of serum-starved cells. Although the basal values of [3H]thymidine incorporation were variable in different passage of cells, the effect of FBS and inhibitors on the [3H]thymidine incorporation was consistent within each passage of cells. Therefore, the changes in [3H]thymidine incorporation produced by FBS and LPC have been presented as percentage above basal levels. The basal PLA2 activity measured by the hydrolysis ofl-[1-14C]phosphatidyl-choline in lysates obtained from different batches of cells was 4962 ± 1202 cpm of 14C per 25 μg of protein per 60 min. The basal CaM kinase II activity measured by the transfer of γ-phosphate group of [γ-32P]ATP to a synthetic substrate in U-937 lysates was 19,442 ± 576 cpm of32P per 15 μg of protein per 30 min. The results are expressed as mean ± S.E. The data were analyzed by one-way analysis of variance. The Newman-Keuls multiple range test was applied to determine the difference among multiple groups, and the unpaired Student's t test was used to determine the difference between two groups. Differences were considered significant at P < 0.05.

Results

Inhibition of CaM Kinase II and cPLA2 Attenuates Proliferation of U-937 Cells.

KN-93 is a selective inhibitor of CaM kinase II (Sumi et al., 1991). Exponentially growing U-937 cells in 10% FBS were treated with different concentrations of KN-93 for longer (72 h) and shorter (4 and 8 h) time durations. Treatment of U-937 cells with KN-93 for 72 h abolished the proliferation of U-937 cells in a concentration-dependent manner (Fig.1A). When treated with KN-93 for 4 and 8 h, cells also reduced FBS-induced [3H]thymidine uptake (Fig. 1B). KN-93 at 5 to 10 μM attenuated 40 to 65% of FBS-stimulated proliferation. KN-92, an inactive analog of KN-93, had no effect on the uptake of [3H]thymidine in U-937 cells. Likewise, treatment of U-937 cells with inhibitors of cPLA2(MAFP and ATK, 1 μM) also attenuated FBS-stimulated [3H]thymidine uptake in U-937 cells (Fig.2).

Figure 1
Figure 1

Effects of inhibitors of CaM kinase II and cPLA2 on proliferation of growing U-937 cells. A, effect of KN-93 on proliferation of U-937 cells. U-937 cells (5000 cells/well) grown in 5% FBS were treated with KN-93 as indicated for 72 h. Proliferation at 24 and 72 h was determined by the MTT dye method as described under Experimental Procedures. Results are mean ± S.E.; n = 6 to 12 values obtained from three independent experiments performed in different batches of cells.†Value significantly different from FBS alone in the presence of vehicle of KN-93 (labeled 0.0) (P < 0.05). B, effects of KN-93 and KN-92 on [3H]thymidine incorporation in growing cells. U-937 cells (5000 cells/well) grown in 5% FBS were treated with 25 μM KN-93 or KN-92 for 4 h. In the last 24 h, cells were labeled with [3H]thymidine (0.5 Ci/ml), and incorporation into DNA was determined.†Value significantly different from vehicle (P < 0.05).

Figure 2
Figure 2

Effects of inhibitors of cPLA2 on FBS-stimulated [3H]thymidine incorporation in arrested U-937 cells. Cells (1 × 105 cells/ml) were made quiescent by culturing them in arresting medium for 48 h, and 4-ml aliquots were collected after 4 h of serum treatment (5% FBS) in the presence and absence of inhibitors of cPLA2 (ATK and MAFP, 1 μM). [3H]thymidine incorporation is presented as cpm per 4 ml of cells. Results are mean ± S.E.;n = 4. *Value significantly different from control without FBS. †Value significantly different from vehicle. Treatment of U-937 cells with ATK or MAFP alone did not alter the basal [3H]thymidine uptake (control).

To corroborate the involvement of cPLA2 in the proliferation of U-937 cells, antisense oligonucleotides directed against the translation initiation sites of cPLA2were used. The cPLA2 antisense approach has been used to reduce the levels of cPLA2 in U-937 cells (Wu et al., 1998). U-937 cells were transfected with either antisense or sense oligonucleotides complexed with lipofectamine for 18 h. Treatment of U-937 cells with antisense but not sense oligonucleotides of cPLA2 decreased the FBS-stimulated proliferation (Fig. 3). Collectively, these data suggest that FBS-stimulated proliferation of U-937 cells is mediated via activation of cPLA2 and CaM kinase II.

Figure 3
Figure 3

Effect of cPLA2 antisense oligonucleotides on FBS-induced proliferation of U-937 cells. U-937 cells were transfected with 1 μM cPLA2-specific antisense and sense oligonucleotides. The transfected cells were then stimulated with FBS (5%) for 24 h. [3H]Thymidine incorporation is presented as percentage above vehicle (Lipo, lipofectamine). [3H]Thymidine incorporation obtained with 5% FBS is calculated as 100%. Results are mean ± S.E.;n = 4 to 15 values obtained from three different experiments. *Value significantly different from vehicle (P < 0.05).

Changes in intracellular Ca2+ concentrations occur at the awakening of cells from quiescence, at the G1/S transition, during the S phase, and at the exit from mitosis (Santella, 1998). It has been reported that activation of CaM kinase II is essential in proliferation of cells by facilitating transition of cells from G1 to S, from G2 to M, and from metaphase to anaphase (Santella, 1998). To study the role of CaM kinase II and cPLA2 in the regulation of G2/M or G1/S phase transitions, a synchronous population of U-937 cells was treated with KN-93, and cell cycle analysis was done. The results are summarized in Table 1. Serum starvation for 72 h (control) resulted in a predominantly G1blockade, whereas treatment with FBS (5%) allowed progression through mitosis and cell division, as evidenced by a greater than 45% distribution of cells in the S phase (Table 1). Treatment of cells with KN-93 resulted in G1 blockade, suggesting that CaM kinase II is important in the cell cycle progression of U-937 cells. Similarly, cells treated with a cPLA2inhibitor also exhibited a partial G1 block (Table 1).

View this table:
Table 1

Effects of inhibitors of CaM kinase II and cPLA2 on cell cycle progression

CaM Kinase II Acts Upstream of cPLA2 through a Mechanism Involving Phosphorylation.

Previous results implicate CaM kinase II and cPLA2 in the proliferation of U-937 cells. We further investigated the effect of FBS (5%) on activation of CaM kinase II and cPLA2. Stimulation of serum-starved U-937 cells with FBS increased CaM kinase II and cPLA2 activities (data not shown). Pretreatment of cells with KN-93 (10 μM) and cPLA2 inhibitors MAFP and AKT (10 μM) blocked FBS-stimulated activation of CaM kinase II and cPLA2, respectively (data not shown).

MAP kinase and other kinases have been reported to activate cPLA2 and to release arachidonic acid in response to various stimuli (Lin et al., 1993). Arachidonic acid metabolites obtained by activation of PLA2 activate MAP kinase and other kinases (Muthalif et al., 1998). To determine the sequence of events in cPLA2 activation in U-937 cells, experiments were conducted to study the effect of FBS on CaM kinase II activation in the presence of cPLA2inhibitors and on cPLA2 activation in the presence of CaM kinase II inhibitors. The CaM kinase II inhibitor KN-93 attenuated the cPLA2 activity elicited by FBS (Fig. 4A), whereas cPLA2 inhibitors did not reduce FBS-stimulated CaM kinase II activation (Fig. 4B). This suggests that cPLA2 and the products generated by its stimulation do not activate CaM kinase II and that CaM kinase II acts upstream of cPLA2 in the FBS-stimulated [3H]thymidine uptake of U-937 cells.

Figure 4
Figure 4

Effects of CaM kinase II inhibitor on cPLA2 activity and cPLA2 inhibitors on CaM kinase II activity. A, effect of KN-93 on FBS-stimulated cPLA2 activity. Quiescent U-937 cells were pretreated with KN-93 (20 μM) for 4 h and then stimulated with 5% FBS for 30 min. Cell homogenates were prepared, and PLA2 activity was measured by hydrolysis ofl-[1-14C]arachidonyl phosphatidylcholine. Data represent the mean ± S.E. of four experiments from two batches of cells. *Value significantly different from basal obtained in the absence of FBS. †Value significantly different from that obtained with vehicle (P < 0.05). B, effects of inhibitors of cPLA2 on CaM kinase II activity. Data represent the mean ± S.E. of four experiments from two batches of cells. *Value significantly different from basal (P< 0.05).

Phosphorylation is essential for enzymatic activation of CaM kinase II and cPLA2 (Lin et al., 1993; Braun and Schulman, 1995); hence, we studied FBS-stimulated phosphorylation of CaM kinase II and cPLA2 in U-937 cells. Treatment of U-937 cells with FBS (5%) stimulated phosphorylation of cPLA2, as measured by incorporation of32P into cPLA2; this phosphorylation was reduced by MAFP and ATK (Fig.5A). Similar to cPLA2 activation, stimulation of serum-starved U-937 cells with FBS (5%) produced a considerable increase in the phosphorylation of CaM kinase II, as evidenced by the increase in phosphorylated CaM kinase II (Fig. 5B). Exposure of these cells to concentrations of KN-93 as low as 1 μM suppressed CaM kinase II phosphorylation. However, FBS-stimulated phosphorylation of CaM kinase II was not reduced by cPLA2 inhibitors.

Figure 5
Figure 5

Effects of inhibitors of cPLA2, CaM kinase II, and MAP kinase on FBS-stimulated phosphorylation of cPLA2 (A) and CaM kinase II (B). A, cPLA2phosphorylation. U-937 cells were prelabeled for 4 h with [32P]orthophosphate (300 μCi/ml) together with inhibitors and then stimulated with 5% FBS for 30 min. cPLA2 was immunoprecipitated with polyclonal cPLA2 antibody overnight and then incubated with protein A-agarose beads for 1 h. The immunoprecipitate was subjected to SDS-PAGE (12% gel) and autoradiography. Lane 1, control; lane 2, FBS (5%); lane 3, KN-93 + FBS; lane 4, MAFP (10 μM) + FBS; lane 5, ATK (10 μM) + FBS. B, CaM kinase II phosphorylation. Western blot using the phospho-specific antibody raised against the phosphorylated CaM kinase II. Cells were incubated for 4 h with inhibitors and then stimulated with FBS for 30 min. The lysates were separated by 10% SDS-PAGE and examined by Western blotting. Another identical blot was probed with CaM kinase II antibody to demonstrate equal loading of proteins. Lane 1, control; lane 2, FBS (5%); lane 3, KN-93 (20 μM) + FBS; lane 4, ATK (10 μM) + FBS; lane 5, MAFP (10 μM) + FBS; lane 6, MAP kinase kinase inhibitor U0126 + FBS.

LPC Contributes to CaM Kinase II/cPLA2-Dependent Proliferation of U-937 Cells.

cPLA2-mediated phosphatidyl choline hydrolysis leads to concomitant generation of arachidonic acid and LPC, which act as second messengers, activating diverse signaling pathways (Leslie, 1997; Dennis, 1997; Kramer and Sharp, 1997). Since inhibitors of cPLA2 and antisense oligonucleotides attenuated [3H]thymidine incorporation of U-937 cells, we explored whether arachidonic acid and LPC, which are produced by PLA2 activation, are involved in the proliferation of U-937 cells. Arachidonic acid (1 μM) in the absence of FBS caused an insignificant reduction in the [3H]thymidine incorporation in U-937 cells (27 ± 10%). On the other hand, LPC increased [3H]thymidine uptake in U-937 cells (Fig.6).

Figure 6
Figure 6

Effects of exogenous LPC on proliferation of U-937 cells. Starved cells were treated with LPC (0.1, 1, 10 μM) for 24 h. [3H]Thymidine incorporation is presented as percentage above vehicle. Results are mean ± S.E.;n = 3 to 9 values obtained from three different batches of cells. *Value significantly different from vehicle (P < 0.05).

Arachidonic acid is metabolized by cyclo-oxygenase, lipoxygenase, and cytochrome P-450 to a variety of biologically potent products, such as prostaglandins and thromboxanes and leukotrienes and hydroxyeicosatetraenoic acids, and hydroxyeicosatetraenoic acid and epoxy-eicosatrienoic acids, respectively (Makita et al., 1996). FBS-stimulated [3H]thymidine incorporation in U-937 cells was not significantly altered in the presence of inhibitors of cyclo-oxygenase (indomethacin, 38 ± 29% above FBS), lipoxygenase (baicalein, 16 ± 22% above FBS), or cytochrome P-450 (17-ODYA, 2 ± 1% above FBS). These observations suggest that LPC but not arachidonic acid or its metabolites generated by cPLA2 activation mediates FBS-induced [3H]thymidine incorporation in U-937 cells.

Discussion

The present study suggests that CaM kinase II is causally involved in the activation of cPLA2 and that this pathway is important in the proliferation of U-937 cells. Moreover, the results demonstrate that FBS-induced proliferation of U-937 cells proceeds via activation of cPLA2 by CaM kinase II through a phosphorylation-dependent mechanism.

Human U-937 cell lines have been used as a model for the study of signaling mechanisms in the regulation of myelopoiesis (Harris and Ralph, 1985). The MAP kinase pathway was previously regarded as critical to this process, as well as in the control of cell growth and differentiation in many cell types. However, it was recently reported that ERKs do not contribute to the proliferation and differentiation of myeloid leukemia cell lines (Ajenjo et al., 2000). The FBS-stimulated proliferation of U-937 cells was minimally reduced by the MAP kinase kinase inhibitor U-0126 (our unpublished data), suggesting that other unidentified kinases are involved in the proliferation of U-937 cells. Ca2+/calmodulin has been implicated in the stimulation of DNA synthesis and cell cycle progression (Rzigalinski et al., 1996). Ca2+/calmodulin is known to activate various enzyme systems, including CaM kinase II. Our finding that the CaM kinase II inhibitor KN-93 abolished FBS-stimulated proliferation implicates CaM kinase II in the cPLA2-regulated proliferation of U-937 cells. At the concentration used in this study, KN-93 is a selective inhibitor of CaM kinase II (Sumi et al., 1991). KN-93 is a useful and convenient pharmacological tool for elucidating physiological functions of CaM kinase II. KN-93 selectively and directly binds to the calmodulin binding site of CaM kinase II or its vicinity and prevents the association of calmodulin and CaM kinase II (Sumi et al., 1991). Moreover, we have demonstrated the selectivity of KN-93 by using its inactive analog KN-92, which failed to alter the CaM kinase II-mediated proliferation of U-937 cells. CaM kinase II has been shown to play an important role in cPLA2activation in vascular smooth muscle cells, and cPLA2 has been implicated in cell proliferation in various cell types (Muthalif et al., 1996; Anderson et al., 1997;Heasley et al., 1997). We demonstrated that the inhibitors of PLA2 (ATK and MAFP) and cPLA2 antisense but not sense oligonucleotides inhibited proliferation of U-937 cells. These data suggest that CaM kinase II-stimulated proliferation of U-937 cells is linked to cPLA2 activation. Supporting this view was our observation that FBS (5%) caused activation and phosphorylation of CaM kinase II and cPLA2. That CaM kinase II acts upstream of cPLA2 was suggested by our findings that the activation and phosphorylation of cPLA2were blocked by the CaM kinase II inhibitor KN-93, whereas cPLA2 inhibitors (ATK and MAFP) did not alter CaM kinase phosphorylation. However, cPLA2 also reduced cPLA2 phosphorylation in U-937 cells caused by FBS. Whether this is due to a nonspecific effect of cPLA2 inhibitors is not known. Our results are in contrast to the findings in intact synaptic nerve endings, wherein membrane depolarization produces a Ca2+- and phosphorylation-dependent inhibition of PLA2activity, and this inhibitory effect results from activation of the multifunctional CaM kinase II (Piomelli and Greengard, 1991). These results could be due to the existence of different isoforms of CaM kinase II and cPLA2 in the nerve endings.

Our studies also suggest that CaM kinase II is required at the G1/S phase transition in U-937 cells. This is consistent with previous studies indicating a requirement for CaM kinase II in the regulation of the cell cycle in sea urchin embryos (Baitinger et al., 1990), Xenopus oocytes (Waldmann et al., 1990), NIH 3T3 cells (Tombes et al., 1995), and HeLa cells (Patel et al., 1999). CaM kinase II inhibitors reduce DNA synthesis in small-cell lung carcinoma cells and in K-562 human chronic myelogenous leukemia cells (Williams et al., 1996). Ca2+ channel blockers and calmodulin inhibitors also reduced the rate of proliferation in HL-60 myeloid leukemia cells (Matsui et al., 1985). The target(s) of CaM kinase II at the G1/S phase transition in U-937 cells, however, remains to be established.

The mechanism by which activation of cPLA2 by CaM kinase II promotes serum-induced proliferation of U-937 cells is not known. U-937 cells contain abundant quantities of cPLA2, and arachidonic acid release in U-937 cells is dependent upon Ca2+ mobilization and is coupled to cPLA2 activation (Ridefelt et al., 1996; Rzigalinski et al., 1996). Lysophospholipids such as LPC and lysophosphatidylethanolamine have been reported to be produced in U-937 cells by the activation of PLA2 (Tsujishita et al., 1994). Activation of cPLA2 promotes hydrolysis of phospholids, phosphatidylcholine to LPC and arachidonic acid. LPC, arachidonic acid, and/or its metabolites have been shown to be involved in proliferation of various cell types (Anderson et al., 1997; Uddin et al., 1998; Yamakawa et al., 1998; Fang et al., 2000). Our finding that LPC, but not arachidonic acid, caused proliferation of U-937 cells suggests that LPC mediates the effect of serum-induced proliferation of these cells via activation of cPLA2 by CaM kinase II. We cannot exclude the contribution of other lysophospholipids, including platelet-activating factor in serum-induced proliferation of U-937 cells. Arachidonic acid in our study produced a slight decrease in proliferation of U-937 cells. Moreover, the inhibitors of cyclo-oxygenase, lipoxygenase and cytochrome P-450, failed to alter serum-induced proliferation of U-937 cells. Since the inhibitors of cPLA2 did not totally block the proliferation of U-937 cells, we cannot exclude the possible contribution of other lipid mediators derived via activation of phospholipases C and D or a mechanism independent of these lipases.

A recent clinical study showed that only 40% of the samples of primary cells extracted from acute myeloid leukemia patients showed activation of ERKs (Towatari et al., 1997), a finding that also supports the existence of alternate mechanisms, such as the CaM kinase II-cPLA2 pathway, in the proliferation of U-937 cells. This pathway provides an attractive target for novel therapeutic intervention in the treatment of myeloid leukemia and other malignancies. Further studies are needed to investigate this pathway and the efficacy of KN-93 in this context. Our studies bring forth CaM kinase II as a key link orchestrating different mitogenic signaling pathways via activation of cPLA2. In conclusion, our study demonstrates a novel pathway by which serum promotes proliferation of U-937 cells, a model of leukemic cells, by causing generation of LPC via activation of cPLA2 by CaM kinase II.

Acknowledgments

We thank Anne Estes for technical assistance, Dr. Lauren Cagen for scientific discussions, Jin Emerson-Cobb for editorial assistance, and the Genetics Institute for providing cPLA2antibody.

Footnotes

  • This work was supported by National Institutes of Health Grant 19134-26 (to K.U.M.), an American Heart Association's Beginners grant-in-aid (to M.M.M.), and a Health Sciences fellowship (to N.P.).

  • Abbreviations:
    MAP
    mitogen-activated protein
    ERK
    extracellular regulated kinase
    cPLA2
    cytosolic phospholipase A2
    CaM kinase II
    Ca2+/calmodulin-dependent kinase II
    FBS
    fetal bovine serum
    ATK
    arachidonyl trifluoromethyl ketone
    MAFP
    methyl arachidonyl fluorophosphonate
    MTT
    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
    PBS
    phosphate-buffered saline
    BSA
    bovine serum albumin
    PAGE
    polyacrylamide gel electrophoresis
    DMEM
    Dulbecco's modified Eagle's medium
    LPC
    lysophosphatidyl choline
    • Received December 26, 2000.
    • Accepted March 27, 2001.

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Research ArticleCELLULAR AND MOLECULAR

Ca2+/Calmodulin-Dependent Protein Kinase II and Cytosolic Phospholipase A2 Contribute to Mitogenic Signaling in Myeloblastic Leukemia U-937 Cells

Mubarack M. Muthalif, Farid Ljuca, J. Brent Roaten, Neelu Pentapaty, Mohammed R. Uddin and Kafait U. Malik
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 272-278;
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