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The Journal of Immunology, 1998, 161: 1390-1398.
Copyright © 1998 by The American Association of Immunologists

The Mitogen-Activated Protein Kinase Pathway Inhibits Ceramide-Induced Terminal Differentiation of a Human Monoblastic Leukemia Cell Line, U9371

Scott J. Ragg*, Shuji Kaga*, Karen A. Berg{dagger} and Atsuo Ochi2,*,{dagger}

* John P. Robarts Research Institute and {dagger} Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myeloid hemopoietic differentiation is a multistage process, with the cells undergoing many phenotypic changes until they reach their final, nonproliferative, terminally differentiated form. The human monoblastic leukemia cell line, U937, has been the subject of studies of hemopoietic cell differentiation in vitro. Various physiologic ligands induce differentiation of this cell line; IFN-{gamma} and IL-6 cause U937 cells to undergo macrophage/monocyte differentiation, whereas granulocyte-macrophage CSF or granulocyte CSF result in differentiated cells with more granulocytic properties (1). In addition to cytokines, nonphysiologic agents are known to induce U937 differentiation. The best studied differentiation inducer of this criteria may be PMA, which stimulates protein kinase C-dependent signaling and transforms U937 cells into a tightly adherent monocytic phenotype (2).

Ceramide, a naturally occurring lipid formed by cleavage of membrane sphingomyelin by sphingomyelinases, has been shown to cause hemopoietic differentiation. Treatment of HL-60 (human promyelocytic leukemia) cells with synthetic, cell-permeable, ceramide analogues (e.g., C2-ceramide) induced maturation and differentiation of these cells with concomitant growth arrest (3). Ceramide appears to induce growth arrest of leukemia cells by halting the cell cycle in the G0/G1 phase and preventing progression into the S phase (4). Likewise, terminal differentiation of these leukemia cells requires that passage from the G0/G1 phase into the S phase be impossible (5-7), such that these cells are locked into the G0 phase and therefore cannot divide further. Recent reports indicate that ceramide may cause cell cycle arrest of the Molt-4 human leukemia cell line by activating the pRb3 tumor suppressor phosphoprotein (8), a protein essential for passage from the G0/G1 to the S phase (5).

We have investigated the signaling and effector mechanisms during differentiation of U937 human monoblastic leukemia cells. The results highlight a MEK-dependent signaling pathway that inhibits terminal differentiation of U937 induced by C2-ceramide. This inhibition pathway was activated by PMA and accompanied by increased activation of ERK, suppression of JNK, and repression of p21CIP1 expression. The data also suggested that distinct pathways regulate differentiation and p21CIP1 expression/cell cycle arrest in ceramide-stimulated cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Synthetic, cell-permeable ceramide (C2-ceramide) and its analogue, dihydro-C2-ceramide, were purchased from BioMol (Plymouth Meeting, PA); D-erythro-sphingosine was obtained from Calbiochem (La Jolla, CA), dissolved in ethanol, and stored at -80°C. PMA (Calbiochem) was dissolved in ethanol and stored at -80°C. The MEK inhibitor PD98059 (BioMol) was dissolved in DMSO and stored at -20°C.

Cells and culture

U937 cells were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 5% FCS (Life Technologies), 2 mM L-glutamine (Sigma), and 0.1 mg/ml gentamicin (Sigma) and incubated at 37°C in a humidified atmosphere of 5% CO2-95% air. For treatment, U937 cells were washed once with PBS, resuspended at 5 x 105/ml in serum-free RPMI 1640 (or RPMI 1640 and 2% FCS when using the MEK inhibitor PD98059) and incubated for an additional 2 h before the addition of reagents. Unless otherwise noted, parental U937 cells were pulsed with the indicated reagents for 8 h in serum-free medium, after which time the medium was aspirated and replaced with fresh medium containing 5% FCS. Zero hour was the point at which reagents were added to the cells.

Immunofluorescence and flow cytometry

Approximately 1 x 106 cells were washed and resuspended in PBS, 2% BSA, and 0.1% NaN3 and incubated with fluorochrome-conjugated anti-human CD11b and CD14 mAbs (Dako, Carpinteria, CA) for 20 min at room temperature. Cells were washed three times with PBS/BSA/NaN3 before fixation. Stained cells were analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson) and CellQuest software. Changes in mean fluorescence intensity of the cell population were determined by comparison to parental cells stained with the Ab of interest.

Cell cycle analysis

Cells (1 x 106) were resuspended in hypotonic propidium iodide staining solution (0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100, and 0.05 mg/ml propidium iodide) and left to stain in the dark at 4°C overnight (9). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and the percentage of cells in each phase of the cell cycle was quantitated using ModFit software (Becton Dickinson). Aggregates were excluded from the analysis by use of the doublet discrimination module and subsequent gating on the linear red fluorescence area and width parameters.

Western blot analysis of pRb and p21CIP1

Cells (2 x 106) were pelleted, resuspended in residual medium, and lysed in 100 µl of lysis buffer (25 mM Tris (pH 7.4), 50 mM NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS, 50 µg/ml aprotonin, 20 µg/ml leupeptin, and 50 mM NaF) as previously described (10). Lysates were immediately clarified by centrifugation at 17,000 x g for 15 min at 4°C. After addition of 20 µl of 6 x Laemmeli sample buffer and boiling for 5 min, 25 µl of each sample was applied to either 6.5% (for pRb analysis) or 14% (for p21CIP1 studies) SDS-polyacrylamide gel. At the completion of electrophoresis, proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH), and the blots were blocked overnight in 5% nonfat dried skim milk in PBS and 0.1% Tween-20 (PBST; Caledon, Georgetown, ON, Canada). Blots were incubated overnight at 4°C with either anti-pRb or anti-p21CIP1 (both from Santa Cruz Biotechnology, Santa Cruz, CA) at 0.5 µg/ml in PBST. After three PBST washes, blots were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary Abs (Santa Cruz Biotechnology) for 2 h at room temperature and washed three times in PBST, and visualization was performed by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

In vitro MAP kinase assays

JNK activity was assayed as described previously (11) with slight modification. Briefly, 1 x 107 cells were lysed in 1 ml of lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM ß-glycerophosphate, 1 mM DTT, 1 mM Na3VO4, 1% Triton X-100, 2 mM leupeptin, and 20 µg/ml aprotonin), incubated on ice for 15 min, and clarified by centrifugation at 17,000 x g for 15 min at 4°C. The supernatant was precleared using normal rabbit IgG and Protein A/G Plus agarose (Santa Cruz Biotechnology). After transferring the supernatant, 1 µg of rabbit anti-JNK1 polyclonal Ab (Santa Cruz Biotechnology) was added, and the sample was rotated at 4°C for 1 h, after which time Protein A/G Plus agarose was added. Immunoprecipitates were collected by centrifugation, washed three times in lysis buffer and twice in kinase buffer (50 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, and 1 mM DTT), and resuspended in 10 µl of kinase buffer. To the bead suspension, 2 µg of gluthione-S-transferase-c-Jun1-79 (Santa Cruz Biotechnology), cold ATP (5 µM final concentration), and 20 µCi [{gamma}-32P]ATP (3000 Ci/mmol; New England Nuclear-DuPont, Boston, MA) were added, and the kinase reaction was allowed to proceed for 20 min at 30°C. The reaction was terminated by addition of Laemmeli buffer, and the products were resolved by 10% SDS-PAGE and visualized by phosphorimagery (Molecular Imager, Bio-Rad, Richmond, CA). For analysis of ERK2 kinase activity, cells were lysed (20 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EGTA, 1 mM Na3VO4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 20 µg/ml aprotonin) and precleared. ERK2 was immunoprecipitated using rabbit polyclonal anti-ERK2 (Santa Cruz Biotechnology) and washed as described above. Kinase reactions (20 µM cold ATP and 10 µCi [{gamma}-32P]ATP) were performed using 10 µg of myelin basic protein as substrate and proceeded for 15 min at 30°C before being terminated by addition of Laemelli buffer. Products were resolved by 14% SDS-PAGE and visualized by phosphorimagery.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMA and C2-ceramide induce dissimilar phenotypes with the PMA phenotype dominant over C2-ceramide

The phenotype of U937 cells induced to differentiate by treatment with 5 µM C2-ceramide, 40 nM (25 ng/ml) PMA, or the combination of these reagents was assessed by both light microscopy (Fig. 1Go) and immunofluorescence (Fig. 2Go). Parental U937 cells were pulsed with the indicated reagents for 8 h in serum-free medium, after which time the medium was aspirated and replaced with fresh medium containing 5% FCS. Cells were analyzed 72 h after the addition of reagents. Compared with the parental and vehicle-treated cells, C2-ceramide-treated cells were larger, more rounded, and uniform in shape, with giant cells occasionally found. Increased adherence to plastic was not noted. The increased size was confirmed during flow cytometric studies by a greater forward light scatter associated with the C2-ceramide-treated cells (data not shown). These cells exhibited increased expression of CD14 and CD11b, consistent with a more mature phenotype (1, 2). Cells treated with dihydro-C2-ceramide were indistinguishable from vehicle-treated or parental U937 cells; thus, dihydro-C2-ceramide is not biologically active in our experimental system. In contrast to C2-ceramide, PMA-treated cells formed tight clusters that were very adherent to plastic and smaller in size than the parental or vehicle-treated U937 cells. The PMA-treated cells had greatly increased levels of CD14 and CD11b expression compared with controls and C2-ceramide-treated cells. When U937 cells were treated simultaneously with C2-ceramide and PMA, the resulting phenotype was indistinguishable from that of cells treated with PMA alone.



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  		FIGURE 1. Morphology of differentiated U937 cells. U937 cells were treated with vehicle (A), 5 µM C2-ceramide (B), 40 nM PMA (C), and 5 µM C2-ceramide plus 40 nM PMA (D) as described in Materials and Methods. Cells were cultured for 72 h after addition of reagents. Magnification, x80.

 


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  		FIGURE 2. Phenotype of differentiated U937 cells. U937 cells were treated with the indicated reagents as described in Materials and Methods. Cells were analyzed for CD14 and CD11b expression after 72 h of culture. Changes in the mean fluorescence intensity of the cell population were determined by comparison with identically stained parental U937 cells. Results are derived from four separate experiments. Error bars represent the SD.

 
Only C2-ceramide rapidly induces cell cycle arrest in U937 cells

To determine whether the phenotypic changes induced by C2-ceramide and PMA were associated with withdrawal from the cell cycle, logarithmically growing cells were treated in a manner identical with that described above and harvested at various time points for cell cycle analysis (Fig. 3GoA). Cells began to accumulate in the G0/G1 phase after approximately 8 h of treatment with C2-ceramide and continued to do so for the duration of the experiment. Consistently, >80% of cells were in the G0/G1 phase at 24 h post-treatment, suggesting that the cells were undergoing cell cycle arrest in response to C2-ceramide. Cells treated with PMA exhibited a very different cell cycle profile; fewer cells were in the G0/G1 phase compared with vehicle-treated controls during the first 16 h of the time course, after which time they paralleled the controls. Of particular note was the fact that cells treated with C2-ceramide and PMA responded in a virtually identical manner as cells treated with PMA alone. Comparison of the distribution of cells in each phase of the cell cycle 24 h after the various treatments (Fig. 3GoB) provides further evidence that U937 cells treated with C2-ceramide are undergoing cell cycle arrest. The greatly reduced proportion of C2-ceramide-treated cells in the S phase is indicative that cells are no longer making the G1 to S phase transition and hence have undergone cell cycle arrest.



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  		FIGURE 3. Cell cycle arrest accompanies C2-ceramide-induced differentiation. A, Kinetics of cell cycle arrest after treatment with the indicated reagents. U937 cells were treated as described in Materials and Methods and subjected to flow cytometric cell cycle analysis. Accumulation of cells in the G0/G1 phase is indicative of cell cycle arrest. B, Distribution of cells in each phase of the cell cycle 24 h after the various treatments. Results shown are representative of four separate experiments.

 
C2-ceramide, but not PMA, induces pRb dephosphorylation and p21CIP1 expression

pRb is a 110-kDa nuclear phosphoprotein that is considered to be a tumor suppressor, as loss or inactivation of both copies of the RB1 gene results in unrestrained malignant growth (12). As hypophosphorylated pRb is the active form, dephosphorylation of the pRb protein has been associated with growth arrest, the G0/G1 phase of the cell cycle, and the differentiated phenotype of hemopoietic cells (10, 13, 14). To obtain further insight about the mechanism of C2-ceramide induced growth and cell cycle arrest in differentiating U937 cells, the phosphorylation status of the pRb protein was determined by taking advantage of the fact that SDS-PAGE and Western blotting can resolve the faster migrating hypophosphorylated forms of pRb from the slower migrating hyperphosphorylated forms. Cells were treated with the indicated reagents and were harvested 24 h later, and lysates were subjected to SDS-PAGE and Western blotting (Fig. 4Go). In vehicle-, PMA-, and C2-ceramide- plus PMA-treated samples, a band of about 120 kDa, representing inactive hyperphosphorylated pRb, was observed. However, in cells treated with C2-ceramide, additional faster migrating bands of Mr ~115kDa accompanied by a simultaneous loss of higher Mr bands were noted, resulting in an obvious downward shift in the pRb band. This shift represents the appearance of hypophosphorylated (active) forms of pRb, thus demonstrating that C2-ceramide induced pRb dephosphorylation. The faster migrating hypophosphorylated pRb band(s) were never observed in cells treated with vehicle, PMA, or C2-ceramide plus PMA.



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  		FIGURE 4. C2-ceramide causes dephosphorylation of the pRb protein. U937 cells were treated with the indicated reagents as described above each lane. Whole cell lysates were prepared after 24 h of culture and were analyzed by 6.5% SDS-PAGE and anti-pRb immunoblotting. The pRbhyper band represents inactive hyperphosphorylated pRb, whereas the pRbhypo band represents hypophosphorylated (activated) pRb. Results are representative of five separate experiments.

 
pRb is phosphorylated by cyclin-dependent kinases whose activity can be negatively regulated by cyclin-dependent kinase inhibitors (5). In particular, the cyclin-dependent kinase inhibitor p21CIP1 has a well-characterized role in mediating cell cycle arrest during differentiation (6, 7), and expression of this protein has been associated with pRb dephosphorylation and growth arrest of leukemic cells in response to differentiation-inducing agents (15-17). To determine whether p21CIP1 was expressed in response to C2-ceramide-induced U937 differentiation, cells were treated as described above with C2-ceramide, PMA, or a combination of these reagents, and p21CIP1 expression was analyzed by Western blotting 24 h after the addition of reagents (Fig. 5GoA). p21CIP1 was strongly expressed in U937 cells undergoing C2-ceramide-induced differentiation, but was not observed in vehicle-, PMA- or C2-ceramide- plus PMA-treated cells at this time point. Time-course studies showed that p21CIP1 was strongly expressed by 4 h postaddition of C2-ceramide and that expression continued after the reagent was removed from the culture at 8 h (Fig. 5GoB). In contrast, such sustained and strong expression of p21CIP1 was not observed in cells treated with PMA or C2-ceramide plus PMA (Fig. 5GoC). Occasional weak and transient expression of p21CIP1 was seen in cells treated with PMA (Fig. 5GoB, 8 and 16 h) or C2-ceramide plus PMA; however, such expression was not as intense or as stable as that observed after treatment with C2-ceramide.



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  		FIGURE 5. C2-ceramide-induces expression of p21CIP1, a cyclin-dependent kinase inhibitor. U937 cells were treated with the indicated reagents as described above each lane. Whole cell lysates were prepared and analyzed by 14% SDS-PAGE and anti-p21CIP1 immunoblotting. A, Induction of p21CIP1 in response to treatment with C2-ceramide. Lysates were prepared 24 h after addition of reagents. B, Time-course study of p21CIP1 expression. C2-ceramide induced strong and stable expression of p21CIP1, whereas PMA induced only weak and transient expression. C, The concomitant stimulation with C2-ceramide and PMA did not induce p21CIP1. D, Sphingosine induced p21CIP1. PMA was used at 160 nM in D. The results shown are representative of three separate experiments.

 
Interestingly, sphingosine is also known as an inducer of G0/G1 cell cycle arrest that is accompanied by pRb dephosphorylation (8). We therefore investigated whether sphingosine, like C2-ceramide, induces p21CIP1 expression in U937 cells. As shown in Figure 5GoD, sphingosine induced p21CIP1, whereas the dose response of this induction was apparently slightly lower than that of C2-ceramide. Sphingosine-induced p21CIP1 expression was negated when PMA was added to the culture (Fig. 5GoD). Importantly, although effective inhibition of cell growth occurred in parallel to the expression of p21CIP1, sphingosine was unable to induce the morphologic changes and CD11b/CD14 expression associated with differentiation (data not shown). The data thus indicate that G0/G1 phase cell cycle arrest is not mandatory for U937 differentiation, while terminal differentiation may result in cell cycle arrest.

C2-ceramide and PMA differentially activate MAP kinases

The MAP kinases are coupled to many diverse extracellular stimuli, including growth factors, hormones, and stress, and appear to regulate proliferation and differentiation (18-22). Differential activation of the MAP kinase cascades is a feature of certain antagonistic stimuli with the preferential activation of a MAP kinase cascade being related to a distinct phenotype or function (21-25). To determine whether the distinct phenotypes that result from differentiation induced by either C2-ceramide or PMA could be associated with preferential activation of either the JNK or ERK cascades, U937 cells were treated with 100 µM C2-ceramide, 160 nM (100 ng/ml) PMA, or a combination of these reagents for the indicated time periods, and immunoprecipitated JNK1 or ERK2 was subjected to in vitro kinase assays (Figs. 6Go and 7). Treatment of U937 cells with C2-ceramide resulted in the strong activation of JNK, as evidenced by phosphorylation of the c-Jun substrate (Fig. 6Go, top left panel). The stimulation of JNK with C2-ceramide was observed in the doses >10 µM (data not shown). Strikingly, this C2-ceramide-induced activation of JNK was abolished by simultaneous addition of PMA. Addition of PMA alone did not activate JNK (Fig. 6Go, top right panel). The converse applied to activation of ERK2 by C2-ceramide and PMA; C2-ceramide did not activate ERK2 (Fig. 7Go, right panel), whereas PMA strongly activated this MAP kinase. Interestingly, simultaneous addition of C2-ceramide plus PMA potentiated ERK2 activation compared with activation achieved by PMA alone (Fig. 7Go, left panel). In additional experiments, we investigated whether JNK activation occurs in cells treated with sphingosine. To our surprise, we consistently observed that sphingosine induced JNK activation in a dose-dependent manner (Fig. 6Go, bottom panel), whereas sphinganine that differs from sphingosine only in that lacks the double bond in the sphingoid backbone did not stimulate JNK or differentiation in U937 cells (data not shown). The data suggest that differentiation induced by C2-ceramide is regulated by a distinct signaling pathway in which JNK activation is unlikely toplay a role.



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  		FIGURE 6. C2-ceramide and sphingosine activate JNK MAP kinase. U937 cells were treated with 100 µM C2-ceramide, 160 nM (100 ng/ml) PMA, or a combination of these reagents for the indicated time periods (shown in top panels). U937 cells were treated with sphingosine at the doses indicated below for 10 min (shown in bottom panel). JNK activation was determined by using immunoprecipitated JNK1 in an in vitro kinase assay with glutathione-S-transferase-c-Jun79 as substrate. Results are representative of three separate experiments.

 


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  		FIGURE 7. PMA, but not C2-ceramide, activates ERK2 MAP kinase. U937 cells were treated with 100 µM C2-ceramide, 160 nM (100 ng/ml) PMA, or a combination of these reagents for the indicated time periods. ERK activation was determined by using immunoprecipitated ERK2 in an in vitro kinase assay with myelin basic protein as substrate. Results are representative of five separate experiments.

 
Inhibition of MEK prevents PMA-induced differentiation of U937 cells

The above data indicate that PMA or C2-ceramide selectively activates the ERK or JNK MAP kinase pathways, respectively, in U937 cells. To determine whether ERK signaling is, in fact, antagonistic to ceramide-induced U937 differentiation, the ERK pathway was inhibited using PD98059, a selective inhibitor of MEK, the ERK-activating kinase (18). Pretreatment of cells with 100 µM PD98059 for 30 min completely blocked the activation of ERK2 by PMA, but did not inhibit ceramide-mediated JNK activation (data not shown), a finding in accordance with other reports (18, 20, 23). To assess the effect of MEK inhibition on U937 differentiation, cells were pretreated with 100 µM PD98059 for 30 min before addition of C2-ceramide, PMA, or C2-ceramide and PMA. Cells were cultured under conditions identical to those described for Figures 1Go and 2Go, except that 100 µM PD98059 was continually present in the medium. Cells were harvested after 3 days and examined by immunofluorescence and flow cytometry (Fig. 8Go). PD98059 caused a slight decrease in CD11b expression in vehicle-treated and control cells; however, C2-ceramide-induced differentiation was not impeded, and the differentiated cells were indistinguishable from those observed in Figures 1Go and 2Go. In contrast, PMA-induced differentiation was prevented by MEK inhibition; the cells were comparable, under the criteria of CD14 and CD11b expression (Fig. 8Go) and morphology (data not shown), to the vehicle-treated cells. Strikingly, PD98059-treated cells induced to differentiate by C2-ceramide plus PMA were indistinguishable from cells treated with C2-ceramide alone; in the absence of MEK inhibition, these cells gain the phenotype associated with PMA treatment. In summary, PD98059 inhibited PMA-induced differentiation, but not that mediated by C2-ceramide, thus linking activation of a particular MAP kinase pathway to a specific differentiation phenotype.



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  		FIGURE 8. Inhibition of ERK signaling prevents PMA-induced, but not C2-ceramide-induced, differentiation. U937 cells were pretreated with 100 µM PD98059, a MEK inhibitor, for 30 min before the addition of reagents. Cells were treated with the indicated reagents as described, with 100 µM PD98059 continuously present. Cells were analyzed for CD14 and CD11b expression after 72 h of culture. Changes in the mean fluorescence intensity of the cell population were determined by comparison with identically stained parental U937 cells. Results are derived from three separate experiments. Error bars represent the SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study examined differentiation of U937 monoblastic leukemia cells induced by the synthetic lipid analogue, C2-ceramide, and compared C2-ceramide differentiation with that induced by the phorbol ester, PMA. Our observations established a clear segregation between PMA and ceramide as they target different signaling pathways. Accordingly, Ca2+-dependent protein kinase Cs are the target of PMA (26), while ceramide appears to activate diverse kinases and phosphatases (27). Furthermore, cell-permeable ceramide is likely to be metabolized to other compounds such as sphingosine 1-monophosphate, glycosylsphingosine, and sphingosine, which might stimulate additional pathways (28). The data presented in this study indicated that JNK activation is unlikely to regulate differentiation by ceramide because sphingosine-induced JNK activation occurred in the absence of morphologic change associated with differentiation. Since dihydroceramide and sphinganine, which lack the 4,5-trans double bond in sphingoid backbone of ceramide and sphingosine, respectively, did not stimulate JNK or differentiation, the double bond in sphingoid backbone must play a key for the JNK activation. On the other hand, to induce the differentiation, both double bond in sphingoid backbone and amide-linked fatty-acyl chain of ceramide appeared to be required. It is therefore probable that ceramide and sphingosine, via double bond in sphingoid backbone, stimulate JNK -> ATF2/E1A/pRb pathway, which may control transcription of the p21CIP1 gene (29-31). Upon elucidating the target pathway(s) of ceramide and sphingosine we will be able to understand the cell differentiation-specific signaling pathway. Ceramide-specific kinases and phosphatase may be considered as the prime candidates that control differentiation.

C2-ceramide-induced differentiation was associated with G0/G1 cell cycle arrest and dephosphorylation (activation) of the pRb tumor suppressor nuclear phosphoprotein. A similar ceramide analogue, C6-ceramide, also induced cell cycle arrest and pRb dephosphorylation in various cell types (4, 8, 32); however, these studies did not link these events with leukemic differentiation. The demonstration in the present study that C2-ceramide rapidly induced stable expression of the cyclin-dependent kinase inhibitor p21CIP1 provides what is believed to be the first report of a ceramide-induced effector mechanism that links cell cycle inhibition and pRb activation with ceramide treatment of cells. The p21CIP1 protein complexes with and inhibits the ability of G1 cyclin-dependent kinases to phosphorylate the pRb protein and mediate the G1-S transition (33, 34). Importantly, C2-ceramide induced p21CIP1 expression in a p53-independent manner as U937 cells lack a functional p53 gene (16, 17, 35), thus implying that ceramide-mediated signaling mechanisms may provide a terminal means for activation of differentiation via p21CIP1 in p53null tumor cells. Since we observed that sphingosine also induced p21CIP1 expression, sphingosine may be the effector that, upon being metabolized from ceramide, coordinates cell cycle arrest. Taken together, these data indicate that C2-ceramide, via multiple pathways, induces molecular changes associated with terminal differentiation of leukemic cells.

Studies have shown that PMA-induced differentiation of U937 cells is unstable and transient, with retrodifferentiation occurring upon withdrawal of PMA (36-38). Chronic treatment of U937 cells with PMA results in G0/G1 cell cycle arrest after approximately 24-36 h (37). Similarly, chronic exposure to PMA can induce p21CIP1 expression, although this expression is transient (17, 39, 40), and PMA-induced cell cycle arrest was reversed upon return of p21CIP1 to basal levels (40). However, there is no reported link between p21CIP1 expression and pRb hypophosphorylation in hemopoietic cells chronically treated with PMA; in fact, PMA is paradoxically reported to induce both pRb hyperphosphorylation (41, 42) and dephosphorylation (14) in hemopoietic cells. The present study used acute exposure to PMA, and the resulting differentiation was not associated with early cell cycle arrest or pRb dephosphorylation, while very weak and transient p21CIP1 expression was observed. This contrasted strongly with the molecular changes associated with acute C2-ceramide induction of U937 differentiation and leads to the suggestion that C2-ceramide initiates a self-perpetuating series of events leading to terminal differentiation, whereas constant stimulation by PMA is required to maintain growth arrest and differentiation.

The question remains as to whether the dissimilar phenotypes that result from differentiation induced by C2-ceramide and PMA is due to differential activation of MAP kinase cascades. PMA may exclusively initiate ERK signaling, whereas JNK appeared to represent one of many effectors activated by C2-ceramide. Furthermore, U937 cells treated simultaneously with C2-ceramide and PMA acquired the phenotype associated with PMA-induced differentiation; this phenotypic dominance by PMA is possibly related to the fact that C2-ceramide- plus PMA-potentiated ERK2 activation while inhibiting JNK activation, thus further supporting the hypothesis that phenotype is related to activation of a particular MAP kinase. Concurrent selective activation and inhibition of JNK and ERK MAP kinase cascades in regulating ceramide-induced cell death (apoptosis) vs growth have been reported (23), and the present study demonstrates that this effect on specific MAP kinase activity can be extended to include C2-ceramide-induced growth arrest and differentiation. Activation of the ERK pathway during PMA-induced U937 differentiation may also account for the fact that the resulting phenotype does not undergo stable growth arrest; activation of ERK is associated with mitogenic signals (24, 25, 43), whereas JNK activation is associated with growth arrest, differentiation, and apoptosis (11, 21, 23-25, 43-45). Accordingly, in the present study, C2-ceramide-induced differentiation associated JNK activation with cell cycle arrest, p21CIP1 expression, and pRb dephosphorylation.

Studies using the MEK-specific inhibitor PD98059 provided striking evidence that MAP kinase cascades are critically involved in determining the phenotype of differentiating leukemic cells. PD98059 completely blocked PMA-induced U937 differentiation, thereby demonstrating the reliance upon the ERK MAP kinase cascade. Conversely, C2-ceramide-induced differentiation was not inhibited by PD98059, thus confirming the independence of ceramide-JNK signaling from the ERK cascade. Cells treated with C2-ceramide plus PMA in the presence of PD98059 acquired the same differentiated phenotype as cells treated with C2-ceramide alone, a finding that represents a reversal of phenotype due to inhibition of MEK. Thus, inhibition of the ERK cascade released the PMA-induced block upon the ceramide-specific differentiation pathway, allowing the C2-ceramide phenotype to be expressed.

If cellular differentiation is blocked at a proliferative stage, then blastic myeloid leukemia results (46). Elucidation of the signal transduction pathways and molecular effector mechanisms that can force the leukemic clone into terminal differentiation will provide new therapeutic strategies for treatment of this disease. All-trans-retinoic acid (ATRA) and 1{alpha},25-dihydroxyvitamin D3 (VD3) have been successfully used in clinical differentiation therapy; in fact, VD3 appears to induce differentiation via intracellular ceramide synthesis (3, 47). However, while differentiation therapy using ATRA or VD3 is encouraging, a high percentage of patients in complete remission induced by ATRA alone relapsed within a few months (48-50), whereas VD3 is limited in its therapeutic use by its hypercalcemic effects. Therefore, synthetic agents that act more selectively on cell growth inhibition and differentiation are required. Ceramide and/or its specific signal transduction pathway(s) may be exploitable for clinical differentiation therapy.

In conclusion, induction of myeloid differentiation by the synthetic lipid C2-ceramide was associated with growth and cell cycle arrest, expression of the cyclin-dependent kinase inhibitor p21CIP1, and activation of the JNK MAP kinase pathway. These results may provide the basis for further investigations into the potential use of ceramide analogues in clinical differentiation therapy of hemopoietic malignancies.


   Footnotes
 
1 This work was supported by grants from the National Cancer Institute of Canada (Grant 006349) and the Leukemia Research Fund of Canada, a Medical Research Fellowship from Merck Frosst, Canada (to S.J.R.), and a Multi Organ Transplant Service Research Fellowship (to A.O.). Back

2 Address correspondence and reprint requests to Dr. Atsuo Ochi, John P. Robarts Research Institute, 1400 Western Rd., London, Ontario, Canada N6G 2V4. E-mail address: Back

3 Abbreviations used in this paper: pRb, retinoblastoma nuclear phosphoprotein; MEK, extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; PBST, phosphate-buffered saline and 0.1% Tween 20; MAP, mitogen-activated protein; ATRA, all-trans-retinoic acid; VD3, 1{alpha},25-dihydroxyvitamin D3. Back

Received for publication July 21, 1997. Accepted for publication March 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bohbot, A., A. Eischen, C. Felden, F. Vincent, F. Oberling. 1993. Transforming growth factor-ß potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines. Exp. Hematol. 21:564.[Medline]
  2. Ways, D. K., K. Posekany, J. de Vente, T. Garris, J. Chen, J. Hooker, W. Qin, P. Cook, D. Fletcher, P. Parker. 1994. Transforming growth factor-ß potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines. Cell Growth Differ. 5:1195.[Abstract]
  3. Okazaki, T., A. Bielawska, R. M. Bell, Y. A. Hannun. 1990. Role of ceramide as a lipid mediator of 1{alpha},25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J. Biol. Chem. 265:15823.[Abstract/Free Full Text]
  4. Jayadev, S., B. Liu, A. E. Bielawska, J. Y. Lee, F. Nazaire, M. Y. Pushkareva, L. M. Obeid, Y. A. Hannun. 1995. Role for ceramide in cell cycle arrest. J. Biol. Chem. 270:2047.[Abstract/Free Full Text]
  5. Grana, X., E. P. Reddy. 1995. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11:211.[Medline]
  6. Parker, S. B., G. Eichele, P. Zhang, A. Rawls, A. T. Sands, A. Bradley, E. N. Olson, J. W. Harper, S. J. Elledge. 1995. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science 267:1024.[Abstract/Free Full Text]
  7. Halevy, O., B. G. Novitch, D. B. Spicer, S. X. Skapek, J. Rhee, G. J. Hannon, D. Beach, A. B. Lassar. 1995. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 267:1018.[Abstract/Free Full Text]
  8. Dbaibo, G. S., M. Y. Pushkareva, S. Jayadev, J. K. Schwarz, J. M. Horowitz, L. M. Obeid, Y. A. Hannun. 1995. Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest. Proc. Natl. Acad. Sci. USA 92:1347.[Abstract/Free Full Text]
  9. Nicoletti, I., G. Migliorati, M. C. Pagliacci, F. Grignani, C. Riccardi. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271.[Medline]
  10. Chao, R., W. Khan, Y. Hannun. 1992. Retinoblastoma protein dephosphorylation induced by D-erythro-sphingosine. J. Biol. Chem. 267:23459.[Abstract/Free Full Text]
  11. Verheij, M., R. Bose, X. H. Lin, B. Yao, W. D. Jarvis, S. Grant, M. J. Birrer, E. Szabo, L. I. Zon, J. M. Kyriakis, A. Haimovitz-Friedman, Z. Fuks, R. N. Kolesnick. 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380:75.[Medline]
  12. Weinberg, R. A.. 1991. Tumor suppressor genes. Science 254:1138.[Abstract/Free Full Text]
  13. Mihara, K., X. R. Cao, A. Yen, S. Chandler, B. Driscoll, A. L. Murphree, A. T’Ang, Y. K. Fung. 1989. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246:1300.[Abstract/Free Full Text]
  14. Chen, P. L., P. Scully, J. Y. Shew, J. Y. Wang, W. H. Lee. 1989. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 58:1193.[Medline]
  15. Jiang, H., J. Lin, Z. Z. Su, F. R. Collart, E. Huberman, P. B. Fisher. 1994. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene 9:3397.[Medline]
  16. Steinman, R. A., B. Hoffman, A. Iro, C. Guillouf, D. A. Liebermann, M. E. el-Houseini. 1994. Induction of p21 (WAF-1/CIP1) during differentiation. Oncogene 9:3389.[Medline]
  17. Zhang, W., L. Grasso, C. D. McClain, A. M. Gambel, Y. Cha, S. Travali, A. B. Deisseroth, W. E. Mercer. 1995. p53-independent induction of WAF1/CIP1 in human leukemia cells is correlated with growth arrest accompanying monocyte/macrophage differentiation. Cancer Res. 55:668.[Abstract/Free Full Text]
  18. Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, A. R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92:7686.[Abstract/Free Full Text]
  19. Kang, C. D., B. K. Lee, K. W. Kim, C. M. Kim, S. H. Kim, B. S. Chung. 1996. Signaling mechanism of PMA-induced differentiation of K562 cells. Biochem. Biophys. Res. Commun. 221:95.[Medline]
  20. Pang, L., T. Sawada, S. J. Decker, A. R. Saltiel. 1995. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270:13585.[Abstract/Free Full Text]
  21. Xia, Z., M. Dickens, J. Raingeaud, R. J. Davis, M. E. Greenberg. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326.[Abstract/Free Full Text]
  22. Campbell, J. S., M. P. Wenderoth, S. D. Hauschka, E. G. Krebs. 1995. Differential activation of mitogen-activated protein kinase in response to basic fibroblast growth factor in skeletal muscle cells. Proc. Natl. Acad. Sci. USA 92:870.[Abstract/Free Full Text]
  23. Cuvillier, O., G. Pirianov, B. Kleuser, P. G. Vanek, O. A. Coso, S. Gutkind, S. Spiegel. 1996. Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381:800.[Medline]
  24. Pyne, S., N. J. Pyne. 1996. The differential regulation of cyclic AMP by sphingomyelin-derived lipids and the modulation of sphingolipid-stimulated extracellular signal regulated kinase-2 in air way smooth muscle. Biochem. J. 315:917.
  25. Pyne, S., L. Steele, J. Chapman, N. J. Pyne. 1996. Sphingomyelin-derived lipids differentially regulate the extracellular signal-regulated kinase-2 (ERK-2) and c-Jun N-terminal kinase (JNK) signal cascades in airway smooth muscle. Eur. J. Biochem. 237:819.[Medline]
  26. Tsutsumi, A., M. Kubo, H. Fujii, J. Freire-Moar, C. W. Turck, J. T. Ransom. 1993. Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells. J. Immunol. 150:1746.[Abstract]
  27. Ballou, L. R., S. J. F. Laulederkind, E. F. Rosloniec, R. Raghow. 1996. Ceramide signalling and the immune response. Biochim. Biophys. Acta 1301:273.[Medline]
  28. Hannun, Y. A.. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125.[Free Full Text]
  29. Horowitz, J. M., D. W. Yandell, S. H. Park, S. Canning, P. Whyte, K. Buchkovich, E. Harlow, R. A. Weinberg, T. P. Dryja. 1989. Point mutational inactivation of the retinoblastoma antioncogene. Science 243:937.[Abstract/Free Full Text]
  30. Kim, S. J., S. Wagner, F. Liu, M. A. O’Reilly, P. D. Robbins, M. R. Green. 1992. Retinoblastoma gene product activates expression of the human TGF-ß2 gene through transcription factor ATF-2. Nature 358:331.[Medline]
  31. Gupta, S., D. Campbell, B. Derijard, R. J. Davis. 1995. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267:389.[Abstract/Free Full Text]
  32. Venable, M. E., J. Y. Lee, M. J. Smyth, A. Bielawska, L. M. Obeid. 1995. Role of ceramide in cellular senescence. J. Biol. Chem. 270:30701.[Abstract/Free Full Text]
  33. Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, S. J. Elledge. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 74:805.
  34. Xiong, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366:701.[Medline]
  35. Liu, M., M. H. Lee, M. Cohen, M. Bommakanti, L. P. Freedman. 1996. Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev. 10:142.[Abstract/Free Full Text]
  36. Meinhardt, G., R. Hass. 1995. Differential expression of c-myc, max and mxi1 in human myeloid leukemia cells during retrodifferentiation and cell death. Leuk. Res. 19:699.[Medline]
  37. Hass, R., H. Gunji, R. Datta, S. Kharbanda, A. Hartmann, R. Weichselbaum, D. Kufe. 1992. Differentiation and retrodifferentiation of human myeloid leukemia cells is associated with reversible induction of cell cycle-regulatory genes. Cancer Res. 52:1445.[Abstract/Free Full Text]
  38. Hass, R.. 1992. Retrodifferentiation: an alternative biological pathway in human leukemia cells. Eur. J. Cell Biol. 58:1.[Medline]
  39. Asiedu, C., J. Biggs, M. Lilly, A. S. Kraft. 1995. Inhibition of leukemic cell growth by the protein kinase C activator bryostatin 1 correlates with the dephosphorylation of cyclin-dependent kinase 2. Cancer Res. 55:3716.[Abstract/Free Full Text]
  40. Zeng, Y. X., W. S. el-Deiry. 1996. Regulation of p21WAF1/CIP1 expression by p53-independent pathways. Oncogene 12:1557.[Medline]
  41. Garcia, P., C. Cales. 1996. Endoreplication in megakaryoblastic cell lines is accompanied by sustained expression of G1/S cyclins and downregulation of cdc25C. Oncogene 13:695.[Medline]
  42. Lee, J. Y., Y. A. Hannun, L. M. Obeid. 1996. Ceramide inactivates cellular protein kinase C{alpha}. J. Biol. Chem. 271:13169.[Abstract/Free Full Text]
  43. Kyriakis, J. M., P. Banerjee, E. Nikolakaki, T. Dai, E. A. Rubie, M. F. Ahmad, J. Avruch, J. R. Woodgett. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156.[Medline]
  44. Westwick, J. K., A. E. Bielawska, G. Dbaibo, Y. A. Hannun, D. A. Brenner. 1995. Ceramide activates the stress-activated protein kinases. J. Biol. Chem. 270:22689.[Abstract/Free Full Text]
  45. Szabo, E., L. H. Preis, M. J. Birrer. 1994. Constitutive c-Jun expression induces partial macrophage differentiation in U-937 cells. Cell Growth Differ. 5:439.[Abstract]
  46. Testa, U., R. Masciulli, E. Tritarelli, R. Pustorino, G. Mariani, R. Martucci, T. Barberi, A. Camagna, M. Valtieri, C. Peschle. 1993. Transforming growth factor-ß potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines. J. Immunol. 150:2418.[Abstract]
  47. Okazaki, T., R. M. Bell, Y. A. Hannun. 1989. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. J. Biol. Chem. 264:19076.[Abstract/Free Full Text]
  48. Glasser, L., G. J. Shamdas, R. Fiederlein, A. R. Brothman. 1994. Functional characteristics of in vivo induced neutrophils after differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid. Cancer 73:1206.[Medline]
  49. Chen, Z. X., C. Li, Y. Q. Xue, W. Wang, R. Zhang, W. Y. Zu, R. F. Tao, X. Z. Yao, X. M. Xia, B. J. Ling. 1991. A clinical and experimental study on all-trans retinoic acid-treated acute promyelocytic leukemia patients. Blood 78:1413.[Abstract/Free Full Text]
  50. Castaigne, S., R. Berger, C. Chomienne, P. Fenaux, M. T. Daniel, L. Degos, P. Ballerini. 1990. All-trans retinoic acid as differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood 76:1710.[Abstract/Free Full Text]



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