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Originally published online as doi:10.1189/jlb.0403142 on August 1, 2003

Published online before print August 1, 2003
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(Journal of Leukocyte Biology. 2003;74:923-931.)
© 2003 by Society for Leukocyte Biology

Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

Richard Karlsson*, Maria Engström*, Maria Jönsson*, Peter Karlberg*, Cornelis J.H. Pronk*, Johan Richter{dagger} and Jan-Ingvar Jönsson*,1

* Division of Molecular Medicine, Department of Laboratory Medicine, Lund University, University Hospital MAS, Malmö, and
{dagger} Department of Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden

1Correspondence: Department of Laboratory Medicine University Hospital MAS, Entrance 78, 3rd floor SE-205 02, Malmö, Sweden. E-mail: Jan-Ingvar.Jonsson{at}molmed.mas.lu.se


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ABSTRACT
 
Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood. Using two myeloid progenitor cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL-mediated survival is dependent on the activation of phosphatidylinositol-3 (PI-3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL, whereas IL-3 and FL were only partially affected. Although all three cytokines induced phosphorylation of protein kinase B (PKB), only KL required PI-3 kinase activity to elicit survival in hematopoietic progenitors. In contrast, pretreatment of cells with inhibitors to the MAP kinase pathway did not affect the survival. We next established if IL-3 and FL activated antiapoptotic Bcl-2 and the related genes Bcl-XL and Mcl-1. By RNA protection assay and Western blot analysis, we show that all three genes are induced by IL-3, whereas FL induces Bcl-2 and to some extent Bcl-XL. Importantly, KL could not sustain their expression. Moreover, use of inhibitors implied that IL-3 was mainly exerting its effect on Bcl-2 at the level of transcription. The addition of LY294002 did not affect the expression of Bcl-2 and Bcl-XL, and thus, we conclude that expression of antiapoptotic Bcl-2 family member genes is not dependent on PI-3 kinase activity. Our results indicate that cytokines exert distinct survival effects and that FL and IL-3 are capable of sustaining progenitor survival by up-regulating the expression of Bcl-2 and related genes.

Key Words: Hematopoiesis • Progenitor • Cytokines • Apoptosis • PKB • Bcl-2


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INTRODUCTION
 
Hematopoiesis is regulated by the combined action of signals from the stromal microenvironment and multiple soluble factors. These regulatory signals control proliferation and survival of hematopoietic stem cells and progenitor cells by overlapping activities. However, multiple regulators are required to provide optimal growth conditions in vitro. To maintain the immature pool of stem cells and progenitors, mechanisms must be activated that suppress apoptosis and permit the cells to respond to other signals for further maturation. Several studies have indicated that certain cytokines are essential to promote survival, including kit ligand (KL; also stem cell factor or steel factor), flt3 ligand (FL), thrombopoietin (Tpo), interleukin-3 (IL-3), IL-6, and IL-11 [1 2 3 4 5 6 7 ].

The ability of cells to escape the apoptotic machinery is mediated by both positive and negative regulators, in part by members of the Bcl-2 gene family. The balance between the intracellular levels of apoptosis-promoting and apoptosis-suppressing proteins regulates the viability or death of many cell types, including hematopoietic cells. Antiapoptotic Bcl-2 is the founding member of this gene family and has been shown to be expressed in both lymphoid and myeloid progenitors [8 ]. Transgenic overexpression of the Bcl-2 gene in hematopoietic cells prevents the induction of apoptosis and leads to accumulation of progenitor cells [9 , 10 ]. Whether Bcl-2 is involved in the regulation of myeloid progenitors is unclear, and it is possible that other antiapoptotic family members may be just as important. For instance, human immature progenitor cells express antiapoptotic Bcl-XL [11 ], and in mice rendered deficient of the Bcl-XL gene by homologous recombination, immature hematopoietic progenitor cells are diminished in cell numbers by massive cell death [12 ]. Moreover, many studies have demonstrated the importance of Bcl-2, Bcl-XL, Mcl-1, and A1 during myeloid differentiation [13 14 15 16 17 18 ].

The activation of phosphatidylinositol-3 (PI-3) kinase is a major intracellular signal transduction pathway by which many cytokines can counteract apoptosis. This leads to the generation of PI-3-phosphorylated lipids in the plasma membrane and the subsequent phosphorylation and activation of the serine-threonine kinase PKB (protein kinase B; also known as Akt) by phosphatidylinositol-dependent kinase 1 (PDK1). PKB in turn has been shown to affect, directly or indirectly, different transcription factor families, e.g., Forkhead and cAMP-response-element binding protein (CREB), but also to phosphorylate and inactivate the proapoptotic Bcl-2 family member Bad (reviewed in [19 , 20 ]).

The interplay between survival and apoptotic signaling is regulated not only by the PI-3 kinase pathway, but also via the induction of the MAP kinase/extracellular signal-regulated kinase (ERK) pathways. Members of these pathways have been implicated in the survival of hematopoietic cells and are activated as a result of cellular stress, but have also been shown to play a role in cytokine-induced proliferation and/or survival. For example, recent reports have demonstrated that both IL-3 and KL can induce phosphorylation of ERK upon ligand binding [21 22 23 24 ]. Thus, it is possible that cytokines induce hematopoietic progenitor survival by dual actions of the PI-3 kinase and the MAP kinase pathways.

As some hematopoietic growth factors and cytokines can affect survival on their own, but when combined, induce strong synergistic proliferative effects, they presumably maintain survival by inhibiting apoptosis by distinct signaling mechanisms. The purpose of the present study was to investigate whether cytokines with effects on stem cells and progenitors, in particular IL-3, KL, and FL, prevent apoptosis by inducing different survival pathways. To investigate their role as antiapoptotic factors, we have used two factor-dependent hematopoietic progenitor cell lines, FDCP-mix [25 ] and FDC-P1 [26 ], expressing the receptor tyrosine kinase c-kit. To be able to study cytokine effects, we also introduced the flt3 gene by retroviral gene transfer into FDC-P1 cells. We show that all three cytokines analyzed activate the PI-3 kinase downstream target PKB. However, by using the PI-3 kinase inhibitor LY294002, we show that this pathway is required for survival mediated by KL. In contrast, both IL-3 and FL can sustain progenitor survival, even in the presence of LY294002. We then demonstrate that IL-3, and to some extent FL, can mediate apoptotic suppressive effects by the induction of several Bcl-2 family member genes.


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MATERIALS AND METHODS
 
Cells, cytokines, and reagents
FDCP-mix and FDC-P1 cells were routinely maintained in Iscove’s modified Dulbecco’s medium (IMDM; PAA Laboratories, Pasching, Austria) supplemented with IL-3-containing supernatant and either 20% horse serum (JRH Biosciences, Lenexa, KS) or 10% fetal calf serum (Gibco, Paisley, UK), respectively. Murine IL-3 was obtained as a culture supernatant from a cell line stably transfected with an expression vector containing the cDNA for IL-3. Recombinant murine KL and recombinant human FL (both from PeproTech, London, UK) were used at 100 ng/ml and 50 ng/ml, respectively. Actinomycin D, cycloheximide, LY294002, PD98059, and U0126 were from Calbiochem (Darmstadt, Germany) or Sigma (Stockholm, Sweden).

Assessment of cell growth
FDC-P1 cells were cultured in 96-well flat-bottom microtiter plates and maintained in IMDM supplemented with 10% fetal calf serum, and IL-3, KL, or FL in the absence or presence of inhibitor in 5% CO2 at 37°C. At the end of the experiment, cultures were labeled for 6 h with 0.5µCi/well [3H]-thymidine, harvested, and counted in a ß-scintillation counter.

Retroviral vector and producer cells
A retroviral vector based on the LXSN vector with an insert in the form of the full-length murine flt3 cDNA was generously obtained from Dr. Olivier Rosnet. This vector, called LF3SN, was transfected into BING cells using standard CaPO4 precipitation. BING cells were cultured in DMEM with 10% FCS and antibiotics and 24 h after transfection supernatants were harvested, filtered through a 0.45 µm low-protein binding filter (Millipore, Bedford, MA), and supplemented with protamine sulfate (Sigma) at a final concentration of 4 µg/ml. These supernatants were used for repeated transduction of GP+E86 cells [27 ], which also were cultured in DMEM with 10% FCS and antibiotics. From this polyclonal producer cell line, individual clones were isolated and viral supernatants from these clones were checked for titer by assessing transfer of G418 resistance to NIH3T3 cells at limiting dilution. Viral supernatants from clone LF3SN6 yielded the highest viral titers (>2 x 106 inf. units/ml), and this producer clone was used for all further experiments. Viral supernatant from LF3SN6 was shown to be free from helper virus by a marker rescue assay and by PCR.

Retroviral infections of hematopoietic cells
Infection of hematopoietic progenitor cell lines was performed by cocultivation on a 50-75% confluent monolayer of irradiated (20 Gy) LF3SN6 cells for 48 h in the presence of protamine sulfate (4 µg/ml), after which they were harvested and allowed to recover before selection in G418 (Gibco; 600 µg/ml active substance). After 1 week, G418-resistant cells were analyzed by FACS for expression of flt3 and c-kit with PE-labeled antibodies (PharMingen, San Jose, CA) and were subsequently used in experiments with FL.

Enrichment of progenitor cells from mouse bone marrow
Bone marrow cells were prepared from femur and tibia from 2-4-month-old female C57BL/6J mice. Low-density cells were isolated on Nycoprep 1.077 animal lymphocyte separation medium (Nycomed, Oslo, Norway) and blocked with mouse IgG before incubating on ice for 30 min with mAbs to B220, CD4, CD8, Gr-1, and Mac-1. After washing, the cells were magnetically depleted of Lin+ cells using the Vario-MACS system (Miltenyi, Cologne, Germany).

Western blot analysis
Cells were lysed in buffer containing 25mM Tris-HCl pH 7,5; 150mM NaCl; 1% Triton X-100; 1mM EDTA; 1 mM DTT; 2 mM Na3VO4; and Complete Protease Inhibitor from Roche, Mannheim, Germany. Protein determination was performed on cleared lysates using the Bradford method and confirmed by Poinceau staining. Lysates corresponding to 20-40 µg total protein were resolved by SDS-PAGE on a 10% minigel and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Antibodies used in this study were hamster antimurine Bcl-2 (PharMingen), rabbit antimurine Bcl-XL (Santa Cruz BioTechnology, Santa Cruz, CA), mouse anti-human Mcl-1 (PharMingen), anti-phospho-ERK (Cell Signaling, Beverly, MA), and anti-phospho-Ser473 PKB (Cell Signaling). Antibody to ERK (Transduction Laboratories, San Jose, CA), GAPDH (Chemicon, Temecula, CA), or PKB (Upstate Biotechnology, Lake Placid, NY) were used as controls. Horseradish peroxidase-conjugated antibodies used were from Jackson (West Grove, PA), Amersham Pharmacia Biotech (Buckinghamshire, UK), and Bio-Rad (Hercules, CA).

Assessment of apoptosis using FACS analysis
An apoptosis detection kit labeling cells with FITC-conjugated annexin V was used as recommended by the manufacturer (R and D Systems, Minneapolis, MN). Briefly, cells were stimulated with IL-3, KL, or FL for 24 h, and then harvested, washed twice with cold PBS, and stained with FITC-conjugated annexin V and propidium iodide. After incubation for 15 min at room temperature, cells were analyzed by flow cytometry.

RNA extraction and RNase protection assay
Total RNA preparations were isolated with Trizol reagent (Gibco). To quantify the expression of the Bcl-2 gene family, we applied the RiboQuantTM kit mAPO-2 from PharMingen and performed RNase protection assays, according to the manufacturer’s recommendations. Briefly, high specific-activity riboprobes were generated by in vitro transcription and these were hybridized overnight with aliquots of 5 µg of each RNA sample. After electrophoresis, the polyacrylamide gel was dried without fixation and transferred to Whatman paper and exposed to Kodak XAR-5 film.


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RESULTS
 
PI-3 kinase mediates phosphorylation of PKB induced by IL-3, KL, and FL
To compare signaling events crucial for cytokine mediated survival via the three major hematopoietic regulators IL-3, KL, and FL, we introduced the flt3 gene into the factor-dependent myeloid progenitor cell line FDC-P1 by retroviral gene transfer. After selection, these cells were shown by FACS analysis to express comparative levels of flt3 and c-kit (Fig. 1A 1B ), indicating that a significant fraction of the cells should be able to respond to both KL and FL. Both the parental cell line FDC-P1, as well as infected FDC-P1/flt3 cells showed little or no proliferation in the presence of KL, whereas introduction of the flt3 gene to FDC-P1 cells render them proliferative in response to FL (Fig. 1C) . Combinations of FL with KL or low amounts of IL-3 were clearly synergistic, and thus, a 1000-fold lower concentration of IL-3, together with FL, induced the same level of proliferation as the high concentration of IL-3 alone.



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  		Figure 1. FDC-P1 and FDC-P1/flt3 cells express c-kit and flt3 and proliferate in the presence of IL-3 or FL. FACS analysis of FDC-P1 and FDC-P1/flt3 cells for expression of (A) flt3 and (B) c-kit. Open histograms indicate staining with control antibodies. FDC-P1 (open bars), and FDC-P1/flt3 cells (black bars) were grown in the presence of IL-3 at either 200 U/ml (IL-3 high) or 0.2 U/ml (IL-3), KL at 100 ng/ml, FL at 50 ng/ml, or (C) in combination of the various cytokines for 48 h. The data presented represent the mean value ± SD from three independent experiments.

Complete block of KL-mediated survival in myeloid progenitor cell lines by the PI-3 kinase inhibitor LY294002
In agreements with previous studies, we found that IL-3 and KL can induce phosphorylation of the PI-3 kinase downstream target PKB, whose activity often is associated with increased rate of survival. We also add the novel finding that FL induces rapid phosphorylation of PKB. The level of phosphorylation was comparable between the three cytokines and appeared in a transient manner and was significantly lower after 120 min compared with the high phosphorylation after 15 min (Fig. 2A ). In all cases, PKB phosphorylation was inhibited by the PI-3 kinase inhibitor LY294002 (50µM). In addition, we found that survival by all three cytokines was strongly inhibited by LY294002. Thus, when FDC-P1/flt3 cells were incubated with cytokines for 24 h and then stained for annexin-V expression to evaluate apoptotic cells, the majority of the cells died when high concentrations of LY29402 (50 µM) were added (Fig. 2B) . However, one major difference was seen. After 24 h, LY294002 reduced the numbers of viable cells from 83% to 40% with IL-3 and to 22% with FL, whereas almost no viable cells were found in cultures with KL. This suggests that the signal-mediating survival via c-kit requires PI-3 kinase activity, whereas IL-3 and FL can activate alternative pathways. Consistently, when we used lower concentrations of LY294002 (5 µM), no significant decrease in the numbers of viable cells was seen after 48 h in IL-3 or FL, whereas in the case of KL, the numbers of viable cells were reduced from 70% to 29% (Fig. 2C) . Thus, despite similar effects on PKB phosphorylation, IL-3 and FL must be able to induce alternative survival pathways not linked to the anti-apoptotic effects mediated via PKB.



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  		Figure 2. PI-3 kinase-dependent phosphorylation of PKB by IL-3, KL, and FL, but only KL-mediated survival requires PI-3 kinase activity. (A) FDC-P1/flt3 cells were deprived of cytokine for 12 h. Thirty min before the addition of cytokine, cells were incubated with LY294002 (50 µM), after which IL-3 (200 U/ml), KL (100 ng/ml), or FL (50 ng/ml) was added for the indicated times, and the level of PKB phosphorylation was determined from whole cell lysates by immunoblotting. (B-C) Effects of LY294002 on survival of FDC-P1/flt3 cells. After 12 h incubation without cytokines, cells were incubated with no cytokine (-), IL-3 (200 U/ml), KL (100 ng/mL), or FL (50 ng/mL), without inhibitor (white bars) or in the presence of LY294002 at 5µM (gray) or 50µM (black) for (B) 24 or (C) 48 h, respectively. In all experiments, the proportion of dead cells was determined after by annexin V staining, followed by FACS analysis. Results presented are from one representative experiment out of three performed.

MAP kinase inhibitors are not able to inhibit IL-3-, KL-, or FL-mediated survival
The MAP kinase/ERK pathway can also induce antiapoptotic mechanisms in hematopoietic progenitors, although the downstream mechanisms have not been as fully elucidated as for the PI-3 kinase pathway. At first, we analyzed if ERK proteins were phosphorylated upon cytokine stimulation. As seen in Fig. 3A , IL-3, KL, and FL induced rapid phosphorylation of both p42 ERK2 and p44 ERK1, which was blocked by the addition of the MAP kinase inhibitors PD98059 and U0126. We then included these inhibitors in the annexin-V assays as described above. In contrast to the addition of LY294002, the numbers of annexin-V positive cells were not increased after 24 h (Fig. 3B) , which was in clear contrast to the complete lack of viable cells with LY294002 (Fig. 2B) . After 48 h, however, a slight decrease of viable cells (25% reduction) was seen with KL (Fig. 3C) . In addition, both PD98059 and U0126 were able to inhibit proliferation of cells regardless of which cytokine that was added (data not shown).



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  		Figure 3. The MAP kinase ERK is phosphorylated by IL-3, KL, and FL but is not required for survival of progenitor cells. Experiments were performed as described in Figure 2 , with the exception that in (A) cytokine-deprived FDC-P1/flt3 cells were preincubated for 30 min with PD98059 or U0126 at 50µM, after which IL-3 (200 U/ml), KL (100 ng/ml), or FL (50 ng/ml) was added, and the level of ERK phosphorylation was determined. (B-C) Effects of MAPK inhibitors on survival of FDC-P1/flt3 cells. Cytokine-deprived cells were incubated with no cytokine (-), IL-3 (200 U/ml), KL (100 ng/mL), or FL (50 ng/mL), without inhibitor (white bars) or in the presence of U0126 at 50µM (gray) or PD98059 at 50 µM (black) for (B) 24 or (C) 48 h, after which dead cells were determined by annexin V staining followed by FACS analysis. Results presented are from one representative experiment out of three performed.

KL does not induce expression of the Bcl-2 or Bcl-XL genes
Previous studies have indicated that some hematopoietic cytokines with the ability to block apoptosis are mediating their effects via the antiapoptotic gene Bcl-2. In addition, Bcl-XL is an important regulator of apoptosis in some types of hematopoietic cells. Thus, the reason for the cells surviving in the presence of LY294002 after stimulation with IL-3 or FL could be due to the activation of Bcl-2 or Bcl-XL. Therefore, we decided to examine the expression of Bcl-2 and Bcl-XL by Western blot analysis after stimulation with IL-3, KL, or FL for 12, 24, and 48 h, respectively, in FDC-P1/flt3 cells. Figure 4A shows that the addition of IL-3 or FL after cytokine deprivation led to a continued expression of Bcl-2 and Bcl-XL. In clear contrast, the expression of these proteins declined rapidly in KL-stimulated cells and was undetectable after 48 h of stimulation.



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  		Figure 4. IL-3 and FL, but not KL, induce expression of the Bcl-2 and Bcl-XL transcripts and sustain protein expression in FDC-P1/flt3 cells. Cells were deprived of cytokine for 12 h, then stimulated with IL-3 (200U/ml), KL (100ng/ml), or FL (50ng/ml). (A) Protein lysates were prepared after 0, 12, 24, and 48 h, and Western blot analysis measuring Bcl-2 and Bcl-XL protein was performed. (B) Total RNA were harvested after 0, 3, 6, 9, 12, and 24 h, and RNase protection assay was performed measuring mRNA levels of the Bcl-2 gene family, according to the manufacturer’s recommendations. Relative expression of the (C) Bcl-2, (D) Bcl-XL, and (E) Bax genes after stimulation with IL-3 (squares), KL (circles), or FL (triangles) were monitored by scanning the autoradiograms and analyzing the intensities of all bands compared with background levels for L32 and GAPDH. The data presented are from one representative experiment out of three.

Using a RNase protection assay, we investigated whether other antiapoptotic Bcl-2 family member genes may be induced by KL. However, a similar pattern emerged at the RNA level as with the Western blot analysis (Fig. 4B) . IL-3 stimulation led to strong and rapid up-regulation of Bcl-2 mRNA, whereas FL led to smaller but still significant up-regulation (Fig. 4C) . When the level of Bcl-XL was analyzed, IL-3 led to enhanced mRNA expression, although not as much as was seen for Bcl-2. In contrast, FL had a smaller effect on Bcl-XL (Fig. 4D) . Importantly, KL was not able to sustain expression of any of these genes. Although it appears from the RNase protection blot that both IL-3 and FL can up-regulate proapoptotic Bcl-2 family members, a quantification of the relative expression of proapoptotic Bax demonstrated very small, if any, up-regulation of its mRNA (Fig. 4E) . Similar results were obtained for another proapoptotic gene, Bak (see Fig. 4B ).

Having demonstrated that IL-3 and FL sustained protein expression, as well as the up-regulation of mRNA transcripts for Bcl-2 and to some extent Bcl-XL, we next wanted to investigate whether the antiapoptotic effects were mediated via transcriptional activation. To study this, FDC-P1/flt3 cells were stimulated with IL-3 together with either cycloheximide (CHX) to block protein synthesis or actinomycin D (Act D) to inhibit mRNA synthesis. We then performed Western blot analysis from these cells and used Bcl-2 as an example. As seen in Fig. 5 , the presence of CHX led to a significant reduction of Bcl-2 protein after 8 h. More important, the addition of Act D resulted in a almost complete loss of Bcl-2. These results suggest that the effects of IL-3 on Bcl-2 is mainly at the level of transcription.



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  		Figure 5. IL-3 induces transcriptional activity of the Bcl-2 and Bcl-XL genes. Western blots were performed on indicated proteins extracted from cytokine-deprived FDC-P1/flt3 cells after IL-3 stimulation (200 U/ml) with cycloheximide (CHX) or actinomycin D (Act D) for 4 and 8 h, respectively. Results presented are from one representative experiment out of three performed.

IL-3 but not KL and FL induces expression of the Mcl-1 gene
It was recently reported that KL can mediate expression of the antiapoptotic Bcl-2 family member gene Mcl-1 in eosinophils [28 ]. Because IL-3 has also been shown to be able to activate Mcl-1 in hematopoietic progenitor cells [29 ], we compared the expression of Mcl-1 after stimulation with IL-3, KL, or FL. We also decided to include another progenitor cell line in this study, FDCP-mix, representing a less differentiated and multipotent progenitor stage [25 , 30 ]. FDCP-mix cells express significant levels of c-kit and are responsive to KL in an equivalent way as FDC-P1/flt3 cells. In these cells, the expression of Bcl-2 and Bcl-XL, as well as for Mcl-1, was sustained by IL-3, whereas Mcl-1 protein levels were rapidly disappearing after KL addition (Fig. 6A ). Unfortunately, we were unable to introduce the flt3 gene into FDCP-mix cells by retroviral gene transfer, and thus, the effects of FL on Mcl-1 protein could only be studied in FDC-P1/flt3 cells. In these, the addition of IL-3 led to an increase in Mcl-1 expression, whereas neither KL nor FL was able to sustain its expression for more than 24 h (Fig. 6B) .



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  		Figure 6. Mcl-1 expression is not induced by KL. Western blot analysis measuring Mcl-1 protein expression in (A) FDCP-mix or (B) FDC-P1/flt3 cells. Cells were grown without cytokine for 12 h, stimulated with IL-3 (200U/ml), KL (100ng/mL), or FL (50ng/mL), and then harvested after time points indicated, and proteins were immunostained with antibodies to Bcl-2, Bcl-XL, Mcl-1, and GADPH as indicated.

IL-3 but not KL sustains expression of Bcl-2 and Bcl-xL protein in primary bone marrow-derived progenitor cells
Because we had not been able to show any effects of KL on the expression of Bcl-2 family members in the progenitor cell lines used, we decided to extend our study to include primary cells. Freshly isolated progenitor cells were obtained from mouse bone marrow by magnetic depletion of Lin+ mature cells. After depletion, Lin- progenitors were shown to be 20-fold enriched for colony-forming progenitors compared with Lin+ cells (not shown). Lin- cells were cultured in IL-3 or KL for 24-48 h, after which Western blot analysis was performed. Although not as pronounced as our results from FDC-P1/flt3 and FDCP-mix cells, KL stimulation led to a gradual loss of expression of both Bcl-2 and Bcl-XL proteins, whereas IL-3 was able to sustain Bcl-2 expression and even up-regulate Bcl-XL (Fig. 7A ). In addition, PI-3 kinase activity was important for KL-, as well as for IL-3-mediated survival, as LY294002 induced more apoptotic cells in both cases (Fig. 7B) . No data on the effects of FL on primary cells could be obtained because the majority of Lin- cells died after 24 h with FL alone.



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  		Figure 7. KL-mediated survival of Lin- progenitor cells is dependent on PI3 kinase activity and does not lead to up-regulation of Bcl-2 and Bcl-XL. Mouse bone marrow cells were magnetically depleted of Lin+ cells (B220, CD4, CD8, Gr-1, and Mac-1) and were then cultured for 24 or 48 h with IL-3 (200U/ml) or KL (100ng/ml). (A) Whole-cell lysates were prepared, and the levels of Bcl-2 and Bcl-XL were determined with immunoblotting. The same blot was reprobed with an anti-GAPDH antibody to ensure equal loading. (B) Lin- cells were cultured with IL-3 (200U/ml) or KL (100ng/ml) with (closed bars) or without (open bars) 20 µM LY294002. After 24 h of treatment, cells were stained with annexin V-PI and analyzed by flow cytometry. The results presented are the mean value ± SD of one of two experiments performed in duplicate.

Expression of Bcl-2 and Bcl-XL via IL-3 or FL is not mediated by PI-3 kinase
Apparently, IL-3 and FL can sustain survival of myeloid progenitors in the presence of LY294002, and so far, our results suggest that this is due to activation of Bcl-2 and Bcl-XL gene expression. Because it has been reported that the PI-3 kinase pathway can regulate transcription of Bcl-2 and Bcl-XL, we decided to examine whether this expression was affected by LY294002 treatment. Upon stimulation with IL-3, KL, or FL in the absence or presence of LY294002, we compared the effects on Bcl-2 and Bcl-XL expression by Western blot analysis. In agreement with previous results, only IL-3 and FL sustained expression of both genes after 24-h stimulation, but importantly, this was not affected by LY294002 addition (Fig. 8 ). We conclude that at least in the progenitor cells used in this study, the PI-3 kinase pathway is not crucial for Bcl-2 and Bcl-XL expression.



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  		Figure 8. Expression of Bcl-2 or Bcl-XL protein by IL-3 or FL is not mediated by PI-3 kinase. FDC-P1/flt3 cells were deprived of cytokine and stimulated with IL-3 (200U/ml), KL (100ng/mL), or FL (50ng/mL) for 24 h in the absence or presence of LY294002 (50µM), after which Western blot analysis measuring Bcl-2 and Bcl-XL protein was performed.


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DISCUSSION
 
Consistent with PI-3 kinase as an important antiapoptotic pathway in several different hematopoietic cell types and PKB being one major downstream target involved, we demonstrate that PI-3 kinase activity is required for KL-mediated survival of the two myeloid progenitor cell lines included in this study. Although phosphorylation of PKB was inhibited by the addition of the PI-3 kinase inhibitor LY294002, a significant fraction of cells stimulated with either IL-3 or FL, but not KL, were able to survive after 48 h in the presence of the inhibitor. We therefore reasoned that IL-3 and FL must be able to utilize alternative survival pathways. Although flt3 signaling and the mechanisms underlying the antiapoptotic effects by FL have not been fully elucidated, IL-3 and KL have been shown to transmit signals into the cell via the MAP kinase pathway. We show that all three cytokines induce strong phosphorylation of ERK. However, when we used inhibitors specific for MAP kinases (PD98059 and U0126), survival of the two cell lines was not affected, indicating that the MAP kinase pathway is not involved in the antiapoptotic control. In contrast, when we compared the induction of antiapoptotic Bcl-2 family members, we found that IL-3 induced up-regulation of mRNA transcripts for both Bcl-2 and Bcl-XL, whereas FL was able to induce Bcl-2 but only low levels of Bcl-XL mRNA. Interestingly, FL induced Bcl-2 mRNA expression with delayed kinetics compared with IL-3. Although the reason for this discrepancy is unknown, it could be due to induction of distinct signaling events. IL-3 is known to be a very potent proliferative and antiapoptotic stimulus of hematopoietic progenitors, whereas FL is more restricted in its ability to support growth and survival. In many cases, FL needs synergistic signals from other cytokines to induce comparable effects.

Despite the changes in mRNA levels for Bcl-2 and Bcl-XL, this was not accompanied by increases in the total level of proteins. Thus, the expression of Bcl-2 and Bcl-XL appears to be controlled both at the level of mRNA and protein. However, treatment with cycloheximide or actinomycin D revealed that IL-3 is exerting its main effect at the level of transcription, since treatment with these reagents induced rapid loss of Bcl-2 protein.

In contrast to IL-3 and FL, KL was unable to induce mRNA transcripts for Bcl-2 and Bcl-XL or to sustain their protein expression. Despite the complete lack of detectable Bcl-2 protein or any of the other family members tested 48 h after KL stimulation, most of the cells were still viable. We have extended these studies up to 9 days and have never been able to detect any Bcl-2 expression (data not shown). Thus, KL is capable of inducing progenitor survival without the contribution of antiapoptotic members of the Bcl-2 gene family. From numerous studies, it has become apparent that activation of specific Bcl-2 family members is dependent on the hematopoietic cell studied and their stage of differentiation. Upon KL stimulation, the Bcl-2 gene has been shown to be up-regulated in progenitors for natural killer cells [31 ] and in human erythroid progenitors [32 ], whereas the induction of Bcl-2 family member genes by FL and IL-3 has previously been reported in different cell lines [29 , 33 , 34 ]. Interestingly, it has been shown that IL-3 can up-regulate Bcl-XL gene expression by a mechanism involving Stat5 [35 ], a signal transduction pathway that also has been implied in the proliferative response of primitive hematopoietic cells to FL [36 ]. The similar response seen in our study with IL-3 and FL in FDC-P1/flt3 cells may in part be explained by these findings, but has to be investigated in future studies.

Survival of hematopoietic cells in different culture systems seems to be optimal in the presence of multiple cytokines, acting in synergy to enhance survival and to induce proliferation. It is likely that synergistic cytokines induce distinct survival-signaling pathways. Previous studies have indicated that KL and FL have synergistic properties that lead to increased survival in human and mouse stem cells and early progenitors [6 , 7 , 37 38 39 ]. The results offer an appealing explanation to these mechanisms. Our study has mainly been performed with myeloid progenitor cell lines, although similar results were obtained from freshly isolated hematopoietic progenitors from mouse bone marrow after KL stimulation. It was recently shown that hematopoietic stem cells need two separate signals to prevent apoptosis. Using transgenic mice overexpressing Bcl-2 in all hematopoietic cells, it was shown that KL in concert with Bcl-2 was able to provide stem cells with signals necessary for optimal survival and growth [40 ].

The question thus arises by what distinct mechanism KL transmits antiapoptosis signals and if KL in parallel to PKB phosphorylation induces a separate pathway or specific targets not affected by IL-3 and FL treatment. A recent study has suggested that the Bcl-2-related protein Mcl-1 is induced by KL in combination with IL-5 in eosinophils [28 ]. However, we could only detect Mcl-1 protein expression after IL-3 stimulation, and it is unlikely that Mcl-1 is involved in KL-mediated survival in myeloid progenitors. In contrast, we have shown that c-kit signaling can inactivate the function of the Forkhead transcription family member FoxO3 in the same progenitor cell lines as used herein, as well as in bone marrow-derived Lin- progenitors [41 ]. Proapoptotic Bad is a target for PKB-mediated survival in hematopoietic cells [42 43 44 ]. Because of the potential involvement of Bad, we have also tried to study its role in survival. However, in the cell lines used in our study, we have not been able to detect any expression of Bad at all.

Because Forkhead transcription factors are involved in apoptosis and cell cycle control, it is possible that IL-3 and KL—although both use the PI-3 kinase pathway—modulate alternative pro- and antiapoptotic proteins as a consequence of additional but distinct signaling pathways. Recently, it was shown that PKB and Bcl-XL block apoptosis by different intracellular mechanisms [45 ], underlying the suggestion that cytokines acting in synergy may induce two separate pathways to provide hematopoietic cells with optimal survival conditions. Further studies are necessary to reveal the alternative mechanisms by which c-kit mediates suppression of apoptosis, and the two cell lines used in our study will provide valuable tools for this assessment.


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ACKNOWLEDGEMENTS
 
RK was a recipient of an award from the order Urania, Landskrona, Sweden. CJH was a holder of an ERASMUS scholarship. This work was supported by grants from the Swedish Cancer Society and the Children’s Cancer Foundation of Sweden, HKH Kronprinsessan Lovisas förening för barnasjukvård, Axel Tielmans Minnesfond, Crafoordska stiftelsen, Stiftelsen för Blodsjukdomars bekämpande, the Malmö University Hospital and its research funds, and the Magnus Bergvalls, the Inga och John Hains, the Hans von Kantzows, and the Anna Lisa och Sven-Eric Lundgrens Stiftelser.

Received April 8, 2003; revised June 19, 2003; accepted July 7, 2003.


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