Mammalian Target of Rapamycin Is Required for Thrombopoietin-Induced Proliferati - 《stemcells》干细胞学杂志干细胞之家



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作者：A. Lyndsay Drayer, Sandra G. M. Olthof, Edo Vellenga作者单位：Department of Research and Education, Sanquin Blood Bank, North East Region, Groningen, The Netherlands, and Division of Hematology, University Medical Centre Groningen, Groningen, The Netherlands . r7 U# L4 M* r' ]1 X2 A- j
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      【摘要】, ?( B1 Z1 m( k% ]
   Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34 , and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival. & x( L; d) Y0 s
      【关键词】 Megakaryopoiesis mTOR TPO CFU-Mk$ Y. T# j! |+ N
   INTRODUCTION( F! \# g, G( K2 J

During thrombopoiesis, megakaryocyte (MK) progenitor cells proliferate and ultimately differentiate into mature MKs, forming large polyploid cells from which platelets are shed. Thrombopoietin (TPO) is the major cytokine regulating MK proliferation and development .

TOR (target of rapamycin) has been implicated in the regulation of cell growth (cell size control) and cell cycle progression in many different cell types (reviewed in .: q: |. f6 O. T3 d- B# E, A

mTOR is activated by different stimuli, including nutrients and growth factors. Recently, the mTOR and PI3-K pathways have been linked through the tumor suppressor complex TSC1/2 . The mechanism by which Rheb activates mTOR, directly or indirectly through other effectors, remains to be elucidated.

As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in TPO-induced proliferation, differentiation, and cell survival in primary human progenitors and in the megakaryoblastic cell line MO7e. Here, we demonstrate that the mTOR pathway is activated by TPO and plays a role in regulating proliferation, without affecting differentiation, in MK progenitors.
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MATERIALS AND METHODS  {2 \. Z& @% Y% s
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RPMI-1640 medium (RPMI) and serum-free hematopoietic progenitor growth medium (HPGM) were purchased from Cambrex (Walkersville, MD, http://www.cambrex.com). Fetal bovine serum (FBS) was obtained from Bodinco B.V. (Alkmaar, The Netherlands, http://www.bodinco.nl). Interleukin-3 (IL-3) was obtained from R&D Systems (ITK Diagnostics, Uithoorn, The Netherlands, http://www.itk.nl). For TPO stimulation, human recombinant pegylated MK growth and differentiation factor (pegMGDF), which was a kind gift from the Kirin Brewery Co. (Tokyo, http://www.kirin.co.jp/english), was used. Stem cell factor (SCF) was obtained from Immunex Corporation (Seattle, http://immunex.com). Rapamycin was obtained from Santa Cruz Biotechnology (SanverTech, Heerhugowaard, The Netherlands), and LY294002 was obtained from Alexis (San Diego). Phycoerythrin (PE)-labeled anti-CD41a and anti-CD42b were purchased from CLB (Amsterdam, The Netherlands). Fluorescein isothiocyanate (FITC)¨Clabeled anti-CD61, IgG controls, and the BrdU (bromodeoxyuridine) Flowcytometry Kit were purchased from BD Biosciences (San Diego, http://www.bdbiosciences.com). Antibodies used for Western blotting were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com; antibodies against Erk2 and p70S6 kinase) and Cell Signaling Technology, Inc. (Beverly, MA, http://www.cellsignal.com; Phospho-S6 ribosomal protein Ser235/236 antibody, Phospho-Akt Ser473, Phospho-FKHRL1 Thr24/32, Phospho-4E-BP1 Thr37/46, Phospho-p70S6 kinase Thr389, phosphomTOR Ser 2448; mTOR and phospho-Erk1/2 Thr202/Tyr204 antibody).

Cell Culture and Purification' ?/ W) J( [/ E8 t& G  N" t
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MO7e cells  were routinely propagated in RPMI supplemented with heat-inactivated FBS (5%, vol/vol) and IL-3 (10 ng/ml). CD34  cells were obtained from healthy donors undergoing G-CSF treatment, in accordance with institutional guidelines. CD34  cells were isolated with magnetic microbead selection using the Isolex-300 method (Baxter, Deerfield, IL) as described by the manufacturer. To generate MK cells, CD34  were grown in HPGM medium supplemented with TPO and SCF (40 ng/ml each). Cells were counted by trypan blue exclusion using a hemocytometer twice weekly and fresh medium plus cytokines, with or without inhibitor. The CD61  cell fraction was purified from primary cultures after 7 days by MoFlow sorting (DakoCytomation, Glustrup, Denmark, http://www.dakocytomation.dk) as indicated. To measure cell viability, the tetrazolium reagent WST-1 (water-soluble tetrazolium¨C1; Boehringer Mannheim GmbH, Mannheim, Germany, http://www.boehringer.com) was added to CD34  cells cultured in TPO for 16 hours in triplicate. Cells were incubated with WST-1 for an additional 4 hours and then shaken thoroughly, and the absorbance at 440 nM was determined. The reading reflects the number of viable cells, based on the cleavage of the WST-1 salt by mitochondrial dehydrogenases.

Colony-Forming Assays of Megakaryocytic Cells! A7 w4 b) _* F- u$ x. r
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A commercially available kit for evaluation of colony-forming units-MK (CFU-MK) was used according to the manufacturer¡¯s instructions (MegaCult-C medium with cytokines; StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com). In short, 4,000 purified CD34  cells were seeded per double-chamber culture slide in serum-free medium containing TPO (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml), and collagen (1.1 mg/ml). Alternatively, cells were seeded in medium (MegaCult-C medium without cytokines) to which only TPO (40 ng/ml) was added. Cultures were incubated 10¨C12 days, followed by dehydration and immunocytochemical staining of the slides. MK colonies were detected using the CD41 and alkaline phosphatase detection system and counterstained in Evan¡¯s Blue. Cultures were scored for the presence of pure MK colonies consisting of at least five nucleated cells expressing CD41.7 F, |# ?6 I9 _& d5 o

Staining with Bromodeoxyuridine0 o+ {% Y9 z6 P$ L  I! F: o

Exponentially growing MO7e cells were washed, and growth factor was depleted for 6 hours. Isolated CD34  and day-7 primary MK cultures were washed and kept in HPGM medium for 3 hours. Cells were preincubated for 30 minutes with 10 nM rapamycin or dimethyl sulfoxide (DMSO) as control and then stimulated with or without TPO (40 ng/ml) as indicated. Cells were cultured for an additional 16 hours and pulsed with 10 µM BrdU for 1 hour, using the BrdU Flowcytometry Kit according to the manufacturer¡¯s instructions. Day-7 MK cultures were washed and stained for 20 minutes with CD41¨CPE antibody. Next, cells were resuspended in Cytofix/Cytoperm buffer for 30 minutes, washed, and incubated with Cytoperm Plus buffer to enhance staining. After refixation, cells were treated for 1 hour with DNAse to expose incorporated BrdU. FITC¨Canti-BrdU was added for 20 minutes at room temperature. Finally, cells were resuspended in phosphate-buffered saline (PBS) buffer containing 7-AAD (7-amino-actinomycin D) and analyzed on a FACS Calibur (BD Biosciences) flow cytometer.# Q$ K* ~; H- [3 S8 N* i
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Polyploidization Analysis3 t& W  H: j, }! A+ I

Primary MK cultures were labeled with CD61-FITC or with an isotype control. Cells were resuspended in 100 µl PBS after which 1 ml of ice-cold ethanol (80% vol/vol) was added drop-wise while mixing the tube. Cells were fixed for at least 1 hour at 4¡ãC, followed by two washes with ice-cold PBS. RNA was digested by treatment with RNAse (10 µg/ml) for 15 minutes at 37¡ãC, and DNA staining was performed by the addition of propidium iodide at a final concentration of 20 µg/ml. Nuclear ploidy was analyzed by flow cytometry.
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Preparation of Cell Lysates and Western Blotting) ^' A- m, ~3 L5 Q0 S

Prior to stimulation, cells were deprived of cytokines overnight (MO7e cells) or for 3 hours (primary cells) in RPMI with 0.5% FBS to allow maximal dephosphorylation of cellular proteins. Inhibitors or an equal volume of DMSO was added 30 minutes prior to TPO stimulation (40 ng/ml). Stimulation was terminated at the indicated time points by placing cells on ice, centrifuging immediately, and resuspending the cell pellets in Laemmli sample buffer. SDS-PAGE and immunoblotting were performed according to standard procedures. Detection was performed according to the manufacturer¡¯s guidelines (ECL, Amersham, Buckinghamshire, U.K.). After detection with phosphospecific antibodies, membranes were stripped and reprobed with an antibody detecting total protein levels.
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Statistical Analysis; _- L! |6 c% o( x$ _/ X

Data were expressed as mean ¡À SEM. Differences between samples were calculated using Student¡¯s t-test. A two-sided p value of

RESULTS
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TPO Activates the mTOR Signaling Pathway in MO7e Cells  S+ L. z" a* ?1 o6 d, A) _9 ]
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We first analyzed the sensitivity of MO7e cells to the mTOR inhibitor rapamycin. Cells, which were serum-and growth factor¨Cdeprived overnight, were preincubated with increasing concentrations of rapamycin for 15 minutes. Subsequently, cells were stimulated with TPO min, and whole-cell lysates were analyzed by Western blotting using an antibody specific for the activated form of p70S6K, phosphorylated on Thr389. As demonstrated in Figure 1A, TPO stimulation induced phosphorylation of p70S6K, which was potently inhibited at low rapamycin concentrations (1¨C10 nM). In addition, TPO stimulated phosphorylation of an additional target recognized by the phospho-specific antibody. This slightly larger protein might be the phosphorylated p85S6K isoform , which is generated by differential splicing; however, this protein was not sensitive to inhibition by rapamycin. Phosphorylation of p70S6K was also sensitive to the presence of the PI3-K inhibitor LY294002 (5¨C10 µM). Treatment with rapamycin or LY294002 was specific and did not affect p70S6K protein levels or activation of the ERK pathway (Fig. 1A, upper and lower panels, respectively).
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Figure 1. TPO-induced activation of mTOR signaling in MO7e cells. (A): Cells were starved of growth factors and serum, preincubated with DMSO vehicle (control) or with the indicated concentration of rapamycin (Rapa) or LY294002 (Ly), and left unstimulated (min) or stimulated with TPO for 30 minutes. (B): Cells were stimulated with TPO for the indicated time points, or were pretreated with DMSO vehicle (control), 10 nM rapamycin, and 10 µM LY294002, and subsequently left unstimulated (min) or stimulated with TPO for 30 minutes. Whole-cell lysates were immunoblotted with phosphospecific antibodies ("Materials and Methods"). Blots were stripped and reprobed with appropriate antibodies (mTOR tot, p70 tot, and ERK tot) to ensure an equal amount of protein in each lane. Data shown are representative of three independent experiments. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular regulated kinase; mTOR, mammalian target of rapamycin; TPO, thrombopoietin.# _+ T! M. |* _3 l  `, Q0 u

Next, we compared activation of the mTOR and PI-3K/PKB signaling pathways. Figure 1B shows that TPO induced rapid phosphorylation of mTOR and PI3-K/PKB with maximal phosphorylation at 10 minutes of TPO stimulation. Activation of PKB was transient, whereas mTOR activation was sustained. Phosphorylation of mTOR at Ser2448 was not inhibited by rapamycin or LY294002. Under these conditions, treatment with LY294002 did affect phosphorylation of PKB whereas treatment with rapamycin did not, as expected. Activation of the mTOR downstream targets p70S6K and S6 was maximal at 30 minutes of TPO stimulation. 4E-BP1 is phosphorylated sequentially on multiple phosphorylation sites after stimulation . The phospho-specific 4E-BP1 antibody detected phosphorylated 4E-BP1 at 10 minutes and the hyperphosphorylated protein at 30 minutes TPO stimulation (Fig. 1B). Hyperphosphorylated 4E-BP1 was reduced in the presence of rapamycin and was completely inhibited by LY294002. Together, these data demonstrate that TPO activates the mTOR signaling pathway in MO7e cells in a rapamycin-dependent way.

Rapamycine-Sensitive mTOR Signaling Is Required for Proliferation of MO7e Cells% r  {6 A8 {# U/ e

To investigate the role of the mTOR pathway in cell proliferation and cell survival, we analyzed cell cycling of MO7e cells in the presence of TPO with and without rapamycin. Therefore, incorporation of BrdU and DNA staining were measured by flow cytometry, as shown by an example in Figure 2A. In the presence of TPO, incorporation of BrdU into cells actively synthesizing DNA was 27% ¡À 3%, whereas in the absence of TPO, incorporation was low (7% ¡À 1%). TPO-induced BrdU uptake was significantly inhibited in the presence of rapamycin (19% ¡À 1%; p = .02 with and without rapamycin, n = 4). From DNA staining, we determined that the reduced cell cycling was due to accumulation of cells in the G0/G1 phase, as shown in Figure 2B (67% ¡À 3% of cells in G0/G1 with TPO rapamycin compared with 58% ¡À 2% of cells with TPO alone; p =.02). Cells cultured in the absence of growth factor for 16 hours underwent apoptosis as determined by the presence of a sub-G0 peak in the DNA staining profile (no TPO in Figure 3B) or by Annexin V staining (not shown). Treatment of cells with rapamycin, however, did not induce apoptosis. Overall, treatment with rapamycin inhibited TPO-induced proliferation of MO7e cells by 35% (Fig. 2C). Therefore, we conclude that, in MO7e cells, inhibition of mTOR blocks TPO-induced cell cycling in G0/G1 and downregulates proliferation but does not induce apoptosis.1 p' M# |1 q$ o$ S
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Figure 2. Effects of rapamycin on cell cycling and proliferation in MO7e cells. Cells were starved of serum and growth factors and then cultured without or with TPO in the absence or presence of rapamycin (10 nM) for 16 hours. (A): Samples were incubated with BrdU for 1 hour, processed for BrdU and DNA staining, and analyzed by flow cytometry. The percentage of cells incorporating BrdU is indicated compared with cells without BrdU. (B): Cell cycle histograms were generated and the percentage of cells in G0/G1, sub-G0 and S phases were determined based on DNA content. (C): MO7e cells were cultured in serum-free medium for 3 days, without or with TPO in the absence or presence of rapamycin (10 nM). Cells were counted manually with a hemocytometer using trypan blue exclusion. Cell expansion was expressed as fold increase compared with input on day 0 (= 1). Data shown are mean values ¡À SEM of three independent experiments. Abbreviations: 7-AAD, 7-amino-actinomycin D; BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; TPO, thrombopoietin.

Figure 3. TPO induces the mTOR signaling pathway in CD34  cells (A) and primary MK cells (B). CD34  cells were purified by immunomagnetic selection. MK cells were isolated from day-7 primary cultures by CD61  flow cytometry sorting. After selection, primary cells were starved of growth factors for 3 hours in serum-free medium and stimulated with TPO for the indicated times or pretreated with dimethyl sulfoxide vehicle (control), rapamycin (Rapa; 10 nM), or LY294002 (Ly; 10 µM), followed by 30 minutes of TPO stimulation. Whole-cell lysates were immunoblotted with phosphospecific antibodies ("Materials and Methods"). Blots were stripped and reprobed with appropriate antibody (p70 tot) to ensure an equal amount of protein in each lane. Data shown are representative of at least two independent experiments. Abbreviations: MK, megakaryocyte; mTOR, mammalian target of rapamycin; TPO, thrombopoietin.' o' s# v. z0 u3 B/ P

TPO Induces Activation of the mTOR/p70S6K Pathway in Primary Human MK Progenitors4 q& k# _, |3 ~! j/ K6 }6 }6 {
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To investigate activation of the mTOR pathway in primary cells, experiments were performed using human CD34  (stem and progenitor) cells and MK cells. Purified MK cells were obtained by culturing CD34  cells in serum-free medium with TPO and SCF for 7 days to induce expansion and megakaryocytic differentiation, followed by isolation of the CD61  cells by flow cytometry. Treatment with TPO induced phosphorylation of the p70S6K and 4E-BP1 pathways, as demonstrated in Figure 3A for CD34  cells and Figure 3B for CD61  cells. The kinetics of activation in primary cells were comparable with stimulation in the MO7e cell line, with maximal phosphorylation levels of p70S6K reached at 30 minutes of TPO stimulation. As a marker for PI3-K activity, we determined phosphorylation of the Fork-head factor FKHRL-1 (Fig. 3A) or phosphorylation of PKB (Fig. 3B) in the cell lysates. In primary human CD34  and CD61  cells, the mTOR inhibitor rapamycin blocked phosphorylation of p70S6K and inhibited hyperphosphorylation of 4E-BP1 but did not affect activation of FKHRL or PKB. In addition, phosphorylations of p70S6K and 4E-BP1 were sensitive to the PI3-K inhibitor LY294002. Thus, in primary MK cells, TPO-induced activation of p70S6K and 4E-BP1 is localized downstream of mTOR and PI3-K.; g: _9 o6 e$ k
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Rapamycin Inhibits Growth of CFU-MK with High Proliferative Capacity
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We used a collagen-based assay to evaluate the influence of rapamycin on CFU-MK formation. Colonies were counted as CFU-MK if they consisted of a minimum of five closely associated CD41  cells and were divided into MK progenitors with low (5¨C20 MK cells/colony), middle (20¨C50 MK cells/colony), and high proliferative capacity (> 50 MK cells/colony). Purified CD34  cells plated in the presence of TPO alone showed poor CFU-MK development; small colonies can be scored as demonstrated in Figure 4. Addition of rapamycin to the cultures did not affect this process (48 ¡À 5 CFU-MK in TPO versus 46 ¡À 7 CFU-MK in TPO plus rapamycin, n = 3). However, when assayed under standard conditions (commercial medium containing TPO, IL-3, and IL-6, indicated as "cytokine mix" in Figure 4), colonies with high as well as low proliferative capacities develop. Addition of rapamycin inhibited the formation of large CFU-MK but not colonies with low proliferative capacity. Indeed, the number of small CFU-MK is increased in the presence of rapamycin, as demonstrated in Figure 4. (Compare control cytokine mix with rapamycin cytokine mix.) It appears that the overall number of CFU-MK is not inhibited by rapamycin, but the size of the individual colonies is reduced.
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Figure 4. Rapamycin inhibits growth of CFU-MK with high proliferative capacity. Purified CD34  cells were plated in semisolid serum-free medium in the absence (control) or presence of rapamycin for the assessment of clonogenic growth. Cytokines TPO, IL-3, and IL-6 were present in the standard assay mix (indicated as "cytokine mix") or TPO was added separately to assay mix without cytokines (lanes marked "TPO"). After incubation, cultures were dehydrated and immunocytochemically stained. The number of CFU-MK were classified according to the number of CD41  cells present in the individual colonies. Black bars: 5¨C20 cells per colony; dark gray bars: 20¨C50 MK cells per colony; and light gray bars: > 50 MK cells per colony. Results are mean ¡À SEM of three experiments. Abbreviations: CFU-MK, colony-forming units-megakaryocyte; IL, interleukin; TPO, thrombopoietin.$ d, d( [$ D3 \: x* C! @% N) |

Rapamycin-Sensitive mTOR Signaling Is Required for Proliferation of Primary MK Progenitors; ?9 c6 ]* A' F3 w
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To further investigate the role of mTOR signaling in proliferation in primary cells, we analyzed cell cycling by BrdU uptake and DNA staining in purified CD34  cells and in MK suspension cultures. As demonstrated in a representative experiment in Figure 5A, stimulation of CD34  cells with TPO for 16 hours resulted in cells actively incorporating BrdU (17% ¡À 4%) compared with cells grown in the absence of TPO (8% ¡À 5%). In the presence of rapamycin, TPO-induced cell cycling was significantly inhibited (9% ¡À 4%; p

Figure 5. Rapamycin inhibits TPO-induced cell cycling in CD34  cells (A) and primary MK cells (B). Purified CD34  cells and day-7 MK cultures were starved of growth factors for 3 hours in serum-free medium and cultured overnight with TPO in the absence (control) or presence of rapamycin. (A): CD34  cells were incubated with BrdU for 1 hour, stained, and analyzed by flow cytometry. The percentage of cells incorporating BrdU is indicated compared with cells without BrdU. (B): After BrdU incubation, day-7 MK cultures were labeled with CD41-PE and further processed for BrdU and DNA staining. Cells expressing CD41 were gated (left panel) and analyzed for incorporation of BrdU in the absence (middle panel) and presence (right panel) of rapamycin (10 nM). Data shown are representative of three independent experiments. Abbreviations: 7-AAD, 7-amino-actinomycin D; BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; MK, megakaryocyte; PE, phycoerythrin; TPO, thrombopoietin.

In another set of experiments, CD34  cells were cultured for 7 days in serum-free medium with TPO SCF to induce MK expansion in the continuous presence or absence of rapamycin. Total cell proliferation was consistently reduced in cultures with rapamycin (Fig. 6A). On average, cells had expanded 4.2 ¡À 0.7¨Cfold in the absence and 1.9 ¡À 0.4¨Cfold in the presence of rapamycin (p
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Figure 6. Rapamycin inhibits expansion of MK cells. CD34  cells were cultured in serum-free medium with TPO SCF for 7 days in the absence (control) or presence of rapamycin. Cells were counted manually with a hemocytometer using trypan blue exclusion and represented as fold expansion relative to input of cells on day 0 (A). Percentage of cells expressing MK markers CD41 (B) and CD42 (C) after 7 days of culture determined by flow cytometry. Results are expressed as mean values of seven (A), six (B), and four (C) separate experiments. Error bars indicate SEM. Abbreviations: MK, megakaryocyte; SCF, stem cell factor; TPO, thrombopoietin.
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MK Maturation Is Not Inhibited by Rapamycin

During maturation, MK cells become larger and undergo endomitosis, thereby increasing their DNA content without cell division. To evaluate the effects of mTOR inhibition on MK maturation, the size of cells and the DNA content were analyzed in cultures with and without rapamycin. Cell size was determined from the forward scatter plots obtained by flow cytometry, as shown by an example in Figure 7A. A number of cells in the cultures display high forward scatter properties, characteristic of the larger MK cells. However, treatment with rapamycin did not affect the overall size of cells (p > .5, n = 5).8 I+ K6 R  b/ h4 ~0 Q6 i

Figure 7. Rapamycin (Rapa) does not affect megakaryocytic maturation. CD34  cells were cultured in serum-free medium with TPO SCF in the absence (control) or presence of rapamycin. (A): Forward and side scatter plots obtained by flow cytometry. Live cells are gated, and the mean values of the forward scatter axis were taken as a measure of cell size. (B): Cells in megakaryocyte cultures were stained with CD61-FITC, subsequently fixed, permeabilized, and stained for DNA with PI. Nuclear ploidy of the CD61  fraction was determined by flow cytometry. The percentage of cells in each ploidy class is indicated. Figures are representative of at least three independent experiments. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; SCF, stem cell factor; TPO, thrombopoietin.' w1 e2 J) j' }1 t6 v

To analyze polyploidization levels in mature MK cells, isolated CD34  cells were first cultured in TPO SCF for 7 days, followed by another 7 days of culture in TPO only (to induce MK polyploidization) in the presence or absence of rapamycin. Cells were stained with anti-CD61¨CFITC antibody to identify the MK cells, and then fixed, and the DNA was stained with propidium iodide, as shown by a representative example in Figure 7B. In control cultures from three independent samples, the percentage of each ploidy class in the CD61  MK cells was determined. DNA content of 8n was 8.1% ¡À 0.6%, the 16n DNA content was 6.6% ¡À 0.2%, and the 32n DNA content 10.2% ¡À 1.6%. Similar values were found for MK cells cultured in the presence of rapamycin; the 8n DNA content in the CD61  cell fraction was 8.7% ¡À 0.9%, the 16n DNA content was 6.2% ¡À 1.0%, and the  32n DNA content was 9.9% ¡À 2.2% (p  .7 for all ploidy levels with and without rapamycin). Therefore, we conclude that treatment with rapamycin did not affect polyploidization levels in MK cells./ Y0 _/ Y% n: ^2 _# d2 u8 }

DISCUSSION

mTOR signaling is emerging as an important regulator in solid tumors and in hematological malignancies . Indeed, in our experiments, MK polyploidization remained low, with few cells reaching ploidy levels higher than 32N. However, we can conclude that early differentiation into MK progenitors and MK cells, as assessed by expression of glycoprotein IIb/IIIa (CD41) and glycoprotein Ib (CD42), is not inhibited by rapamycin.4 b! f! u( a: v8 h% V# _

The downstream effectors of mTOR, p70S6K/S6 and 4E-BP1, require activation by PI3-K in MK cells. Previous studies have demonstrated an important role for the PI3-K pathway in regulating the cell cycle progression in MK cells. PI3-K activity was shown to regulate G1-to-S-phase transition in both mitotically (2N MKs) and endomitotically (4N MKs) cycling cells in response to TPO . Here, we demonstrate a role for the mTOR pathway in the mitotic cell cycle. The effects of PI3-K on the endomitotic cell cycle are likely independent of mTOR, as we did not observe a difference in MK polyploidization levels in the presence of rapamycin. In addition, PI-3K/PKB signaling is known to play an important role in promoting cell survival. We did not observe an increase in apoptotic cells during rapamycin treatment in either MO7e cells or primary cultures, whereas culture of cells in the presence of the PI3-K inhibitor LY294002 resulted in complete cell death (unpublished observations). Therefore, we propose that at least part of the effects of PI3-K in promoting cell cycling is mediated through the mTOR pathway.
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mTOR activity is potently inhibited by rapamycin; however, recent publications have demonstrated that mTOR is found in two separate complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 contains mTOR in association with raptor and GßL proteins and is the target of rapamycin . Instead, the rictor/mAVO3¨CmTOR complex signals through Protein Kinase C (PKC) and regulates the actin cytoskeleton. The mechanisms by which mTORC2 is regulated are unknown as yet, as nutrients and growth factors do not regulate PKC phosphorylation or the mTOR¨Crictor/mAVO3 association. In addition, it is not clear if the PI3-K pathway regulates mTORC2 function. Therefore, the role of mTOR is likely to be more comprehensive than previously appreciated from studies employing rapamycin.+ F( }! x8 d: @; Y0 \- q
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CONCLUSION9 l- c0 @" M4 C1 h) W' ^9 k
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We have shown that the rapamycin-sensitive mTOR pathway is required for proliferation of human MK progenitors. In addition, the results of this study provide evidence for separate signaling routes controlling proliferation versus differentiation of MK cells.
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ACKNOWLEDGMENTS, |' K$ _& T8 q4 R( I# T' |

We wish to thank Geert Mesander and Henk Moes for assistance and advice with cell sorting and FACS (fluorescence-activated cell sorter) analysis. We are thankful to Kirin Brewery Co. for the gift of recombinant TPO. This work was supported by a grant from the Sanquin Blood Supply Foundation (PPOC-03-013).( @8 p  P! ~* w. u

DISCLOSURES9 Y* |! a: r1 N5 c5 D

The authors indicated no potential conflicts of interest.
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