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TISSUE-SPECIFIC STEM CELLS

Timing and Expression Level of Protein Kinase C Regulate the Megakaryocytic Differentiation of Human CD34 Cells

^aDepartment of Anatomy, Pharmacology & Forensic Medicine, Human Anatomy Section, University of Parma, Ospedale Maggiore, via Gramsci, Parma, Italy;
^bCellular Signalling Laboratory, Department of Anatomical Sciences, University of Bologna, Bologna, Italy;
^cIstituto di Genetica Molecolare-Consiglio Nazionale delle Ricerche, Unit of Bologna, c/o Istituti Ortopedici Rizzoli, Bologna, Italy

Key Words. Megakaryocytopoiesis • Stem cells • Cytokines • Thrombopoietin • Tumor necrosis factor-related apoptosis-inducing ligand

Correspondence: Marco Vitale, M.D., Department of Anatomy, Pharmacology & Forensic Medicine, Human Anatomy Section, University of Parma, Ospedale Maggiore, Via Gramsci, 14, I-43100 Parma, Italy. Telephone: +39.0521.033032; Fax: +39.0521.033033; e-mail: marco.vitale{at}unipr.it

Received on December 29, 2006; accepted for publication on June 5, 2007.

First published online in STEM CELLS EXPRESS  June 14, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Protein kinase C (PKC)-mediated intracellular signaling participates in several key steps of hematopoietic cell differentiation. The isoform of PKC has been associated with erythroid differentiation as well as with the early phases of megakaryocytic (MK) lineage commitment. Here, we worked on the hypothesis that PKC expression levels might be modulated during MK differentiation, with a specific role in the early as well as in the late phases of thrombopoiesis. We demonstrate that—at variance with the erythroid lineage development—PKC is completely downmodulated in TPO-induced CD34 cells from day 6 onward. The forced expression of PKC in the late phases of MK differentiation delays the phenotypic differentiation of progenitors likely via Bcl-xL upregulation. Moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), known as a negative regulator of early erythroid expansion, is not apoptogenic for thrombopoietin-induced CD34 cells, but rather accelerates their maturation. However, PKC levels negatively interfere also with the effects of TRAIL in MK differentiation. PKC can therefore be considered a signaling intermediate whose expression levels are finely tuned, with a virtually opposite kinetic, in erythroid versus megakaryocytic lineages, to adequately respond to the signaling requirements of the specific hematopoietic lineage.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Platelets, produced by the cytoplasmic fragmentation of bone marrow megakaryocytes (MK), are essential for primary hemostasis, to repair microvascular damages, and to initiate thrombus formation. The major regulator of megakaryocytopoiesis and thrombopoiesis is thrombopoietin (TPO), an acidic glycoprotein produced by the liver, kidney, and bone marrow, that interacts with its receptor, c-Mpl, a member of the type-1 of hematopoietic growth factor receptor family [1].

Studies on TPO activity have demonstrated that this cytokine—although able to promote virtually all the aspects of MK maturation and platelet generation—has more general, nonredundant effects on hematopoiesis, particularly on the survival of hematopoietic stem cells [2, 3]. Genetic defects of TPO or c-Mpl production and expression lead not only to thrombocytopenia, but also to a generalized cytopenia of all hematopoietic lineages [4, 5].

Upon binding to c-Mpl, TPO generates the phosphorylation of Janus tyrosine kinase (JAK)-2 [6] and the consequent activation of signal transducer and activator of transcription (STAT)3 and STAT5 transcription factors [7, 8] that migrate to the nucleus and activate several genes, such as cyclin D1 and Bcl-xL [9]. In addition to STATs, phosphorylated JAK-2 activates two other main downstream signaling cascades: (a) the Raf/Ras/mitogen-activated protein kinase (MAPK) pathway, which involves both ERK1/2 and p38 MAPKs [10], with consequent phosphorylation of transcription factors like CREB and ATF1 [11, 12]; (b) by forming a complex with p85, the PI3 kinase pathway [13, 14]. The downstream activation of Akt is then responsible for the majority of the multiple biological effects of PI3 kinase activation in MK cells, like cyclin D1 and c-fos gene transcription. However, PI3 kinase also induces c-myc expression independently from Akt [15].

Of the several known isoforms of Protein kinase C (PKC), we have demonstrated that PKC is modulated during erythropoiesis [16], being specifically upregulated during erythropoietin (EPO)-dependent erythroid differentiation of human CD34 cells. Earlier observations demonstrated that phorbol esters (PMA) induce the DAMI human MK cell line to become polyploid and to express platelet-specific markers. Moreover, PMA induces the translocation of PKC isoforms , , and from the cytosolic to the membrane fraction [17]. Both erythroblasts and MK express PKC, although at different levels [18]. More recently, Goldfarb et al. showed that PKC specifically participates in MK lineage commitment through the functional cooperation with GATA-1 in the activation of megakaryocytic promoters, and that the downregulation of PKC and MAPK inhibits the megakaryocytic differentiation of K562 cells [19]. We recently demonstrated that PKC protects differentiating erythroid cells from the apoptogenic effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which are—on the contrary—exposed in the earlier phases of differentiation [20]. However, TRAIL does not significantly modulate the survival of MK precursors [21]: on the contrary, TRAIL has been recently proposed as a promoter of the maturation of megakaryocytes [22]. In this respect, it must be noted that de Botton and coworkers demonstrated that caspase activation is causal to proplatelet formation, showing a localized caspase 3 activation with no DNA fragmentation [23]. Given this complex background, we decided to study the modulation of PKC levels along the TPO-induced MK differentiation of human CD34 cells and its potential role in regulating cell differentiation and overall bone marrow platelet production.

	MATERIALS AND METHODS

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CD34⁺ Cell Purification
Primary CD34⁺ cells were isolated from peripheral blood of normal donors by immunomagnetic positive selection using the CD34⁺ cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) in the magnetic field of a Vario-MACS apparatus (Miltenyi Biotech) according to manufacturer's protocol. Human material was obtained according to the Helsinki declaration of 2004, and informed consent was obtained from the donors. Purity of CD34⁺ cell preparations was immediately checked by a R-phycoerythrin (RPE)-conjugated anti-CD34 monoclonal antibody (mAb) (Immunotech, Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp) and flow cytometry. Only samples exceeding 95% purity were used for subsequent experiments.

Cell Cultures and Treatment
Purified human CD34⁺ cells were cultured up to 20 days, at an optimal cell density of 1 x 10⁶ cells per milliliter, in serum-free X-vivo medium (BioWhittaker, Walkersville, MD, http://www.cambrex.com) supplemented with 3 ng/ml recombinant human interleukin-3 (IL-3) and 40 ng/ml recombinant human stem cell factor (SCF) in the presence or absence (controls) of 100 ng/ml TPO. Cells were reseeded in complete fresh medium every 3 days. In some experiments, after 3, 6, 9, and 14 days of culture in the presence of TPO, the CD61⁺ cells were purified by immunomagnetic positive selection using the CD61⁺ cell isolation kit (Miltenyi Biotech) in the magnetic field of a Vario-MACS apparatus according to manufacturer's protocol. Purity of CD61⁺ cell preparations was immediately checked by anti-CD61-RPE mAb (Immunotech) and flow cytometry. Only samples exceeding 95% purity were used for subsequent experiments.

Cell Morphology
Cell morphology was analyzed by light microscopy at day 5 and day 15 of culture and by fluorescent microscopy at day 0 and day 14 of culture. Aliquots of cultured cells were centrifuged with a StatSpin CytoFuge (StatSpin, Norwood, MA, http://www.statspin.com) at 600 rpm for 4 minutes. For the light microscopy analysis, slides were stained with hematoxylin/eosin and examined by a Coolscope digital light microscope (Nikon Corporation, Tokyo, http://www.nikon.co.jp/main/eng). For immunofluorescence analysis, cells were fixed with methanol for 5 minutes. Cells were then washed with phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100, and incubated with a fluorescein isothiocyanate (FITC)-conjugate anti-CD62p mAb (4.7 µg/ml; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) or with a FITC-conjugated isotype-matched irrelevant mAb (negative control) for 1 hour. Unspecific binding was prevented by 30 minutes incubation of cells in 2% bovine serum albumin. We performed 4,6-diamidino-2-phenylindole staining (3.3 µg/ml) to visualize cell nuclei. Slides were analyzed by an ECLIPSE 80i fluorescence microscope (Nikon) and the ACT-2U software (Nikon).

Flow Cytometric Analysis
To assess megakaryocytic differentiation, aliquots of 0.3 x 10⁶ cells per experimental point were labeled with RPE-conjugated anti-CD61 (BD Pharmingen), RPE-conjugated anti-CD41 (Chemicon, Temecula, CA, http://www.chemicon.com), Cyanin-5 (Cy5)-conjugated anti-CD42b (BD Pharmingen), FITC-conjugated anti-CD62p (BD Pharmingen), RPE-conjugated anti-Glycophorin A (Dako, Glostrup, Denmark, http://www.dako.com), FITC-conjugated anti-CD14, or anti-CD15 (Exalpha Corporation, Boston, http://www.exalpha.com). Working dilutions of all reagents were previously optimized by serial dilution tests.

In some experiments, aliquots of 0.3 x 10⁶ cells per experimental point were labeled by a panel of anti-TRAIL-Rs MoAbs (Alexis Biochemical, San Diego, http://www.axxora.com). Expression of TRAIL-receptor (R)1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was analyzed by indirect staining using 1 µg of HS101 anti-human TRAIL-R1, HS201 anti-human TRAIL-R2, HS301 anti-human TRAIL-R3, and HS401 anti-human TRAIL-R4 monoclonal antibodies, followed by RPE-labeled goat anti-mouse IgG (Immunotech) as a second reagent.

Analysis of the samples was performed by an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) and the Expo ADC software (Beckman Coulter). In some experiments, the absolute number of surface antigens expressed per cell was calculated. To this purpose, the flow cytometer was calibrated with a set of standardized beads (Dako), each with a known amount of fluorochrome (FITC, RPE, or Cy5) expressed in units of molecules of equivalent soluble fluorescein (MESF). Thus, a standard curve was constructed by plotting MESF values for the beads against the median channel in which the peak was displayed [24].

Flow cytometry was also used to study the ploidy of the differentiating cell populations on PKC-transfected and wild-type cells. For DNA analysis, aliquots of cells were fixed by 0.5% paraformaldehyde (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 15 minutes and subsequently permeabilized by a 4-minute treatment with PBS containing 0.2% Triton X-100 (Sigma). Washed cells were then incubated in PBS containing 50 µg/ml propidium iodide (PI) and 100 µg/ml RNase A for 1 hour. CD34⁺ cells at day 0 were used as a marker for the identification of the diploid (2N) peak.

Finally, to quantify the platelet production in culture, fixed volumes of the culture supernatants were collected, incubated with anti-CD41-RPE and calcein AM (to exclude fragments; Sigma), and added to a fixed volume of calibration beads (Dako) at known concentration. Both the platelets and bead populations were simultaneously identified in flow cytometry on the forward scatter versus logarithmic side scatter dot plot. The absolute platelet count was performed on the gated CD41⁺/calcein AM⁺ platelet population.

Small Interfering RNA Design and Cell Transfection
PKC expression levels were downregulated in primary MK progenitors at day 3 of culture by transfection of double-stranded small interfering (si)RNAs designed to target sequences corresponding to nucleotides 223–244, 429–450, 942–963, and 1,158–1,179 on human PKC mRNA (NM005400). The target sequences were 5'-AAGAT CAAAA TCTGC GAGGCC-3', 5'-AAGAT CGAGC TGGCTG TCTTT-3', 5'-AACTA CAAGG TCCCT ACCTTC-3', and 5'-AAAAA GCTCA TTGCT GGTGCC-3'.

The respective sense and antisense RNA sequences were synthesized by Silencer siRNA Construction Kit (Ambion, Austin, TX, http://www.ambion.com) [25]. Nonspecific siRNA duplexes containing the same nucleotides, but in irregular sequence (i.e., scrambled PKC siRNA), were prepared according to manufacturer's protocol and used as controls.

PKC was overexpressed in primary MK progenitors at day 8 of culture. The green fluorescent protein (GFP)-PKC expression and control plasmids were kindly provided by Professor Peter Parker (Cancer Research UK, London Research Institute). The murine GFP-tagged PKC and the murine GFP-K552M mutated PKC constructs were cloned in the pCDNA3.1 hygro vector fused with green fluorescence protein [26]. To maximize transfection efficiency, siRNAs (100 nM each) and GFP-PKC plasmids (1 µg per transfection) were delivered using the amaxa nucleofection technology (amaxa Inc., Gaithersburg, MD, http://www.amaxa.com) according to manufacturer's protocols [25].

Western Blot
Cultured cells were counted, and 1.5 x 10⁶ cells were collected at specific time points, washed in PBS, and centrifuged at 200g for 10 minutes. Pellets were resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1 mM Na₃VO₄; 1 mM NaF) supplemented with fresh protease inhibitors, and protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, http://www.piercenet.com). Thirty µg of proteins from each sample were then migrated in 5% SDS-acrylamide gels and blotted onto nitrocellulose filters.

Blotted filters were blocked and incubated with specific primary antibodies diluted as described in manufacturers' protocols. Specifically, rabbit polyclonal anti-PKC and anti-phospho-PKC antibodies (Upstate, Charlottesville, VA, http://www.upstate.com) were used at the concentration of 1 µg/ml. Anti-ß-actin (Sigma) MoAb was diluted 1:5,000, whereas anti-Bcl-xL polyclonal antibody (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) was diluted 1:1,000 before use.

Filters were washed and further incubated for 1.5 hours at room temperature with 1:5,000 peroxidase-conjugated anti-rabbit or with 1:2,000 peroxidase-conjugated anti-mouse IgG (Pierce) in the primary antibody working solution at room temperature. Specific reactions were revealed with the ECL SuperSignal West Pico Chemiluminescent Substrate Detection System (Pierce).

Assessment of Apoptosis
Cell culture viability was assessed by trypan blue exclusion. Apoptotic cells were identified by flow cytometry either as subdiploid peaks generated by DNA fragmentation or by Annexin V/PI staining. Specifically, cells were either permeabilized by ethanol in the presence of RNase H buffer and stained with 50 µg/ml PI or phosphatidylserine was stained by FITC conjugate Annexin V (ACTIPLATE, Valter Occhiena, Torino, Italy, http://www.valterocchiena.com) in Ca²⁺ and PI staining buffer, following manufacturer's protocol.

	RESULTS

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PKC Levels Are Modulated During MK Differentiation of CD34 Cells
Based on the previous demonstration of PKC activity in the early phases of megakaryopoiesis [19], we decided to study its expression along the TPO-induced MK differentiation of primary human CD34⁺ cells. We first set up the culture conditions to induce MK differentiation. We therefore assayed PKC levels by Western blot analysis of purified CD34 samples isolated from five unrelated healthy donors. Purified CD34⁺ cells were cultured up to 14 days in serum-free medium supplemented with IL-3 and SCF in the presence or absence (control) of TPO. In the time period of cell culture, TPO induced the MK differentiation of CD34 cells, detected by the early and stable expression of GPIIIa (CD61) and the progressive expression of the late GPIb (CD42b) surface antigens (Fig. 1A). In the absence of TPO, neither CD61 nor CD42b were expressed in the time period studied. Other myeloid lineage-specific differentiation markers (Glycophorin A, CD14, and CD15) were all negative (data not shown). The analysis of the DNA content in cells treated with TPO for 14 days shows the polyploidization with a prevalence of cell accumulation in 16N (Fig. 1B). Light microscopy analysis of CD34⁺ cells cultured with TPO showed an enlarged and multinucleated morphology [27] (Fig. 1C). Indeed, fluorescence microscopy analysis at day 14 of culture with TPO showed a strong cytoplasmic staining with anti-CD62p, indicating the presence of -granules (Fig. 1D). At day 9, the number of platelets in the culture had increased by 40% (Fig. 1E).

TPO induces PKC in the early phases of MK differentiation (Fig. 2A), which is in agreement with previous data [19] demonstrating a functional role for PKC at the beginning of megakaryopoiesis. However, from day 6 onward, PKC levels decrease dramatically, notwithstanding the continuous presence of TPO in the cell cultures. The cumulative densitometric measurements of the samples tested are shown in Figure 2B. To demonstrate that the decrease of PKC expression effectively took place around day 6 of culture in the presence of TPO, we purified from our cell cultures the CD61⁺ and the CD61^– cells at 3, 6, 9, and 14 days of culture and analyzed the expression of PKC by Western blot. Figure 2C shows that, in the CD61⁺ population, PKC is already completely downmodulated at day 9, whereas in the CD61^– cells there is still a residual expression of the kinase, which, however, disappears at day 14. Of note, the kinetic of PKC expression induced by TPO in CD34 cells is opposite to what has been previously described in erythroid precursors [20] and confirmed by parallel experiments where CD34 cells were induced to MK differentiation by TPO or to erythroid differentiation by EPO (Fig. 2D).

View larger version (39K): [in this window] [in a new window]   Figure 1. Protein kinase C levels are modulated during megakaryocytic (MK) differentiation. (A): TPO-induced differentiation of human CD34 cells. The starting population (T = 0) is CD34+ and does not express MK antigens. At the indicated times, CD34-derived megakaryoblastic cells were stained with specific monoclonal antibodies to CD61 and CD42b, as described in Materials and Methods. Specific fluorescence histograms (gray) are superimposed on negative controls (isotype-matched irrelevant monoclonal antibodies, empty histograms). Glycophorin A, CD14, and CD15 were not expressed during the culture period (not shown). In the absence of TPO, neither CD61 nor CD42b was expressed in the time period studied. (B): Analysis of DNA content in CD34+ cells treated with TPO for 14 days (right). CD34+ cells at day 0 (left) were used as control. (C): Light microscopy analysis of the cells in culture shows that CD34+ cells treated with TPO progressively acquired an enlarged and multinucleated morphology, typical of differentiating megakaryocytes (hematoxylin/eosin staining; original magnification, x40). (D): Immunofluorescence analysis of CD34+ cells at day 0 (Dd1–Dd4) and at day 14 (Dd5, Dd6) of treatment with TPO. Nuclei were counterstained with 4,6-diamidino-2-phenylindole, whereas p-selectin was specifically stained by a fluorescein isothiocyanate (FITC)-conjugated anti-CD62p monoclonal antibody (Dd4, Dd6). Immunofluorescence negative controls were stained with a FITC-conjugated isotype-matched irrelevant monoclonal antibody (Dd2) (original magnification, x40). (E): Relative number of platelets in the culture of TPO-treated CD34 cells at days 0, 6, and 9. The number of platelets was calculated on the gated CD41+/calcein AM+ population in combination with a known number of calibration beads. Data from three unrelated experiments (means ± SD; * p < .05). Abbreviations: T, time; TPO, thrombopoietin.

View larger version (29K): [in this window] [in a new window]   Figure 2. The kinetic of PKC induced by TPO in CD34 cells. (A): Western blot detection of total PKC protein expression in CD34 cells cultured in serum-free medium with interleukin-3 + stem cell factor in the presence (+TPO) or absence (–TPO) of thrombopoietin. ß-Actin was monitored for protein loading. (B): Levels of PKC protein expression during megakaryocytic differentiation of CD34 cells. Densitometric measurements of Western blots from five unrelated healthy donors (means ± SD). (C): Western blot detection of total PKC protein expression in CD61+ and CD61– purified cells. CD34+ cells were cultured in the presence of TPO and CD61+ cells were selectively isolated at days 3, 6, 9, and 14. ß-Actin was monitored for protein loading. (D): Time course of PKC expression levels along the first 14 days of TPO-induced megakaryocytic differentiation (TPO, dotted line) as compared with EPO-induced erythroid differentiation (EPO, continuous line, [20]). Abbreviations: EPO, erythropoietin; OD, optical density; PKC, protein kinase C; TPO, thrombopoietin.

View larger version (35K): [in this window] [in a new window]   Figure 3. Differentiating megakaryocytes are resistant to TRAIL. (A): Purified CD34 cells were differentiated for 20 days in serum-free medium with interleukin (IL)-3 and stem cell factor (SCF) in the presence of TPO (+TPO). At the indicated time intervals, TRAIL-R expression was evaluated by specific monoclonal antibody staining and immunophenotyping. Gray histograms: specific fluorescence; empty histograms: isotype-matched irrelevant antibody control. (B): Flow cytometric analysis of CD34 cells (TPO, SCF, and IL-3) treated with 100 ng/ml TRAIL for 24 hours: residual cell viability was evaluated by staining apoptotic cells with Annexin V-FITC and propidium iodide. TPO-treated CD34 cells are resistant to apoptosis. (C): Aliquots of CD34 cells were differentiated with EPO in parallel to TPO and used as positive controls for TRAIL. Differently from megakaryoblasts, erythroblasts were efficiently killed by TRAIL. Negative controls were cultured in the absence of TRAIL. Data from five unrelated healthy donors are expressed as means ± SD (* p < .05). (D): Effect of TRAIL on TPO-treated CD34 cells. CD34+ purified cells were cultured for 14 days in serum-free medium with IL-3, SCF, and TPO with or without 25 ng/ml TRAIL. Data from five experiments are expressed as means ± SD (* p < .05). (Data are indicated as absolute values [MESF]. The CD61 histograms are shown at a lower scale because surface CD42b molecules expressed at day 14 largely outnumber CD61 molecules). Abbreviations: EPO, erythropoietin; FITC, fluorescein isothiocyanate; MESF, molecules of equivalent soluble fluorescein; T, days; TPO, thrombopoietin; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL-R, tumor necrosis factor-related apoptosis-inducing ligand receptor.

PKC Levels Interfere with MK Differentiation
Given the modulation of PKC along MK differentiation, we started searching for a role of PKC in MK precursors. We therefore overexpressed PKC in TPO-induced primary CD34 cells from day 8 onward, when its levels are physiologically downmodulated, using as negative control an inactive PKC K522M mutated (PKCm) kinase [26]. Overexpression of PKC, but not of PKCm, induced a significant downmodulation of the expression of CD61, CD41, and CD42b surface markers and of CD62p cytoplasmic maturation marker, as evaluated by quantitative flow cytometry (Fig. 4A). Moreover, PKC overexpression selectively reduced both the cell polyploidization (Fig. 4B) and the platelet production (Fig. 4C). When MK precursors were treated with TRAIL to boost their differentiation, overexpression of PKC also abrogated its promoting effects on MK precursors (Fig. 4D).

View larger version (31K): [in this window] [in a new window]   Figure 4. PKC levels interfere with megakaryocytic (MK) differentiation. (A): PKC overexpression reduces the density of both surface and intracellular MK markers in thrombopoietin (TPO)-treated CD34 cells. CD34 cell cultures from three independent healthy donors were transfected (at day 8 of culture) with PKC (black histograms) or mutated PKC (gray histograms) plasmids and cultured for a further 5 days in the presence of TPO. Data are expressed as means ± SD of absolute numbers of surface antigens expressed per cell (* p < .05). (B): PKC overexpression impairs the polyploidization of differentiating CD34 cells. CD34 cell cultures from three independent healthy donors were transfected (at day 8 of culture) with PKC (black circles) or mutated PKC (gray circles) plasmids and cultured for a further 5 days. Nontransfected cells (open squares) were used as controls. Whereas untreated or mock-transfected cells accumulate at 16N, PKC-overexpressing cells accumulate at 4N. Data are expressed as means ± SD. (C): PKC overexpression impairs the release of platelets in the culture. CD34 cell cultures from three independent healthy donors at day 8 of culture with TPO (control: white histogram) were transfected with PKC (black histogram) or mutated PKC (gray histogram) plasmids. Transfected cells (black and gray histograms) were then cultured for a further 5 days in the presence of TPO. Data are expressed as means ± SD of relative numbers of platelets (* p < .05). (D): Effects of TRAIL on CD42b expression in CD34-derived megakaryoblasts transfected with PKC or mutated PKC. CD34 cells from three healthy donors were transfected at day 8 and cultured for 5 days with or without 25 ng/ml TRAIL. Data are expressed as means ± SD of absolute numbers of surface antigens expressed per cell (* p < .05). Abbreviations: MESF, molecules of equivalent soluble fluorescein; PKC, protein kinase C; PKCm, mutated protein kinase C; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

PKC Modulates Bcl-xL in MK Progenitors

View larger version (38K): [in this window] [in a new window]   Figure 5. PKC modulates Bcl-xL in CD34-derived megakaryocytic precursors. (A): CD34-derived megakaryoblasts differentiated with thrombopoietin (TPO) up to 14 days. Total Bcl-xL and PKC protein expression levels were monitored at the indicated times by Western blot. (B): Levels of PKC and Bcl-xL protein expression during megakaryocytic differentiation of CD34 cells. Densitometric measurements of Western blots from five unrelated healthy donors (means ± SD). (C): Western blot analysis of PKC and Bcl-xL in TPO-induced CD34 cells. Cells were transfected with the PKC-GFP or PKCm-GFP plasmids at day 8 of culture in presence of TPO and then cultured for a further 5 days with TPO. Samples were immunoblotted with anti-PKC and Bcl-xL antibodies. Bcl-xL levels are upregulated by overexpression of PKC. As control, CD34 cells were transfected with PKC-specific siRNA or control siRNA at day 3 of culture with TPO and maintained for a further 2 days in the presence of TPO. (D): Levels of Bcl-xL protein expression during megakaryocytic differentiation of CD34 cells overexpressing PKC or mutated PKC or transfected with PKC-specific siRNA or control. Densitometric measurements of Western blots from two unrelated healthy donors (means ± SD; * p < .05). Abbreviations: CTRL, control; GFP, green fluorescent protein; OD, optical density; PKC, protein kinase C; PKCm, mutated protein kinase C; PKC(ov), overexpressed protein kinase C or mutated protein kinase C; PKC(wt), endogenous protein kinase C; siRNA, small interfering RNA.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

We demonstrate here that, in differentiating MK progenitors, PKC levels are regulated by TPO with a kinetic that is opposite to that found in erythroid precursors, being specifically downmodulated by TPO from day 6 onward. The physiological relevance of this observation is demonstrated by the fact that the persistence of elevated PKC levels interferes with the late phases of MK differentiation, likely via the downstream modulation of Bcl-xL.

PKC is important for growth factor and cytokine receptor signaling, and loss of PKC results in a severely attenuated response of macrophages to lipopolysaccharide and interferon (IFN) in PKC^–/– animals as well as in impaired platelet-derived growth factor receptor signaling [33, 34]. It has also been demonstrated that PKC acts as a link between the integrin and IFN signaling pathways [35]. More specifically, a role for PKC in MK development has been proposed by Goldfarb and coworkers, who demonstrated that PKC activity is essential in the early phases of MK differentiation of various cell lines, when it cooperates with the GATA-1 transcription factor in the activation of megakaryocytic promoters like the IIb promoter [19]. Our data parallel those from Goldfarb and coworkers, since we found elevated levels of PKC in CD34 cells in the first 6 days of treatment with TPO. However, we demonstrate that, from day 6 onward, it is essential that MK precursors downmodulate PKC; forced expression of PKC later than day 6 delays the further differentiation of MK precursors, likely via Bcl-xL upregulation. It is in fact well-known that Bcl-xL levels must be downmodulated in the later phases of thrombopoiesis. We show that PKC is upstream Bcl-xL, since (a) both are modulated with a similar kinetic during MK differentiation; (b) PKC overexpression induces Bcl-xL; and (c) the accumulation of Bcl-xL induced by TPO depends on PKC expression. We cannot exclude, however, that PKC might modulate intermediates other than Bcl-xL also able to counteract the full maturation of MK precursors. Elevated PKC levels, however, also interfere with the MK differentiation promoted by TRAIL, which acts as a cytokine in the human thrombopoiesis. The forced expression of PKC in TPO-treated CD34 cells is, in fact, sufficient to abrogate the differentiating effects of TRAIL on MK precursors.

Altogether, our data show that, in CD34 hematopoietic progenitors, TPO rapidly induces high levels of PKC, which are downmodulated from day 6 onward, likely to decrease downstream Bcl-xL levels that would impair the later phases of MK differentiation. When comparing the erythroid and megakaryocytic differentiation lineages, it emerges that PKC is carefully modulated, and its levels are kept under a precise—and opposite—kinetic control. The initial "low PKC period" exposes early erythroid precursors to the death-ligand-induced apoptosis (the "TRAIL window") with the likely purpose of a negative regulation of red-cell expansion. Subsequent PKC upregulation then protects late erythroid progenitors from apoptosis induced by extracellular death ligands as TRAIL. On the contrary, the early "high PKC period," which likely may contribute to redirect the entire transcriptional program of the cell toward megakaryopoiesis, must be followed by a final low PKC period that—decreasing downstream Bcl-xL expression—allows the correct conclusion of the entire process of thrombopoiesis.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We are grateful to Luciana Cerasuolo, Vincenzo Palermo, Domenico Manfredi, and Davide Dallatana for technical support. We are grateful to Instrumentation Laboratory, Italy, for technical support. This work was supported by MUR—COFIN 2006 and FIRB, Fondazione Cariparma AIL and AIRC grants.

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