Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells.

OBJECTIVE: During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non-redundant complexes of mTOR have been described; mTORC1 containing rapamycin-associated TOR protein (Raptor) and mTORC2 containing Rapamycin-insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study. METHODS: We have used an siRNA approach to selectively knock down either Rictor or Raptor expression in MO7e megakaryoblastic cells. Using flow cytometry, nuclear ploidity, and cell cycling as assessed by BrdU incorporation were investigated. Electron microscopy and cotransductions with GFP-LC3 were used to quantify autophagy. Activation of intracellular signal transduction pathways was studied by Western blot analysis. RESULTS: We observed a reduced cell cycling upon Rictor siRNA transduction, resulting in decreased numbers of polypoid cells. Knocking down Raptor expression resulted in a reduced expansion and a reduced cell size. In addition, increased autophagy was observed in Raptor siRNA-transduced cells, in correspondence with an attenuation of activation of the p70S6K/S6, and 4E-BP pathways. CONCLUSIONS: The current study shows that the mTORC1 and mTORC2 complexes have distinct, non-redundant functions in MO7e MK cell proliferation, and development. The mTOR/Rictor complex affects megakaryopoiesis by regulating nuclear division and subsequent cell cycle progression, whereas Raptor signaling protects MK cells from autophagic cell death, enabling normal megakaryopoiesis to take place.

Downregulation of signal transducer and activator of transcription 5 (STAT5) in CD34+ cells promotes megakaryocytic development, whereas activation of STAT5 drives erythropoiesis.

Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.

Plant sterols cause macrothrombocytopenia in a mouse model of sitosterolemia.

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

Reduced activation of protein kinase B, Rac, and F-actin polymerization contributes to an impairment of stromal cell derived factor-1 induced migration of CD34+ cells from patients with myelodysplasia.

Patients with myelodysplasia (MDS) show a differentiation defect in the multipotent stem-cell compartment. An important factor in stem-cell differentiation is their proper localization within the bone marrow microenvironment, which is regulated by stromal cell-derived factor (SDF-1). We now show that SDF-1-induced migration of CD34(+) progenitor cells from MDS patients is severely impaired. In addition, these cells show a reduced capacity to polymerize F-actin in response to SDF-1. We demonstrate a major role for Rac and phosphatidylinositol 3-kinase (PI3K) and a minor role for the extracellular signal-regulated kinase (ERK)1/2 signaling pathway in SDF-1-induced migration of normal CD34(+) cells. Furthermore, SDF-1-stimulated activation of Rac and the PI3K target protein kinase B is impaired in CD34(+) cells from MDS patients. Lentiviral transduction of MDS CD34(+) cells with constitutive active Rac1V12 results in a partial restoration of F-actin polymerization in response to SDF-1. In addition, expression of constitutive active Rac increases the motility of MDS CD34(+) cells in the absence of SDF-1, although the directional migration of cells toward this chemoattractant is not affected. Taken together, our results show a reduced migration of MDS CD34(+) cells toward SDF-1, as a result of impaired activation of the PI3K and Rac pathways and a decreased F-actin polymerization.

The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation.

Patients with myelodysplasia (MDS) show an impaired reactive oxygen species (ROS) production in response to fMLP stimulation of GM-CSF-primed neutrophils. In this study, we investigated the involvement of lipid rafts in this process and showed that treatment of neutrophils with the lipid raft-disrupting agent methyl-beta-cyclodextrin abrogates fMLP-induced ROS production and activation of ERK1/2 and protein kinase B/Akt, two signal transduction pathways involved in ROS production in unprimed and GM-CSF-primed neutrophils. We subsequently showed that there was a decreased presence of Lyn, gp91(phox), and p22(phox) in lipid raft fractions from neutrophils of MDS. Furthermore, the plasma membrane expression of the lipid raft marker GM1, which increases upon stimulation of GM-CSF-primed cells with fMLP, was reduced significantly in MDS patients. By electron microscopy, we showed that the fMLP-induced increase in GM1 expression in GM-CSF-primed cells was a result of de novo synthesis, which was less efficient in MDS neutrophils. Taken together, these data indicate an involvement of lipid rafts in activation of signal transduction pathways leading to ROS production and show that in MDS neutrophils, an impaired lipid raft formation in GM-CSF-primed cells results in an impaired ROS production.

Mammalian target of rapamycin is required for thrombopoietin-induced proliferation of megakaryocyte progenitors.

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.

Stem cell factor synergistically enhances thrombopoietin-induced STAT5 signaling in megakaryocyte progenitors through JAK2 and Src kinase.

Stem cell factor (SCF) has a potent synergistic effect during megakaryopoiesis when administered in combination with the major megakaryocytic cytokine, thrombopoietin (TPO). In this study we analyzed the underlying mechanisms with regard to STAT5 activity. TPO stimulation of MO7e cells resulted in STAT5 transactivation, which could be enhanced 1.6-fold by costimulation with SCF, whereas SCF alone did not induce STAT5 transcriptional activity. This costimulatory effect of SCF was reflected in an increase in TPO-induced STAT5 DNA binding and increased and prolonged STAT5 tyrosine phosphorylation in both MO7e cells and primary human megakaryocyte progenitors. In contrast, serine phosphorylation of STAT5 was constitutive and associated with an inhibitory effect on STAT5 transactivation. Signal transduction pathways that might synergize in TPO-mediated STAT5 transactivation were analyzed using specific pharmacological inhibitors and indicated an essential role for Janus-activated kinase 2 (JAK2) and a partial role for Src-family kinases. Costimulation with SCF was found to increase and prolong tyrosine phosphorylation of JAK2 and the TPO receptor c-mpl. In addition, the Src kinase inhibitor SU6656 partially downregulated the additional effect of SCF costimulation on STAT5 tyrosine phosphorylation. SCF-induced enhancement of JAK2 phosphorylation was not affected by inhibition of Src kinase, suggesting that both JAK2 and Src kinase mediate STAT5 tyrosine phosphorylation. Synergistic activation of JAK2 and Src kinase may thus contribute to the enhanced STAT5 signaling in the presence of TPO and SCF.

Impaired interleukin-8- and GROalpha-induced phosphorylation of extracellular signal-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia.

Patients with myelodysplasia suffer from recurrent bacterial infections as a result of differentiation defects of the myeloid lineage and a disturbed functioning of neutrophilic granulocytes. Important physiological activators of neutrophils are the cytokines interleukin-8/CXC chemokine ligand 8 (IL-8/CXCL8), which activates CXC chemokine receptor 1 and 2 (CXCR1 and CXCR2), and growth-related oncogene (GROalpha)/CXCL1, which stimulates only CXCR2. In this study, we show that migration toward IL-8/GROalpha gradients is decreased in myelodysplastic syndrome (MDS) neutrophils compared with healthy donors. We investigated the signal transduction pathways involved in IL-8/GROalpha-induced migration and showed that specific inhibitors for extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol-3 kinase (PI-3K) abrogated neutrophil migration toward IL-8/GROalpha. In accordance with these results, we subsequently showed that IL-8/GROalpha-stimulated activation of ERK1/2 was substantially diminished in MDS neutrophils. Activation of the PI-3K downstream target protein kinase B/Akt was disturbed in MDS neutrophils when cells were activated with IL-8 but normal upon GROalpha stimulation. IL-8 stimulation resulted in higher migratory behavior and ERK1/2 activation than GROalpha stimulation, suggesting a greater importance of CXCR1. We then investigated IL-8-induced activation of the small GTPase Rac implicated in ERK1/2-dependent migration and found that it was less efficient in neutrophils from MDS patients compared with healthy donors. In contrast, IL-8 triggered a normal activation of the GTPases Ras and Ral, indicating that the observed defects were not a result of a general disturbance in CXCR1/2 signaling. In conclusion, our results demonstrate a disturbed CXCR1- and CXCR2-induced neutrophil chemotaxis in MDS patients, which might be the consequence of decreased Rac-ERK1/2 and PI-3K activation within these cells.

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia.

The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K-Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.

Reduced expression of flavocytochrome b558, a component of the NADPH oxidase complex, in neutrophils from patients with myelodysplasia.

OBJECTIVE: Patients with myelodysplasia (MDS) show a disturbed production of ROS in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils. Because generation of ROS is mediated by the NADPH oxidase complex, a component of which is flavocytochrome b558, we investigated whether the expression of flavocytochrome b558 in neutrophils from MDS patients is affected. MATERIAL AND METHODS: Neutrophils were stimulated with fMLP and GM-CSF, and plasma membrane expression of flavocytochrome b558 and specific granule markers were assessed by fluorescence-activated cell sorting analysis. Protein levels of the flavocytochrome b558 subunits gp91phox and p22phox in whole neutrophil lysates were detected by Western blotting. RESULTS: Stimulation of neutrophils with GM-CSF and fMLP increased the flavocytochrome b558 plasma membrane expression. The fMLP-induced translocation of flavocytochrome b558 was reduced in neutrophils from MDS patients (140%+/-9% vs 180%+/-13%, p<0.05). Analysis of cell surface expression of markers of flavocytochrome b558 containing granules (CD35 and CD66b) indicated that exocytosis of these granules in response to fMLP stimulation was not affected in MDS patients. Western blot analysis demonstrated a decreased protein expression level of the flavocytochrome b558 subunits gp91phox and p22phox in neutrophils from MDS patients. CONCLUSION: Our results indicate both a lower basal protein level and a disturbed fMLP-induced increase in plasma membrane expression of flavocytochrome b558 in neutrophils from MDS patients, which together might play a role in decreased ROS production.

Stem cell factor enhances erythropoietin-mediated transactivation of signal transducer and activator of transcription 5 (STAT5) via the PKA/CREB pathway.

OBJECTIVE: To define whether the observed synergistic effects of erythropoietin (EPO) and stem cell factor (SCF) on erythroid cells can, in part, be mediated by the signal transducer and activator of transcription 5 (STAT5). METHODS: STAT5 activation was examined in erythroid cell lines by analyzing the effects of EPO and SCF on STAT5 tyrosine phosphorylation, serine phosphorylation, DNA binding, and STAT5-mediated gene transactivation. RESULTS: EPO induced a 5.0-fold+/-0.4-fold increase in STAT5 transactivation, which could be further enhanced by SCF. SCF pretreatment followed by EPO stimulation resulted in a 9.0-fold+/-0.9-fold increase in STAT5 transactivation, while SCF alone did not increase STAT5 transactivation. This costimulatory effect of SCF was not mediated by increased STAT5 tyrosine or serine phosphorylation or increased STAT5 DNA binding. In addition, enhanced STAT5 transactivation was independent of the phosphatidyl inositol 3-kinase and MAPK(p42/p44) pathways. Instead, the protein kinase A (PKA) inhibitor protein PKI and the PKA inhibitor H89 prevented the costimulatory SCF effect. Furthermore, the PKA target CREB showed a strongly increased and prolonged serine-133 phosphorylation after costimulation with SCF + EPO. The involvement of CREB in STAT5 transactivation was demonstrated by overexpression of serine-133-mutated CREB, which completely blocked the SCF effect. In addition, the CREB-binding protein CBP/p300 was shown to be essential for EPO- and SCF-mediated STAT5 transactivation. CONCLUSION: SCF enhances the EPO-mediated STAT5 transactivation by triggering a PKA/CREB-dependent pathway.

Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia.

Neutrophils from patients with myelodysplastic syndrome (MDS) show a disturbed differentiation pattern and are generally dysfunctional. To study these defects in more detail, we investigated reactive-oxygen species (ROS) production and F-actin polymerization in neutrophils from MDS patients and healthy controls and the involvement of N-formyl-L-methionyl-L-lucyl-L-phenylaline (fMLP) and granulocyte macrophage-colony-stimulating factor (GM-CSF)-stimulated signal transduction pathways. Following fMLP stimulation, similar levels of respiratory burst, F-actin polymerization, and activation of the small GTPase Rac2 were demonstrated in MDS and normal neutrophils. However, GM-CSF and G-CSF priming of ROS production were significantly decreased in MDS patients. We subsequently investigated the signal transduction pathways involved in ROS generation and demonstrated that fMLP-stimulated ROS production was inhibited by the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the MAPK/ERK kinase (MEK) inhibitor U0126. In contrast, ROS production induced by fMLP stimulation of GM-CSF-primed cells was inhibited by LY294002 and U0126. This coincides with enhanced protein kinase B (PKB/Akt) phosphorylation that was PI3K dependent and enhanced extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation that was PI3K independent. We demonstrated higher protein levels of the PI3K subunit p110 in neutrophils from MDS patients and found that though the fMLP-induced phosphorylation of PKB/Akt and ERK1/2 could also be enhanced by pretreatment with GM-CSF in these patients, the degree and kinetics of PKB/Akt and ERK1/2 phosphorylation were significantly disturbed. These defects were observed despite a normal GM-CSF-induced signal transducer and activator of transcription 5 (STAT5) phosphorylation. Our results indicate that the reduced priming of neutrophil ROS production in MDS patients might be caused by a disturbed convergence of the fMLP and GM-CSF signaling routes.

cAMP/PKA-mediated regulation of erythropoiesis.

The role of cyclic AMP (cAMP) as second messenger in erythropoiesis has been suggested in the early 1980s. However, careful analysis showed that cAMP is not generated in direct response to the main erythropoiesis-controlling cytokines such as erythropoietin (Epo). As a result, cAMP disappeared from the central stage in research of erythropoiesis. Instead, other signal transduction pathways, including the Ras/extracellular regulated kinase (ERK)-pathway, the phosphatidylinositol 3-kinase (P13K) and the signal transducer and activator of transcription (STAT5)-pathways, have been found and explored. In concert, these signaling pathways control the transcriptional machinery of erythroid cells. Although cAMP is not directly generated in response to Epo stimulation, it has recently been demonstrated that increased cAMP-levels and in particular the cAMP-dependent protein kinase A (PKA) can modulate erythroid signal transduction pathways. In some cases, like the ERK-signaling pathway, PKA affects signal transduction by regulating the balance between specific phosphatases and kinases. In other cases, such as the STAT5 pathway, PKA enhances Epo signaling by inducing recruitment of additional co-regulators of transcription. In addition to STAT5, PKA also activates other transcription factors that are required for erythroid gene expression. This review discusses the impact of cAMP/PKA on Epo-mediated signaling pathways and summarizes the role of cAMP in malignant erythropoiesis.

Prostaglandin-E2 enhances EPO-mediated STAT5 transcriptional activity by serine phosphorylation of CREB.

Erythroid colony formation in response to erythropoietin (EPO) stimulation is enhanced by costimulating the cells with prostaglandin-E2 (PGE2). The present study further analyzed the underlying mechanisms and demonstrated that EPO-mediated STAT5 transactivation in the erythroid AS-E2 cell line was enhanced 6-fold by PGE2 (10 microM), without affecting the STAT5 tyrosine phosphorylation or STAT5-DNA binding. Moreover, the PGE2-enhancing effect was independent of STAT5 serine phosphorylation. In AS-E2 cells STAT5 is constitutively phosphorylated on Ser780 (STAT5A) and EPO-dependently phosphorylated on Ser726/731 (STAT5A/STAT5B), but overexpression of STAT5 serine mutants did not affect STAT5 transactivation. In addition, PGE2 did not affect STAT5 serine phosphorylation. Instead, the stimulatory effect of PGE2 on STAT5 signaling could be mimicked by dibutyryl-cyclic adenosine monophosphate (cAMP) and the phosphodiesterase inhibitor IBMX, suggesting that the effect was mediated by cAMP. Activation of the cAMP pathway resulted in cAMP-response element binding protein (CREB) phosphorylation, which was sustained in the presence of EPO plus PGE2 and transient on EPO stimulation alone. The costimulatory effect of PGE2 on EPO-mediated STAT5 transactivation was inhibited by overexpression of serine-dead CREB or protein kinase A (PKA) inhibitor (PKI), in contrast to EPO-mediated transactivation, which was PKA independent. Furthermore, CREB-binding protein (CBP)/p300 was shown to be involved in EPO-mediated STAT5 transactivation, and a CBP mutant with increased affinity for CREB resulted in an additional enhancement of the PGE2 effect. Finally, we demonstrated that the STAT5 target genes Bcl-X, SOCS2, and SOCS3 were up-regulated by costimulation with PGE2. In summary, these studies demonstrate that PGE2 enhancement of EPO-induced STAT5 transactivation is mediated by the cAMP/PKA/CREB pathway.

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

Effects of overexpression of the SH2-containing inositol phosphatase SHIP on proliferation and apoptosis of erythroid AS-E2 cells.

Previous studies have demonstrated that SH2-containing inositol phosphatase (SHIP) is involved in the control of B cell, myeloid cell and macrophage activation and proliferation. The goal of the present study was to examine the role of SHIP during proliferation and apoptosis in cells of the erythroid lineage. Wild-type and catalytically inactive SHIP proteins were overexpressed in the erythropoietin (EPO)-dependent cell line AS-E2. Stable overexpression of catalytically inactive SHIP decreased proliferation and resulted in prolonged activation of the extracellular signal-regulated protein kinases ERK1/2 and protein kinase B (PKB), while wild-type SHIP did not affect EPO-mediated proliferation or phosphorylation of ERK and PKB. When AS-E2 cells were EPO deprived a significant increase in apoptosis was observed in clones overexpressing wild type. Mutational analysis showed that this increase in apoptosis was independent of the enzymatic activity of SHIP. The enhanced apoptosis due to overexpression of SHIP was associated with an increase in caspase-3 and -9 activity, without a distinct effect on caspase-8 activity or mitochondrial depolarization. Moreover, in cells overexpressing SHIP apoptosis could be reduced by a caspase-3 inhibitor. These data demonstrate that in the erythroid cell line AS-E2 overexpression of catalytically inactive SHIP reduced proliferation, while overexpression of wild-type SHIP had no effect. Furthermore, overexpression of SHIP enhanced apoptosis during growth factor deprivation by inducing specific caspase cascades, which are regulated independently of the 5-phosphatase activity of SHIP.

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors.

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- (> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.

The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity.

Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates. SHIP1 is the 145-kDa SH2 domain-containing inositol 5-phosphatase expressed in haematopoietic cells. SHIP2 is a related but distinct gene product. We report here that SHIP2 can be expressed in an active form both in Escherichia coli and in COS-7 cells. A truncated 103-kDa recombinant protein could be purified from bacteria that display both inositol 1,3,4,5-tetrakisphosphate (InsP4) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) phosphatase activities. COS-7 cell lysates transfected with SHIP2 had increased PtdIns(3,4,5)P3 phosphatase activity as compared to the vector alone.

Tyrosine phosphorylation and relocation of SHIP are integrin-mediated in thrombin-stimulated human blood platelets.

The SH2 domain-containing inositol 5-phosphatase, SHIP, known to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate has recently been shown to be expressed in a variety of hemopoietic cells. This 145-kDa protein is induced to associate with Shc by multiple cytokines and may play an important role in the negative regulation of immunocompetent cells mediated by FcgammaRIIB receptor. We report here that SHIP is present in human blood platelets and may be involved in platelet activation evoked by thrombin. Platelet SHIP was identified by Western blotting as a single 145-kDa protein. Both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4, 5-tetrakisphosphate 5-phosphatase activities could be demonstrated in anti-SHIP immunoprecipitates of platelet lysate. Thrombin stimulation induced a tyrosine phosphorylation of SHIP, this effect being prevented if platelets were not shaken or if RGD-containing peptides were present, indicating an aggregation-dependent, integrin-mediated event. Moreover, although the intrinsic phosphatase activity of SHIP did not appear to be significantly increased, tyrosine-phosphorylated SHIP was relocated to the actin cytoskeleton upon activation in an aggregation- and integrin engagement-dependent manner. Finally, the striking correlation observed between phosphatidylinositol 3,4-bisphosphate production and the tyrosine phosphorylation of SHIP, as well as its relocation to the cytoskeleton upon thrombin stimulation, suggest a role for SHIP in the aggregation-dependent and GpIIb-IIIa-mediated accumulation of this important phosphoinositide.