A. Lyndsay Drayer

Impaired interleukin-8- and GROα-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 (GROα)/CXCL1, which stimulates only CXCR2. In this study, we show that migration toward IL‐8/GROα gradients is decreased in myelodysplastic syndrome (MDS) neutrophils compared with healthy donors. We investigated the signal transduction pathways involved in IL‐8/GROα‐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/GROα. In accordance with these results, we subsequently showed that IL‐8/GROα‐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 GROα stimulation. IL‐8 stimulation resulted in higher migratory behavior and ERK1/2 activation than GROα 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.

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.

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.

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.

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 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‐β‐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, gp91phox, and p22phox 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.

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.

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.

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.

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.