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 reactiveoxygen 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. (Blood.

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

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

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.

Erythroid colony formation in response to erythropoietin (EPO) stimulation is enhanced by costimulating the cells with prostaglandin-E 2 (PGE 2 ). The present study further analyzed the underlying mechanisms and demonstrated that EPOmediated STAT5 transactivation in the erythroid AS-E2 cell line was enhanced 6-fold by PGE 2 (10 M), without affecting the STAT5 tyrosine phosphorylation or STAT5-DNA binding. Moreover, the PGE 2 -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, PGE 2 did not affect STAT5 serine phosphorylation. Instead, the stimulatory effect of PGE 2 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 PGE 2 and transient on EPO stimulation alone. The costimulatory effect of PGE 2 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 PGE 2 effect. Finally, we demonstrated that the STAT5 target genes Bcl-X, SOCS2, and SOCS3 were up-regulated by costimulation with PGE 2 . In summary, these studies demonstrate that PGE 2 enhancement of EPO-induced STAT5 transactivation is mediated by the cAMP/PKA/CREB pathway. (Blood. 2002;100:467-473)

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.Plant sterols structurally differ from cholesterol only by the presence of an additional methyl or ethyl group at C-24 (campesterol and sitosterol, respectively), in some cases with an additional double bond at C-22 (brassicasterol and stigmasterol, respectively). These structural differences appear to have a major impact on their handling in the body. Despite the fact that regular Western diets contain similar amounts of cholesterol and plant sterols (intakes of ϳ200 -400 mg/day for both) (1), plasma levels of plant sterols are Ͼ100 times lower than those of cholesterol in healthy individuals (2), indicating the presence of effective systems to prevent plant sterol accumulation in the human body. It has become clear that the ATPbinding cassette half-transporters ABCG5 and ABCG8, most abundantly present in intestine and liver, are critically involved herein. ABCG5 and ABCG8 function as an obligate heterodimer and mediate cholesterol and plant sterol transport out of intestinal and hepatic cells (3, 4). The functional heterodimer is present at the apical membranes of enterocytes and hepatocytes, where it transports (plant) sterols back into the intestinal lumen or bile, respectively (4, 5). Expression of ABCG5 and ABCG8 in other organs and tissues is relatively low (6 -8), and functionality at sites other than intestine and liver has not been reported.Mutations in either the ABCG5 or ABCG8 gene cause sitosterolemia (9, 10). Sitosterolemia is a relatively rare autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues. Patients frequently develop tendon and cutaneous xanthomas and, most important, are at risk of developing premature coronary atherosclerosis (11)(12)(13)(14). A ...

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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.