PURPOSE OF REVIEWHematopoietic stem cell (HSC) transplantation is the most powerful treatment modality for a variety of hematological disorders. Successful hematopoietic recovery after ...transplantation depends on optimal homing of HSCs to the bone marrow and subsequent lodging in the HSC niche. The molecular mechanisms underlying bone marrow homing are, thus far, incompletely understood. This review focuses on recent studies that extended our understanding of how the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB/c-akt) signaling module can regulate migration and homing of HSCs.
RECENT FINDINGSIn addition to regulation of HSC maintenance and lineage development, it has recently become apparent that the PI3K/PKB signaling module plays a critical role in regulation of migration and adhesion of hematopoietic stem and progenitor cells. Activation of this signaling pathway enhances firm adhesion, reduces migration and inhibits bone marrow homing, whereas inhibition of PKB conversely induces bone marrow homing.
SUMMARYThese findings clearly implicate the PI3K/PKB signaling module in playing a critical role in regulation of bone marrow homing, suggesting that pharmacological modulation of this signaling molecule prior to transplantation may provide a clinical means of improving engraftment levels and accelerating hematopoietic recovery.
A plethora of extracellular stimuli regulate growth, survival, and differentiation responses through activation of the MEK-ERK MAPK signaling module. Using CD34+ hematopoietic progenitor cells, we ...describe a novel role for the MEK-ERK signaling module in the regulation of proliferation, survival, and cytokine production during neutrophil differentiation. Addition of the specific MEK1/2 inhibitor U0126 resulted in decreased proliferation of neutrophil progenitors. Conversely, transient activation of a conditionally active MEK1 mutant resulted in the expansion of progenitor cells, which thereafter differentiated normally into mature neutrophils. In contrast, chronic MEK1 activation was found to induce cell death of CD34+ neutrophil progenitors. Microarray analysis of CD34+ progenitor cells showed that activation of MEK1 resulted in changes in expression of a variety of cell-cycle modulating genes. Furthermore, conditional activation of MEK1 resulted in a dramatic increase in the expression of mRNA transcripts encoding a large number of hematopoietic cytokines, chemokines, and growth factors. These findings identify a novel role for MEK-ERK signaling in regulating the balance between proliferation and apoptosis during neutrophil differentiation, and they suggest the need for tight control of MEK-ERK activation to prevent the development of bone marrow failure.
Inhibitor of DNA binding (Id) proteins function as inhibitors of members of the basic helix-loop-helix family of transcription factors and have been demonstrated to play an important role in ...regulating lymphopoiesis. However, the role of these proteins in regulation of myelopoiesis is currently unclear. In this study, we have investigated the role of Id1 and Id2 in the regulation of granulopoiesis. Id1 expression was initially up-regulated during early granulopoiesis, which was then followed by a decrease in expression during final maturation. In contrast, Id2 expression was up-regulated in terminally differentiated granulocytes. In order to determine whether Id expression plays a critical role in regulating granulopoiesis, Id1 and Id2 were ectopically expressed in CD34+ cells by retroviral transduction. Our experiments demonstrate that constitutive expression of Id1 inhibits eosinophil development, whereas in contrast neutrophil differentiation was modestly enhanced. Constitutive Id2 expression accelerates final maturation of both eosinophils and neutrophils, whereas inhibition of Id2 expression blocks differentiation of both lineages. Transplantation of β2-microglobulin-/- nonobese diabetic severe combined immunodeficient (NOD/SCID) mice with CD34+ cells ectopically expressing Id1 resulted in enhanced neutrophil development, whereas ectopic expression of Id2 induced both eosinophil and neutrophil development. These data demonstrate that both Id1 and Id2 play a critical, although differential role in granulopoiesis.
1 Molecular Immunology Lab, Department of Immunology, UMC, Utrecht;
2 Department of Physiological Chemistry, Centre for Biomedical Genetics, UMC Utrecht, Utrecht;
3 Department of Hematology, UMC, ...Groningen and
4 Department of Pediatric Immunology, UMC Utrecht, Utrecht, The Netherlands
Correspondence: Miranda Buitenhuis, Dept. of Immunology, University Medical Center Utrecht, Lundlaan 6, 3584 CX Utrecht, The Netherlands. E-mail: m.buitenhuis{at}umcutrecht.nl
Background: The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis.
Design and Methods: An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis.
Results: Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34 + lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling.
Conclusions: Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34 + hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.
Key words: mammalian target of rapamycin, mTOR, rapamycin, myeloid, hematopoiesis, CD34 + .
Although a number of genetic defects in myelodysplastic progenitor cells have been described, the intracellular signaling pathways underlying aberrant regulation of hematopoiesis remain relatively ...undefined. The findings of this study suggest that targeting the ID1 and C/EBPα transcriptional regulators may be of benefit in the design of novel therapies for low-risk myelodysplastic syndromes.
Abstract
Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced “inside-out” activation of integrins, little is known about how basal levels of ...integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of α4β1 integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca2+ and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal α4 integrin activity in nonactivated eosinophils. Basal α4 integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca2+. However, depletion of intracellular Ca2+ in CD34+ hematopoietic progenitor cells abolished RapV12-mediated induction of α4 integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca2+-depleted cells is not sufficient to induce α4 integrin activation. These data suggest that activation of functional α4 integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca2+ is at a normal homeostatic concentration.
Background and Objectives
Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). ...Despite the favorable prognosis associated with ETV6-RUNX1 positive ALL, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. Leukemic cells reside in the bone marrow microenvironment, where they are nurtured and protected against chemotherapy. In this study, we investigated novel ways to disrupt this leukemic niche by targeting signaling pathways contributing to the migration of ETV6-RUNX1 positive leukemic cells.
Results
Gene expression profiling and subsequent pathway analysis of leukemic blasts of 654 ALL patients revealed a significant enrichment of genes involved in regulation of cellular movement and cell morphology in ETV6-RUNX1 positive BCP-ALL patients compared with ETV6-RUNX1 negative BCP-ALL patients (p < 0.001). In correspondence, the same pathways were significantly upregulated in cord blood-derived hematopoietic progenitor cells (CB-CD34+) ectopically expressing ETV6-RUNX1 (p < 10E-06). LARG (ARHGEF12) was identified to be the most important regulator of this pro-migratory signature. This gene encodes for the G-protein-regulated Rho Guanine Exchange Factor 12, a specific activator of the GTPase RhoA. LARG expression was 5.7-fold higher in ETV6-RUNX1 positive BCP-ALL cells than in ETV6-RUNX1 negative BCP-ALL cells (p < 10E-06). Similarly, LARG was upregulated 5.4-fold in CB-CD34+ cells expressing ETV6-RUNX1 compared with empty vector controls (p = 0.03).
To determine the importance of the LARG/RhoA pathway in the induction of this migratory phenotype, we used two recently identified small molecule inhibitors of the LARG/RhoA pathway: Y16, a specific LARG inhibitor, and G04 (Rhosin), a specific RhoA inhibitor. Both inhibitors significantly reduced the migration of ETV6-RUNX1 positive leukemic cells towards a gradient of CXCL12 (75%, p < 0.01) whereas the migration potential of ETV6-RUNX1 negative leukemic cells remained unaffected. Migration of ETV6-RUNX1 positive leukemic cells towards patient derived mesenchymal stromal cells was inhibited to a similar extent (75-85%, p < 0.01). In contrast, LARG/RhoA inhibition in ETV6-RUNX1 negative cells resulted in an induction of migration towards MSCs. While LARG/RhoA inhibitors reduced migration of ETV6-RUNX1 positive cells in a targeted manner, inhibition of CXCR4 by the CXCR4 antagonist AMD3100 (Plerixafor) reduced the migration of both ETV6-RUNX1 positive cells and ETV6-RUNX1 negative BCP-ALL cells.
Next, we studied the additional cellular effects of LARG/RhoA inhibition on leukemic cells. shRNA-mediated silencing of LARG modestly reduced the proliferation rate of both ETV6-RUNX1 positive and ETV6-RUNX1 negative cell lines (p<0.05). This reduced proliferation rate was accompanied by a moderate induction of apoptosis (p < 0.05). Using a multiplexed cell signalling assay, we further show that silencing of LARG moderately reduced the phosphorylation level of CREB (S133, 25%, p<0.05), while the phosphorylation state of other key signaling proteins was unaffected (including PI3K/PKB, MAPK/ERK, JAK/STAT and NFκB). In line with these data, inhibition of LARG/RhoA signaling with small molecule inhibitors did not affect cell survival of primary ETV6-RUNX1 positive BCP-ALL cells or resistance of these cells to Prednisolone or L-Asparaginase.
Conclusion
Together, our data indicate that small molecule inhibition of LARG/RhoA signaling affects the migration potential of ETV6-RUNX1 positive cells. In contrast to inhibition of the CXCR4/CXCL12 axis that reduces the migration of both leukemic and healthy hematopoietic cells, inhibition of the LARG/RhoA signaling pathway appears to specifically inhibit ETV6-RUNX1 positive cell migration. Our results warrant further studies to establish the clinical benefit of disrupting the leukemic niche in ETV6-RUNX1 positive BCP-ALL patients using small molecule inhibitors of LARG/RhoA signaling.
No relevant conflicts of interest to declare.
Eosinophil differentiation is a complex series of events regulated by cytokines at multiple levels, including proliferation, survival, and maturation. The development of an ex vivo eosinophil ...differentiation model, using the current knowledge on factors involved in this process, has facilitated efforts to understand the molecular mechanisms underlying human eosinophil development. Differentiation of human hematopoietic progenitor cells, isolated by density centrifugation and immunomagnetic cell separation, towards mature eosinophils, involves a 17-day culture period in the presence of a mixture of cytokines. At early stages of differentiation, these cells can be retrovirally transduced resulting in modulation of the expression of genes of interest to examine their role in eosinophil development. Eosinophil maturation can be analyzed by combining three different methods: histochemical analysis, flow cytometric analysis, and Luxol Fast Blue staining. In addition to this ex vivo differentiation model, human hematopoietic progenitors can be transplanted into immune-deficient mice resulting in the development of all human hematopoietic lineages in the mouse bone marrow, including eosinophils. Although the ex vivo differentiation model can be used separately, combining it with the transplantation model will give insight into not only regulation of human eosinophil development but also hematopoiesis in general.
To explore whether and how T cells can affect myelopoiesis, we investigated myeloid differentiation in a model for T cell-mediated immune activation. We found that CD70-transgenic (CD70TG) mice, ...which have elevated numbers of interferon-γ (IFN-γ)–producing effector T cells in the periphery and bone marrow, are almost devoid of eosinophilic granulocytes. Induction of allergic airway inflammation in these mice failed to induce eosinophilia as well as airway hyperresponsiveness. CD70TG mice also have strongly reduced numbers of eosinophil lineage-committed progenitors, whereas granulocyte/macrophage progenitors from these mice are unable to generate eosinophils in vitro. We found that granulocyte/macrophage progenitors express IFN-γR1 and that IFN-γ is sufficient to inhibit eosinophil differentiation of both murine and human progenitor cells in vitro. We demonstrate that inhibition of eosinophil development in CD70TG mice is IFN-γ–dependent and that T cell–derived IFN-γ is sufficient to inhibit eosinophil formation in vivo. Finally, we found that IFN-γ produced on anti-CD40 treatment and during viral infection can also suppress eosinophil formation in wild-type mice. These data demonstrate that IFN-γ inhibits the differentiation of myeloid progenitors to eosinophils, indicating that the adaptive immune system plays an important role in orchestrating the formation of the appropriate type of myeloid cells during immune activation.
Plasmacytoid dendritic cells (pDCs) are considered potential tools or targets for immunotherapy. However, current knowledge concerning methodologies to manipulate their development or function ...remains limited. Here, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB)–mammalian target of rapamycin (mTOR) axis in human pDC development, survival, and function. In vitro pDC generation from human cord blood–derived CD34+ hematopoietic progenitors was reduced by pharmacologic inhibition of PI3K, PKB, or mTOR activity, and peripheral blood pDCs required PI3K-PKB-mTOR signaling to survive. Accordingly, activity of this pathway in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active PKB or pharmacologic inhibition of negative regulator phosphatase and tensin homolog (PTEN) resulted in increased pDC numbers in vitro and in vivo. Furthermore, MHC class II and costimulatory molecule expression, and production of IFN-α and TNF-α, were augmented, which could be explained by enhanced IRF7 and NF-κB activation. Finally, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced PI3K-PKB-mTOR activity. In conclusion, intact PI3K-PKB-mTOR signaling regulates development, survival, and function of human pDCs, and pDC development and functionality can be promoted by PI3K-PKB hyperactivation. Manipulation of this pathway or its downstream targets could be used to improve the generation and function of pDCs to augment immunity.
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