Sub theme 2.1.1
Regulation of proliferation and differentiation of hematopoietic stem cells

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Workgroup leaders   Department
Dr.  T.  Cupedo   Hematology
Dr.  S.J.  Erkeland   Hematology
prof. dr  I.P.  Touw   Hematology

Goals of research: general outline

Hematopoiesis requires maintenance of mostly quiescent stem cells, transient amplification of progenitor cells and adequate production of mature cells in the blood. Our research aims to unravel how the balance between transient amplification and maturation is controlled by growth factors, and how stem- and progenitor compartments are regulated when DNA damage results in bone marrow aging.

With respect to the role of growth factors we study how granulocytes are produced in response to granulocyte-colony stimulating factor (G-CSF), and how erythrocyte production is controlled by stem cell factor (SCF) and erythropoietin (Epo). We aim to understand the molecular and biochemical mechanisms that control signaling specificity and kinetics, and the signaling pathways acting at distinct cellular compartments to control self-renewal, lineage commitment and differentiation, proliferation and survival and migration.

Aging of bone marrow reduces its production capacity, which compromises the quality of life, but also presents a risk for leukemia development. We aim to understand the molecular and cellular changes that occur in premature aging of progenitors resulting from a DNA repair deficiencies. These studies provide insights in the control of normal blood cell development, but they are also crucial to our understanding of the pathogenesis of hematopoietic disorders, specifically neutropenia, anemia, myelodysplasia and leukemia.

Scientific achievements

Signaling function of G-CSF receptor in the control of neutrophil development depends on  intracellular routing of  the receptor

  • Previously, we have unraveled signaling mechanisms coupled to the G-CSF receptor that are essential for G-CSF-induced expansion of myeloid progenitor and the appropriate balance of proliferation and differentiation during G-CSF-induced neutrophil development. These mechanisms involve the association of specific signaling substrates (STAT and SOCS proteins, Ras signalling intermediates, Src-like kinases and SH2-containing protein tyrosine phosphatases) to distinct domains of the G-CSF-R cytoplasmic domain. In addition, we had found that receptor endocytosis and intracellular routing has a major impact on the signaling function of G-CSF receptor. All these studies are directly relevant to the role of G-CSF receptor mutations found in severe congenital neutropenia, which are strongly associated with the leukemic transformation of this disorder (ref. 3). These mutant receptors are severely affected in their signaling function and intracellular routing. We have further elucidated the mechanisms involved in lysosomal routing of the G-CSF receptor and found that the suppressor of cytokine signaling 3 (SOCS3), a protein with E3 ligase activity,  has a major role in lysosomal targeting of G-CSFR via a mechanism involving ubiquitination of a highly conserved juxtamembrane lysine residue (Ref. 1 and 4). Another group of SOCS-related E3 ligases, the Wsb proteins, were found to be involved in controlling the forward routing of G-CSFR from the ER/Golgi towards the cell surface (Ref 2).   Finally, we found that also the assembly of JAK2/receptor complexes plays an important role in the forward routing of G-CSFR (Meenhuis et al, Biochem. J. 2009).

Generation of Cre knockin strains for lineage tracing and to study cell stage specific effects of gene manipulations in myeloid progenitors

  • Despite recent advances in the identification of transcription factors involved in specification of lymphoid and erythroid progenitor cells, little is known about the initial events in myeloid cell development. We generated Cebpa-Cre and Csf3r-Cre knockin mice and crossed these with Rosa-LacZ and Rosa-YFP reporter strains, allowing to trace the fate of stem and progenitor cells in steady-state hematopoiesis. Thus far, we have analyzed the Cebpa-Cre  model in greatest detail. We found that expression of C/EBPa, a factor indispensable for granulocyte formation, defines a population of multipotent progenitors (MPPs) with predominant myeloid potential. In the thymus, C/EBPa expression also identifies progenitor cells with robust myeloid potential with a significant fraction of thymic dendritic cells. These results indicate that a C/EBPa-driven transcriptional program already determines myeloid lineage instruction in MPPs as well as early thymic progenitors.

Control of erythropoiesis by Epo and SCF

  • Previous studies indicated that activation of the PI3K/PKB pathway is crucial to sustain proliferation and inhibit differentiation of erythroid progenitors. One of the targets of this pathway is Forkhead transcription factors class O (Foxo). Foxo3a phosphorylation results in inhibition by retention in the cytoplasm. Surprisingly, Foxo3a target gene identification by gene expression profiling showed that only part of all Foxo3a targets is controlled by PKB, suggesting that different Foxo3a activating stimuli (DNA damage, oxidative stress) may elicit distinct cellular responses (ref. 7, Bakker, 2007). The PI3K/PKB pathway also controls the mTOR/eIF4E pathway. Comparing SCF-induced gene expression in total and polysome bound mRNA we found that expression of multiple genes is controlled at the level of polysome recruitment and not by factor-controlled gene transcription (ref 9,  Blazquez, 2005; ref 5, Grech, 2008). Constitutive expression of some of the translationally regulated genes (Igbp1, Use1) appeared to be sufficient to inhibit erythroid differention (ref 5, Grech, 2008).

Bone marrow aging and leukemic transformation  in response to DNA damage

  • Mice harboring a defect in the nucleotide excision enzyme Ercc1 are unable to resolve interstrand crosslinks in the DNA. Sensitivity to interstrand cross links is a hallmark of Fanconi Anemia. Both Ercc1 and Fanconi deficient cells undergo premature differentiation in culture and competitive in vivo assays show a gradual depletion of Ercc1-deficient cells and increase of wt cells over time. As a result the bone marrow of Ercc1 deficient mice displays marked exhaustion of stem- and progenitor cells (ref 10).
  • More recent studies have demonstrated that this exhaustion is p53, but not p16 dependent. We currently employ the Ercc1 deficient model to study mechanisms of leukemia development in a background of bone marrow aging and genomic instability. Challenging the expansion of Ercc1-deficient bone marrow by transplantation did result in leukemia development, the leukemia’s are being characterized. 

Future plans: special goals and approach

Future studies will continue to address questions related to the signaling function of G-CSF receptor. One of the role of receptor internalization and intracellular trafficking for the appropriate proliferation and differentiation signaling function of the G-CSF receptor. We have obtained evidence for a crucial role of the most C-distal 20 amino acids for sorting of the receptor into late endosomes and lysosomes. Importantly, deletion of this region has a negative impact on differentiation signaling. Using a combination of biochemical, proteomics and imaging technology, we will try to identify the regulatory molecules involved in G-CSF-R trafficking and to elucidate the spatio-temporal dynamics of G-CSF-R signaling complexes in relation to signaling function. In addition, we will continue to apply our newly generated mouse models (Cebpa-Cre, Csf3r-Cre) that will allow us to determine the consequences of conditional gene modulation at distinct stages of myeloid cell development.

Spatiotemporal control of the signaling function of G-CSF receptor

  • Our current and future experiments will continue to focus on key aspects of receptor trafficking and its impact on the signaling output of  G-CSFR that is essential for a balanced proliferation and differentiation of myeloid progenitors under steady state conditions and during episodes of bacterial infections, a state also referred to as “emergency” granulopoiesis. To this end we combine biochemical approaches and proteomics strategies with live cell imaging. We have introduced several new imaging technologies in our research program that are particularly suited for spatio-temporal imaging of G-CSFR complexes, including total internal reflection fluorescence (TIRF) microscopy that can be combined with fluorescence recovery after photobleaching (FRAP). This instrument is equipped with a dual view system that allows simultaneous real time imaging of receptor complexes with  signaling molecules at the cell surface and  in endocytotic vesicles. 

Control of erythropoiesis by Epo and SCF

We will focus on PI3K-dependent mRNA translation and investigate:

  • The role of genes that are regulated by SCF dependent mRNA translation
  • The sequences and protein-RNA interactions involved in SCF-dependent mRNA translation of these genes
  • Whether the same or different genes are affected in ‘ribosome diseases’ such as Diamond Blackfan Anemia, characterized by mutations in ribosomal proteins, or Shwachman Diamond Syndrome, characterized by mutation in a protein that is crucial to release 60S ribosomal units in the cytoplasm.

Bone marrow aging in response to DNA damage

  • We will analyze the genetic and epigenetic alterations that take place during aging and leukemic transformation of Ercc1-deficient bone marrow stem- and progenitor populations. These studies will e.g., involve extensive gene expression array experiments and chromatin immune precipitation (ChIP) combined with promoter chip hybridizations, as well as DNA methylation profiling on purified stem and progenitor cell subsets. 


Most recent publications
  1. Irandoust MI, Aarts LH, Roovers O, Gits J, Erkeland SJ, Touw IP. Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor.  EMBO J. 2007 Apr 4;26(7):1782-93.
  2. Stefan J. Erkeland1, Lambertus H. Aarts1, Mahban Irandoust1, Onno Roovers1, Alexandra Klomp1, Marijke Valkhof1, Judith Gits1, Sven Eyckerman2, Jan Tavernier2 and Ivo P. Touw1. Novel role of WD40 and SOCS box protein-2 (Wsb-2) in steady state distribution of granulocyte colony-stimulating factor (G-CSF) receptor and G-CSF-controlled proliferation and differentiation signaling. Oncogene 2007, Mar 29;26(14):1985-94.
  3. Touw IP, Bontenbal M: Granulocyte colony-stimulating factor: key (f)actor or innocent bystander in the development of secondary myeloid malignancy? J Natl Cancer Inst. 2007 Feb 7;99(3):183-6.
  4. Wölfler A, Irandoust M, Meenhuis A, Gits J, Roovers, Touw IP. Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the G-CSF receptor. Traffic 2009 Aug;10(8):1168-79
  5. Grech G, Blazquez-Domingo M, Kolbus A, Bakker, WJ, Mullner, EW, Beug H, von Lindern, M. (2008) Igbp1 is part of a positive feedback loop in stem cell factor-dependent, selective mRNA translation initiation inhibiting erythroid differentiation. Blood 112:2750-2760.
  6. Grosso AR, Gomes AQ, Barbosa-Morais NL, Caldeira S, Thorne NP, Grech G, von Lindern M, Carmo-Fonseca M. (2008) Tissue-specific splicing factor gene expression signatures. Nucleic Acids Res.36:4823-4832.
  7. Bakker WJ, van Dijk TB, Parren-van Amelsvoort M, Kolbus A, Yamamoto K, Steinlein P, Verhaak RG, Mak TW, Beug H, Lowenberg B, and von Lindern M. (2007). Differential regulation of Foxo3a target genes in erythropoiesis. Mol Cell Biol. 27:3839-3854.
  8. Ferreira R, Wai A, Shimizu R, Gillemans N, Rottier R, von Lindern M, Ohneda K, Grosveld F, Yamamoto M, Philipsen S. (2007). Dynamic regulation of Gata factor levels is more important than their identity. Blood. 109:5481-90.
  9. Blazquez-Domingo M, Grech G, von Lindern M. (2005) Translation initiation factor 4E inhibits differentiation of erythroid progenitors. Mol Cell Biol. 25:8496-8506
  10. Joanna M. Prasher, Astrid S. Lalai, Claudia Heijmans-Antonissen, Robert E. Ploemacher, Jan H.J. Hoeijmakers, Ivo P. Touw, Laura J. Niedernhofer. Reduced hematopoietic reserves in DNA interstrand crosslink repair deficient-Ercc1-/- mice. EMBO J. 2005, 24(4):861-71