Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

Erythrozyten zählen zu den am häufigsten vorkommenden terminal differenzierten Zelltypen des menschlichen Körpers. Durchschnittlich werden täglich ca. 2 x 1011 von ihnen im Körper eines erwachsenen Menschen produziert. Die reifen Erythrozyten entstehen aus multipotenten hämatopoetischen Stammzellen, die über Stadien von erythroiden Vorläuferzellen, erst den sogenannten burst forming units-erythroid (BFU-E) und später den colony forming units-erythroid (CFU-E), zu kernlosen hämoglobinisierten Zellen differenzieren. <br /> <br /> Für die Untersuchung der molekularen Mechanismen der humanen Erythropoese ist die effektive in vitro Amplifizierung einer weitgehend homogenen Population der Vorläuferzellen der einzelnen Entwicklungsstadien notwendig. Den Wachstumsfaktoren stem cell factor (SCF) und Erythropoietin (Epo) fällt dabei eine entscheidende Rolle zu. Unter ihrem synergistischen Einfluß lassen sich Epo-abhängige Zellpopulationen, die sich aus BFU-E und CFU-E Typ Zellen zusammensetzen, ausreichend amplifizieren (Panzenböck et al., 1998). Freyssinier et al., 1999 beschrieb erstmals die Isolierung einer Epo-unabhängigen Population von Vorläuferzellen (CD36+ Vorläuferzellen), die ebenfalls erythroide Eigenschaften aufweisen.<br /> <br /> Ziel dieser Arbeit war die Isolierung und Charakterisierung von Epo-unabhängigen Vorläuferzellen, die eine frühe erythroide und möglichst homogene Vorläuferzellpopulation darstellen und möglicherweise ein höheres Proliferationspotential aufweisen.<br /> <br /> Für die Identifizierung der Epo-unabhängigen Vorläuferzellen, wurden CD34+ Zellen aus Nabelschnurblut aufgereinigt und unter serumfreien Kulturbedingungen und unter Zusatz der Wachstumsfaktoren SCF, Interleukin 3 (IL-3) und eines Fusionsproteins aus IL-6 und löslichem IL-6 Rezeptor (hyper-IL-6) über einen Zeitraum von 8 Tagen kultiviert. Anschließend wurde eine Population von E-cadherin positiven (E-cad+) Zellen über immunomagnetische Selektion isoliert. Diese neu gewonnenen Epo-unabhängigen E-cad+ Vorläuferzellen wurden hinsichtlich ihres proliferativen Potentials und ihrer Differenzierungseigenschaften mit SCF/Epo-Vorläuferzellen und CD36+ Vorläuferzellen verglichen. Von allen drei Zelltypen wurden des weiteren detailierte molekulargenetische Analysen mittels DNA microarray Technologie durchgeführt und die resultierenden Genexpressionsmuster miteinander verglichen.<br /> <br /> Die Ergebnisse zeigen, dass die E-cad+ Vorläuferzellen eine frühe, weitgehend homogene Epo-unabhängige Population vom BFU-E Typ darstellen und durch entsprechende Änderungen der Kulturbedingungen zu einer in vitro Differenzierung angeregt werden können. Die E-cad+ Vorläuferzellen sind hinsichtlich ihres proliferativen Potentials, ihrer Reaktion auf verschiedene Wachstumsfaktoren, der Expression spezifischer Oberflächenmoleküle und ihrer Genexpressionsmuster mit SCF/Epo-Vorläuferzellen und CD36+ Vorläuferzellen vergleichbar.<br /> <br /> Aufgrund der Identifizierung unterschiedlich exprimierter Gene zwischen den Epo-unabhängigen E-cad+ und den Epo-abhängigen SCF/Epo Vorläuferzellen konnten Kanditatengene wie Galectin-3, Cyclin D1, der Anti-Müllerian Hormonrezeptor, Prostata-Differenzierungsfaktor und insulin-like growth factor binding protein 4 identifiziert werden, die als potentielle Regulatoren der Erythropoese in Betracht kommen könnten. Es konnte weiterhin gezeigt werden, dass CD36+ Vorläuferzellen, die aus der selben Zellpopulation wie die E-cad+ Vorläuferzellen immunomagnetisch selektioniert wurden, eine heterogene Population darstellen, die sowohl E-cadherin positive als auch negative Zellen enthält. Die Analyse der Genexpressionsmuster zeigte, dass in den CD36+ Vorläuferzellen zwar auch die Expression erythroid-spezifischen Gene nachgewiesen werden kann, hier aber im Gegensatz zu den E-cad+ Vorläuferzellen auch für Megakaryozyten spezifische Gene stark exprimiert sind.<br /> <br /> Die Ergebnisse dieser Arbeit tragen zu einem neuen Modell der in vivo Abläufe der Entwicklung roter Blutzellen bei und werden der weiteren Untersuchung der molekularen Mechanismen der Erythropoese dienen. / Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent.<br /> <br /> To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique.<br /> <br /> Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features.<br /> <br /> These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.

Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

Das Eukaryotische-Initiations faktor-4E Bindungsprotein (4E-BP) ist ein Inhibitor der Translationsinitiation. Nicht-phosphoryliertes 4E-BP bindet an den eukaryotischen Initiationsfaktor 4E (eIF4E). Diese Bindung blockiert die Rekrutierung des Initiationskomplexes eIF4F an die Cap-Struktur des 5´Endes von eukaryotischen zellulären mRNAs, was die Initiation der Translation verhindert. Phosphorylierung von 4E-BP durch die mTOR Kinase führt zur Dissoziation des 4E-BP/eIF4E Komplexes und erhöht die Verfügbarkeit von eIF4E, dies wird mit Zellproliferation assoziiert. Die Aktivität von eIF4E wird nicht nur von 4E-BP, sondern auch durch Phosporylierung reguliert, welche wiederum durch die "MAP-Kinase-Interacting-Protein-Kinase" (MNK) reguliert wird. Drei Isoformen von 4E-BP sind bekannt: 4E-BP1, 4E-BP2 and 4E-BP3. 4E-BP1 und 4E-BP2 sind an oxidativem und adipogenetischen Stress beteiligt. Beide Proteine werden im h?matopoetischen System gleich exprimiert, wohingegen 4E-BP3 nicht detektiert wird. 4E-BP1 wird während der Erythroblasten-Proliferation phosphoryliert. Aus diesem Grund habe ich die Hämatopoese und die durch Phenylhydrazine (PHZ) induzierte Stress-Erythropoese in 4E-BP1 und 4E-BP2 Knock-Out Mäusen und 4E-BP1,2 Doppel-Knock-Out Mäusen analysiert. Ich konnte zeigen, dass die Hämatopoese in 4E-BPs defizienten Mäusen nicht beeinflusst wird. Allerdings zeigten 4E-BP1,2-/- und 4E-BP2-/- Mäuse eine verspätete Antwort auf Phenylhydrazin (PHZ) induzierten erythropoetischen Stress. Gleichzeitig war die mRNA Translation von GATA-1, ein essentieller erythropoetischer Transkriptionsfaktor in Erythroblasten runterreguliert. Die Signaltransduktionswege mTOR und MNK1 waren bei erythropoetischen Stress aktiviert. Diese Daten zeigen, dass 4E-BP2, aber nicht 4E-BP1, notwendig ist um auf erythropoetischen Stress zu reagieren und deuten an, dass die 4E-BP gesteuerte translations-regulierende Maschinerie eine Rolle in der Stress-Erythropoese spielt. / Translational regulation allows an organism to generate fast responses to environmental changes quickly. Eukaryotic initiation factor 4E binding protein (4E-BP) is an inhibitor of translation initiation. Unphosphorylated 4E-BP binds to eukaryotic initiation factor 4E (eIF4E) blocking recruitment of the initiation complex eIF4F to the cap structure at the 5´ terminus of eukaryotic cellular mRNAs. Thus initiation of translation is blocked. Phosphorylation of 4E-BP by the mTOR kinase causes disassociation of the 4E-BP/eIF4E complex and increases the availability of eIF4E. EIF4E activity is not only regulated by 4E-BP, but also phosphorylation which is regulated by MAP kinase - interacting protein kinase (MNK). Three isoforms of 4E-BP are known, termed 4E-BP1, 4E-BP2 and 4E-BP3. 4E-BP1 and 4E-BP2 are involved in oxidative and adipogenetic stresses in vivo. They are equally expressed in hematopoietic system, whereas 4E-BP3 is not detected. 4E-BP1 is phosphorylated during erythroblast proliferation. Erythroid differentiation is blocked by overexpresssion of eIF4E in tissue culture. These studies implied that 4E-BPs might play role in response to erythropoietic stress. I examined hematopoiesis and phenylhydrazine (PHZ) induced stress erythropoiesis in 4E-BP1 and 4E-BP2 individual knock out mice and 4E-BP1,2 compound knock out mice. I found that the hematopoiesis of 4E-BPs deficient mice were unaffected. However, 4E-BP1,2-/- and 4E-BP2-/- mice showed delayed response to phenylhydrazine (PHZ) induced erythropoietic stress. Simultaneously, the mRNA translation of GATA-1, which is the essential erythroid transcription factor, was downregulated in their erythroblasts. The signaling pathways through the mTOR and MNK1 were activated in erythropoietic stress. These data showed that 4E-BP2 but not 4E-BP1 was required for the response to erythropoietic stress and suggested that 4E-BP related translation regulatory machinery played a role in stress erythropoiesis.