Hematopoiesis starts from stem-cells in the bone marrow which gradually differentiate towards the well-known specific lineages of blood cells. The process ends with late-stage committed progenitors undergoing terminal maturation. Our group focused on molecules critically involved in reaching the balance between sustained proliferation versus terminal differentiation of progenitors, with particular emphasis on erythropoiesis.

As systems, we used fetal liver- or bone marrow-derived mouse erythroblasts as well as myeloid progenitors from various genetically modified mouse strains. Moreover, we employed cells from cord- or peripheral blood of healthy or diseased human donors. These cell types could be kept immature in culture for extended periods and terminally differentiated in a highly synchronous manner.

These cellular tools were mainly used to study

• Signalling pathways emanating from extracellular ligands like growth factors (stem cell factor, Wnt, erythropoietin) or steroid hormones (thyroid hormone T3, glucocorticoid, androgen)
• Cell-type specific features in the regulation of iron metabolism during erythropoiesis and
• Cell size control. This topic was originally triggered by the decrease in cell volume accompanying erythroid maturation.

Background
The impact of research on haematopoiesis in terms of immunology and leukaemogenesis can hardly be overemphasised. But also the more "humble" process of erythropoiesis – besides its relevance for blood formation – is a fascinating topic in terms of molecular cell biology. It allows to study a developmental processes from long term self renewing stem cells in the bone marrow to mature erythrocytes in the blood. Along this axis, multiple signalling pathways govern the relevant developmental decisions before the erythroblast stage, in which we are mainly interested, is reached.
 
Sustained proliferation of erythroid progenitors (in vitro) requires signals from erythropoieitin, stem cell factor and glucocorticoids in a process resembling stress erythropoiesis in vivo. Removal of these factors and replacement by high levels of erythropoietin and insulin induces highly synchronous terminal differentiation.

This final developmental step is accompanied by a massive accumulation of hemoglobin, which ultimately reaches a higher concentration than any other protein in any other cell type. In parallel, the cells consume 80% of total body iron, necessitating highly specific regulatory processes to escape the toxicity of this metal. These steps are accompanied by a gradual decrease of cell size by about 80%, while the cells are still rapidly cycling, a phenomenon called "differentiation divisions" and in some aspects reminding of earliest embryogenesis. Ultimately, the nucleus gets extremely condensed and extruded from the cells, which assume the characteristic donut-shaped morphology of mature erythrocytes. As mentioned above, a highly specialized cell fate and a truly fascinating topic ...

Current Projects

Jak-Stat signalling: Sufficient but not necessary for erythropoiesis

The erythroid phenotype of mice completely lacking both isoforms of Stat5 (Stat5a and Stat5b)

Erythropoiesis strictly depends on signal transduction through the erythropoietin receptor (EpoR) – Janus kinase 2 (Jak2) – signal transducer and activator of transcription 5 (Stat5) axis, regulating proliferation, differentiation and survival. While the knockout of Jak2 and EpoR are embryonic lethal, the exact role of the transcription factor Stat5 in erythropoiesis had remained puzzling, since the first Stat5-deficient mice carried a hypomorphic Stat5 allele (N-terminal truncation), impeding full phenotypical analysis.
 
Most fully Stat5-/- embryos develop to term but suffer from severe microcytic anemia,. We could attribute this phenotype to multiple defects in erythropoiesis: In addition to a massive increase in apoptosis of erythroid cells in the fetal liver, we also uncovered an unexpected defect on cellular iron metabolism due to Stat5 deficiency.

Despite the observed lethality, Stat5-/- embryos did form red blood, cells albeit in reduced numbers. This underscored the hypothesis that the requirement for this transcription factor along the Epo / EpoR / Jak2 / Stat5 axis in red cell development is not absolute and that lack of Stat5 can be (at least partially) compensated by other pathways.
 
Thus it was surprising to observe that constitutive activation of a mutated Stat5 variant was sufficient to enable proliferation and terminal maturation of EpoR-/- as well as Jak2-/- erythroid progenitors.
 
Apparently, like in other systems, erythroid development is based on a complex signaling network with a remarkable degree of plasticity and redundancy.

Constitutive activation of Stat5 enables erythropoiesis in the absence of EpoR and Jak2

Expression of the constitutively active cS5 variant of Stat5a (harboring a S710F point mutation) was sufficient to rescue the self renewal defect of erythroid progenitors from Jak2- as well as EpoR-deficient embryos. Furthermore, expression of cS5 rendered WT, Jak2-/- and EpoR-/- erythroblasts Epo-independent for self renewal and cS5-transduced Jak2-/- and EpoR-/- cells displayed a marked increase in CFU-E and BFU-E colony formation. In addition, we discovered mechanistic insights how Jak2 activation can contribute to early hematopoiesis downstream of c-Kit signaling. Jak2-deficient cells also exhibited less lymphopoiesis and myelopoiesis (and not only erythropoiesis) upon SCF stimulation and had a severe reduction in hematopoietic progenitor cell numbers. All these defects could be rescued by cS5 transduction both in vitro as well as in vivo. Surprisingly, also growth factor receptors such as c-Kit appeared to use the Jak2-Stat5 axis in conjunction with cytokines to promote efficient hematopoiesis. This receptor cross-talk was not reported previously.  [PubMed]

The anti-apoptotic protein Mcl1 as a relevant Stat5 target in erythroblasts
Up-regulation of the anti-apoptotic protein Bcl-xL during terminal erythropoiesis was described as a major event required to protect late-stage erythroid cells from apoptosis. Furthermore, it was suggested that Bcl-xL is the main target gene of Stat5 in these cells. To our surprise we found only a 50% reduction in Bcl-xL expression in Stat5-/- cells but an almost complete loss in expression of the anti-apoptotic protein Mcl-1.

We could show that Mcl-1 indeed is an Epo-induced, Stat5-dependent target gene and that ectopic expression of Mcl-1 was able to fully rescue primary wild-type and Stat5-/- erythroblasts from apoptosis induced by Epo withdrawal. Thus we add Mcl-1 as a new important player to the anti-apoptotic machinery of erythroid cells.

Regulation of Iron Metabolism in Erythroid Cells

Stat5 is involved in erythroid iron metabolism
Maturing erythroid progenitors require large amounts of bio-available iron, provided by iron-loaded serum transferrin, to ensure the necessary high rates of heme synthesis. This iron uptake is mediated by transferrin receptor-1 (TfR-1), whose mRNA stability is modulated by the specific mRNA-binding proteins IRP-1 and –2 (IRP = iron regulatory protein).
 
Erythropoiesis strictly depends on signal transduction through the erythropoietin receptor (EpoR) – Janus kinase 2 (Jak2) – signal transducer and activator of transcription 5 (Stat5) axis, regulating proliferation, differentiation and survival. The exact role of the transcription factor Stat5 in erythropoiesis had remained puzzling, however, since the first Stat5-deficient mice carried a hypomorphic Stat5 allele, impeding full phenotypical analysis.
 
When studying the phenotype of mice completely lacking Stat5 – displaying perinatal lethality – we observed that these animals suffer from microcytic anemia. Besides reduced expression of the anti-apoptotic proteins Bcl-xL and Mcl-1 (followed by enhanced apoptosis; see section on Jak/Stat signalling), the erythroid Stat5-/- cell phenotype could be ascribed to a significant down regulation of TfR-1 cell surface expression in vivo, which led to massive reduction of iron uptake. We further demonstrated that in particular the levels of IRP-2 are strongly decreased upon loss of Stat5. IRP-2, the dominant regulatory factor of TfR-1 in erythroid cells could be shown by chromatin immuno-precipitation to be a direct target gene of Stat5. Moreover, also the promoter of TfR-1 itself was found by us and others to contain functional binding sites for Stat5.

Thus, the dual down-regulation of IRP-2 and TfR-1 mRNAs in the absence of Stat5 causes reduced TfR-1 cell surface expression leading to the severe iron deficiency observed in Stat5-/- animals. Interestingly, with regard to regulation of IRP-2 and TfR-1 expression, Stat5 knock out mice pheno-copy the IRP-2 knock out, further underscoring the importance of Stat5 in iron metabolism.

Frataxin in Friedreich's Ataxia (FRDA)

FRDA is the most common human autosomal recessive neurodegenerative disease (1/30.000 in the European population). The incurable disease has its onset around puberty and is caused by a large (GAA)n repeat in the frataxin gene resulting in reduced protein expression (5-30% of normal). Although studies in yeast showed a role for frataxin in mitochondrial iron metabolism its exact role in this process remains controversial. One plausible role is its involvement in heme synthesis. Cellular heme biosynthesis occurs in eight sequential steps last being the insertion of ferrous iron into protoporphyrin IX (PPIX) by the mitochondrial enzyme ferrochelatase. Multiple publications suggest a role of frataxin in this process either by helping in FeS cluster synthesis for ferrochelatase function and/or by donating iron to ferrochelatase for heme formation.

To analyze a possible role of frataxin in erythropoiesis we used a human erythroid cell culture system developed in our lab [Link] for in vitro long-term expansion of human erythroid progenitors from peripheral blood. These cultures could be kept in sustained proliferation for ¡Ý4 weeks properly responding to physiological signals active in stress erythropoiesis (low erythropoietin [Epo], stem cell factor, glucocorticoid) and could be induced to undergo highly synchronous terminal maturation into erythrocytes (high Epo, insulin, transferrin). So far, we analyzed erythroid progenitors of 4 FRDA patients and compared them to cells from healthy volunteers. We characterized erythroid proliferation and differentiation by cytospins, cell size profiles, cell viability and heme assays. Moreover, we monitored erythroid specific cell surface marker expression like stem cell factor receptor (c-Kit), transferrin receptor (CD71) and glycophorin A (GPA). Differentiating erythroid cells of FRDA patients accumulated significantly less hemoglobin, though all other differentiation parameters were equal to cells expressing normal levels of frataxin. These findings suggest a role of frataxin in heme synthesis during erythroid differentiation at least in stress erythropoiesis. Thus thid system appears well suited to study the function of frataxin in diseased and healthy erythroid cells in molecular detail.