B IOLOGICAL FUNCTION

Erythrocyte production is dynamic and tightly regulated. A total number of 2-3 x 10¹³ erythrocytes is maintained by healthy adults, which are approximately 5 million erythrocytes per microlitre blood.

The life span of an erythrocyte is about 120 days. Therefore, 1% of the circulating erythrocytes is replaced each day (Torbett and Friedman 2009).

Erythropoiesis is the term for the regulated process of proliferation and differentiation of

haematopoietic progenitor cells into mature red blood cells (figure 17). The process can be divided into three major steps (Baron and Fraser 2005, McGrath and Palis 2008):

o Generation of erythroid committed blast cells from multipotent haematopoietic progenitors o Division and differentiation of erythroid progenitor cells

o Terminal cellular morphologic changes (including enucleation) to produce reticulocytes and eventually mature red cells

Figure 17: Schematic overview of the role of EPO in erythropoiesis

Erythroblasts are produced in adults from committed progenitors in the bone marrow. During this process, the multipotency is lost and there is an increased lineage restriction (Baron and Fraser 2005,

24 McGrath and Palis 2008). The stages in erythrocyte formation are defined by their ability to form colonies in semisolid medium supplemented with specific cytokines (CFU, colony-forming units). At early stages, the cells respond to a broad variety of cytokines, but as differentiation progresses, the cytokine responses become more specific to erythroid progenitors (Migliaccio and Migliaccio 1988, Koury and Bondurant 1990, McGrath and Palis 2008).

A common myeloid progenitor gives rise to bipotential progenitors restricted to either the erythroid/megakaryocyte or the granulocyte/macrophage pathways. In vitro, this stage of development is represented by the colony-forming unit – granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) precursor (Debili, Coulombel et al. 1996, Akashi, Traver et al. 2000).

Only the erythroid/megakaryocyte-restricted progenitors express the erythropoietin receptor (EPOR) and are responsive to EPO. The most immature erythroid-restricted progenitor is the burst-forming unit – erythroid (BFU-E) (Stephenson, Axelrad et al. 1971, Heath, Axelrad et al. 1976). They are highly proliferative blast-like cells, express EPOR only moderately and give rise to CFU-E progenitors, which are highly EPO-responsive. On these cells, EPOR is expressed in high amounts. CFU-E progenitors begin to express haemoglobin and generate smaller colonies. It was found that EPO stimulate division and prevent apoptosis (Heath, Axelrad et al. 1976).

Several stages of morphologically identifiable nucleated precursors arise from CFU-E to reticulocytes (Stephenson, Axelrad et al. 1971). Important cellular processes take places during this development (Torbett and Friedman 2009):

o Accumulation of haemoglobin o Decrease in cell size

o Nuclear condensation o Final enucleation

Erythroblasts mature in the erythroblastic island, which is a specialised microenvironmental niche in the bone marrow (Manwani and Bieker 2008). These islands consist of a central macrophage that extends cytoplasmic protrusions to a ring of surrounding erythroblasts (Gifford, Derganc et al. 2006).

The macrophage serves as a source for nutrients, survival and proliferative signals to the erythroblasts. Finally, the reticulocytes mature into erythrocytes with the help of the central macrophage (Manwani and Bieker 2008).

1.7.2.2. Hypoxic regulation of EPO

The regulation of EPO production during normoxia and hypoxia is depicted in figure 18. Hypoxia is primarily sensed in the kidneys and will lead to an increase in EPO production. The renal produced EPO stimulates the maturation of the erythroid progenitors in the bone marrow. The increased number of red blood cells carrying oxygen, results in a corrected oxygen state of the tissue.

Therefore, the concentration of EPO in blood serum is inverse proportional to the haematocrit as an indicator for hypoxia (Torbett and Friedman 2009).

In adult mammals, peritubular interstitial fibroblasts in the kidney are the major EPO production site (Lacombe, Da Silva et al. 1988, Bachmann, Le Hir et al. 1993). At the molecular level, EPO expression is coupled to prolyl hydroxylase domain (PHD) proteins, which are oxygen sensors, and to the

25 transcription factor hypoxia inducible factor (HIF). HIF is a heterodimeric transcription factor consists of a labile α- and a constitutively expressed β-subunit (Wang and Semenza 1993, Wang and Semenza 1993). Under normoxic conditions, PHD proteins constitutively hydroxylate two specific proline residues in HIF-α, which then, can be bound by the von Hippel-Lindau protein. Subsequently, this leads to ubiquitination and proteosomal degradation of HIF-α (Semenza 2001). However, under hypoxia conditions, HIF-α is stabilised by HIF-β and subsequently, the transcription of the EPO gene and other hypoxia response genes is activated. The α-subunit is rate-limiting in the transcription complex and its destruction is controlled by the amount of cellular oxygen (Torbett and Friedman 2009).

Figure 18: Schematic overview of the regulation of EPO production during normoxia and hypoxia; figure is inspired by (Torbett and Friedman 2009)

1.7.2.3. EPO receptor signalling processes

Figure 19 depicts an overview of EPOR signalling processes. EPOR is a member of the cytokine-receptor superfamily, which is characterised by an extracellular-binding region, a transmembrane region and an intracellular domain (Youssoufian, Longmore et al. 1993). Upon binding of EPO to EPOR, a tighter connection of the two homodimers of EPOR is induced, due to a conformational change (Cheetham, Smith et al. 1998) and two Janus kinase 2 (JAK2) tyrosine kinase molecules are activated (Witthuhn, Quelle et al. 1993, Remy, Wilson et al. 1999). This leads to phosphorylation of several tyrosine residues in the intracellular region of EPOR, which is a docking site for signalling proteins with phospho-tyrosine binding motifs of several pathways including STAT5, phosphatidyl-inositol 3-kinase (PI3K/Akt) and Ras/MAPK (Richmond, Chohan et al. 2005, Watowich 2011). Finally, these pathways lead to transcription of genes for survival, proliferation and differentiation of the cell (Jelkmann 2004). The signal transduction is terminated by the haematopoietic cell phosphatase, which catalyses the dephosphorylation of JAK2 (Klingmuller, Lorenz et al. 1995). The EPO/EPOR complex is internalised after dephosphorylation of the receptor. The duration of EPO signalling is

26 controlled by the proteasome, which inhibits the renewal of receptor molecules on the cell surface (Verdier, Walrafen et al. 2000).

Figure 19: Schematic overview of intracellular signalling processes upon EPO receptor binding; figure is inspired by (Jelkmann 2004)

1.7.3. EPO as a pharmaceutical