Changes in the cell surface phenotype that accompany erythroid differentiation and maturation

The cell-surface phenotypes of erythroid progenitors and precursors are quite distinctive, reflecting the different signalling programmes of the cells as they differentiate. These markers are also of value in the analysis of erythroid progenitors and precursors as they can be used to identify and purify subpopulations of cells. CD34 is present on nearly all multipotent progenitors and committed BFU-E but is lost on later erythroid progenitors (CFU-E) and all precursors.

A similar pattern of expression is shown for the receptor c-Kit. Epo receptor (EpoR, see below) first appears in small numbers (20-50 copies per cell) on late BFU-Es, increases in CFU-Es and pronormoblasts (~1000 copies per cell) and subsequently declines and disappears in later erythroid precursors. CD71 (transferrin receptor, TfR) allows transferrin-bound iron to be taken into the cell and is present on early haemopoietic cells but is considerably upregulated on cells that are actively synthesizing haemoglobin, reaching a peak of 800 000 molecules

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Polychromatic Examples

Figure 2.4 (a) Examples of pronormoblasts (i), basophilic and polychromatic erythroblasts (ii) and polychromatic and orthochromatic erythroblasts (iii and iv). All of these different cell types can also be conveniently viewed at: http://hsc.virginia.edu/medicine/clinical/pathology/educ/innes/ text/nh/mature.html. (b) An example of early (pronormoblasts), intermediate (polychromatic erythroblasts) and late (orthochromatic erythroblasts) separated on the basis oftheir cell-surface markers (CD71 and GPA).

Figure 2.4 (a) Examples of pronormoblasts (i), basophilic and polychromatic erythroblasts (ii) and polychromatic and orthochromatic erythroblasts (iii and iv). All of these different cell types can also be conveniently viewed at: http://hsc.virginia.edu/medicine/clinical/pathology/educ/innes/ text/nh/mature.html. (b) An example of early (pronormoblasts), intermediate (polychromatic erythroblasts) and late (orthochromatic erythroblasts) separated on the basis oftheir cell-surface markers (CD71 and GPA).

per cell on polychromatic normoblasts. CD71 levels diminish in the late phase of terminal differentiation and the receptor is not detectable on mature erythrocytes. Glycophorin A (GPA) is a membrane sialoglycoprotein whose expression is highly upregu-lated as erythroid progenitors mature from pronormoblasts. Combinations of these cell-surface markers can be used to distinguish early, intermediate and late erythroid precursors (Figure 2.4b). Developing erythroid cells express cell-surface adhesion molecules that interact with the extracellular matrix; these include ICAM-1 (a member of the immunoglobulin superfamily) and integrin a4P1 VLA4 (CD29, CD49d), which interacts with fibronectin. These adhesion molecules are most highly expressed in the early precursors and lost as maturation proceeds, freeing erythroid cells to exit the bone marrow.

As cells go through the final divisions of erythropoiesis and postmitotic maturation there is progressive condensation of chromatin accompanied by complex changes in gene expression. When assessed by microarray analysis, many mRNAs are downregulated as multipotent progenitors enter terminal differentiation, reflecting the commitment of multipotent cells to a single specialized lineage. A subset of general mRNAs associated with proliferation, replication and cell cycle control show alterations as the growth characteristics of the cells change. mRNAs encoding proteins that characterize the red cell phenotype are, in general, upregu-lated. Examples include blood group antigens, red cell membrane proteins (e.g. spectrin, ankyrin, actin, protein 4.1), red cell glycolytic pathway enzymes, carbonic anhydrase and enzymes of the haem synthesis pathway (e.g. S-aminolaevulinic acid synthase, ALA synthase). A full catalogue of these changes in gene expression can be found at http://hembase.niddk.nih.gov.

The main purpose of erythropoiesis is to synthesize large amounts of haemoglobin (Figures 2.3-2.5). Globin mRNA sequences are first expressed in pronormoblasts and early basophilic erythroblasts. Globin chain synthesis parallels accumulation of globin mRNA, increasing at the polychromatophilic and ortho-chromatic stages. The amount of globin mRNA reaches 20 000

Figure 2.5 The coordination of globin synthesis, haem synthesis and iron regulation. Blue lines indicate some of the known regulatory feedback systems. The red shaded box indicates molecules per cell in late polychromatic orthochromatic erythro-blasts. During the later stages of erythroid cell maturation, the amount of RNA per cell and the rate of total protein synthesis declines, but the relative stability of globin mRNA ensures that globin becomes the predominant polypeptide made in late ery-throblasts and reticulocytes.

The individual components of the haemoglobin synthetic pathway (iron, free porphyrins, haem and monomeric globin chains) are all extremely toxic to the cell, and consequently many positive and negative feedback loops have evolved and been incorporated into this process. The synthesis of globin must be very accurately matched with the synthesis of haem in which some steps take place in the cytoplasm and others occur in the mitochondria (Figure 2.5). mRNAs encoding many components of the haem biosynthetic pathway (e.g. ALA synthase and porphobilinogen deaminase, PBGD) are coordinately upregulated in terminal erythroid differentiation and their genes contain similar cis-regulatory elements.

Continued translation of globin chains from mRNA only occurs in the presence of adequate haem. Reduced levels of haem rapidly trigger the formation of the 'haem regulated inhibitor' (HRI), a kinase that interacts with the translation initiating factor eIF-2a and prevents translation of a- and P-globin mRNA. The synthesis of haem itself is also regulated at many points and is particularly sensitive to the levels of available iron. Via a well-characterized pathway involving the iron-regulatory proteins (IRP1 and IRP2), binding to iron response elements (IREs) in the mRNA transcripts of ferritin, TfR and ALA synthase, the level of intracellular iron thus controls the reactions occurring in the mitochondria. Rate-limiting controls of haem synthesis are shown in black boxes.

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