No abstract

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes vErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal dierentiation under appropriate conditions, were established from fetal livers of p537/7 mice. Expression and activation of the EGF-receptor (HER-1/ c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal dierentiation. Upon inhibition of ErbB function, dierentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5-and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms. Oncogene (2001) 20, 3651 ± 3664.

During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c‐Kit and c‐ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self‐renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self‐renew, even if c‐Kit, c‐ErbB and the estrogen receptor were activated simultaneously. To induce self‐renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF‐2 transactivation domain are required for proliferation stimulation and differentiation arrest. c‐myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that Δc‐Myb, an activated c‐Myb protein, can functionally replace the GR

Expression profiling with cDNA arrays is an excellent tool for molecular analysis of complex processes such as terminal erythroid differentiation. The shortcomings of the currently available erythroid in vitro differentiation models, however, severely impaired the usefulness of this approach to study erythropoiesis. Here, we describe a novel, murine erythroid cell system closely corresponding to in vivo erythroid progenitors. Mortal, long‐term proliferating erythroid progenitors of fetal liver or immortal strains of p53‐deficient erythroblasts were established in culture. Both cell types proliferated in serum‐free medium and were strictly dependent on physiologically relevant cytokines and hormones, stably retaining a diploid set of chromosomes. If exposed to physiological differentiation factors (erythropoietin plus insulin), cells synchronously recapitulated the normal in vivo differentiation program to mature terminally into enucleated erythrocytes and expressed stage‐specific erythroid transcription factors in the expected temporal order. Using cDNA arrays, we found a large number of genes differentially expressed at time points during differentiation. Already 6 h after differentiation induction, 17% of the expressed genes showed significant alterations in mRNA abundance, increasing to 53% (12% up‐regulated, 41% down‐regulated genes) by 48 h. Cluster analysis of mRNA expression kinetics during differentiation identified six distinct expression patterns. All genes on the array with a known function in erythropoiesis showed the expected variations in expression. The genes identified also allowed first insights into the sequence of events within the regulatory network responsible for erythroid maturation. In mortal wild‐type as well as immortal p53‐/‐ erythroblasts, changes in mRNA abundance of several well‐regulated gene products was verified at the protein level. Taken together, this novel hematopoietic cell system faithfully executes essential steps of normal erythropoiesis and allows us to dissect and characterize molecular mechanisms involved in erythropoiesis.

During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X(L). This allows "default" terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals.