The Regulation of mRNA Translation Controls the Balance between Expansion and Differentiation of Erythroid Progenitors.

Erythroid progenitors can be expanded in vitro in the presence of erythropoietin (Epo), stem cell factor (SCF) and dexamethasone, while they differentiate to enucleated erythrocytes in presence of Epo only. Our study aims to identify (i) signaling pathways that control expansion of erythroid progenitors and (ii) genes regulated by these signaling pathways. Since SCF strongly activates phosphotidylinositol 3 kinase (PI3K) and inhibition of PI3K with LY294002 induces terminal differentiation of erythroid progenitors under Epo and SCF stimulation, SCF seems to enhance renewal divisions of erythroid progenitors via a PI3K-dependent mechanism. An important PI3K-dependent mechanism is the regulation of cap-binding factor eIF4E (eukaryotic Initiation Factor 4E), a rate limiting step in translation initiation. eIF4E is sequestered by 4E-BP (4E-Binding Protein), but released when 4E-BP is phosphorylated by mTOR (mammalian target of rapamycin), a target of the PI3K/PKB pathway. We show that moderate overexpression of eIF4E (up to 2-fold) markedly delays Epo-induced differentiation and allows for an increased number of renewal divisions instead. Moreover, eIF4E overexpressing cells are unresponsive to the PI3K inhibitor LY294002 under progenitor expansion conditions. These data suggest that mRNAs with a structured untranslated region (UTR), that are only efficiently translated at optimal eIF4E levels, contribute to maintain proliferation of hematopoietic progenitors. To identify the genes whose expression is regulated by signaling-induced polysome recruitment, we analysed gene-expression profiles on micro-arrays (Affymetrix, MGU74), in which we compared total and polysome-bound mRNA from factor deprived and Epo-, SCF- or Epo plus SCF restimulated erythroid progenitors. Profiling was done with 4 biological replicates and candidate genes were selected using ANOVA. In subsequent cluster analysis we combined these data with (polysomal) expression profiles of differentiating erythroid cells and identified a cluster containing genes, upregulated in response to Epo/SCF specifically at the level of mRNA polysome recruitment and downregulated during erythroid differentiation. A selection of gene transcripts was validated by RT-PCR and Northern blot analysis using sub-polysomal and polysomal mRNA fractions isolated from cells stimulated with Epo and SCF in presence and absence of the PI3K inhibitor LY294002. Polysome recruitment of 10 transcripts tested so far, appeared to be PI3K dependent. In cells overexpressing eIF4E, polysome-association of these mRNAs was increased under conditions of suboptimal PI3K/PKB activation. In conclusion, we have demonstrated that regulation of translation initiation by eIF4E is an important pathway stimulated by SCF to delay differentiation and to maintain expansion of erythroid progenitors. We identified gene-transcripts of which polysome recruitment is regulated by SCF-induced PI3K/PKB activity. Currently we are evaluating the role of selected genes involved in signal transduction mechanisms (acp1, igbp1) and DNA binding (rbms1, YB1) in the control of expansion and differentiation of erythroid progenitors.