Abstract

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 (i) genes regulated by these signaling pathways. Since (i) SCF strongly activates phosphotidylinositol 3 kinase (PI3K) and (ii) 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 process in cell proliferation is regulation of mRNA translation. PI3K controls the activity of mTOR (mammalian target of rapamycin), whose activation results in phosphorylation of eIF4E (eukaryote Initiation Factor 4E)-binding protein (4E-BP). Fully phosphorylated 4E-BP releases eIF4E, which can subsequently bind eIF4G, the scaffolding protein of the eIF4F cap-binding and scanning complex. In particular mRNAs with a structured UTR (untranslated region) require optimal availability of eIF4E to be translated. SCF, but not Epo can induce full phosphorylation of 4E-BP and efficient formation of the eIF4F cap-binding complex. Overexpression of eIF4E inhibited erythroid differentiation as if SCF were present, indicating that SCF-induced release of eIF4E from 4E-BP is an important mechanism regulating the balance between progenitor expansion and differentiation. A major step in mRNA translation controlled by eIF4F is polysome recruitment. To identify genes whose expression is regulated by signaling-induced polysome recruitment, we compared total and polysome-bound mRNA from factor deprived and Epo-, SCF- or Epo plus SCF restimulated progenitors on gene-expression micro-arrays (Affymetrix). The profiling was performed 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. Thus we identified a cluster containing genes, upregulated in part or completely at the level of mRNA polysome recruitment and downregulated during erythroid differentiation. Targets involved in signal transduction and gene expression were analyzed in more detail. Polysome recruitment of 15/17 targets tested so far appeared to be dependent on PI3K activation and eIF4E expression. Constitutive expression of these targets in erythroid progenitors revealed that one target in particular was able to inhibit erythroid differentiation comparable to overexpression of eIF4E. This target was IGBP1 (Immunoglobulin binding protein 1). IGBP1 binds to and modulates the activity of the catalytic subunit of PP2A, a major cellular serine/threonine phosphatase, which also dephosphorylates 4E-BP. Overexpression of IGBP1 does not inhibit 4E-BP dephosphorylation in absence of factor, but enhances phosphorylation of 4E-BP in presence of Epo. Nevertheless, constitutive expression of IGBP1 does not increase polysome association of structured mRNAs. The multiple functions of PP2A suggest that the potent inhibition of erythroid differentiation by IGBP1 may be due to deregulation of several cellular mechanisms.

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