PI3K/Akt‐dependent Epo‐induced signalling and target genes in human early erythroid progenitor cells

Abstract

erythroid progenitors stimulated with Epo in the presence or absence of LY294002 were subjected to gene expression profiling. Several novel target genes of Epo were identified, and the majority were regulated in a PI3K-dependent manner, including KIT (CD117) and CDH1 (E-cadherin). FACS analysis of Epo-stimulated erythroid progenitors showed that the increased mRNA expression of KIT and CDH1 was accompanied by an induction of the corresponding proteins CD117 and E-cadherin.Keywords: Erythropoietin, phosphatidylinositol 3-kinase, KIT, erythropoiesis, microarray. molecules. The lipid products of PI3K activate a plethora of targets, including the protein kinase B/Akt. The PI3K-Akt pathway has been shown to play a central role in regulation of apoptosis and proliferation in several systems, including normal erythroid progenitors (Haseyama et al, 1999;Uddin et al, 2000). In the context of Epo signalling it has been shown that Akt kinase, which is activated by PI(3,4,5)P3 or PI(3,4)P2, phosphorylates both GATA-1 and Foxo3a, transcription factors of crucial importance in erythropoiesis (Uddin et al, 2000;Bouscary et al, 2003;Kadri et al, 2005).In the present study we have investigated how Epo, by activating PI3K, exerts its effects on cell cycle progression and differentiation in human CD34+ progenitor cells. By use of genome wide expression profiling of Epo-stimulated CD34 + CD71 + CD45RA) GPA ) erythroid progenitors, we identified novel Epo target genes and confirmed their differential expression by fluorescent-activated cell sorting (FACS) analysis, as well as hypothesise novel roles for signalling pathways that have no previously established function in erythropoiesis. Materials and methods DNA expression vectorsExpression vectors for HA-tagged wild type (WT-Akt) or myristylated Akt1 (Myr-Akt) created in pCMV6, were provided as a gift (Ahmed et al, 1997). pcDNA3 without insert was used as control vector (Control). Reagents and antibodiesRecombinant human (rh) Epo was obtained from Boehringer Mannheim (GmbH, Germany) and rh Scf was from R&D Systems (Abingdon, UK). Cytokines were used at predetermined optimal concentrations: 5 U/ml for Epo and 50 ng/ml for Scf or as specified. LY294002 was from Calbiochem (Darmstadt, Germany). The following antibodies (mAbs, antihuman) were used in flow-cytometry analysis: anti-CD34 phycoerythrin (PE), anti-CD117 PE-cyanin 5 (CY5) and anti-CD45RA PE were from Becton Dickinson (San Jose, CA, USA), anti-glycophorin A-PE (GPA PE), and anti-CD71 fluorescein isothiocyanate (FITC) were from DakoCytomation AS (Glostrup, Denmark). Anti E-cadherin was from Calbiochem (San Diego, CA, USA; mouse IgG1, #205601) and anti mouse IgG1 PE (Southern Biotech, Birmingham, AL, USA) was used as secondary layer. The following antibodies were used in immunoblot analysis: anti-cyclin D 3 , anti-cyclin A (sc-596), anti-cyclin E (sc-247), anti p21 (sc-397), anti-p27 (sc-528), anti-pRB (Ser780) and from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti-Akt (#9272) and anti phospho Akt1 (Ser473, #9271) were ...

A mouse model of adult-onset anaemia due to erythropoietin deficiency

“…Expression profiles of the 3.3-Epo3 0 transgene appeared to be affected by The rHuEPO has been used widely as a powerful drug for anaemic patients [20][21][22] , but its downstream target genes are largely unknown. In this regard, previous studies mainly used primary culture systems for global gene expression analyses in which the erythroid precursors were stimulated with Epo 34,35 . Our in vivo microarray analysis employing ISAM revealed the Epo-responsive genes rapidly induced in the bone marrow as a result of Epodeficient conditions.…”

“…rHuEpo administration increased the expression of genes related to membrane cytoskeletons, transporters, glycolytic enzymes and transcription factors, which have key roles in erythroid differentiation. The upregulated group contained Cish, Gdf3, Pim1, Tfrc, Egr1 and Podxl genes, which were known as Epo-responsive erythroid genes 34,35 (circles in Fig. 4a).…”

Lysophosphatidic Acid Induces Erythropoiesis through Activating Lysophosphatidic Acid Receptor 3

“…These results suggest the role of LPARs and b-catenin in human erythropoiesis and translocation of b-catenin is important for stem cell differentiation. However, quercetin also blocks the PI3K/AKT pathway which modulates the early-stage of EPO-dependent erythroid development [33,34]. In addition, activation of LPA 3 has been suggested to regulate proliferation and migration in several cell types via PI3K/AKT pathway [35,36].…”

Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop

“…Proliferation of Epo-independent proerythroblastic cells (called HS2 cells), derived from transgenic mice overexpressing Spi-1/ PU.1, requires active PI3K/AKT and mitogen-activated protein kinase pathways (Barnache et al, 2001). Consistently, Epo initiates a transcriptional program that leads to significant change of expression levels in over 580 genes, the majority of which are apparently regulated in a PI3K-dependent manner (Sivertsen et al, 2006).…”