Stem cell factor enhances erythropoietin-mediated transactivation of signal transducer and activator of transcription 5 (STAT5) via the PKA/CREB pathway

Objective. To define whether the observed synergistic effects of erythropoietin (EPO) and stem cell factor (SCF) on erythroid cells can, in part, be mediated by the signal transducer and activator of transcription 5 (STAT5).Methods. STAT5 activation was examined in erythroid cell lines by analyzing the effects of EPO and SCF on STAT5 tyrosine phosphorylation, serine phosphorylation, DNA binding, and STAT5-mediated gene transactivation.Results. EPO induced a 5.0-fold +/- 0.4-fold increase in STAT5 transactivation, which could be further enhanced by SCF. SCF pretreatment followed by EPO stimulation resulted in a 9.0-fold +/- 0.9-fold increase in STAT5 transactivation, while SCF alone did not increase STAT5 transactivation. This costimulatory effect of SCF was not mediated by increased STAT5 tyrosine or serine phosphorylation or increased STAT5 DNA binding. In addition, enhanced STAT5 transactivation was independent of the phosphatidyl inositol 3-kinase and MAPK(p42/p44) pathways. Instead, the protein kinase A (PKA) inhibitor protein PKI and the PKA inhibitor H89 prevented the costimulatory SCF effect. Furthermore, the PKA target CREB showed a strongly increased and prolonged serine-133 phosphorylation after costimulation with SCF + EPO. The involvement of CREB in STAT5 transactivation was demonstrated by overexpression of serine-133-mutated CREB, which completely blocked the SCF effect. In addition, the CREB-binding protein CBP/p300 was shown to be essential for EPO- and SCF-mediated STAT5 transactivation.Conclusion. SCF enhances the EPO-mediated STAT5 transactivation by triggering a PKA/CREB-dependent pathway. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.