Stem cell factor synergistically enhances thrombopoietin-induced STAT5 signaling in megakaryocyte progenitors through JAK2 and Src kinase.

Stem cell factor (SCF) has a potent synergistic effect during megakaryopoiesis when administered in combination with the major megakaryocytic cytokine, thrombopoietin (TPO). In this study we analyzed the underlying mechanisms with regard to STAT5 activity. TPO stimulation of MO7e cells resulted in STAT5 transactivation, which could be enhanced 1.6-fold by costimulation with SCF, whereas SCF alone did not induce STAT5 transcriptional activity. This costimulatory effect of SCF was reflected in an increase in TPO-induced STAT5 DNA binding and increased and prolonged STAT5 tyrosine phosphorylation in both MO7e cells and primary human megakaryocyte progenitors. In contrast, serine phosphorylation of STAT5 was constitutive and associated with an inhibitory effect on STAT5 transactivation. Signal transduction pathways that might synergize in TPO-mediated STAT5 transactivation were analyzed using specific pharmacological inhibitors and indicated an essential role for Janus-activated kinase 2 (JAK2) and a partial role for Src-family kinases. Costimulation with SCF was found to increase and prolong tyrosine phosphorylation of JAK2 and the TPO receptor c-mpl. In addition, the Src kinase inhibitor SU6656 partially downregulated the additional effect of SCF costimulation on STAT5 tyrosine phosphorylation. SCF-induced enhancement of JAK2 phosphorylation was not affected by inhibition of Src kinase, suggesting that both JAK2 and Src kinase mediate STAT5 tyrosine phosphorylation. Synergistic activation of JAK2 and Src kinase may thus contribute to the enhanced STAT5 signaling in the presence of TPO and SCF.