Distinct Functions of Erythropoietin and Stem Cell Factor Are Linked to Activation of p70S6/mTOR Kinase Signaling Pathway in Primary Erythroid Progenitors.

Erythropoietin (Epo) and stem cell factor (SCF) guide erythroid cell maturation by exerting their effects at various stages of differentiation. Distinct and overlapping functions of these two growth factors have been well characterized. However, signaling pathways responsible for the antiapoptotic function of Epo and the proliferative function of SCF has not been fully characterized. Especially activation of common upstream signaling elements PI3-kinase, Akt kinase and phosphorylation/inactivation of forkhead transcription factors by both Epo and SCF bring about distinct functional outcomes have not been understood. In the present study we examined the activation of p70S6/mTOR pathway by Epo and SCF in CD34-derived primary erythroid progenitors. Our results provide evidence for activation of p70S6 kinase and mTOR by SCF but not by Epo or IGF-1 (insulin-like growth factor-1). We also show that only SCF phosphorylates protein translational regulatory proteins, 4E-BP1 and S6 ribosomal protein suggesting its involvement in promoting protein translation. Furthermore, we demonstrate that inhibition of mTOR by rapamycin results in reduction in erythroid cell proliferation and colony-formation under steady state culture conditions demonstrating the involvement of downstream signaling elements in the PI3/Akt kinase pathway in cell proliferation apart from its antiapoptotic signal. The reduction of both BFU-E and CFU-GM colony formation indicated that rapamycin also affects early hematopoietic cells. Examination of a parallel pathway involving signaling element Mnk1 showed that both Mnk1 and its downstream target eIF4E are not phosphorylated in response to SCF or Epo. However, these protein were constitutively phosphorylated in primary erythroid progenitors. Interestingly, we also found that during the proliferative phase of erythroid differentiation mTOR is mostly detected in the cytoplasmic fraction of the cells whereas during terminal phase of differentiation mTOR is detected in the nuclear fraction. These results suggest that mTOR may have additional functions associated with chromatin remodeling in erythroid progenitors, which occur prior to enucleation. Taken together, our data provide a mechanism for how distinct functions of Epo and SCF are accomplished through selective use of a common signaling pathway explaining in part how functional diversity is acheived.