Mechanisms of Epo-mediated hematopoietic progenitor cell proliferation and survival: Roles forc-Kit, Jak2, Pim1 kinase and Y343 of the Epo receptor

During red blood cell development, the glycoprotein hormone erythropoietin (Epo) acts via its lineage- and stage-specific cell surface receptor (EpoR) to promote erythroid progenitor cell survival and growth. Presently, three sets of investigations were performed to identify minimal EpoR domains and associated effectors that are important for proliferative signaling. First, a cell line (FDCW2) was identified which was useful for EpoR structure-function studies, including its co-signaling with the transmembrane receptor for stem cell factor (SCF), c-Kit. Second, the activities of two minimal EpoR forms in mediating Epo-dependent mitogenesis and survival were tested: ER-Bx1 (lacking all cytoplasmic tyrosines but retaining box 1 site for Jak2 kinase binding) and ER-J2KK (extracellular and transmembrane domains of the EpoR fused directly to the kinase and kinase-like domains of Jak2). Third, the ability of Pim1 (a cytoplasmic serine/threonine kinase whose expression is induced via minimal EpoR forms) to promote progenitor cell survival upon its ectopic expression in FDCW2ER372 cells was tested. Also, a role for the STAT5 binding site Y343 of the EpoR in Epo-induced pim1 and bcl-x transcription (as well as Epo-induced mitogenesis and inhibition of apoptosis) was assessed.; In murine myeloid FDCW2 cells, mitogenic synergy with c-Kit was observed for ectopically expressed wild-type EpoR (wtER), an EGFR/EpoR chimera, and ER-Bx1 receptor forms. Thus, the EpoR cytoplasmic box1 subdomain, in part, mediates this co-signaling with c-Kit. In studies of potential effectors of this effect, Jak2 tyrosine phosphorylation was shown to be induced by Epo, but not by SCF.; ER-Bx1 was shown to support mitogenesis (65% of maximal vs. wtER), survival and Pim1 expression, while ER-J2KK was efficient only in mitogenesis (45% of maximal vs. ER-Bx1).; Upon its stable expression in FDCW2ER372 cells, Pim1 uniformly inhibited cell death due to cytokine withdrawal, gamma-irradiation, adriamycin, and dexamethasone. In addition, Y343 of the Epo receptor was required for efficient pim1 and bcl-x gene expression, as well as for mediating Epo-induced inhibition of PCD and mitogenesis. Overall, these investigations serve to define roles for c-Kit, Jak2, Pim1 kinase and Y343 of the EpoR in mediating hematopoietic progenitor cell proliferation and survival.