Epo is expressed in response to hypoxia and upon binding to its transmembrane receptor on erythroid progenitor cells acts as an essential inhibitor of programmed cell death (PCD). Upstream effectors that dock at Epo-receptor complexes are relatively well defined (Jak2, Syp, HCP, STAT5, PLC-gamma, P13K, SHIP, Shc, Grb2, Crk-l, Cbl, Vav) and for many, links to mitogenic response pathways established. Yet, beyond the observations that EPO sustains Bcl-xl expression and protects against p53-induced PCD in a cell cycle phase-independent fashion, little is understood about effectors of PCD. Studies aim to advance an understanding of mechanisms of this key response.
Aim #1 : In Epo, SCF-, IL-3 dependent FDCW2 cell lines expressing EpoR deletion and point- mutants, the extend to which Epo-dependent pathways to P13K/Akt kinase activation and Bad phosphorylation (versus hypothesized alternative pathways) inhibit PCD will be studied. hEGFR-EpoR chimeras with defined mutations in cytoplasmic domains have been expressed in erythroid progenitor cells in transgenic mice. Using this novel in vivo model (and thiamphenicol to induced splenic CFUe) essential signals for PCD inhibition also will be investigated. In purified CFUe Bcl2-related, - associated factors whose expression is modulated by EPO also will be defined.
Aim #2 : Expression of Pim1 kinase is induced via minimal EpoR forms that efficiently inhibit PCD, and ectopically expressed Pim1 inhibits PCD. Based on these observations, mechanisms of Pim1-inhibited PCD will be investigated. Evidence for targets upstream of APAF1 will be sought; the ability of Pim1 to protect against PCD as induced by radiation, dexamethasone and/or Myc-induced PCD will be tested; and possible effects of Pim1 on the expression of Bcl2-associated, related factors will be studied. Also, cis-elements and transfactors that regulate Pim1 gene expression in response to Epo versus SCF will be investigated in folate-deficiency and Diamond-Blackfan anemias, erythroid failure is characterized by PCD and EpoR mutations are associated with erythrocytosis and polycythemias. Insight into regulation of PCD gained from studies of Epo signaling should extend to related clinically important cytokine systems.
