Epo Receptor Circuits to Irs2, Foxo3a, Trb3 and Bim1 Expression, and Erythroblast Survival.

Epo cytoprotection of developing erythroblasts mechanistically is thought to involve 1] PI3K and Akt pathways (eg. mTOR, Foxo3A inhibition) as engaged via a distal EpoR PY479 site, and 2] anti-apoptotic effects as exerted by EpoR proximal box1 and PY343 signals (eg. Bcl-xl transcription). To globally assess Epo-regulation of survival- associated genes, a maximally Epo-responsive cohort of bone-marrow derived Kit^posCD71^high erythroblasts was prepared, purified and used in Affymetrix 430–2.0 array based profiling of Epo responsive genes. Quadruplicate array analyses and RT-QPCR revealed selective modulation of PI3K pathway associated factors Irs2, p85-alpha, Trb3, and Foxo3a as well as the Bcl2-related factor Bim1. Irs2, p85-alpha and Trb3 were induced three- to five-fold, while Foxo3a was rapidly down-modulated. As studied using knocked-in minimal EpoR alleles, induction of Irs2 and Trb3 (a pseudo-kinase Akt inhibitor) depended sharply upon an EpoR PY343 Stat5 binding site. Epo down-modulation of Foxo3a (as well as Trb2 and Bim1), in contrast, occurred via EpoR PY-independent routes. Unexpectedly, the EpoR allele, EpoR-H-Y343 also protected erythroblasts from LY294002 and wortmanin induced apoptosis, while an EpoR-HM-F343 allele was hyper-sensitive. For EpoRH, this proved to correlate specifically with Y343 dependent phosphorylation of GAB2. PY343 couples specifically to Stat5, and phospho-Stat5 therefore is proposed to recruit GAB2 and enable its PY452 phosphorylation. GAB2 then provides a shunt to PI3K activation. Findings provide new insight into Epo-regulation of a select set of PI3K-associated genes, and core EpoR cytoprotective mechanisms.