Core Epo Receptor Signaling Circuits in Adult Bone Marrow-Derived Primary Pro-Erythroblasts.

Knock-in experiments have revealed that Epo receptor-dependent steady-state erythropoiesis is supported by a Jak2-coupled cytoplasmic Epo receptor (EpoR) box-1 domain. However, signals that are transduced by this cytoplasmic phosphotyrosine-null EpoR-HM allele (and by a related EpoR-H allele with a restored PY343-STAT5 binding site) are not well understood. To address this basic question, conditions have been optimized for the serum-free expansion of adult bone marrow- derived primary erythroid progenitor cells, and core signaling circuits for EpoR-HM, EpoR-H and wt-EpoR receptor forms have been defined. As an important baseline consideration, each receptor form proved to activate Jak2 at an essentially equivalent time-course and intensity (but EpoR-HM did not detectably activate Stat5, or appreciably stimulate CIS transcription). In addition, EpoR-HM failed to stimulate AKT, and this outcome discounts possible EpoR-HM plus Jak2 coupling to P13K (eg. via IRS-1/2 and/or GAB-1/2). AKT was stimulated at moderate, yet sustained levels via EpoR-H (and a PY343/Stat5 axis). EpoR-HM, however, did couple to p60Src and ERK1, 2. While p60-Src stimulation was somewhat diminished, ERK1/ 2 activation via EpoR-HM was sharp and sustained (as compared directly to EpoR-H and the wt-EpoR). EpoR coupling to apoptotic factors also was assessed. In EpoR-HM erythroblasts, Bcl-xl expression was clearly (and reproducibly) deficient, but occurred at wild-type levels in Stat5-coupled EpoR-H erythroblasts. Bax expression was less affected, but was reproducibly increased 1.5 to 2-fold in both EpoR-HM and EpoR-H erythroblasts. Finally, expression of an under-studied pro-apoptotic DAPK-1 orthologue, DAPK2 (death associated protein kinase-2) was discovered to be markedly elevated in EpoR-HM erythroblasts, but to be repressed to normal levels in EpoR-H cells. With regards to bioactivity, EpoR-HM pro-erythroblasts faltered in their ex vivo development specifically at a transition from a Ter119^negCD71^low to Ter119^posCD71^high stage, and were visibly deficient in their levels of hemoglobinization. Overall, these first-time detailed studies of Epo signaling in primary adult pro-erythroblasts from minimal EpoR allele knock-in mice reveal: 1/ that EpoR plus Jak2 dependent erythropoiesis may depend primarily upon enhanced ERK1/2 stimulation (and not upon collateral Stat5 or AKT circuits); 2/ that EpoR-HM retains coupling to p60-Src; 3/ that an EpoR-H/PY343/Stat5 axis (but not an EpoR/Jak2 only axis) can link to AKT; 4/ that EpoR/PY343/Stat5 signals regulate not only Bcl-xl, but also pro-apoptotic DAPK2 expression at a key stage of Epo-dependent pro-erythroblast development; and 5) that EpoR-PY coupled signals contribute in several important ways to red cell development.