Signals for stress erythropoiesis are integrated via an erythropoietin receptor–phosphotyrosine-343–Stat5 axis
Madhu P. Menon, Vinit Karur, Olga Bogacheva, Oleg Bogachev, Bethany Cuetara, Don M. Wojchowski
Madhu P. Menon, Vinit Karur, Olga Bogacheva, Oleg Bogachev, Bethany Cuetara, Don M. Wojchowski
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Research Article Hematology

Signals for stress erythropoiesis are integrated via an erythropoietin receptor–phosphotyrosine-343–Stat5 axis

Abstract

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor–phosphotyrosine-343–Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit–CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343–mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

Authors

Madhu P. Menon, Vinit Karur, Olga Bogacheva, Oleg Bogachev, Bethany Cuetara, Don M. Wojchowski

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At a Kit+CD71high stage, EpoR-HM erythroblasts exhibit proliferation and...
At a Kit+CD71high stage, EpoR-HM erythroblasts exhibit proliferation and survival defects that are corrected by EpoR-H PY343 signals. (A) Erythroid progenitor cells from WT-EpoR mice were expanded in SP34-EX. At 72 hours of culture, Kit+CD71high erythroblasts were isolated by FACS and plated in methylcellulose. Colonies with CFUe morphologies uniformly formed as shown in the right panels (and as confirmed by benzidine staining, not shown). Magnification, ×250. (B) Isolated Kit+CD71high WT-EpoR, EpoR-HM, and EpoR-H erythroblasts were cultured in SP34-EX in the presence of Epo or SCF at the concentrations indicated. At 20 hours, cytokine-induced 3HdT incorporation rates were determined. For Epo, mean 3HdT incorporation rates ± SD (n = 3) are graphed and are normalized for SCF responsiveness. Results are representative of 2 independent experiments (n = 3 WT-EpoR, EpoR-HM, and EpoR-H mice per experiment). (C) For Kit+CD71high and Kit–CD71high WT-Epo, EpoR-HM, and EpoR-H erythroblasts, frequencies of annexin V–positive cells were assayed by flow cytometry. Values are means ± SD of triplicate analyses (*P < 0.01 vs. WT-EpoR and EpoR-H).