ROLES FOR AN EPO-RECEPTOR PY343-STAT5 SIGNALING AXIS IN STRESS ERYTHROPOIESIS

Open Access
- Author:
- Menon, Madhu Parameswar
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 18, 2006
- Committee Members:
- Richard John Frisque, Committee Chair/Co-Chair
Don M Wojchowski, Committee Chair/Co-Chair
Pamela Correll, Committee Member
Robert Paulson, Committee Member
Robert Bruce Mitchell, Committee Member - Keywords:
- stress erythropoiesis
Epo
EpoR
erythropoietin receptor
erythropoietin
EpoR-H
EpoR-HM - Abstract:
- Erythropoietin (Epo) is a cytokine essential to the process of erythropoiesis and is currently in use as a therapeutic drug in human beings for treatment of anemia. Signals for erythroblast development are conducted via activation of JAK-2 and subsequently the cytoplasmic tyrosine sites in the erythropoietin receptor (EpoR). However, steady-state erythropoiesis seems to be controlled effectively by EpoR alleles that are either completely lacking, or truncated in terms of distal cytoplasmic phosphotyrosine sites. Present studies ascribe specific stress erythropoietic roles to the phosphotyrosine PY343 linked STAT-5 pathway in the Erythropoietin receptor (EpoR). Knock-in mice expressing a phosphotyrosine-null Epo receptor allele (EpoR-HM) exhibit defects in recovery from anemia due to hemolysis (Phenylhydrazine) or bone marrow suppression (5-Fluorouracil). In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently rescue recipient mice from irradiation induced bone marrow ablation. In addition, Epo induced reticulocyte formation was attenuated in EpoR-HM mice. In each context, stress erythropoiesis was restored to wild-type levels upon the selective restoration of EpoR PY343 STAT-5 binding site (EpoR-H allele). In a newly developed ex vivo erythroid expansion system (SP34-EX), EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. However, this defect was not observed in EpoR-H derived erythroid progenitor cells (EPCs). In order to characterize the downstream pathways employed by EpoR-HM and EpoR-H forms, signaling studies in response to Epo were conducted in bone-marrow derived EPCs. As an initial step, uniformity of JAK2 signaling was confirmed amongst the three alleles. As expected, STAT-5 was found to be activated via EpoR-H and wt-EpoR but not via EpoR-HM. For both EpoR-HM and EpoR-H, AKT and p70S6-kinase activation was decreased significantly. For Map kinases, JNK activation was minimal in the truncated alleles. Interestingly, ERK was found to be hyperactivated uniquely via EpoR-HM allele. Also, in vitro studies with bone marrow derived EPCs demonstrate defects in EpoR-HM erythroblast differentiation (based on enucleation analysis, forward-angle light scatter profiles, and hemoglobinization) which were corrected upon MEK1,2 inhibition. These EpoR-HM–specific differentiation defects were not observed in EpoR-H EPCs. In addition, Epo-induction of the STAT-5 target genes Pim-1 and oncostatin-M was found to be deficient in EpoR-HM derived erythroblasts. However a previously reported STAT-5 target gene Bcl-x, was not found to be modulated by Epo at the transcript level via wt-EpoR, EpoR-HM or EpoR-H alleles. Renal Epo transcripts were found to be slightly elevated in EpoR-HM kidney during steady-state erythropoiesis but were several fold higher than wt-EpoR or EpoR-H during stress erythropoiesis. In maturing KitnegCD71pos erythroblasts, oncostatin-M exerted anti-apoptotic effects, which likewise depended upon EpoR-PY343- mediated events. Stress erythropoiesis therefore requires EpoR-PY343-STAT-5 mediated pathways, certain of which converge and synergize with that of SCF and oncostatin-M. In addition, predominant signals emanating from EpoR-HM allele seems to involve ERK but is independent of STAT-5, STAT-1, p70S6-kinase, and JNK.