Abstract
Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.
Highlights
As committed erythroid progenitors transit through a CFU-e stage, proerythroblast formation becomes dependent upon key signals transduced by EPO’s cell surface receptor (EPOR)
To broaden insight into EPO action mechanisms, we presently report on global transcript response events that EPO regulates within primary bone marrow CFUe- like progenitors
Development beyond the CFUe stage fails due to disrupted expression of Epo, or the EpoR [27]
Summary
As committed erythroid progenitors transit through a CFU-e stage, proerythroblast formation becomes dependent upon key signals transduced by EPO’s cell surface receptor (EPOR). Interest in better understanding EPO effects (and EPOR action mechanisms) recently has intensified. This is based, in part, on the clinical emergence of new EPO orthologues and mimetics [1], and on EPO’s ability to cytoprotect select non-hematopoietic tissues from ischemic injury [2]; to regulate select immune responses [3]; and to modulate susceptibility to diabetes [4]. EPO binding conformationally alters EPOR complexes [8] This leads to Jak activation, and the phosphorylation of up to eight cytoplasmic EPOR PY sites [1]. PYdeficient EPOR forms that retain only a box-1,2 Jak binding domain can support erythropoiesis at steady-state, but are markedly defective during anemia [12,13]. Candidate necessary-and-sufficient roles for Erk’s have been discounted by the recent observation that erythropoiesis can be bolstered when Erk is disrupted [14]
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