Arvind Dev

EPO receptor circuits for primary erythroblast survival

EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPO-regulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wild-type EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPO-dependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit(pos)CD71(high)Ter119(neg) erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus FoxO3a, and EPOR/PY343/STAT5-dependent stimulation of Pim1, Pim3, Irs2 plus Serpina-3G, and Trb3 as new antiapoptotic effectors.

Erythropoietin receptor response circuits

Purpose of reviewIn 1985–1989, erythropoietin (EPO), its receptor (EPOR), and janus kinase 2 were cloned; established to be essential for definitive erythropoiesis; and initially intensely studied. Recently, new impetus, tools, and model systems have emerged to re-examine EPO/EPOR actions, and are addressed in this review. Impetus includes indications that EPO affects significantly more than standard erythroblast survival pathways, the development of novel erythropoiesis-stimulating agents, increasing evidence for EPO/EPOR cytoprotection of ischemically injured tissues, and potential EPO-mediated worsening of tumorigenesis. Recent findingsNew findings are reviewed in four functional contexts: (pro)erythroblast survival mechanisms, new candidate EPO/EPOR effects on erythroid cell development and new EPOR responses, EPOR downmodulation and trafficking, and novel erythropoiesis-stimulating agents. SummaryAs Current Opinion, this monograph seeks to summarize, and provoke, new EPO/EPOR action concepts. Specific problems addressed include: beyond (and before) BCL-XL, what key survival factors are deployed in early-stage proerythroblasts? Are distinct EPO/EPOR signals transduced in stage-selective fashions? Is erythroblast proliferation also modulated by EPO/EPOR signals? What functions are subserved by new noncanonical EPO/EPOR response factors (e.g. podocalyxin like-1, tribbles 3, reactive oxygen species, and nuclear factor kappa B)? What key regulators mediate EPOR inhibition and trafficking? And for emerging erythropoiesis-stimulating agents, to what extent do activities parallel EPOs (or differ in advantageous, potentially complicating ways, or both)?

DYRK3 Dual-specificity Kinase Attenuates Erythropoiesis during Anemia

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

Defining an EPOR- Regulated Transcriptome for Primary Progenitors, including Tnfr-sf13c as a Novel Mediator of EPO- Dependent Erythroblast Formation

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.

Spry1 as a novel regulator of erythropoiesis, EPO/EPOR target, and suppressor of JAK2

Sprouty proteins are established modifiers of receptor tyrosine kinase (RTK) signaling and play important roles in vasculogenesis, bone morphogenesis, and renal uteric branching. Little is understood, however, concerning possible roles for these molecular adaptors during hematopoiesis. Within erythroid lineage, Spry1 was observed to be selectively and highly expressed at CFU-e to erythroblast stages. In analyses of possible functional roles, an Mx1-Cre approach was applied to conditionally delete Spry1. At steady state, Spry1 deletion selectively perturbed erythroid development and led to reticulocytosis plus heightened splenic erythropoiesis. When challenged by hemolysis, Spry1-null mice exhibited worsened anemia and delayed recovery. During short-term marrow transplantation, Spry1-null donor marrow also failed to efficiently rescue the erythron. In each anemia model, however, hyperexpansion of erythroid progenitors was observed. Spry function depends on phosphorylation of a conserved N-terminal PY motif. Through an LC-MS/MS approach, Spry1 was discovered to be regulated via the erythropoietin receptor (EPOR), with marked EPO-induced Spry1-PY53 phosphorylation observed. When EPOR signaling pathways were analyzed within Spry1-deficient erythroid progenitors, hyperactivation of not only Erk1,2 but also Jak2 was observed. Studies implicate Spry1 as a novel regulator of erythropoiesis during anemia, transducer of EPOR signals, and candidate suppressor of Jak2 activity.