STAT3 Is Differentially Activated Downstream of the Erythropoietin Receptor in Primitive Compared to Definitive Erythroblasts
Authors: Malik Jeffrey , England Samantha J , Kingsley Paul D , Palis James 
Published: October 2019

Abstract
Abstract 2383
Erythropoietin (EPO) acting through its receptor (EPOR), is a critical regulator of definitive erythropoiesis. The EPOR signaling network has been extensively studied in cell lines overexpressing various forms of EPOR. Together with genetic models, these studies have defined an EPOR-JAK2-STAT5 pathway that is critical for the formation of red blood cells. However, the hematopoietic phenotype of mice with a truncated EPOR incapable of activating STAT5 (EPOR-HM) suggests that STAT5 activation is not the only pathway mediating the erythropoietic effects of EPO-EPOR signaling. Our recent studies indicate that EPO-EPOR signaling also regulates primitive erythroblast maturation in the mammalian embryo. To better understand EPOR signaling in the context of primary erythroid cells, we utilized primitive erythroblasts from wild-type and EPOR-HM murine embryos. Robust phosphorylation of STAT5 was detected by western blot in freshly isolated primitive erythroblasts from wild-type embryos. As expected, there is no STAT5 phosphorylation evident in primitive erythroblasts from EPOR-HM embryos. Surprisingly, EPOR-HM embryos produce nearly normal numbers of primitive erythroblasts and we detected equivalent induction of STAT target genes following EPO stimulation in wild-type and EPOR-HM primitive erythroblasts. To address the discrepancy between these data and the accepted model of EPOR-STAT5 signaling, we searched for compensatory signaling via other STAT family members. We found that STAT3, but not STAT1, was phosphorylated in primitive erythroblasts from EPOR-HM embryos, suggesting that phospho-STAT3 compensates for the loss of phospho-STAT5 in the EPOR-HM mice. We also detected a lower level of STAT3 phosphorylation in wild-type primitive erythroblasts that was increased upon in vitro EPO stimulation. These results indicate that EPOR-STAT3 signaling normally occurs in primitive erythroblasts. Furthermore, our preliminary data show that STAT3 transcript levels are higher in primitive erythroblasts compared to definitive erythroblasts from the fetal liver or bone marrow, suggesting that STAT3 activation during erythropoiesis may be lineage-dependent. To test this hypothesis, we utilized a small molecule inhibitor of STAT3 to examine the function of STAT3 signaling both in primitive and in definitive erythroblasts during in vitro maturation. The terminal maturation of definitive erythroblasts is unaffected by STAT3 inhibition. However, inhibition of STAT3 causes a 40% reduction in primitive erythroblast proliferation in a novel 2-step ex vivo culture system. We conclude that, in contrast to definitive erythroblasts, STAT3 is activated downstream of EPOR in primitive erythroid cells. EPOR-STAT3 signaling has been reported in non-erythroid cells as well as in several cancer cell types. Therefore the study of EPOR signaling in primary embryonic erythroblasts may provide novel insights regarding the function of EPO in these cells.

Disclosures:
No relevant conflicts of interest to declare.