Stat5 and Stat3 Differentially Regulate Early and Late Stages of Primary Embryonic Erythroid Cell Maturation

Erythropoietin is the critical regulator of adult (definitive) red cell production, acting through its cognate receptor (EpoR) and downstream Janus kinase (Jak)2 and signal transducer and activator of transcription (Stat)5 signaling. Mutant JAK2 activation causes hyperproliferation of erythroid precursors in polycythemia vera, highlighting the importance of Jak2-STAT signaling in pathologic states in addition to normal development. EpoR-null mouse embryos die at midgestation of profound anemia and we have recently determined that EpoR signaling is also specifically required for the survival and terminal maturation of primitive erythroblasts. Unlike definitive erythropoiesis, Stat3 is significantly expressed in primitive erythroblasts but its function in erythropoiesis remains poorly understood. We find that Stat3, in addition to Stat5, is phosphorylated downstream of EpoR in primitive erythroblasts. Interestingly, phospho-Stat5 intensity decreases during primitive erythroblast maturation while phospho-Stat3 levels remain constant, resulting in an increase in relative phospho-Stat3 in late-stage primitive erythroblasts. Knockdown of Jak2 and Stat5 each results in accelerated maturation, decreased cell size, and increased apoptosis of primary primitive erythroblasts. In contrast, Stat3 knockdown results in delayed maturation and increased cell size of late-stage primary primitive erythroblasts, consistent with an increased role of phospho-Stat3 once phospho-Stat5 signal intensity has diminished. Stat3 knockdown, both in vitro and in vivo, also increased apoptosis of late-stage primitive erythroblasts. While Stat3 and Stat5 bind many apoptosis-related genes in common, Stat3 specifically binds several caspase genes and their regulators. We find that activated caspase3 levels increase in late-stage primitive erythroblasts, and that knockdown of Stat3 results in decreased levels of caspases and their regulators. In addition, caspase inhibition, like Stat3 knockdown, results in delayed maturation of late-stage primitive erythroblasts. Taken together, our data support a model where Stat3 and Stat5 differentially regulate erythroid maturation and Stat3 preferentially regulates terminal stages of primitive erythropoiesis through its activation and regulation of apoptotic machinery.