Stat5 Signaling Specifies Basal versus Stress Erythropoietic Responses through Distinct Binary and Graded Dynamic Modalities

Stat5 signaling in erythroblasts can assume either a binary, low-intensity form, essential for basal erythropoiesis, or a graded, high-intensity response, restricted to early erythroblasts and to erythropoietic stress. Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a “knocked-in” EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and graded modalities combine to generate high-fidelity Stat5 signaling over the entire basal and stress Epo range. They suggest that dynamic behavior may encode information during STAT signal transduction. Author Summary: Hormone signaling through the erythropoietin (Epo) pathway is required both for the continuous replacement of red blood cells (RBCs) that are lost through aging (a process known as "basal erythropoiesis") and to boost tissue oxygen when bleeding, in anemia or at high altitude ("stress erythropoiesis"). A key challenge lies in understanding how extracellular Epo concentration is translated into different intracellular signals that promote transcription of proteins that are specific to basal versus stress erythropoiesis. Binding of Epo to its receptor EpoR on the surface of an erythroblast (the precursors of RBCs) triggers the addition of phosphates to a target protein Stat5; the phosphorylated Stat5 becomes activated and induces transcription. We show that the dynamic properties of the Stat5 activation signal convey additional information that specifies either basal or stress responses. During basal conditions, the Stat5 signal is low and binary in nature—an on/off switch-like response. Stress, on the other hand, triggers a distinct Stat5 response consisting of a highintensity signal that increases in a graded fashion with rising Epo concentration. We found that a mouse bearing a truncated EpoR is restricted to the low-intensity binary Stat5 signal and correspondingly fails to initiate stress erythropoiesis. Ultimately, it is the Stat5 protein level in erythroblasts that determines their ability to generate the high-intensity graded Stat5 signal in response to high Epo. These findings have therapeutic potential: targeting Stat5's high-intensity graded signal may inhibit its aberrant function in blood cell cancers without affecting its important binary response in normal cells.