GSBS Dissertations and Theses

Approval Date

8-16-2011

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Program in Immunology and Virology

Subjects

Dissertations, UMMS; Erythropoiesis; Erythropoietin; Receptors, Erythropoietin; STAT5 Transcription Factor

Abstract

Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts.

We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal.

The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling.

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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