Negative Autoregulation by Fas Stabilizes Adult Erythropoiesis and Accelerates Its Stress Response Miroslav Koulnis, Ying Liu, Kelly Hallstrom, Merav Socolovsky* Department of Pediatrics and Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Abstract Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, ‘EryA’, negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune- deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response. Citation: Koulnis M, Liu Y, Hallstrom K, Socolovsky M (2011) Negative Autoregulation by Fas Stabilizes Adult Erythropoiesis and Accelerates Its Stress Response. PLoS ONE 6(7): e21192. doi:10.1371/journal.pone.0021192 Editor: Jianming Qiu, University of Kansas Medical Center, United States of America Received March 28, 2011; May 22, 2011; Published July 8, 2011 Copyright: 2011 Koulnis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by NIH/NHLBI RO1 HL084168 (M.S.), and by the American Cancer Society grant RSG06-051-01 (M.S). Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: merav.socolovsky@umassmed.edu Introduction The production of red blood cells (Erythropoiesis) is continuous throughout life, maintaining an optimal number of circulating red cells and tissue oxygen tension (pO2). A decrease in tissue pO2, as may occur in anemia, bleeding, high altitude or respiratory disease, drives erythropoiesis up to 10 fold its basal rate. This response is regulated through a negative feedback loop in which decreasing tissue pO2 increases synthesis of the hormone Epo ([1,2,3], Fig 1A). Epo-mediated activation of its receptor, EpoR [4], on erythroid progenitors increases their number and consequently, erythropoi- etic rate and tissue pO2. During accelerated rates of erythropoiesis, Epo cooperates with additional factors, notably glucocorticoid hormones and stem cell factor [5,6,7,8]. Epo controls an early cellular compartment within the ‘erythroblastic island’, the developmental niche on the surface of a macrophage that supports erythroid maturation [9] (Fig 1A). Three to five cell divisions must elapse before cells in this early compartment mature into red blood cells. This inherent delay in the pO2/Epo-regulated feedback loop is likely to compromise the stability and rapid stress response of the erythropoietic system. Additional control mechanisms may therefore exist to compensate for this inherent delay. The earliest Epo-dependent progenitor is the ‘Colony-forming Unit-erythroid’ or CFU-e, giving rise to colonies of at least 8 red cells within 48–72 hours in vitro [10]. Its erythroblast progeny are classified by their morphology [11]. We previously developed a flow-cytometric approach to identify erythroblasts directly in freshly-harvested mouse hematopoietic tissue, using cell surface markers CD71, Ter119 and cell size, measured by flow-cytometric forward scatter (FSC). We classify increasingly mature erythroid precursors subsets as ‘ProE’ (CD71highTer119med), ‘EryA’ (CD71highTer119highFSChigh), ‘EryB’ (CD71highTer119highFSClow) and ‘EryC’ (CD71lowTer119highFSClow)[12] (Fig 1A–B). We found that, in addition to the well documented increase in CFU-e [13,14], the early erythroblast subsets ProE and EryA are responsive to EpoR signaling in vivo [12]. Epo promotes erythroblast survival in vitro [15], suggesting this mechanism may underlie its regulation of erythropoietic rate. Our recent experiments in vivo [12] confirm this hypothesis. During basal erythropoiesis, the majority of ProE and EryA undergo apoptosis, particularly in spleen, the murine organ of erythropoi- etic reserve [16]. During stress, high Epo decreases their apoptosis, increasing ProE and EryA number [12]. The reasons for this apparently wasteful mechanism of erythropoietic rate regulation have not been addressed experimentally. PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e21192