Dissection and Manipulation of the Cellular Response to Iron Restriction

描述（由申请人提供）：细胞对铁可用性降低的反应，铁限制，是全球超过10亿人贫血的基础。这种反应损害了促红细胞生成素（Epo）驱动的骨髓红细胞祖细胞的发育，并导致与慢性疾病、衰老以及缺铁性贫血（IDA）相关的贫血。总的来说，与限铁有关的贫血会对健康和经济产生重大影响。因此，了解铁限制反应的机制和设计针对这种反应的治疗是至关重要的。红系铁限制反应涉及参与柠檬酸盐到异柠檬酸盐代谢的乌头酸酶的谱系选择性失活。在细胞培养和贫血动物模型中，用异柠檬酸盐补充缺铁细胞显著地挽救了红细胞的发育。反之，药理抑制乌头酸酶足以阻断红细胞祖细胞的分化，诱导正常小鼠贫血，纠正真性红细胞增多症小鼠红细胞增多。乌头酸酶活性通过代谢和Epo信号之间复杂且鲜为人知的相互作用调控红细胞生成，影响PKC、ERK和AKT。我们最近发表的一篇文章强调了这一途径的翻译重要性，该文章显示异柠檬酸盐治疗可以改善大鼠关节炎模型中慢性炎症的贫血。投资。， 123:3614- 23,2013)。这些研究导致NIH STTR资助异柠檬酸盐作为慢性疾病和炎症（ACDI）人类贫血的新疗法的临床前开发。异柠檬酸盐的治疗效果源于其阻断铁限制反应的能力，否则会使红系祖细胞对炎症细胞因子的抑制敏感。这种致敏是由特异性铁限制和炎症信号通路之间的协同相互作用引起的转录因子PU.1的超诱导。通常在红细胞发育早期下调，PU.1是一个主要的调节因子，其水平决定造血祖细胞的髓细胞与红细胞的命运。目的1将描述与铁限制性贫血临床相关的体内模型相关的红细胞PU.1失调。关于红系铁限制的近端信号异常，令人兴奋的新数据暗示支架蛋白Scribble是乌头酶活性的关键靶点。Scribble通常是细胞极性的决定因素，也是多种磷酸酶和激酶的关键组装平台。我们发现乌头酸酶抑制会扰乱发育中的红母细胞的极性形态。此外，Scribble控制的信号通路受到乌头碱酶抑制或铁限制的失调。最令人信服的是，乌头碱酶抑制诱导Scribble及其伴侣SGT1的显著下调。铁限制也强烈下调Scribble，异柠檬酸治疗阻断这种下调。因此，Aim 2将通过乌头酸酶抑制和铁限制来确定红系Scribble下调的机制和后果。

DESCRIPTION (provided by applicant): The cellular response to decreased iron availability, iron restriction, serves as the basis for anemia in over a billion people worldwide. This response impairs erythropoietin (Epo) driven development of bone marrow erythroid progenitors and contributes to anemias associated with chronic diseases, aging, as well as iron deficiency anemia (IDA). In aggregate, anemias associated with iron restriction have major health and economic impacts. Therefore, understanding the mechanisms underlying the iron restriction response and designing therapies to target this response are critically important. The erythroid iron restriction response involves lineage-selective inactivation of the aconitase enzymes involved in metabolism of citrate to isocitrate. Supplementation of iron-deprived cells with isocitrate strikingly rescues erythroid development in cell culture and animal models of anemia. Conversely, pharmacologic inhibition of aconitase suffices to block differentiation of erythroid progenitors, inducing anemia in normal mice and correcting erythrocytosis in mice with polycythemia vera. The regulation of erythropoiesis by aconitase activity arises through complex and poorly-understood interplay between metabolism and Epo signaling, affecting PKC, ERK, and AKT. The translational importance of this pathway was highlighted in our recent publication showing isocitrate treatment to ameliorate anemia of chronic inflammation in a rat arthritis model (J. Clin. Invest., 123:3614-23, 2013). These studies have led to NIH STTR funding for pre-clinical development of isocitrate as a novel therapy for human anemias of chronic disease and inflammation (ACDI). The therapeutic efficacy of isocitrate derives from its capacity to block the iron restriction response, which otherwise sensitizes erythroid progenitors to inhibition by inflammatory cytokines. This sensitization arises from superinduction of the transcription factor PU.1 by cooperative interplay between specific iron restriction and inflammatory signaling pathways. Normally downregulated early in erythroid development, PU.1 is a master regulator whose levels dictate myeloid versus erythroid cell fate in hematopoietic progenitors. Aim 1 will characterize erythroid PU.1 dysregulation associated with clinically-relevant in vivo models of iron restricted anemia. Regarding proximal signaling abnormalities in erythroid iron restriction, exciting new data implicate the scaffold protein Scribble as a key target of aconitase activity. Scribble normally functions as a determinant of cell polarity, as well as a critical assembly platform for multiple phosphatases and kinases. We have found that aconitase inhibition perturbs the polar morphology of developing erythroblasts. Furthermore, signaling pathways controlled by Scribble are dysregulated by either aconitase inhibition or iron restriction. Most compellingly, aconitase inhibition induces dramatic downregulation of Scribble and of its chaperone SGT1. Iron restriction also strongly downregulates Scribble, and isocitrate treatment blocks this downregulation. Accordingly, Aim 2 will determine the mechanism and consequences of erythroid Scribble downregulation by aconitase inhibition and iron restriction.