Dissection and Manipulation of the Cellular Response to Iron Restriction

描述(由申请人提供)：细胞对铁供应减少的反应，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)驱动的骨髓红系祖细胞的发育，并导致与慢性病、衰老和缺铁性贫血(IDA)相关的贫血。总体而言，与铁限制相关的贫血会对健康和经济产生重大影响。因此，了解铁限制反应的潜在机制并设计针对这种反应的治疗方法至关重要。红细胞铁限制反应包括参与柠檬酸代谢为异柠檬酸的乌头酸酶的谱系选择性失活。用异柠檬酸补充缺铁细胞能显著挽救细胞培养和贫血动物模型中的红系发育。相反，药物抑制乌头酸酶足以阻断红系祖细胞的分化，导致正常小鼠贫血，纠正真性红细胞增多症小鼠的红细胞增多症。乌头酸酶活性通过代谢和EPO信号之间复杂而鲜为人知的相互作用来调节红细胞生成，影响PKC、ERK和AKT。这一途径的翻译重要性在我们最近的出版物中得到了强调，表明异柠檬酸治疗可以改善大鼠关节炎模型中慢性炎症的贫血(J.Clin。投资，123：3614-23,2013)。这些研究已经为NIH STTR提供了资金，用于临床前开发异柠檬酸作为一种治疗人类慢性病和炎症贫血(ACDI)的新疗法。异柠檬酸的治疗效果来自于其阻断铁限制反应的能力，否则，铁限制反应会使红系祖细胞对炎性细胞因子的抑制敏感。这种敏化是通过特定的铁限制和炎症信号通路之间的协同相互作用而过度诱导转录因子PU.1的。正常情况下，PU1在红系发育早期下调，是一种主控调节因子，其水平决定了造血祖细胞中髓系与红系细胞的命运。目的1将表征与临床相关的体内铁限制性贫血模型相关的红系PU.1失调。关于红系铁限制的近端信号异常，令人兴奋的新数据表明支架蛋白Scribble是乌头酸酶活性的关键靶点。Scrible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，乌头酸酶的抑制作用扰乱了发育中的红细胞的极地形态。此外，Scribble调控的信号通路受到乌头酸酶抑制或铁限制的失调。最引人注目的是，抑制乌头酸酶导致Scribble及其伴侣SGT1的显著下调。铁限制也强烈下调Scribble，异柠檬酸治疗阻止了这种下调。因此，目标2将通过抑制乌头酸酶和限制铁来确定红系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.