Feline Leukemia Virus C Receptor Heme Export and Erythroid Differentiation

猫白血病病毒 C 受体血红素输出和红细胞分化

• 批准号: 7885549
• 负责人: Sioban Bridget KEEL
• 金额: $ 12.68万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885549
• 关键词: Antibodies; Apoptosis; Biological Assay; Breeding; Catabolism; Cell Surface Receptors; Cell physiology; Cells; Development; Disease; Down-Regulation; Equilibrium; Erythrocytes; Erythroid Cells; Erythropoiesis; Feline Leukemia Virus; Felis catus; Goals; Hematopoietic; Heme; Human; K-562; Knockout Mice; Luciferases; Measures; Medicine; Mentors; Methods; Mus; Physiology; Pronormoblasts; Pure Red-Cell Aplasia; Research Personnel; Safety; Scientist; Small Interfering RNA; Subgroup; System; Testing; Toxic effect; Training Programs; base; career; career development; design; erythroid differentiation; heme 1; heme receptor; human disease; in vivo; insight; programs; tool; transcription factor

DESCRIPTION (provided by applicant): This application describes a five-year mentored training program to develop an independent clinician- scientist whose career in academic medicine will focus on furthering our understanding of human red blood cell development. Cats naturally or experimentally infected with feline leukemia virus, subgroup C (FeLV-C) develop profound pure red cell aplasia (PRCA) or on rare occasion ineffective erythropoiesis. Our lab previously demonstrated that the cell surface receptor for feline leukemia virus, subgroup C (FLVCR) exports cytoplasmic heme, and is required for CFU-E/proerythroblast survival or differentiation. My goal in these studies is to determine if these observations connect, and more broadly, to use FLVCR as a tool to study early erythroid differentiation. I plan three specific aims to evaluate this: 1) to determine if intracellular free heme increases when FLVCR expression or function is downregulated in erythroid cells; 2) to determine if erythroid differentiation is blocked or apoptosis is induced when FLVCR function or expression is downregulated in primary human cells and in vivo in mice; 3) to determine if reducing intracellular free heme in cells where FLVCR is downregulated (restoring heme balance) will allow erythroid differentiation. These studies will test my hypothesis that FLVCR acts as a mandatory safety (overflow) valve that protects early erythroid cells from heme toxicity, will provide insight into the physiology responsible for PRCA in cats viremic with FeLV-C, and will further our understanding of normal and disordered erythropoiesis. I will accomplish these specific aims by the following methods: 1) design a dual luciferase assay based on the transcription factor Bach1 to directly measure intracellular free heme during the erythroid differentiation of K562 and primary human hematopoietic cells and determine the effects of downregulation of FLVCR function (inhibitory antibody) or expression (siRNA) using this assay system; 2) characterize erythroid differentiation in primary human hematopoietic cells when FLVCR is downregulated by antibody and breed a viable Flvcr(null/null) mouse and characterize its erythropoiesis; 3) attempt to restore intracellular free heme balance when FLVCR is downregulated (by either blocking heme synthesis or increasing its catabolism) and thereby permit erythroid differentiation of human and murine cells. These studies focus on basic red blood cell physiology and may lend insight into human diseases where red blood cell development is disturbed.

描述（由申请人提供）： 本申请描述了一个为期五年的指导培训计划，以发展一个独立的临床医生-科学家，其职业生涯在学术医学将侧重于进一步了解人类红细胞的发展。 自然或实验感染猫白血病病毒C亚组（FeLV-C）的猫会出现严重的纯红细胞再生障碍性贫血（PRCA）或罕见的无效红细胞生成。我们的实验室以前证明，猫白血病病毒的细胞表面受体，亚群C（FLVCR）出口细胞质血红素，并需要CFU-E/proerythroblast生存或分化。我在这些研究中的目标是确定这些观察结果是否相关，更广泛地说，使用FLVCR作为研究早期红细胞分化的工具。我计划用三个具体目标来评估这一点： 1)以确定当FLVCR表达或功能在红系细胞中下调时细胞内游离血红素是否增加; 2）确定当FLVCR功能或表达在原代人细胞中和在小鼠体内下调时红系分化是否被阻断或诱导凋亡; 3）确定减少FLVCR下调的细胞中的细胞内游离血红素（恢复血红素平衡）是否将允许红系分化。 这些研究将检验我的假设，即FLVCR作为一种强制性安全（溢流）阀，保护早期红系细胞免受血红素毒性的影响，将深入了解FeLV-C病毒血症猫PRCA的生理学，并将进一步了解正常和紊乱的红细胞生成。 本论文主要通过以下几个方面的研究来实现上述目的：1）设计一种基于转录因子Bach 1的双荧光素酶检测方法，直接检测K562细胞和原代造血细胞红系分化过程中细胞内游离血红素的变化，并检测FLVCR功能下调的影响（抑制性抗体）或表达（siRNA）; 2）表征当FLVCR被抗体下调时原代人造血细胞中的红系分化，并培育活的Flvcr（null/null）小鼠并表征其红细胞生成; 3）当FLVCR下调时（通过阻断血红素合成或增加其催化剂）尝试恢复细胞内游离血红素平衡，从而允许人和鼠细胞的红系分化。 这些研究侧重于基本的红细胞生理学，并可能有助于了解红细胞发育受到干扰的人类疾病。