Regulation/function of the neuropeptide neuromedin U during hematopoiesis

神经肽神经调节素 U 在造血过程中的调节/功能

• 批准号: 8067810
• 负责人: SUSAN SHETZLINE
• 金额: $ 20万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8067810
• 关键词: Anemia; Animal Model; BFU-E; Blood Cell Count; Blood Cells; CD34 gene; CFU-E; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Coculture Techniques; Complex; Data; Dominant-Negative Mutation; Embryo; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Erythropoietin; Figs - dietary; Frequencies; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Transcription; Growth; Hematopoiesis; Hematopoietic; Hematopoietic System; Hemoglobin; Human; In Vitro; K562 Cells; MAPK3 gene; MYB gene; Messenger RNA; MicroRNAs; Modeling; Molecular Profiling; Mus; Neuromedin U; Neuropeptides; Pregnancy; Process; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-myb; Regulation; Relative (related person); Reporting; Response Elements; Small Interfering RNA; Stem Cell Factor; Testing; Text; Time; Transactivation; Transcript; Transcriptional Regulation; Translations; Transplantation; Untranslated Regions; Xenograft procedure; autocrine; base; bone marrow hyperplasia; in vivo; inhibitor/antagonist; member; myb Genes; novel; novel therapeutics; particle; progenitor; promoter; public health relevance; receptor; reconstitution; small hairpin RNA; transcription factor; Anemia; Animal Model; BFU-E; Blood Cell Count; Blood Cells; CD34 gene; CFU-E; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Coculture Techniques; Complex; Data; Dominant-Negative Mutation; Embryo; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Erythropoietin; Figs - dietary; Frequencies; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Transcription; Growth; Hematopoiesis; Hematopoietic; Hematopoietic System; Hemoglobin; Human; In Vitro; K562 Cells; MAPK3 gene; MYB gene; Messenger RNA; MicroRNAs; Modeling; Molecular Profiling; Mus; Neuromedin U; Neuropeptides; Pregnancy; Process; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-myb; Regulation; Relative (related person); Reporting; Response Elements; Small Interfering RNA; Stem Cell Factor; Testing; Text; Time; Transactivation; Transcript; Transcriptional Regulation; Translations; Transplantation; Untranslated Regions; Xenograft procedure; autocrine; base; bone marrow hyperplasia; in vivo; inhibitor/antagonist; member; myb Genes; novel; novel therapeutics; particle; progenitor; promoter; public health relevance; receptor; reconstitution; small hairpin RNA; transcription factor

DESCRIPTION (provided by applicant): Erythropoiesis is a process by which hematopoietic cells become red blood cells (RBC). Mice that do not express the proto-oncogene c-myb, a transcription factor expressed predominantly in hematopoietic cells and functions to regulate their proliferation, survival, and differentiation, die at embryonic day 15 of severe anemia, strongly suggesting that c-Myb through the transactivation of its target genes is essential during erythropoiesis. Using a microarray approach to identify Myb-gene targets, we observed the greatest change in the expression of the neuropeptide neuromedin U (NmU) in hematopoietic cells which also express NmU's cognate receptor type-1 (NMUR1). Transient inhibition of NmU gene expression by mature siRNA in primary human CD34+ cells impaired the formation of erythroid progenitors even when erythropoietin (EPO) was present in the culture. Co-culturing primary human CD34+ in which NmU was transiently inhibited with EPO and exogenous NmU restored the frequency of erythroid progenitor generation to that observed with control siRNA treated cells. It is important to note that NmU alone could not induce eyrthroid progenitor formation in primary human CD34+ cells. But, without NmU expression in primary human CD34+ cells, EPO and stem cell factor (SCF), two indispensable factors that support erythropoiesis, could not effectively induce erythroid progenitor formation. Combined, these data strongly indicate that during erythropoiesis NmU is critical for erythroid progenitor formation from primary human CD34+ cells. Based on our novel findings, we hypothesize that sustained inhibition of NmU in primary CD34+ cells will impair erythropoiesis in vivo. To test this hypothesis, we will study NmU's in vivo function and gene expression regulation with the following specific aims: Aim #1 - Determine the effect of sustained NmU inhibition in primary human CD34+ cells on their ability to provide full human hematopoietic reconstitution in NOD-SCID-gcnull (NSG) mice. Once we have achieved sustained inhibition of NmU in primary human CD34+ cells, we will transplant them into the NSG mice and evaluate the impact of NmU inhibition on the generation of erythroid progenitor cells. Aim #2 - Investigate the transcriptional regulation of NmU in hematopoietic and erythroid progenitor cells. We will identify the components of the c-Myb transcription factor complex that induce NmU expression and determine the ability of Elk-1 to activate NmU transcription. Aim #3 - Determine the regulation of NmU protein synthesis by microRNA (miRNA) in hematopoietic and erythroid progenitor cells. We will identify miRNA molecules that interact with NmU's 3'-UTR and determine the ability of c-Myb to regulate these miRNA molecules through a regulatory loop. The results from these proposed studies may provide a new therapeutic avenue for anemia. PUBLIC HEALTH RELEVANCE: Anemia is a condition marked by a deficiency in red blood cell number or of hemoglobin expressed in red blood cells. The neuropeptide neuromedin U has emerged as a novel factor that functions during erythropoiesis which is the process by which immature blood cells differentiate into red blood cells. Understanding neuromedin U's function during erythropoiesis may offer a new therapeutic avenue for anemia.

描述（由申请人提供）：红细胞生成是造血细胞成为红细胞（RBC）的过程。不表达原始癌基因C-MYB的小鼠，这是一种转录因子，主要在造血细胞中表达，并在严重贫血的胚胎第15天死亡，以调节其增殖，存活和分化，这强烈表明C-MYB通过其靶基因的交易是在欧洲抗体中必不可少的。使用微阵列方法来识别Myb-Gene靶标，我们观察到造血细胞中神经肽神经蛋白U（NMU）表达的最大变化，这些细胞也表达了NMU的同源受体1型（NMUR1）。即使在培养物中存在促红细胞生成素（EPO）时，在原代人CD34+细胞中对成熟siRNA对NMU基因表达的瞬时抑制也会抑制红细胞祖细胞的形成。共同培养的原代人CD34+，其中NMU用EPO和外源性NMU瞬时抑制，将红细胞祖细胞产生的频率恢复为对照siRNA处理的细胞观察到的频率。重要的是要注意，单独的NMU不能在原代人CD34+细胞中诱导眼科祖细胞的形成。但是，在原代人CD34+细胞中没有NMU表达，EPO和干细胞因子（SCF）（支持红细胞生成的两个必不可少的因素）无法有效诱导红细胞祖细胞的形成。这些数据合并强烈表明，在红细胞生成期间，NMU对于原代人CD34+细胞的红细胞祖细胞形成至关重要。根据我们的新发现，我们假设在原代CD34+细胞中持续抑制NMU会损害体内红细胞生成。为了检验这一假设，我们将研究NMU的体内功能和基因表达调节，其特定目的：目标＃1-确定原代人CD34+细胞中持续的NMU抑制作用对它们提供全部人类造血性造血性重新构造的能力的影响。一旦我们实现了原代人CD34+细胞中NMU的持续抑制作用，我们将将它们移植到NSG小鼠中，并评估NMU抑制对红细胞祖细胞产生的影响。 AIM＃2-研究造血和红细胞祖细胞中NMU的转录调节。我们将确定诱导NMU表达的C-MYB转录因子复合物的成分，并确定ELK-1激活NMU转录的能力。 AIM＃3-确定造血和红系祖细胞中microRNA（miRNA）对NMU蛋白合成的调节。我们将确定与NMU的3'-UTR相互作用的miRNA分子，并确定C-MYB通过调节环调节这些miRNA分子的能力。这些拟议的研究的结果可能为贫血提供了新的治疗途径。 公共卫生相关性：贫血是以红细胞数量或红细胞表达的血红蛋白缺乏为标志的疾病。神经肽神经蛋白U已成为一种新的因素，在红细胞生成过程中起作用，这是未成熟血细胞分化为红细胞的过程。了解神经蛋白在红细胞生成期间的功能可能会为贫血提供新的治疗途径。