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: 7897207
• 负责人: SUSAN SHETZLINE
• 金额: $ 23.98万
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897207
• 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; 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变成红细胞（RBC）的过程。不表达原癌基因 c-myb（一种主要在造血细胞中表达并具有调节其增殖、存活和分化功能的转录因子）的小鼠会在胚胎第 15 天因严重贫血而死亡，这强烈表明 c-Myb 通过其靶基因的反式激活在红细胞生成过程中至关重要。使用微阵列方法来识别 Myb 基因靶标，我们观察到造血细胞中神经肽神经调节蛋白 U (NmU) 的表达发生了最大的变化，这些细胞也表达 NmU 的同源受体 1 型 (NMUR1)。即使培养物中存在促红细胞生成素 (EPO)，原代人 CD34+ 细胞中成熟 siRNA 短暂抑制 NmU 基因表达也会损害红系祖细胞的形成。共培养原代人CD34+，其中NmU被EPO和外源NmU瞬时抑制，将红系祖细胞生成频率恢复到用对照siRNA处理的细胞观察到的频率。值得注意的是，单独的 NmU 不能诱导原代人 CD34+ 细胞中红细胞祖细胞的形成。但是，如果原代人 CD34+ 细胞中没有 NmU 表达，EPO 和干细胞因子 (SCF) 这两个支持红细胞生成不可或缺的因子就不能有效诱导红系祖细胞形成。综合起来，这些数据强烈表明，在红细胞生成过程中，NmU 对于原代人 CD34+ 细胞的红系祖细胞形成至关重要。基于我们的新发现，我们假设在原代 CD34+ 细胞中持续抑制 NmU 将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控，具体目标如下： 目标 #1 - 确定原代人 CD34+ 细胞中持续 NmU 抑制对其在 NOD- 中提供完全人类造血重建的能力的影响SCID-gcnull (NSG) 小鼠。一旦我们在原代人 CD34+ 细胞中实现了 NmU 的持续抑制，我们将把它们移植到 NSG 小鼠中，并评估 NmU 抑制对红系祖细胞生成的影响。目标#2 - 研究 NmU 在造血和红系祖细胞中的转录调控。我们将鉴定诱导 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。目标 #3 - 确定造血和红系祖细胞中 microRNA (miRNA) 对 NmU 蛋白质合成的调节。我们将鉴定与 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。这些拟议研究的结果可能为贫血提供新的治疗途径。 公共卫生相关性：贫血是一种以红细胞数量或红细胞中表达的血红蛋白缺乏为特征的疾病。神经肽神经调节素 U 已成为一种在红细胞生成过程中发挥作用的新因子，红细胞生成是未成熟血细胞分化为红细胞的过程。了解神经调节素 U 在红细胞生成过程中的功能可能为贫血提供新的治疗途径。