Pharmacological Control Of Human Hemoglobin Gene Expression

人血红蛋白基因表达的药理学控制

• 批准号: 8553398
• 负责人: Alan Schechter
• 金额: $ 11.53万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553398
• 关键词: Accounting; Adult; Affect; Age; Antibodies; Biological Models; Birth; Blood; Blood Circulation; Blood Vessels; Bone Marrow; Bone Marrow Cells; Butyrates; CD34 gene; CD34+ precursor; Cells; Child; Clinical; Complex; Control Groups; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cysteine; Cytostatics; Data; Development; Developmental Biology; Disease; Disease remission; Endothelial Cells; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Factor Analysis; Fetal Hemoglobin; Free Radicals; Functional disorder; Funding; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Globin; Growth; Guanylate Cyclase; Heme; Hemoglobin; Hemoglobin concentration result; Hereditary Disease; High Pressure Liquid Chromatography; Human; Impairment; Ions; K562 Cells; Laboratories; Manuscripts; Measurement; Measures; Messenger RNA; Methods; Metric; Molecular Genetics; Myeloproliferative disease; Newborn Infant; Nitric Oxide; Nitric Oxide Donors; Nitrites; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Population; Process; Production; Proteasome Inhibition; Proteins; Proto-Oncogene Proteins c-akt; Publishing; RNA Interference; Reporting; Research; Reticulocytes; Reticulocytosis; Role; Severities; Sickle Cell; Sickle Cell Anemia; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Symptoms; Syndrome; System; Thalassemia; Therapeutic; Time; United States National Institutes of Health; Work; cGMP production; erythroid differentiation; gamma Globin; human NOS3 protein; hydroxyurea; improved; infancy; inhibitor/antagonist; interest; iron nitrosyl; macrophage; novel strategies; paracrine; polymerization; precursor cell; progenitor; programs; research clinical testing; transcription factor

The ontogeny of the human globin genes is an important focus of study both with respect to the fundamental developmental biology and molecular genetics of the control of expression of this complex gene system, but also because modifying this developmental control would be of therapeutic value in the treatment of the prevalent genetic diseases of hemoglobin, such as sickle cell anemia and thalassemia. We are studying this problem from several aspects. First, we have improved culture methods for human erythroid precursors, by antibody selection, so as to obtain purified populations of these cells to establish at which developmental stage drugs change their phenotype. Second, we have developed real-time, quantitative PCR methods to measure globin gene expression in single cells and have used these methods to analyze the mechanisms of the effects of chemically useful drugs, such as hydroxyurea and butyrate, on globin gene expression. Third, we have shown that nitric oxide donors, such as cysteine-NO, can induce fetal hemoglobin expression in K562 cells as well as the purified erythroid (CD34) precursor cells described above. Induction of fetal hemoglobin was demonstrated both by quantitative PCR of gamma-globin mRNA and by HPLC of fetal hemoglobin protein. Further, we have shown that both NO-donors and hydroxyurea acts by stimulating guanylyl cyclase and increasing cyclic GMP levels; conversely, inhibitors of guanylyl cyclase block the action of both agents. Agents to increase fetal hemoglobin by this mechanism are being developed in several laboratories and being readied for clinical testing. In more recent studies we have shown that hydroxyurea induces NO production in macrophages and endothelial cells-in part through inhibition of proteasome degradation of the eNOS protein-and that this effect may allow a mechanism for a paracrine efect of NO on erythroid progenitors from these signalling molecules produced by stromal and other bone marrow cells. In current studies of globin production-at the mRNA and protein levels-we are studying the mechanisms of such signalling in hemoglobin ontogeny and its therapeutic implications. Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cAMP levels, but cGMP levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in erythroid progenitor cells (EPC). The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 fmol cGMP/min/ng of purified sGC, similar to induction by 1 ?M of DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPC. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF. Overall, these results raise the possiblity that agents which affect this cyclic nucleotide pathway, or other signaling pathways, can be used to increase fetal hemoglobin levels in patients with sickle cell anemia and thalassemia. Such therapeutic advances have been the focus of this research program for almost three decades. In other recent studies we have been analyzing the change in fetal hemoglobin levels in normal newborns with age and find-using both quantitative protein and mRNA measurements-that there appear to be two distinct silencing mechanisms for fetal hemoglobin-one primarily cellular and one primarily with respect to transcription mechanisms. We are now studying these processes in sickle cell children and will examine the effects of drugs on these silencing mechanisms. An important recent result is the appreciation that high levels of reticulocytosis in infancy in children with sickle cell anemia is correlated with a subsequent more severe clinical course. We are now examining whether this metric may be used to judge needs for specific therapies. Further analyses of erythroid cells in culture are underway with respect to gene expression patterns in these cells and several manuscripts on this work have recently been submitted or published. One such study shows that many transcription factors change during ontogeny of human CD34+ erythroid cells in culture, especially related to the JAK-STAT and AKT pathways. We are also now analyzing the role of several specific RNAi species which appear to correlate with levels of HbF and may explain some aspects of the developmental control of this important gene. In addition to changes in fetal hemoglobin we find that changes in reticulocytes and other blood parameters may contribute to the onset of symptoms in sickle cell children during the first years after birth.

人类球蛋白基因的个体发育是研究这种复杂基因系统表达的基本发育生物学和分子遗传学的重要重点，而且还因为修改这种发育控制在治疗流行的遗传性遗传疾病的治疗是具有治疗价值的，例如诸如小心细胞和thalsamia的流行遗传疾病。 我们正在从几个方面研究这个问题。首先，通过抗体选择，我们对人类红细胞前体进行了改进的培养方法，以便获得这些细胞的纯化群体，以确定发育阶段药物改变其表型的哪些细胞。其次，我们开发了实时的定量PCR方法来测量单个细胞中的球蛋白基因表达，并使用这些方法分析了化学有用药物（例如羟基脲和丁酸酯对球蛋白基因表达）的作用的机制。第三，我们已经表明，一氧化氮供体（例如半胱氨酸-NO）可以诱导K562细胞以及上述纯化的红细胞（CD34）前体细胞中的胎儿血红蛋白表达。通过γ-球蛋白mRNA的定量PCR和胎儿血红蛋白蛋白的HPLC诱导胎儿血红蛋白的诱导。此外，我们已经表明，没有donor和羟基脲通过刺激鸟叶叶烯酸酯和增加环状GMP水平来起作用。相反，鸟类环化酶的抑制剂阻止了两种药物的作用。通过这种机制增加了通过这种机制增加胎儿血红蛋白的药物，正在在几个实验室中开发，并准备进行临床测试。在最近的研究中，我们表明羟基脲通过抑制eNOS蛋白的蛋白酶体降解而诱导巨噬细胞和内皮细胞的一部分，并且这种作用可以允许从这些信号分子产生的骨膜和其他骨骼和其他骨骼元素产生的红细胞祖细胞的旁氨基脱氨基的机制。 在当前对球蛋白生产的研究中，mRNA和蛋白质水平 - 我们正在研究血红蛋白个体发育中这种信号的机制及其治疗意义。 羟基脲是一种广泛用于治疗脊髓增生性疾病的药物，也已通过培养胎儿血红蛋白（HBF）批准治疗镰状细胞疾病。我们现在证明，在红细胞分化期间，内皮NO合酶mRNA和蛋白质水平稳步下降，无衍生物和cAMP水平的产生也稳定，但是CGMP水平稳定。羟基脲提高了细胞内CGMP水平和红细胞祖细胞（EPC）的cAMP水平。 No捐助者Deanonoate诱导了CGMP更高的水平，但营地水平降低。羟基脲（1 mM）诱导的纯化SGC的大约45 fmol CGMP/min/ng的产生，类似于1？m的deanonoate诱导。我们发现羟基脲和脯氨酸酸盐产生了SGC的亚硝基衍生物。因此，我们确认羟基脲可以直接与SGC的脱氧 - 血红素相互作用，这可能是通过自由基硝酸途径并激活CGMP的产生。这些数据增加了人们对羟基脲作为EPC中NO/CGMP途径的诱导剂的作用的不断扩展。这些机制也可能参与羟基脲的细胞抑制作用以及HBF的诱导。总体而言，这些结果提高了影响这种环状核苷酸途径或其他信号通路的药物的可能性，可用于增加镰状细胞贫血和丘疹性疾病患者的胎儿血红蛋白水平。 近三十年来，这种治疗性进步一直是该研究计划的重点。 在最近的其他研究中，我们已经分析了随着年龄的年龄的正常新生儿的胎儿血红蛋白水平的变化，并使用定量蛋白和mRNA测量结果 - 似乎有两种胎儿的不同沉默机制 血红蛋白一个主要是细胞，主要是基于转录机制。 我们现在正在研究镰状细胞儿童中的这些过程，并将检查药物对这些沉默机制的影响。最近的一个重要结果是欣赏，镰状细胞贫血儿童婴儿期的高水平网状细胞增多症与随后更严重的临床病程相关。我们现在正在研究是否可以使用该指标来判断特定疗法的需求。 关于这些细胞中基因表达模式，对培养中红细胞细胞的进一步分析正在进行中，最近提交或发表了有关这项工作的一些手稿。一项这样的研究表明，在培养中人类CD34+红细胞的个体发育过程中，许多转录因子在培养中发生了变化，尤其是与JAK-STAT和AKT途径有关的。现在，我们还分析了几种与HBF水平相关的几种特定RNAi物种的作用，并可能解释了该重要基因的发育控制的某些方面。 除了胎儿血红蛋白的变化外，我们发现网状细胞和其他血液参数的变化可能会导致镰状细胞儿童出生后的第一年的症状发作。