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: 7967220
• 负责人: Alan Schechter
• 金额: $ 9.92万
• 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/7967220
• 关键词: Adult; Affect; Age; Antibodies; Biological Models; Blood Circulation; Blood Vessels; Bone Marrow; Bone Marrow Cells; Butyrates; 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; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Factor Analysis; Fetal Hemoglobin; Free Radicals; Functional disorder; Funding; Gene Expression; 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; Molecular Genetics; Myeloproliferative disease; Newborn Infant; Nitric Oxide; Nitric Oxide Donors; Nitrites; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Population; Process; Production; Proteasome Inhibition; Proteins; Publishing; Reporting; Research; 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; inhibitor/antagonist; interest; iron nitrosyl; macrophage; novel strategies; paracrine; polymerization; precursor cell; progenitor; programs; research clinical testing

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 efect may allow a mechanism for a paracrine efect of NO on erythroid progenitors from these signalling molecules produceed by stromal and other bone marrow cells. In current studies of globin production-at the mRNA and protein levels-we are studying the mechansims of such singaling 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 mechansims 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. 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.

人类珠蛋白基因的个体发育是研究的一个重要焦点，不仅涉及该复杂基因系统表达控制的基本发育生物学和分子遗传学，而且还因为修改这种发育控制对于治疗常见的血红蛋白遗传疾病（如镰状细胞性贫血和地中海贫血）具有治疗价值。 我们正在从几个方面来研究这个问题。首先，我们通过抗体选择改进了人红系前体细胞的培养方法，以获得这些细胞的纯化群体，以确定药物在哪个发育阶段改变其表型。其次，我们开发了实时定量PCR方法来测量单细胞中的珠蛋白基因表达，并使用这些方法来分析化学有用药物（例如羟基脲和丁酸盐）对珠蛋白基因表达的影响机制。第三，我们已经证明一氧化氮供体，例如半胱氨酸-NO，可以诱导 K562 细胞以及上述纯化的红系 (CD34) 前体细胞中胎儿血红蛋白的表达。通过γ-珠蛋白 mRNA 的定量 PCR 和胎儿血红蛋白蛋白的 HPLC 证明了胎儿血红蛋白的诱导。此外，我们还表明，NO 供体和羟基脲均通过刺激鸟苷酸环化酶和增加环 GMP 水平发挥作用。相反，鸟苷酸环化酶抑制剂会阻断这两种药物的作用。几个实验室正在开发通过这种机制增加胎儿血红蛋白的药物，并准备进行临床测试。在最近的研究中，我们已经表明，羟基脲在巨噬细胞和内皮细胞中诱导 NO 产生，部分是通过抑制 eNOS 蛋白的蛋白酶体降解，并且这种作用可能允许一种机制，通过基质细胞和其他骨髓细胞产生的这些信号分子，NO 对红系祖细胞产生旁分泌作用。 在目前对球蛋白产生（mRNA 和蛋白质水平）的研究中，我们正在研究血红蛋白个体发育中这种信号传导的机制及其治疗意义。 羟基脲是一种广泛用于治疗骨髓增生性疾病的药物，也已被批准用于通过提高胎儿血红蛋白 (HbF) 来治疗镰状细胞病。我们现在证明，在红细胞分化过程中，内皮NO合酶mRNA和蛋白质水平稳步下降，NO衍生物的产生和cAMP水平也稳定下降，但cGMP水平稳定。羟基脲增加了红系祖细胞 (EPC) 的细胞内 cGMP 水平和 cAMP 水平。 NO 供体 DEANONOate 诱导更高的 cGMP 水平，但降低了 cAMP 水平。羟基脲 (1 mM) 诱导产生约 45 fmol cGMP/min/ng 的纯化 sGC，与 1 µM DEANONOate 的诱导相似。我们发现羟基脲和 ProliNONOate 产生 sGC 的亚硝酰铁衍生物。因此，我们证实羟基脲可以直接与 sGC 的脱氧血红素相互作用（可能是通过自由基硝基氧途径），并激活 cGMP 的产生。这些数据进一步加深了对羟基脲作为 EPC 中 NO/cGMP 途径诱导剂的作用的认识。这些机制也可能与羟基脲的细胞抑制作用以及 HbF 的诱导有关。总体而言，这些结果提出了影响这种环核苷酸途径或其他信号传导途径的药物可用于增加镰状细胞性贫血和地中海贫血患者的胎儿血红蛋白水平的可能性。 近三十年来，此类治疗进展一直是该研究项目的重点。 在最近的其他研究中，我们分析了正常新生儿胎儿血红蛋白水平随年龄的变化，并使用定量蛋白质和 mRNA 测量结果发现，胎儿血红蛋白似乎存在两种不同的沉默机制。 血红蛋白——一种主要与细胞有关，另一种主要与转录机制有关。 我们现在正在研究镰状细胞儿童的这些过程，并将检查药物对这些沉默机制的影响。 对培养中的红系细胞的基因表达模式的进一步分析正在进行中，并且最近已提交或出版了有关这项工作的几篇手稿。