Pharmacological Control Of Human Hemoglobin Gene Expression

人珠蛋白基因的个体发生是研究的重要焦点，这不仅是关于控制该复杂基因系统表达的基础发育生物学和分子遗传学，而且还因为修饰该发育控制将在治疗血红蛋白的流行遗传疾病（例如镰状细胞性贫血和地中海贫血）中具有治疗价值。 我们正在从几个方面研究这个问题。首先，我们通过抗体选择改进了人类红系前体细胞的培养方法，从而获得这些细胞的纯化群体，以确定在哪个发育阶段药物改变其表型。第二，我们已经开发了实时，定量PCR方法来测量珠蛋白基因在单细胞中的表达，并使用这些方法来分析化学有用的药物，如羟基脲和丁酸，珠蛋白基因表达的影响的机制。第三，我们已经表明，一氧化氮供体，如半胱氨酸-NO，可以诱导胎儿血红蛋白在K562细胞以及上述纯化的红系（CD34）前体细胞的表达。胎儿血红蛋白的诱导通过γ-珠蛋白mRNA的定量PCR和胎儿血红蛋白蛋白的HPLC来证明。此外，我们已经表明，NO-供体和羟基脲的行为，刺激鸟苷酸环化酶和增加环GMP水平;相反，鸟苷酸环化酶的抑制剂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，并准备进行临床试验。在最近的研究中，我们已经表明，在巨噬细胞和内皮细胞中，羟基脲诱导NO的产生，部分通过抑制eNOS蛋白的蛋白酶体降解，这种作用可能允许一个机制，从这些信号分子产生的基质和其他骨髓细胞的红系祖细胞的旁分泌效应的NO。 在目前的珠蛋白生产的研究-在mRNA和蛋白质水平-我们正在研究的机制，这种信号在血红蛋白个体发育及其治疗意义。 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和蛋白水平稳步下降，NO衍生物和cAMP水平的生产，但cGMP水平是稳定的。在红系祖细胞（EPC）中，羟基脲增加细胞内cGMP水平和cAMP水平。NO供体DEANONOate诱导更高的cGMP水平，但降低cAMP水平。羟基脲（1 mM）诱导生产约45 fmol cGMP/min/ng的纯化sGC，类似于1？Deanonoate的M。我们发现，羟基脲和ProliNONOate产生sGC的铁-亚硝酰基衍生物。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，大概是通过自由基氮氧化物途径，并激活cGMP的生产。这些数据增加了一个不断扩大的升值的作用，羟基脲作为一个诱导剂的NO/cGMP途径在EPC。这些机制也可能参与了羟基脲的细胞抑制作用以及HbF的诱导。总之，这些结果提高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，这种治疗进展一直是该研究计划的重点。 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，并发现-使用定量蛋白质和mRNA测量-胎儿血红蛋白的表达似乎有两种不同的沉默机制。 血红蛋白-一种主要是细胞的，一种主要是关于转录机制。 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。最近一个重要的结果是认识到镰状细胞性贫血儿童婴儿期高水平的网织红细胞增多症与随后更严重的临床病程相关。我们现在正在研究这个指标是否可以用来判断特定疗法的需求。 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，最近已经提交或发表了几篇关于这项工作的手稿。一项这样的研究表明，许多转录因子在培养的人CD34+红系细胞的个体发育过程中发生变化，特别是与JAK-STAT和AKT途径相关。我们现在还在分析几种特定RNAi的作用，这些RNAi似乎与HbF水平相关，并可能解释这一重要基因发育控制的某些方面。 除了胎儿血红蛋白的变化，我们发现网织红细胞和其他血液参数的变化可能有助于镰状细胞患儿出生后最初几年内症状的发作。

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.