Pharmacological Control Of Human Hemoglobin Gene Expression

人类珠蛋白基因的个体发育是控制这一复杂基因系统表达的基础发育生物学和分子遗传学的重要研究重点，也是因为修改这一发育调控机制将在治疗常见的血红蛋白遗传性疾病，如镰状细胞性贫血和地中海贫血方面具有治疗价值。我们正在从几个方面研究这个问题。首先，我们改进了人红系前体细胞的培养方法，通过抗体选择，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，我们发展了实时定量聚合酶链式反应方法来测量单细胞中珠蛋白基因的表达，并用这些方法分析了化学有用的药物，如羟基脲和丁酸盐，对珠蛋白基因表达的影响的机制。第三，我们已经证明了一氧化氮供体，如半胱氨酸-NO，可以诱导K562细胞和上述纯化的红系(CD34)前体细胞表达胎儿血红蛋白。通过定量聚合酶链式反应和高效液相色谱检测胎儿血红蛋白，证实了胎儿血红蛋白的诱导。此外，我们还表明，NO供体和羟基脲都通过刺激鸟苷酸环化酶和增加环状GMP水平来发挥作用；相反，鸟苷酸环化酶的抑制剂可以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，羟基尿素诱导巨噬细胞和内皮细胞产生NO-部分是通过抑制蛋白酶体对eNOS蛋白的降解-这种效应可能允许一种机制，即通过基质细胞和其他骨髓细胞产生的这些信号分子对红系祖细胞产生旁分泌作用。在目前关于珠蛋白产生的研究中--在信使核糖核酸和蛋白质水平上--我们正在研究这种信号在血红蛋白个体发育中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)来治疗镰状细胞疾病。我们现在证明，在红系分化过程中，内皮细胞一氧化氮合酶mRNA和蛋白水平稳步下降，一氧化氮衍生物的产生和cAMP水平也下降，但cGMP水平稳定。羟基脲增加红系祖细胞(EPC)内cGMP水平和cAMP水平。NO供体DEANONOate诱导cGMP水平升高，但cAMP水平降低。羟基脲(1 MM)可诱导约45fmolcGMP/min/ng的sGC产生，与1 mM二硝酸酯的诱导作用相似。我们发现羟基脲和普罗诺酯产生sGC的亚硝基铁衍生物。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，可能是通过自由基氮氧化物途径，并激活cGMP的产生。这些数据增加了对羟基脲在EPC中作为NO/cGMP途径的诱导者的作用的更广泛的认识。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，这些结果提高了影响这一环核苷酸途径或其他信号途径的药物可用于提高镰状细胞性贫血和地中海贫血患者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，我们一直在分析正常新生儿的胎儿血红蛋白水平随年龄的变化，并发现--使用定量蛋白质和信使核糖核酸测量--胎儿似乎有两种不同的沉默机制 血红蛋白-一种主要是细胞的，一种主要是与转录机制有关的。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。最近的一个重要结果是认识到，镰状细胞性贫血儿童在婴儿期高水平的网织红细胞增多与随后更严重的临床病程相关。我们现在正在研究这一指标是否可以用来判断特定治疗的需要。 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，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，在培养的人CD34+红系细胞的个体发育过程中，许多转录因子发生了变化，特别是与JAK-STAT和AKT通路有关。我们现在还在分析几个特定的RNAi物种的作用，这些物种似乎与HBF水平相关，并可能解释这个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，我们还发现网织红细胞和其他血液参数的变化可能导致镰状细胞儿童在出生后的第一年出现症状。

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.