Intracelllar Pathways That Silence the Fetal Globin Gene

描述（由申请人提供）： 大量的关注和努力已经指向新的胎儿血红蛋白（Hb F）诱导剂的开发。羟基脲（HU）用于治疗镰状细胞性贫血（SCA），但SCA患者对HU的反应程度显著不同。HU治疗β-地中海贫血的临床疗效尚不清楚。而不是研究顺式作用的调控元件，我们的研究一直专注于澄清细胞内途径，调节珠蛋白基因的表达。我们最近发现，包括可溶性鸟苷酸环化酶（sGC）和cGMP依赖性蛋白激酶（PKG）的细胞内途径在原代成红细胞中γ-珠蛋白基因表达中起着重要作用。我们关于sGC-PKG通路在Hb F表达中起重要作用的假设最近被一对意大利兄弟的发现所证实，他们在β-珠蛋白基因座中没有突变，但表达25 - 30%的Hb F。发现它们具有高水平的原卟啉IX，这是sGC的强激活剂。为了“用新的眼光”研究γ-珠蛋白基因沉默的分子机制，我们关注的现象是，γ-和β-珠蛋白基因的表达在围产期受到“相互作用的方式”的调节。这使我们推测，γ-珠蛋白基因在成年阶段被激活β-珠蛋白基因所需的机制沉默。我们将在这个应用程序中测试这个假设。我们的初步研究表明：1）cAMP依赖性蛋白激酶（PKA）活性对于成人红系细胞如MEL细胞和原代成红细胞中β-珠蛋白基因的诱导是必需的，2）γ-珠蛋白基因的表达通过激活K562细胞中cAMP依赖性途径而降低，和3）通过抑制PKA活性，HS 2和γ-珠蛋白基因启动子的活性显著增加。这些结果表明，cAMP依赖的途径可能发挥负面作用的γ-珠蛋白基因的表达。这项申请有三个具体目标。在第一个目标中，我们将表征激活β-珠蛋白基因表达所需的细胞内途径。第二个目标将集中在β-珠蛋白基因表达的细胞内途径沉默γ-珠蛋白基因的分子机制，在第三个目标中，我们将检查是否γ-珠蛋白基因的表达被诱导在β-珠蛋白疾病的初级成红细胞和具有低活性的β-珠蛋白基因表达的细胞内途径的成年小鼠。这一应用不仅可以阐明γ-珠蛋白基因沉默的机制，而且还可以为β-珠蛋白基因的发育调控机制提供线索。本研究为开发新型Hb F诱导剂提供了重要信息。

DESCRIPTION (provided by applicant): Considerable concerns and efforts have been directed to the development of new fetal hemoglobin (Hb F) inducers. Hydroxyurea (HU) is being used for treating sickle cell anemia (SCA), but the magnitude of response to HU significantly varies between SCA patients. The clinical efficacy of HU for beta-thalassemia remains unclear. Rather than studying cis-acting regulatory elements, our research has been focusing on clarifying intracellular pathways that regulate globin gene expression. We recently showed that an intracellular pathway comprising soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays an important role in gamma-globin gene expression in primary erythroblasts. Our hypothesis that the sGC-PKG pathway plays an important role in Hb F expression was recently substantiated by a discovery of Italian brothers who have no mutations in beta-globin locus but express 25 to 30 % Hb F. They were found to have high levels of protoporphyrin IX, which is a strong activator of sGC. To study "with fresh eyes" the molecular mechanisms for gamma-globin gene silencing, we were concerned with a phenomenon that expression of the gamma- and beta-globin genes is regulated "in a reciprocal manner" at the perinatal period. This has led us to speculate that the gamma-globin gene is silenced in the adult stage by the mechanisms required for activating the beta-globin gene. We will test this hypothesis in this application. Our preliminary studies suggested that 1) cAMP-dependent protein kinase (PKA) activity is necessary for the induction of the beta-globin gene in adult erythroid cells such as MEL cells and primary erythroblasts, that 2) expression of the gamma-globin gene is decreased by activating cAMP-dependent pathways in K562 cells, and that 3) activity of HS2 and a gamma-globin gene promoter is markedly increased by suppressing PKA activity. These results suggest that cAMP-dependent pathways might play a negative role in gamma-globin gene expression. This application has three specific aims. In the first aim, we will characterize intracellular pathways that are required for activating beta-globin gene expression. The second aim will focus on the molecular mechanisms by which intracellular pathways for beta-globin gene expression silence the gamma-globin gene, In the third aim, we will examine whether expression of the gamma-globin gene is induced in primary erythroblasts of the beta-globin disorders and adult mice which have low activities of intracellular pathways for beta-globin gene expression. This application should not only shed much light on the mechanisms that silence the gamma-globin gene, but also provide a clue to the mechanisms underlying the developmental regulation of beta-like globin genes. Furthermore, important information to develop novel Hb F inducers will be disclosed by this study.