Intracelllar Pathways That Silence the Fetal Globin Gene

描述(由申请人提供)： 新的胎儿血红蛋白(HbF)诱导剂的开发已经引起了相当大的关注和努力。羟基脲(HU)正被用于治疗镰状细胞性贫血(SCA)，但对HU的反应在不同的SCA患者之间存在显著差异。HU治疗β-地中海贫血的临床疗效尚不清楚。我们的研究不是研究顺式作用的调控元件，而是专注于澄清调控珠蛋白基因表达的细胞内途径。我们最近发现，由可溶性鸟苷环化酶(SGC)和cGMP依赖的蛋白激酶(PKG)组成的细胞内通路在原代红细胞中γ-珠蛋白基因的表达中起着重要作用。我们关于sGC-PKG途径在HbF表达中起重要作用的假说最近被意大利兄弟证实，他们没有β-珠蛋白基因突变，但表达25%到30%的HbF。他们被发现有高水平的原卟啉IX，这是sGC的强激活剂。为了用新的眼光研究伽马珠蛋白基因沉默的分子机制，我们关注的是在围产期伽马珠蛋白和β珠蛋白基因的表达“以交互方式”调节的现象。这导致我们推测，在成年阶段，伽马珠蛋白基因被激活β-珠蛋白基因所需的机制沉默。我们将在此应用程序中测试这一假设。我们的初步研究表明，1)cAMP依赖的蛋白激酶(PKA)活性是诱导成人红系细胞(如MEL细胞和原代红细胞)β-珠蛋白基因所必需的，2)激活K562细胞中cAMP依赖的通路降低了γ-珠蛋白基因的表达，3)抑制PKA活性显著提高了HS2和γ-珠蛋白基因启动子的活性。这些结果表明，cAMP依赖的通路可能在伽马珠蛋白基因的表达中起到负面作用。这个应用程序有三个具体目标。在第一个目标中，我们将描述激活β-珠蛋白基因表达所需的细胞内途径。第二个目标将集中于β-珠蛋白基因表达的细胞内途径沉默的分子机制，在第三个目标中，我们将检查在β-珠蛋白疾病的原代红细胞和细胞内β-珠蛋白基因表达途径活性较低的成年小鼠中是否诱导了γ-珠蛋白基因的表达。这一应用不仅应该对使伽马珠蛋白基因沉默的机制有很大的了解，而且还可以为β-样珠蛋白基因的发育调控机制提供线索。此外，本研究还将为开发新型Hb-F诱导剂提供重要信息。

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.