Cyclic Nucleotides and Fetal Globin Gene Expression

描述（由申请方提供）：对胎儿的药理学刺激 血红蛋白（Hb F）多年来一直引起人们的极大关注，但 诱导G-珠蛋白基因表达的分子机制， 成虫期仍不清楚。 该提案的长期目标是 通过阐明细胞内途径开发“新型Hb F诱导剂”， 调节G-珠蛋白基因表达。 我们发现G-珠蛋白的表达 基因在红白血病细胞以及原代成红细胞中被诱导 通过激活包括可溶性鸟苷酸环化酶的细胞内途径 (sGC)和cGMP依赖性蛋白激酶（PKG）。 这一途径也被发现， 对于氯化血红素诱导的G-珠蛋白基因表达是必需的，或 丁酸盐。 在第一个具体的目标，我们将研究的分子机制， sGC-PKG途径诱导g-珠蛋白基因表达。 首先，我们将确定和 在b-珠蛋白基因座内表征顺式作用元件和反式作用元件 介导G-珠蛋白基因途径的分子效应。 接下来我们 将检查sGC-PKG通路是否有助于表达 β-地中海贫血中的γ-珠蛋白基因。 在第二个具体目标中，我们将使用转基因小鼠来测试假设 G-珠蛋白基因的表达是在成年阶段被过度诱导的， 表达或激活sGC，sGC是a-和B-的专性异二聚体， 亚单位。 我们将首先用DNA构建体制造转基因小鼠， 由b-珠蛋白基因启动子和LCR驱动的sGC亚基基因， 携带人类b-珠蛋白基因位点的小鼠进行实验。 第二，我们将研究 是否可以通过表达sGC来缓解镰状细胞小鼠的表型 在高水平的亚单位。 最近，我们发现两个正常人没有 b-珠蛋白基因座突变，表达30% Hb F， 卟啉，如原卟啉IX（PPIX）和ZnPP，两者都是sGC 活化剂。 第三，我们将研究g-珠蛋白基因表达是否可以被 通过增加红细胞中PPIX浓度诱导。 这将是 通过将携带人类b-珠蛋白基因座的小鼠与 亚铁螯合酶缺乏症 如果成功实施，这一建议不仅应加强我们的 了解调节基因表达的分子机制， G-珠蛋白基因在发育过程中的作用，而且还提供了重要的信息， 开发用于治疗b-珠蛋白疾病的新型Hb F诱导剂。

DESCRIPTION (provided by applicant): Pharmacological stimulation of fetal hemoglobin (Hb F) has been attracting great concerns for many years, but the molecular mechanisms by which expression of the g-globin gene is induced in the adult stage still remain unclear. The long-term goal of this proposal is to develop "novel Hb F inducers" by clarifying intracellular pathways that regulate g-globin gene expression. We showed that expression of the g-globin gene is induced in erythroleukemic cells as well as in primary erythroblasts by activating an intracellular pathway comprising soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). This pathway was also found to be essential for the induced expression of the g-globin gene by hemin or butyrate. In the first specific aim, we will study the molecular mechanisms by which the sGC-PKG pathway induces g-globin gene expression. First, we will identify and characterize within the b-globin locus cis-acting and trans-acting elements that mediate molecular effects of the pathway to the g-globin gene. Next, we will examine whether the sGC-PKG pathway contributes to the expression of the gamma-globin gene in beta-thalassemia. In the second specific aim, we will test using transgenic mice the hypothesis that expression of the g-globin gene is induced in the adult stage by over- expressing or activating sGC, which is an obligate heterodimer of a- and b- subunits. We will first create transgenic mice with DNA constructs carrying sGC subunit genes driven by the b-globin gene promoter and the LCR, and breed them with mice carrying the human b-globin locus. Second, we will examine whether the phenotype of sickle cell mice can be alleviated by expressing sGC subunits at high levels. Recently we found two normal subjects with no mutations in the b-globin locus who express 30% Hb F and have high levels of porphyrins such as protoporphyrin IX (PPIX) and ZnPP, both of which are sGC activators. Third, we will examine whether g-globin gene expression can be induced by increasing PPIX concentrations in red cells. This will be performed by breeding mice carrying the human b-globin locus with those of ferrochelatase deficiency. If successfully implemented, this proposal should not only enhance our understanding of the molecular mechanisms that regulate the expression of the g-globin gene during development, but also provide important information to develop novel Hb F inducers for treating the b-globin disorders.