Hormonal Regulation of Surfactant Protein mRNA Stability

描述(申请人提供)：肺表面活性物质的合成，一种降低肺泡表面张力的脂蛋白复合体，在胎儿肺组织中受到发育和激素的调节。表面活性蛋白B(SP-B)在表面活性物质的作用中起着关键作用；当SP-B水平下降到正常水平的25%以下时，肺容易受到损伤和衰竭。缺乏足够的表面活性物质的早产儿可能会患上呼吸窘迫综合征，这是新生儿发病率和死亡率的主要原因。产前应用糖皮质激素可加速胎肺成熟，并通过增加人SP-B mRNA的稳定性增强SP-B的表达，其机制未知。由于糖皮质激素在临床上用于治疗早产儿的RDS，了解糖皮质激素调节肺表面活性蛋白基因表达的分子机制(S)是很重要的。我们假设SP-B mRNA稳定性的调节是由特定的mRNA介导的：定位于SP-B mRNA 3‘-非翻译区(UTR)的蛋白质相互作用。我们已经确定了调节糖皮质激素诱导的SP-B mRNA稳定所必需的和充分的区域，这些区域仅限于3‘-UTR。胞浆蛋白与SP-B 3‘-UTR的126个核苷酸特异地独立结合。最近，我们在该区域发现了一个小的30个核苷酸的信使核糖核酸元件，预测形成一个茎环结构，足以介导糖皮质激素诱导的信使信使RNA的稳定，并降低SP-B信使信使的内在稳定性。这种成分可能提供了一个靶点，在不使用糖皮质激素的情况下提高SP-B的mRNA水平。本研究的目的是更全面地阐明糖皮质激素通过3‘-非编码区元件增强SP-B基因稳定性的分子机制，以及这些元件介导SP-B基因内在稳定性的分子机制。本应用的目的如下：(1)鉴定人SP-BmRNA3‘-UTR中足以介导体内糖皮质激素稳定和/或SP-BmRNA内在稳定性的元件，(2)鉴定通过与SP-BmRNA3’-UTR元件相互作用而介导糖皮质激素调节或SP-B mRNA稳定性内在调节的蛋白质，以及(3)鉴定可能通过与SP-BmRNA3‘-UTR元件相互作用而介导糖皮质激素调节和SP-B mRNA稳定性内在调节的microRNAs(MiRNAs)。这项拟议的研究将通过鉴定特定的mRNA序列、蛋白质和miRNAs来确定糖皮质激素调节SP-B mRNA稳定性的分子机制，这些序列、蛋白质和miRNAs可能参与激素和内在调节mRNA稳定性的机制。这些成分的鉴定和表征将勾勒出激素调节肺内基因表达的复杂机制。对这些机制的了解可能会导致改进的治疗策略的发展，以促进肺成熟和预防RDS及其后果。项目简介：糖皮质激素广泛用于治疗早产儿，因此有必要了解激素调节表面活性蛋白mRNA的机制。最终，这些信息可能允许设计治疗方案，其中保留了糖皮质激素治疗的优势，避免了糖皮质激素的有害影响。

DESCRIPTION (provided by applicant): The synthesis of pulmonary surfactant, a lipoprotein complex that acts to reduce lung alveolar surface tension, is developmentally and hormonally regulated in fetal lung tissue. Surfactant protein B (SP-B) is critical in the function of surfactant; the lung is susceptible to injury and failure when SP-B levels decrease below 25% of normal. Prematurely-born infants that lack adequate surfactant can develop Respiratory Distress Syndrome, a leading cause of neonatal morbidity and mortality. Antenatal administration of glucocorticoids accelerates fetal lung maturity and enhances SP-B expression by increasing human SP-B mRNA stability by unknown mechanisms. Since glucocorticoids are used clinically in the treatment of premature infants against RDS, it is important to understand the molecular mechanism(s) by which glucocorticoids act to regulate surfactant protein gene expression in the lung. We hypothesize that regulation of SP-B mRNA stability is mediated by specific mRNA:protein interactions localized to the SP-B mRNA 3'-untranslated (UTR) regions. We have identified regions necessary and sufficient for mediating glucocorticoid-induced stabilization of SP-B mRNA which are restricted to the 3'-UTR. Cytosolic proteins specifically and independently bind to a 126 nt long region of the SP-B 3'-UTR. Recently, we have identified a small 30 nt mRNA element in this region predicted to form a stem-loop structure that is sufficient for mediating glucocorticoid-induced stabilization of mRNA and reduces intrinsic stability of SP-B mRNA. This element may provide a target to increase SP-B mRNA levels without the use of glucocorticoids. The objective of this application is to more completely define the molecular mechanisms whereby glucocorticoids enhance SP-B mRNA stability through elements in the 3'- UTR and the molecular mechanisms by which these elements mediate intrinsic SP-B mRNA stability. The following specific aims are proposed in this application: (1) to identify elements of the human SP-B mRNA 3'-UTR that are sufficient for mediating in vivo glucocorticoid stabilization and/or intrinsic stability of SP-B mRNA, (2) to identify proteins that may mediate glucocorticoid regulation or intrinsic regulation of SP-B mRNA stability through interaction with elements of the SP-B mRNA 3'-UTR, and (3) to identify microRNAs (miRNAs) that may mediate glucocorticoid regulation and intrinsic regulation of SP-B mRNA stability through interaction with elements of the SP-B mRNA 3'-UTR. The proposed research will define molecular mechanisms by which SP-B mRNA stability is regulated by glucocorticoids through identification of specific mRNA sequences, proteins and miRNAs that may be involved in hormonal and intrinsic regulation of mRNA stability. Identification and characterization of these components will delineate the complex mechanisms whereby hormones regulate gene expression in lung. The understanding of these mechanisms may lead to development of improved therapeutic strategies that enhance lung maturation and prevent RDS and its consequences. PROJECT NARRATIVE: The widespread use of glucocorticoids for treatment of prematurely-born infants makes it necessary to understand the mechanisms of regulation of surfactant protein mRNA by hormones. Ultimately, the information may allow the design of treatment regimes where the advantages of glucocorticoid treatment are retained and the deleterious effects of glucocorticoids are avoided.