Hormonal Regulation of Surfactant Protein mRNA Stability

描述（由申请人提供）：肺表面活性剂的合成是一种脂蛋白复合物，可降低肺泡表面张力，在胎儿肺组织中受发育和激素调节。表面活性剂蛋白B （SP-B）在表面活性剂的功能中起关键作用；当SP-B水平低于正常水平的25%时，肺容易受到损伤和衰竭。缺乏足够表面活性剂的早产婴儿可发展为呼吸窘迫综合征，这是新生儿发病率和死亡率的主要原因。产前给药糖皮质激素加速胎儿肺成熟，并通过增加人SP-B mRNA的稳定性来增强SP-B的表达，其机制尚不清楚。由于糖皮质激素在临床上用于治疗早产儿RDS，因此了解糖皮质激素调节肺表面活性剂蛋白基因表达的分子机制非常重要。我们假设SP-B mRNA稳定性的调节是由特定mRNA介导的：定位于SP-B mRNA 3'-未翻译（UTR）区域的蛋白质相互作用。我们已经确定了介导糖皮质激素诱导的SP-B mRNA稳定的必要和充分的区域，这些区域仅限于3'-UTR。胞质蛋白特异性和独立结合SP-B 3'-UTR的126 nt长区域。最近，我们在该区域发现了一个小的30 nt mRNA元件，预计该元件可形成一个茎环结构，足以介导糖皮质激素诱导的mRNA稳定化，并降低SP-B mRNA的内在稳定性。该元件可能提供一个不使用糖皮质激素而增加SP-B mRNA水平的靶标。本应用程序的目的是更完整地定义糖皮质激素通过3'- UTR中的元件增强SP-B mRNA稳定性的分子机制，以及这些元件介导SP-B mRNA固有稳定性的分子机制。本申请的具体目的如下：(1)鉴定人SP-B mRNA 3′-UTR中足以介导SP-B mRNA体内糖皮质激素稳定和/或内在稳定性的元件；(2)鉴定可能通过与SP-B mRNA 3′-UTR元件相互作用介导SP-B mRNA稳定性的糖皮质激素调节或内在调节的蛋白；(3)鉴定可能通过与SP-B mRNA 3′-UTR元件相互作用介导糖皮质激素调控和SP-B mRNA稳定性内在调控的microRNAs （miRNAs）。本研究拟通过鉴定可能参与mRNA稳定性激素调控和内在调控的特定mRNA序列、蛋白和mirna，明确糖皮质激素调控SP-B mRNA稳定性的分子机制。鉴定和表征这些成分将描绘复杂的机制，即激素调节基因表达在肺。对这些机制的理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。项目描述：糖皮质激素在早产儿治疗中的广泛应用，使得有必要了解表面活性剂蛋白mRNA受激素调节的机制。最终，这些信息可能允许设计治疗方案，其中保留糖皮质激素治疗的优点，并避免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.