Hormonal Regulation of Surfactant Protein mRNA Stability

描述（由申请方提供）：肺表面活性物质（一种降低肺泡表面张力的脂蛋白复合物）的合成在胎肺组织中受到发育和生殖调控。表面活性蛋白B（SP-B）在表面活性剂的功能中是关键的;当SP-B水平降低到正常的25%以下时，肺易于损伤和衰竭。缺乏足够表面活性剂的早产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。产前应用糖皮质激素可通过增加人SP-B mRNA的稳定性加速胎肺成熟并增强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稳定性的调节是由定位于SP-B mRNA 3 '-非翻译（UTR）区域的特异性mRNA：蛋白质相互作用介导的。我们已经鉴定了对于介导糖皮质激素诱导的SP-B mRNA稳定化所必需和足够的区域，其限于3 '-UTR。胞质蛋白特异性且独立地结合SP-B 3 '-UTR的126 nt长的区域。最近，我们已经确定了一个小的30 nt的mRNA元件在该区域预测形成茎环结构，足以介导糖皮质激素诱导的mRNA的稳定性，并降低SP-B mRNA的内在稳定性。该元件可以提供靶点以在不使用糖皮质激素的情况下增加SP-B mRNA水平。本申请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稳定性的分子机制和这些元件介导内在SP-B mRNA稳定性的分子机制。在本申请中提出了以下具体目标：（1）鉴定足以介导SP-B mRNA的体内糖皮质激素稳定性和/或内在稳定性的人SP-B mRNA 3 ′-UTR的元件，（2）鉴定可通过与SP-B mRNA 3 ′-UTR的元件相互作用介导糖皮质激素调节或SP-B mRNA稳定性的内在调节的蛋白质，和（3）鉴定可通过与SP-B mRNA 3 ′-UTR元件相互作用介导糖皮质激素调节和SP-B mRNA稳定性内在调节的微小RNA（miRNAs）。拟议的研究将通过鉴定可能参与mRNA稳定性的激素和内在调节的特定mRNA序列、蛋白质和miRNA来确定糖皮质激素调节SP-B mRNA稳定性的分子机制。这些成分的鉴定和表征将阐明激素调节肺基因表达的复杂机制。对这些机制的理解可能会导致改善治疗策略的发展，促进肺成熟和预防RDS及其后果。项目叙述：糖皮质激素在早产儿治疗中的广泛应用使得了解激素对表面活性蛋白mRNA的调节机制成为必要。最终，这些信息可以允许设计治疗方案，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作用。

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.