REGULATION OF SURFACTANT PROTEIN B GENE EXPRESSION

肺表面活性物质蛋白B （Pulmonary surfactant protein B, SP-B）是维持肺正常功能的关键表面活性成分，参与II型肺泡细胞板层体的形成，参与表面活性物质的吸收和膜的稳定性。在人类婴儿和敲除小鼠中，SP-B发育缺陷导致表面活性剂缺乏是一种新生儿致命疾病。此外，获得性SP-B缺乏在各种急性肺损伤动物模型中都存在，并可能导致人类肺部疾病涉及炎症。甲状腺转录因子-1 （TTF-1）是SP-B启动子活性的关键转录因子。在过去资助的体外研究中发现，phorbol酯和TGFbeta都能降低SP-B基因的转录，对这两种药物的反应性都映射到SP-B启动子的近端包含TTF-1和HNF3结合位点的区域，并且在处理细胞的细胞核中存在两种转录因子的丢失。最近的初步数据表明，激素诱导的人胎儿肺上皮细胞表面活性剂系统的分化与TTF-1基因表达的增加有关。这项更新项目的总体目标是进一步阐明参与SP-B基因表达的正调控和负调控的因素和机制，至少部分是通过调节TTF- 1的表达或功能来介导的。大多数实验将在一个独特的原代肺细胞培养系统中进行，在这个系统中，表面活性剂系统的分化是由糖皮质激素和cAMP治疗诱导和维持的。目的1将在体外培养的胎儿肺上皮细胞中检测TTF-1的激素诱导及其与SP-B基因表达的关系，检测TTF-1的剂量和时间过程、转录率和TTF-1亚型。Aim 2将研究TTF-1在SP-B基因表达中的作用，Aim 3将研究tgf - β对SP-B的下调，研究Smad信号、TTF-1和磷酸化的作用。目的4将利用小鼠博来霉素肺损伤模型和两种消除tgf - β信号通路的方法，研究tgf - β和TTF-1在SP-B体内表达中的作用。本研究是对近期研究的合理延伸，将为这一关键的表面活性剂蛋白和转录因子提供新的信息。该项目涉及到与PPG大多数其他项目的交互和协作，并利用每个核心。

Pulmonary surfactant protein B (SP-B) is a critical surfactant component for normal lung function, involved in formation of lamellar bodies in type II alveolar cells and both surfactant absorption and film stability. Developmental deficiency of SP-B contributes to surfactant deficiency in both human infants and knock-out mice is a newborn lethal condition. In addition, acquired deficiency of SP-B occurs in various animal models of acute lung injury and may contribute to human lung diseases involving inflammation. Thyroid transcription factor-1 (TTF-1) is a key transcription factor for activity of the SP-B promoter. In vitro studies during the past funding found that both phorbol ester and TGFbeta decrease SP-B gene transcription, that responsiveness to both agents maps to a region of the proximal SP-B promoter containing TTF-1 and HNF3 binding sites, and that there was a loss of both transcription factors from nuclei of treated cells. Recent preliminary data indicate that hormone induced differentiation of the surfactant system in human fetal lung epithelial cells is associated with increased TTF-1 gene expression. The overall goal of this renewal project is to further elucidate factors and mechanisms involved in both positive and negative regulation of SP-B gene expression is mediated, at least in part, through modulation of TTF- 1 expression or function. Most experiments will be carried out in a unique primary lung cell culture system in which differentiation of the surfactant system is induced and maintained by glucocorticoid plus cAMP treatment. Aim 1 will characterize hormonal induction of TTF-1 and its relationship to SP-B gene expression in cultured fetal lung epithelial cells examining dose and time course, transcription rate and TTF-1 isoforms. Aim 2 will examine the role of TTF-1 in SP-B gene expression using Aim 3 will address down regulation of SP-B by TGFbeta, investigating the role of Smad signaling, TTF-1 and phosphorylation. Aim 4 will examine the roles of TGFbeta and TTF-1 in SP-B expression in vivo using the mouse bleomycin lung injury model and two approaches to abrogation to TGFbeta signaling. The proposed studies are a logical extension of recent research and will provide new information related to this critical surfactant protein and transcription factor. This project involves interactions and collaborations with most of the other PPG projects and utilizes each core.