Regulatory elements controlling elastin transcription

弹性蛋白是人体主要的弹性蛋白，为肺组织、血管和皮肤提供拉伸和后坐力。弹性蛋白主要在胎儿发育和新生儿时期合成，并由这些组织中的特定细胞亚群表达。通常情况下，弹性纤维是身体中最持久的结构之一，但弹性纤维的破坏是几种慢性肺病的核心。早产儿肺部的弹性纤维发育不充分，无法承受机械通气的压力，可能会受损或成团沉积，导致肺部发育异常。在成人中，当肺弹性纤维因肺气肿等疾病而受损或破坏时，几乎没有有效的修复方法。因此，肺气肿中弹性纤维的丧失是肺功能丧失的主要原因。尽管弹性蛋白在肺中起着关键作用，但以组织特异性和发育调节的方式控制弹性蛋白合成的机制仍在很大程度上是未知的。先前的研究发现弹性蛋白“启动子”在瞬时转染中较弱，并且细胞类型特异性表达的基础尚不清楚。为了寻找一种类似增强子的元件，这种元件可以激活弹性蛋白启动子，新的研究发现了弹性蛋白基因的一个名为T-Exl的调节区域，该区域位于转录开始和延伸到编码区的第一个外显子之间。在表达构建体中包含T-Exl使弹性蛋白在瞬时转染中具有细胞类型特异性表达，并使肺肌成纤维细胞的启动子活性增加6倍。在凝胶转移实验中，表达弹性蛋白的肺肌成纤维细胞的核提取物特异性地结合了这个调节区域。进一步的研究已经确定核蛋白是SP-1转录因子家族的成员。本建议概述的研究将集中于：1)通过一系列缺失和诱变构建体的瞬时转染识别和描述控制细胞类型特异性基因表达的重要调控位点；2)表征、识别和测试结合该调控区域的转录因子的作用；3)确定慢性炎症期间抑制弹性蛋白合成的机制。这些方法将揭示控制弹性蛋白合成的关键机制，并确定调节早产儿和肺气肿肺部弹性蛋白合成的策略靶点。

Elastin is the primary elastic protein in humans, and provides stretch and recoil to lung tissue, blood vessels, and skin. Elastin is primarily synthesized during fetal development and the neonatal period, and is expressed by specific subsets of cells in these tissues. Normally, elastic fibers are among the most durable structures in the body, but destruction of elastic fibers is central to several chronic lung diseases. Elastic fibers in the lungs of very premature infants are not sufficiently developed to withstand the strain of mechanical ventilation, and can be damaged or deposited in clumps, contributing to abnormal lung development. In adults, when lung elastic fibers are damaged or destroyed in diseases such as emphysema, there is little effective repair. Consequently, loss of elastic fibers in emphysema is central to loss of lung function. Despite a pivotal role in the lung, the mechanisms that control elastin synthesis in a tissue-specific and a developmentally regulated way are still largely unknown. Previous studies found the elastin "promoter" to be weak in transient transfections, and the basis for cell type-specific expression was unknown. In a search for an enhancer-like element that would confer strong activation of the elastin promoter, new studies identified a regulatory region of the elastin gene named T-Exl, found between the start of transcription, and extending into the first exon of the coding region. Inclusion of T-Exl in expression constructs confers cell type-specific expression of elastin in transient transfections, and increases promoter activity in lung myofibroblasts 6-fold. Nuclear extracts from elastin expressing lung myofibroblasts specifically bind this regulatory region in gel shift assays. Further studies have identified the nuclear proteins as members of the SP-1 family of transcription factors. The studies outlined in this proposal will focus on 1) Identifying and delineating the important regulatory sites controlling cell type-specific gene expression using transient transfections with a series of deletion and mutagenized constructs, 2) Characterizing, identifying, and testing the role(s) of transcription factors binding this regulatory region, and 3) Determining the mechanisms that suppress elastin synthesis during chronic inflammation. These approaches will reveal key mechanisms controlling elastin synthesis and identify targets for strategies to modulate elastin synthesis in the lungs of premature infants and in pulmonary emphysema.