REGULATION OF PULMONARY ELASTIN PRODUCTION BY RETINOIDS

肺部疾病，如： 肺气肿和支气管扩张可以不可逆地改变弹性 通过结构蛋白弹性蛋白的降解来改变肺的性质。 由于几乎所有的弹性蛋白在正常的肺是产生过程中 早期喜欢，研究调节弹性蛋白合成的因素， 沉积，并最终修复弹性蛋白网络， 在疾病中受损。 假设：围产期肺含有供应 类维生素A，它可以使用在最大的肺泡期 中隔弹性蛋白合成。 类视色素，特别是视黄酸 (RA)，可能促进间质性肺成纤维细胞合成弹性蛋白 (LIF)调节弹性蛋白合成的增加， 正常的肺泡间隔形成 初步研究表明， RA和视黄酸受体-γ mRNA的量， 新生大鼠肺成纤维细胞中蛋白质的时间变化 这表明它们可以帮助启动弹性蛋白合成的增加， 这些细胞。 其他研究表明，RA增加弹性蛋白 通过培养的新生儿LIF产生，并在 转录。 本研究的主要目的是研究 RA可能影响弹性蛋白合成的分子机制 在正常的肺泡发育过程中。 获得和代谢 将检查肺组织和分离的LIF中类维生素A的含量， 内源性肺储备的利用。 基础和RA- 诱导各种视黄酸受体（RAR）的表达， 将在培养的大鼠LIF中研究维甲酸-X受体（RXR）基因 和从RAR-γ缺失小鼠中分离的LIF。 RAR和RXR 将使用核糖核酸酶保护定量mRNA和蛋白质 测定和免疫印迹。 一个支配者的影响 将在以下患者中检查弹性蛋白表达的阴性RAR突变： 培养细胞 弹性蛋白mRNA，不溶性弹性蛋白积聚，以及 将在携带基因缺失的小鼠中研究肺泡生长， RAR-γ和/或RXR-α。 影响的分子细节 RA对弹性蛋白基因的影响将通过缺失分析来阐明， 两个潜在的RA反应元件（RARE）的突变， 大鼠弹性蛋白基因的5'侧翼区。 电泳迁移率 将使用偏移试验来证明RA应答性增加， 核蛋白与这些元件的结合，在培养的LIF中， 发育中的肺 连接介导的聚合酶链反应 将用于评估蛋白质与这些推定的RARE的结合， 在肺发育过程中体内的弹性蛋白基因，以及对 外源性RA 阐明了弹性蛋白合成的机制， 将提供新的信息， 适用于特发性肺纤维化和支气管肺炎 发育不良

Destructive inflammatory lung diseases lung diseases such as emphysema and bronchiectasis can irreversibly alter the elastic properties of the lung by degradation of the structural protein elastin. Since virtually all of the elastin in the normal lung is produced during early like, studying the factors which regulate elastin synthesis and deposition, and to ultimately repair the elastin network that is damaged in disease. Hypothesis: The perinatal lung contains a supply of retinoids, that it may use during the period of maximal alveolar septal elastin synthesis. Retinoids, and in particular retinoic acid (RA), may promote elastin synthesis by interstitial lung fibroblasts (LIF) and modulate the increase in elastin synthesis that is required for normal alveolar septal formation. Preliminary studies show that the quantities of RA and retinoic acid receptor-gamma mRNA and protein in neonatal rat lung fibroblasts change in a temporal pattern that suggests they could help initiate an increase in elastin synthesis by these cells. Additional studies show that RA increases elastin production by cultured neonatal LIF and acts at the level of transcription. The major goal of the proposed research is to examine the molecular mechanisms by which RA may influence elastin synthesis during normal alveolar development. The acquisition and metabolism of retinoids by lung tissue and isolated LIF will be examined to assess the utilization of endogenous pulmonary stores. The basal and RA- induced expression of the various retinoic acid receptor (RAR) and retinoid-X receptor (RXR) genes will be studied in cultured rat LIF and in LIF isolated from RAR-gamma null mice. RAR and RXR mRNA and protein will be quantitated using ribonuclease protection assays and immunoblotting, respectively. The effects of a dominant negative RAR mutation of elastin expression will be examined in cultured cells. Elastin mRNA, insoluble elastin accumulation, and alveolar growth will be studied in mice bearing gene deletions for RAR-gamma and/or RXR-alpha. The molecular details of the effects of RA on the elastin gene will be elucidated by deletional analysis and mutagenesis of two potential RA response elements (RARE) within the 5' flanking region of the rat elastin gene. Electrophorectic mobility shift assays will be used to demonstrate RA-responsive increases in the binding of nuclear proteins to these elements, in cultured LIF and in the developing lung. A ligation-mediated polymerase chain reaction will be used to evaluate binding of proteins to these putative RARE in the elastin gene in vivo during lung development, and in response to exogenous RA. Elucidation of mechanisms whereby elastin synthesis is initiated in the alveoli would provide novel information that may also be applicable to idiopathic pulmonary fibrosis and bronchopulmonary dysplasia.