Molecular regulation of GM-CSF expression in alveolar epithelial cells

描述(由申请人提供)： 肺泡上皮是人体与外界环境相互作用的最大表面。肺泡上皮细胞的正常功能损伤或破坏 (AEC)对肺气体交换(导致急性肺损伤)、确定肺损伤后正常或异常的肺修复以及肺天然免疫有重要影响。粒细胞巨噬细胞集落刺激因子(GM-CSF)是由正常AEC产生的一种内源性肺细胞因子，现已成为肺的重要捍卫者。GM-CSF对氧化应激所致的急性肺损伤具有保护作用，是AEC的抗凋亡因子。暴露于博莱霉素等可导致肺纤维化的损害与GM-CSF在AEC中的表达减少有关；经GM-CSF治疗后纤维化减轻。GM-CSF对于肺泡巨噬细胞的正常功能是必不可少的，肺泡巨噬细胞是肺天然免疫的关键组成部分，也负责维持正常的表面活性物质的动态平衡。AEC在肺损伤中经历的氧化应激导致AEC GM-CSF表达受损，并增加了对致死性肺炎的易感性。这一过程可通过GM-CSF治疗而逆转。尽管有这些保护作用，GM-CSF仍占据着一个复杂的利基市场，提示不适当的表达可能会导致类风湿性关节炎或COPD等病理炎症状态。我们的数据表明，在肺泡上皮细胞中对GM-CSF表达的调节在根本上不同于在其他细胞中对这种生长因子的调节。因此，了解这种多能细胞因子在肺泡上皮细胞中的调节细节是至关重要的。由于肺上皮细胞系不能复制原代AEC表达GM-CSF的模式，因此研究原代AEC中GM-CSF的表达规律并将其推广到完整的肺组织中具有重要意义。我们假设AEC GM-CSF的表达在转录后和转录水平上都受到调控。这项提案将审查 这两个部分都有详细的说明。我们的初步研究表明，在氧化应激的背景下，mRNA的稳定性是GM-CSF表达的一个重要决定因素。我们已经鉴定出一组microRNAs(MiRNAs)，它们的行为表明它们是参与氧化应激背景下GM-CSF mRNA失稳的候选分子，并证实了我们使用慢病毒转导在原代小鼠AEC中操纵miRNA的能力。该提案的特定目标1将使用GM-CSF3‘非翻译区的报告构建以及miRNA模拟和敲除来确定这些miRNA的调节作用。我们也有证据表明，锌指蛋白ZFP 36或Tristetraprolin(TTP)可能有助于GM-CSF mRNA的降解。具体目标2将确定TTP在调节AEC中GM-CSF mRNA稳定性中的作用，特别是参考miRNA对TTP表达的影响。具体目标3将确定染色体可及性和组蛋白乙酰化在确定正常AEC中GM-CSF转录模式以及在损伤和修复环境中的作用。这些研究将确定一种关键细胞类型-肺泡上皮细胞中GM-CSF表达调节的关键特征，并对于在人类肺部防御和疾病的背景下理解GM-CSF的生物学至关重要。它们将提供有针对性地操纵肺泡腔内内源性GM-CSF表达的机会，以促进肺损伤后的正常修复，并恢复或增强肺宿主对肺炎的防御。因此，它们有可能提供新的治疗方法来恢复肺泡上皮完整性，同时限制意外的不良反应。

DESCRIPTION (provided by applicant): The pulmonary alveolar epithelium forms the largest surface of interaction of the human body with the external environment. Injury or disruption of normal function in alveolar epithelial cells (AEC) has important consequences for pulmonary gas exchange (resulting in acute lung injury), for determining normal or aberrant lung repair following lung injury, and for pulmonary innate immunity. Granulocyte-macrophage colony stimulating factor (GM-CSF) is an endogenous pulmonary cytokine produced by normal AEC that has emerged as a key defender of the lung. GM-CSF is protective against acute lung injury induced by oxidative stress and is an anti-apoptotic factor for AEC. Insults, such as exposure to bleomycin, that result in pulmonary fibrosis are associated with decreased AEC expression of GM-CSF; fibrosis is reduced following treatment with GM-CSF. GM-CSF is essential for normal function of alveolar macrophages, critical components of the pulmonary innate immunity that are also responsible for maintenance of normal surfactant homeostasis. Oxidative stress experienced by AEC in the setting of lung injury leads to impaired AEC GM-CSF expression and increased susceptibility to lethal pneumonia. This process is reversed by treatment with GM-CSF. Despite these protective effects, GM-CSF occupies a complex niche, with suggestions that inappropriate expression may contribute to pathologic inflammatory states such as rheumatoid arthritis or COPD. Our data demonstrate that regulation of GM-CSF expression in the alveolar epithelium differs in fundamental ways from regulation of this growth factor in other cells. Thus it is essential to understand the details of regulation of this pluripotent cytokine in the alveolar epithelium. Because lung epithelial cell lines poorly replicate the pattern of expression of GM-CSF by primary AEC, it is important to study the regulation of GM-CSF expression in primary AEC and to extend these studies to the intact lung. We hypothesize that AEC GM-CSF expression is regulated both post-transcriptionally and at the level of transcription. This proposal will examine both of these components in detail. Our preliminary studies indicate that mRNA stability is an important determinant of GM-CSF expression in the setting of oxidative stress. We have identified a group of microRNAs (miRNAs) whose behavior suggests that they are candidates to participate in GM-CSF mRNA destabilization in the setting of oxidative stress and have confirmed our ability to manipulate miRNA in primary murine AEC using lentviral transduction. Specific Aim 1 of this proposal will determine the regulatory roles of these miRNA, using a reporter construct for the GM-CSF 3' untranslated region and miRNA mimics and knockdown. We also have evidence that a zinc finger protein, ZFP36 or tristetraprolin (TTP), may contribute to GM-CSF mRNA degradation. Specific Aim 2 will define the role of TTP in the regulation of GM-CSF mRNA stability in AEC, with particular reference to miRNA effects on TTP expression. Specific Aim 3 will define the contribution of chromosomal accessibility and histone acetylation in determining the pattern of GM-CSF transcription in normal AEC and in the setting of injury and repair. These studies will determine critical features of the regulation of GM-CSF expression in a key cell type, the alveolar epithelial cell, and are essential for understanding th biology of GM-CSF in the contexts of defense of, and diseases of the human lung. They will offer the opportunity for targeted manipulation of endogenous GM-CSF expression within the alveolar space to promote normal repair following lung injury and restore or augment pulmonary host defense against pneumonia. Thus, they have the potential to provide new therapeutic approaches to restore alveolar epithelial integrity while limiting unintended adverse effects.