Molecular regulation of GM-CSF expression in alveolar epithelial cells

描述（由申请人提供）： 肺泡上皮形成人体与外部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）、确定肺损伤后的正常或异常肺修复以及肺先天性免疫具有重要影响。粒细胞-巨噬细胞集落刺激因子（GM-CSF）是由正常AEC产生的内源性肺细胞因子，已成为肺的关键防御者。GM-CSF对由氧化应激诱导的急性肺损伤具有保护作用，并且是AEC的抗凋亡因子。导致肺纤维化的损伤，如暴露于博来霉素，与GM-CSF的AEC表达降低相关;用GM-CSF治疗后纤维化减少。GM-CSF对于肺泡巨噬细胞的正常功能是必需的，肺泡巨噬细胞是肺先天免疫的关键组分，也负责维持正常的表面活性剂稳态。AEC在肺损伤的情况下经历的氧化应激导致AEC GM-CSF表达受损和对致死性肺炎的易感性增加。这一过程可通过GM-CSF治疗逆转。尽管有这些保护作用，GM-CSF占据了一个复杂的生态位，提示不适当的表达可能导致病理性炎症状态，如类风湿性关节炎或COPD。我们的数据表明，在肺泡上皮细胞中的GM-CSF表达的调节在根本上不同于在其他细胞中的这种生长因子的调节。因此，了解肺泡上皮细胞中这种多能细胞因子的调节细节是至关重要的。由于肺上皮细胞系很少复制原代AEC表达GM-CSF的模式，因此研究原代AEC中GM-CSF表达的调节并将这些研究扩展到完整肺是重要的。 我们推测AEC GM-CSF的表达在转录后和转录水平上都受到调节。该提案将审查 这两个部分的细节。我们的初步研究表明，mRNA的稳定性是一个重要的决定因素，GM-CSF表达的设置氧化应激。我们已经鉴定了一组microRNA（miRNA），其行为表明它们是参与氧化应激环境中GM-CSF mRNA不稳定的候选者，并且已经证实了我们使用慢病毒转导在原代鼠AEC中操纵miRNA的能力。本提案的具体目标1将确定这些miRNA的调控作用，使用GM-CSF 3'非翻译区和miRNA模拟物和敲除的报告构建体。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将定义TTP在AEC中GM-CSF mRNA稳定性调节中的作用，特别是miRNA对TTP表达的影响。具体目标3将定义染色体可及性和组蛋白乙酰化在确定正常AEC中以及在损伤和修复的情况下GM-CSF转录模式中的贡献。这些研究将确定GM-CSF在关键细胞类型肺泡上皮细胞中表达调控的关键特征，并且对于理解GM-CSF在人类肺部防御和疾病背景下的生物学是必不可少的。它们将提供在肺泡空间内靶向操纵内源性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.