IL8-induced Post-transcriptional Regulation of the MUC5AC mucin gene

描述(申请人提供)：分泌型粘蛋白糖蛋白是肺粘液的主要大分子成分，包裹和保护呼吸道。在肺部疾病中，粘蛋白过度分泌和过度产生，从而导致呼吸道粘液阻塞，并导致哮喘、囊性纤维化和慢性阻塞性肺疾病患者的疾病发病率和死亡率。MUC5AC是一种主要的呼吸道粘蛋白，定位于传导气道上皮中的杯状细胞，在肺部疾病中过表达。我们的长期目标是在分子水平上了解MUC5AC粘蛋白基因表达的调控，以便为开发新的药物以减少粘蛋白过度生产提供更好的基础。MUC5AC基因在转录和转录后水平受特定炎症介质的调节。对MUC5AC基因转录上调的机制进行了研究。然而，对MUC5AC基因转录后调控机制的研究还很少，但可以预见到是由RNA结合蛋白(RBPs)和/或microRNA(MiRNAs)介导的，这是一种新发现的转录后调控机制。炎症介质IL8稳定肺细胞MUC5AC的mRNA丰度，并在转录后水平调节MUC5AC。在这个R21项目中，我们将专注于确定IL8诱导的MUC5AC基因表达的转录后调控是如何通过限制性商业惯例，特别是PTB1和候选限制性商业惯例，以及let-7 miRNA与MUC5AC mRNA 3‘UTR中的靶序列的相互作用来介导的。我们将在分化的人支气管上皮细胞和A549肺细胞系中研究这些事件的潜在机制。在目标1中，我们将从功能上评估PTB1在IL8诱导的MUC5AC稳定中的作用，并鉴定和研究额外的候选限制性商业惯例，以确定它们是否与IL8暴露后MUC5AC的3‘非编码区中的同源顺式序列结合，以提高MUC5AC mRNA的稳定性。在目标2中，我们将确定let-7miRNA/微核糖核蛋白(MRNP)复合体是否针对MUC5AC mRNA的3‘UTR，并招募其他miRNP来增加MUC5AC转录本的稳定性。该项目将为确定炎症介质调节MUC5AC转录本稳定性的分子机制奠定基础，以维持炎症期间粘蛋白的过度生产和高分泌。公共卫生相关性：分泌性粘蛋白是一种大的、粘弹性的糖蛋白，在肺部疾病中过度生产和过度分泌。它们会导致呼吸道粘液阻塞，并导致哮喘、囊性纤维化和其他慢性阻塞性肺疾病患者的发病率和死亡率。MUC5AC是一种主要的肺粘蛋白，MUC5AC基因受患者肺部分泌物中存在的炎症介质的调节。本项目将研究炎症介质IL8如何在转录后水平调节MUC5AC基因的表达，以增加MUC5AC的稳定性，从而在炎症状态下维持肺细胞中MUC5AC的产生。了解在暴露于炎症因子后细胞因子稳定MUC5AC mRNA表达的机制，对于了解粘蛋白如何在病变的呼吸道中持续产生是重要的。这将是制定针对以粘蛋白过度产生为表现的肺部疾病的治疗干预措施的基础。

DESCRIPTION (provided by applicant): Secretory mucin glycoproteins are the major macromolecular component of lung mucus, which coats and protects the respiratory tract. Mucins are hypersecreted and overproduced in lung diseases, thereby contributing to airway mucus obstruction and to disease morbidity and mortality in patients with asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. MUC5AC, a major airway mucin, is localized to goblet cells in the conducting airway epithelium and overexpressed in lung diseases. Our long term objective is to understand regulation of MUC5AC mucin gene expression at the molecular level in order to provide a better foundation for developing novel pharmaceutical agents to diminish mucin overproduction. The MUC5AC gene is regulated at the transcriptional and post-transcriptional level by specific inflammatory mediators. Mechanisms of transcriptional upregulation of the MUC5AC gene expression have been studied. However, post-transcriptional mechanisms of MUC5AC gene regulation are markedly understudied, but predictably are mediated by RNA binding proteins (RBPs) and/or microRNA (miRNAs), a newly identified mechanism for post- transcriptional regulation. The inflammatory mediator IL8 stabilizes MUC5AC mRNA abundance in lung cells and regulates MUC5AC at the post-transcriptional level. In this R21 project, we will focus on defining how the IL8-induced post-transcriptional regulation of MUC5AC gene expression is mediated by interactions induced by binding of RBPs, specifically PTB1 and candidate RBPs, and of Let-7 miRNA to target sequences in the 3'UTR of the MUC5AC mRNA. We will investigate mechanisms underlying these events in differentiated human bronchial epithelial cells and in the A549 lung cell line. In Aim 1 we will functionally evaluate the role of PTB1 in the IL8-induced stability of MUC5AC and identify and study additional candidate RBPs to determine whether they bind to cognate cis-sequences in the 3'UTR of MUC5AC after IL8 exposure to increase MUC5AC mRNA stability. In Aim 2 we will determine whether a Let-7 miRNA/micro-ribonucleprotein (mRNP) complex targets the 3'UTR of MUC5AC mRNA and recruits other miRNP to increase the stability of the MUC5AC transcript. This project will lay the groundwork to identify molecular mechanisms that regulate the stability of the MUC5AC transcript by inflammatory mediators to sustain mucin overproduction and hypersecretion during inflammation. PUBLIC HEALTH RELEVANCE: Secretory mucins are large, viscoelastic glycoproteins that are overproduced and hypersecreted in lung diseases. They contribute to airway mucus obstruction and to disease morbidity and mortality in patients with asthma, cystic fibrosis, and other chronic obstructive pulmonary diseases. MUC5AC is a predominant lung mucin and the MUC5AC gene is regulated by inflammatory mediators present in the lung secretions of patients. This project will investigate how the inflammatory mediator, IL8, mediates MUC5AC gene expression at the post-transcriptional level to increase MUC5AC stability and thus sustain MUC5AC production in lung cells during inflammatory states. Understanding the mechanisms whereby MUC5AC mRNA expression is stabilized by cellular factors following exposure to inflammatory factors will be important for understanding how mucin production is sustained n diseased airways. This will be fundamental for formulating therapeutic interventions for lung diseases that manifest with mucin overproduction.