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

性状（由申请方提供）：分泌性粘蛋白糖蛋白是肺粘液的主要大分子组分，其覆盖并保护呼吸道。粘蛋白在肺部疾病中是高分泌和过度产生的，从而导致气道粘液阻塞以及哮喘、囊性纤维化和慢性阻塞性肺病患者的疾病发病率和死亡率。MUC 5AC是一种主要的气道粘蛋白，定位于传导气道上皮中的杯状细胞，并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MUC 5AC粘蛋白基因表达的调控，以便为开发新的药物制剂以减少粘蛋白过度产生提供更好的基础。MUC 5AC基因在转录和转录后水平受特异性炎症介质调节。已经研究了MUC 5AC基因表达的转录上调机制。然而，MUC 5AC基因调控的转录后机制明显未被充分研究，但可预测的是由RNA结合蛋白（RBP）和/或微小RNA（miRNA）介导，这是一种新鉴定的转录后调控机制。炎症介质IL 8稳定肺细胞中MUC 5AC mRNA丰度，并在转录后水平调节MUC 5AC。在这个R21项目中，我们将集中于确定IL 8诱导的MUC 5AC基因表达的转录后调节是如何通过RBP（特别是PTB 1和候选RBP）和Let-7 miRNA与MUC 5AC mRNA的3 'UTR中的靶序列结合诱导的相互作用介导的。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。在目的1中，我们将从功能上评估PTB 1在IL 8诱导的MUC 5AC稳定性中的作用，并鉴定和研究其他候选RBP，以确定它们是否在IL 8暴露后结合MUC 5AC的3 'UTR中的同源顺式序列以增加MUC 5AC mRNA稳定性。在目标2中，我们将确定Let-7 miRNA/微核糖核蛋白（mRNP）复合物是否靶向MUC 5AC mRNA的3 'UTR并募集其他miRNP以增加MUC 5AC转录物的稳定性。该项目将为确定炎症介质调节MUC 5AC转录物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分泌过多。公共卫生相关性：分泌性粘蛋白是一种大的粘弹性糖蛋白，在肺部疾病中过度产生和分泌。在哮喘、囊性纤维化和其他慢性阻塞性肺病患者中，它们导致气道粘液阻塞和疾病发病率和死亡率。MUC 5AC是主要的肺粘蛋白，并且MUC 5AC基因受存在于患者肺分泌物中的炎性介质调节。该项目将研究炎症介质IL 8如何在转录后水平介导MUC 5AC基因表达，以增加MUC 5AC稳定性，从而在炎症状态下维持肺细胞中MUC 5AC的产生。了解MUC 5AC mRNA表达在暴露于炎性因子后通过细胞因子稳定的机制对于了解粘蛋白产生如何在患病气道中持续将是重要的。这将是制定治疗干预措施的基础，表现为粘蛋白过度生产的肺部疾病。

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.