Transcriptional regulation of goblet cell metaplasia

杯状细胞化生的转录调控

• 批准号: 8744367
• 负责人: Vladimir Kalinichenko
• 金额: $ 39万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-05 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8744367
• 关键词: Allergens; Applications Grants; Asthma; Binding; Biological Assay; Boxing; Cell Differentiation process; Chronic; Chronic Obstructive Airway Disease; Clara cell; Clinical; Cystic Fibrosis; Data; Disease; Doxycycline; Embryonic Development; Epithelial; Epithelial Cells; Family; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Goblet Cells; Human; Immunohistochemistry; In Vitro; Interleukin-13; Laboratories; Lasers; Lung Inflammation; Malignant Neoplasms; Mediating; Mediator of activation protein; Metaplasia; Modeling; Molecular; Morbidity - disease rate; Mucous body substance; Mus; Pathogenesis; Pathway interactions; Patients; Peptides; Phenotype; Play; Production; Promoter Regions; Proteins; Pyroglyphidae; Regulation; Regulatory Element; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Testing; Therapeutic; Time; Transcriptional Regulation; Transgenes; Transgenic Mice; Up-Regulation; Western Blotting; airway epithelium; airway hyperresponsiveness; chromatin immunoprecipitation; efficacy testing; forkhead protein; gain of function; in vivo; inhibitor/antagonist; loss of function; lung injury; methacholine; mortality; mouse model; new therapeutic target; novel; prevent; promoter; public health relevance; research study; response; small hairpin RNA; small molecule

DESCRIPTION (provided by applicant): Goblet cell metaplasia, associated with increased mucus production, is a key pathogenic feature of chronic airway disorders, such as asthma, COPD and cystic fibrosis, all of which contribute to significant morbidity and mortality worldwide. Pharmacological targeting of goblet cell metaplasia represents a significant clinical challenge. Therefore, identification of new molecular mechanisms in airway epithelial differentiation will provide novel therapeutic targets for treatment of chronic airway disorders. Our grant proposal focuses on novel molecular mechanisms in goblet cell differentiation that are regulated by FoxM1, a transcription factor from the Forkhead box (FOX) family. While FoxM1 plays an important role in embryonic development and pathogenesis of various cancers, the role of FoxM1 in chronic airway diseases is unknown. In our preliminary data, FoxM1 was induced in airway epithelial cells of mice with asthma-like diseases caused by OVA, IL-13 and house dust mite extract (HDM). FoxM1 was also induced in airway epithelial cells of human patients with asthma and COPD. Genetic deletion of the Foxm1 gene (CCSP-Cre) or pharmacological inhibition of the FoxM1 protein (ARF peptide) in HDM-challenged airway epithelium effectively diminished goblet cell metaplasia, reduced lung inflammation and decreased airway hyper-responsiveness to methacholine. While these data suggest that FoxM1 plays a key role in asthma pathogenesis, molecular mechanisms regulated by FoxM1 remain uncharacterized. We propose to test the hypothesis that FoxM1 acts downstream of the IL-13/Stat6 pathway to induce expression of goblet cell-specific genes in airway epithelial cells. In Aim I, we will use transgenic mice with FoxM1 "gain-of-function" and "loss-of-function" in airway Clara cells to identify downstream FoxM1 target genes critical for allergen-mediated differentiation of Clara cells into goblet cells. Furthermore, we provide preliminary data demonstrating that IL-13 induces FoxM1 expression in cultured human airway epithelial cells and airway epithelium of transgenic mice. Knockdown of FoxM1 in vitro inhibited differentiation of airway epithelial cells toward goblet cell phenotype in response to IL-13 stimulation. In Aim II, we will determine if FoxM1 is required for IL-13/Stat6 signaling to induce goblet cell differentiation in vivo. The IL-13/Stat6 signaling pathway will be activated using intratracheal administration of IL-13 and a Doxycycline-inducible IL-13 transgene. FoxM1 inhibition will be achieved by a genetic approach (IL-13/ CCSP- Cre/ Foxm1-/- mice) and a pharmacological approach (ARF peptide and novel small molecule FoxM1 inhibitors). These experiments will determine whether inactivation of FoxM1 will prevent or decrease IL- 13/Stat6 signaling in airway epithelial cells in vivo. Altogether, these studies will identify molecular mechanisms regulated by FoxM1 in airway epithelial cells and determine the therapeutic benefit of FoxM1 inhibitors in mouse asthma models.

描述（由申请人提供）：杯状细胞化生与粘液产生增加相关，是慢性气道疾病的一个关键致病特征，例如哮喘、慢性阻塞性肺病和囊性纤维化，所有这些疾病都会导致全世界显着的发病率和死亡率。 杯状细胞化生的药理学靶向是一项重大的临床挑战。因此，鉴定气道上皮分化的新分子机制将为治疗慢性气道疾病提供新的治疗靶点。我们的资助提案重点关注由叉头盒 (FOX) 家族的转录因子 FoxM1 调节的杯状细胞分化的新分子机制。虽然 FoxM1 在胚胎发育和各种癌症的发病机制中发挥着重要作用，但 FoxM1 在慢性气道疾病中的作用尚不清楚。在我们的初步数据中，FoxM1 在患有由 OVA、IL-13 和屋尘螨提取物 (HDM) 引起的哮喘样疾病的小鼠的气道上皮细胞中被诱导。 FoxM1 也在人类哮喘和慢性阻塞性肺病患者的气道上皮细胞中被诱导。在受到 HDM 攻击的气道上皮中，Foxm1 基因 (CCSP-Cre) 的遗传缺失或 FoxM1 蛋白（ARF 肽）的药理学抑制有效地减少了杯状细胞化生、减少了肺部炎症并降低了气道对乙酰甲胆碱的高反应性。虽然这些数据表明 FoxM1 在哮喘发病机制中发挥关键作用，但 FoxM1 调节的分子机制仍不清楚。我们建议检验 FoxM1 在 IL-13/Stat6 通路下游作用以诱导气道上皮细胞中杯状细胞特异性基因表达的假设。在目标 I 中，我们将使用气道 Clara 细胞中具有 FoxM1“功能获得”和“功能丧失”的转基因小鼠来鉴定对过敏原介导的 Clara 细胞分化为杯状细胞至关重要的下游 FoxM1 靶基因。此外，我们提供的初步数据表明，IL-13 在培养的人气道上皮细胞和转基因小鼠气道上皮中诱导 FoxM1 表达。体外敲低 FoxM1 可抑制气道上皮细胞响应 IL-13 刺激而向杯状细胞表型分化。在目标 II 中，我们将确定 IL-13/Stat6 信号传导是否需要 FoxM1 来诱导体内杯状细胞分化。 IL-13/Stat6 信号通路将通过气管内施用 IL-13 和多西环素诱导型 IL-13 转基因来激活。 FoxM1 抑制将通过遗传方法（IL-13/CCSP-Cre/ Foxm1-/- 小鼠）和药理学方法（ARF 肽和新型小分子 FoxM1 抑制剂）来实现。这些实验将确定 FoxM1 失活是否会阻止或减少体内气道上皮细胞中的 IL-13/Stat6 信号传导。总之，这些研究将确定 FoxM1 在气道上皮细胞中调节的分子机制，并确定 FoxM1 抑制剂在小鼠哮喘模型中的治疗效果。