Department of Medicine, Section of Pulmonary, Critical Care and Sleep Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and.
Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma.
Am J Respir Cell Mol Biol. 2021 Apr;64(4):426-440. doi: 10.1165/rcmb.2020-0302OC.
Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory ("goblet") cells, defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD were differentiated on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using ) the NOTCH/γ-secretase inhibitor dibenzazepine (DBZ), ) lentiviral overexpression of the NICD3 (NOTCH3-intracellular domain), or ) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Overexpression of NICD3 increased the expression of goblet cell-associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.
慢性阻塞性肺疾病（COPD）是美国的第三大致死原因，主要由吸烟引起。产生更多黏液的分泌细胞（“杯状”），定义为杯状细胞化生或增生（GCMH），对 COPD 的病理生理学有重要贡献。本研究的目的是确定 NOTCH 信号是否调节杯状细胞分化以响应香烟烟雾。来自不吸烟和 COPD 吸烟者的原代人支气管上皮细胞（HBECs）在气液界面上分化，并暴露于香烟烟雾提取物（CSE）中 7 天。使用）NOTCH/γ-分泌酶抑制剂二苯并氮杂（DBZ），）NICD3（NOTCH3-细胞内结构域）的慢病毒过表达，或）NOTCH3 特异性 siRNA 来调节 NOTCH 信号活性。通过定量 PCR、Western 印迹、免疫染色和 RNA 测序评估细胞分化和对 CSE 的反应。我们发现，CSE 暴露于非吸烟者气道上皮细胞中诱导了具有 GCMH 特征的杯状细胞分化。DBZ 处理抑制了 CSE 依赖性诱导的杯状细胞分化。此外，CSE 诱导了 NOTCH3 的激活，这表现为 NOTCH3 核定位增加和 NICD3 蛋白水平升高。NICD3 的过表达增加了与杯状细胞相关的基因 SPDEF 和 MUC5AC 的表达，而 NOTCH3 的敲低抑制了 CSE 介导的 SPDEF 和 MUC5AC 的诱导。最后，CSE 暴露于 COPD 气道上皮细胞中以 NOTCH3 依赖性方式诱导杯状细胞分化。这些结果表明 NOTCH3 激活是香烟烟雾诱导杯状细胞分化的重要机制之一，从而为控制吸烟者和 COPD 患者的 GCMH 相关病变提供了一种新的潜在策略。
