Epithelial Cell Cycle Regulation by Elastase

弹性蛋白酶对上皮细胞周期的调节

• 批准号: 7099872
• 负责人: BERNARD M FISCHER
• 金额: $ 23.31万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7099872
• 关键词: DNA damage; DNA repair; cell cycle; cell line; cell proliferation; cytogenetics; elastases; enzyme activity; flow cytometry; free radical oxygen; gene expression; immunocytochemistry; kinase inhibitor; neutrophil; oxidative stress; oxidoreductase; quinones; respiratory epithelium; small interfering RNA; western blottings

DESCRIPTION (provided by applicant): Respiratory morbidity and mortality in patients with cystic fibrosis (CF), chronic bronchitis (CB), and pollution- or viral-triggered asthma results from chronic neutrophil-dominant inflammation. These patients are plagued by progressive airway obstruction, parenchyma! damage and scarring. Neutrophil elastase (NE), a serine protease released by neutrophils is present in the airways of these patients and injures the airway epithelium. We have reported that as part of the mechanism of NE-regulation of MUC5AC mucin gene expression, NE triggers the generation of intracellular reactive oxygen species (ROS). We examined the epithelial response to NE - mediated oxidative injury. Although, NE did not cause cell death, it did cause a G1 cell cycle arrest corresponding to a marked decrease in epithelial DNA synthesis and proliferation. Further, we demonstrate that NE- generated ROS mediate the decrease in DNA synthesis. In this proposal, we establish a previously unrecognized mechanism employed by the airway epithelium to reestablish homeostasis following NE-induced injury. The Hypothetical Schema to be addressed is: NE triggers the generation of intracellular reactive oxygen species (ROS) by NAD(P)H:quinone oxidoreductase 1 (NQ01), specifically superoxide and hydrogen peroxide. As a result of oxidant stress and consequential DNA damage, there is increased expression of the Cip/Kip family of mitotic inhibitors. Increased expression of these Cip/Kip proteins results in G1 arrest and inhibition of cell cycle progression and epithelial proliferation. This pause in the cell cycle allows for subsequent DNA repair followed by restoration of epithelial proliferation. The Specific Aims are: 1) To determine whether or not NE - induced DNA damage and G1 cell cycle arrest are mediated by reactive oxygen species; 2) To determine whether or not NE - induced molecular regulation and expression of the Cip/Kip family of mitotic inhibitors, p21, p27 and p57, is mediated by reactive oxygen species; 3) To determine whether or not NE - induced inhibition of DNA synthesis and cell cycle arrest are mediated by the Cip/Kip family of mitotic inhibitors - p21, p27, and p57. The information derived from these experiments will provide fundamental insights into epithelial response to injury, and provide a foundation to investigate normal epithelial regeneration versus pathologic remodeling.

描述（由申请人提供）：囊性纤维化（CF）、慢性支气管炎（CB）和污染或病毒引发的哮喘患者的呼吸系统发病率和死亡率是由慢性中性粒细胞为主的炎症引起的。这些患者都受到进行性气道实质梗阻的困扰！损伤和疤痕。中性粒细胞弹性蛋白酶（NE）是中性粒细胞释放的一种丝氨酸蛋白酶，存在于这些患者的气道中并损伤气道上皮。我们报道，作为 NE 调节 MUC5AC 粘蛋白基因表达机制的一部分，NE 触发细胞内活性氧 (ROS) 的产生。我们检查了上皮细胞对 NE 介导的氧化损伤的反应。虽然 NE 不会导致细胞死亡，但它确实会导致 G1 细胞周期停滞，从而导致上皮 DNA 合成和增殖显着减少。此外，我们证明 NE 产生的 ROS 介导 DNA 合成的减少。在这个提案中，我们建立了一种以前未被认识的机制，气道上皮在 NE 诱导的损伤后重建稳态。要解决的假设方案是：NE 通过 NAD(P)H:醌氧化还原酶 1 (NQ01)，特别是超氧化物和过氧化氢触发细胞内活性氧 (ROS) 的产生。由于氧化应激和随之而来的 DNA 损伤，有丝分裂抑制剂 Cip/Kip 家族的表达增加。这些 Cip/Kip 蛋白表达的增加导致 G1 期停滞并抑制细胞周期进程和上皮增殖。细胞周期的这种暂停允许随后的 DNA 修复，然后恢复上皮增殖。具体目标是： 1) 确定 NE 诱导的 DNA 损伤和 G1 细胞周期停滞是否由活性氧介导； 2)确定NE诱导的有丝分裂抑制剂Cip/Kip家族p21、p27和p57的分子调节和表达是否由活性氧介导； 3)确定NE-诱导的DNA合成抑制和细胞周期停滞是否由Cip/Kip家族有丝分裂抑制剂-p21、p27和p57介导。从这些实验中获得的信息将为上皮对损伤的反应提供基本见解，并为研究正常上皮再生与病理重塑提供基础。