Epithelial Cell Cycle Regulation by Elastase

描述（由申请人提供）：慢性中性粒细胞显性炎症导致囊性纤维化（CF）、慢性支气管炎（CB）和污染或病毒引发哮喘患者的呼吸道发病率和死亡率。这些患者的困扰是进行性气道梗阻，实质！伤害和疤痕。中性粒细胞弹性蛋白酶（NE）是一种由中性粒细胞释放的丝氨酸蛋白酶，存在于这些患者的气道中，并损伤气道上皮。我们已经报道了NE调控MUC5AC粘蛋白基因表达的部分机制，NE触发细胞内活性氧（ROS）的产生。我们检测了上皮细胞对NE介导的氧化损伤的反应。虽然NE不引起细胞死亡，但它确实引起G1细胞周期阻滞，相应的上皮DNA合成和增殖明显减少。此外，我们证明了NE产生的ROS介导DNA合成的减少。在这个建议中，我们建立了一个以前未被认识到的机制，气道上皮在ne诱导损伤后重建稳态。要解决的假设图式是：NE通过NAD(P)H：醌氧化还原酶1 （NQ01）触发细胞内活性氧（ROS）的产生，特别是超氧化物和过氧化氢。由于氧化应激和随之而来的DNA损伤，有丝分裂抑制剂的Cip/Kip家族的表达增加。这些Cip/Kip蛋白的表达增加导致G1阻滞，抑制细胞周期进程和上皮细胞增殖。细胞周期的暂停允许随后的DNA修复，随后是上皮细胞增殖的恢复。具体目的是：1)确定NE诱导的DNA损伤和G1细胞周期阻滞是否由活性氧介导；2)确定NE诱导的Cip/Kip家族有丝分裂抑制剂p21、p27和p57的分子调控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阻滞是否由Cip/Kip家族的有丝分裂抑制剂p21、p27和p57介导。从这些实验中获得的信息将为研究上皮细胞对损伤的反应提供基本的见解，并为研究正常上皮细胞再生与病理性重塑提供基础。

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.