Epithelial Cell Cycle Regulation by Elastase

描述(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道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呼吸道阻塞的困扰，实质！损伤和伤疤。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，存在于这些患者的呼吸道中，损伤呼吸道上皮。我们已经报道，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。我们检测了内皮细胞对去甲肾上腺素介导的氧化损伤的反应。尽管去甲肾上腺素没有引起细胞死亡，但它确实引起了与上皮DNA合成和增殖显著减少相对应的G1期细胞周期停滞。此外，我们证明了去甲肾上腺素产生的ROS介导了DNA合成的减少。在这项建议中，我们建立了一种先前未知的机制，用于在NE诱导的损伤后重建气道上皮细胞的动态平衡。要解决的假设模式是：NE通过NAD(P)H：Quone氧化还原酶1(NQ01)触发细胞内活性氧物种(ROS)的产生，特别是超氧化物和过氧化氢。由于氧化应激和相应的DNA损伤，有丝分裂抑制因子Cip/KIP家族的表达增加。这些Cip/Kip蛋白的表达增加会导致G1期停滞，并抑制细胞周期进展和上皮细胞增殖。细胞周期的这种暂停允许随后的DNA修复，随后是上皮细胞增殖的恢复。其具体目的是：1)确定NE诱导的DNA损伤和细胞周期停滞是否由活性氧介导；2)确定NE诱导的CIP/KIP家族有丝分裂抑制物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停滞是否由CIP/KIP家族有丝分裂抑制物p21、p27和p57介导。从这些实验中获得的信息将为上皮细胞对损伤的反应提供基本的见解，并为研究正常的上皮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.