Epithelial Cell Cycle Regulation by Elastase

弹性蛋白酶对上皮细胞周期的调节

• 批准号: 7386704
• 负责人: BERNARD M FISCHER
• 金额: $ 22.72万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386704
• 关键词: Address; Antimitotic Agents; Antioxidants; Asthma; Biological Assay; Bronchitis; CDKN1A gene; CDKN1C gene; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Proliferation; Chronic; Chronic Bronchitis; Cicatrix; Comet Assay; Complex; Cyclin-Dependent Kinase Inhibitor; Cyclin-Dependent Kinases; Cyclins; Cystic Fibrosis; DNA Damage; DNA biosynthesis; DNA chemical synthesis; DNA strand break; Deoxyguanosine; Dicumarol; Disease; Elastases; Epithelial; Epithelial Cells; Epithelium; Failure; Family; Flow Cytometry; Foundations; G1 Arrest; G1 Phase; G2 Phase; Gene Expression; Generations; Genes; Histone H3; Homeostasis; Human; Hydrogen Peroxide; Hydroxyl Radical; Immunoblotting; Immunohistochemistry; Immunoprecipitation; In Vitro; Inflammation; Inflammatory; Injury; Leukocyte Elastase; Lung; MUC5AC gene; Mediating; Messenger RNA; Mitosis; Mitotic Activity; Molecular; Morbidity - disease rate; Mucins; NAD(P)H dehydrogenase (quinone) 1, human; NQO1 gene; Natural regeneration; Nuclear; Oxidants; Oxidative Stress; Palliative Care; Pancreatic Elastase; Pathologic; Pathway interactions; Patients; Phase; Phosphotransferases; Plague; Ploidies; Pollution; Population; Population Distributions; Post-Transcriptional Regulation; Process; Production; Propidium Diiodide; Proteins; RNA Stability; Reactive Oxygen Species; Recurrence; Regulation; Reporter; Reporting; Research Personnel; Ribonucleases; Running; S Phase; Serine Protease; Small Interfering RNA; Staining method; Stains; Stress; Superoxides; Thymidine; Viral; airway epithelium; airway obstruction; base; cystic fibrosis patients; inhibitor/antagonist; injured airway; insight; member; mortality; neutrophil; novel; oncoprotein p21; oxidative DNA damage; p27 Cell Cycle Protein; p27 Enzyme Inhibitor; programs; promoter; protein expression; repaired; research study; respiratory; response; response to injury; restoration; uptake

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-inducedinjury. 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 DNArepair 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) Todetermine 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) Todetermine whether or not NE- induced inhibition of DNAsynthesis andcell cycle arrest are mediatedby 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.

囊性纤维化（CF）、慢性支气管炎（CB）和慢性支气管炎患者的呼吸系统发病率和死亡率 污染或病毒引发的哮喘是由慢性嗜中性粒细胞为主的炎症引起的。这些患者 受到进行性气道阻塞、软组织阻塞的困扰！损伤和疤痕。中性粒细胞弹性蛋白 (NE)，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， 气道上皮我们已经报道，作为MUC 5AC粘蛋白NE调节机制的一部分， 基因表达，NE触发细胞内活性氧（ROS）的产生。我们研究 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，但它确实会导致细胞死亡。 导致G1细胞周期停滞，对应于上皮DNA合成的显著减少， 增殖此外，我们证明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的机制， 上皮细胞在NE诱导的损伤后重建稳态。 要解决的假设图式是： NE通过NAD（P）H：醌触发细胞内活性氧（ROS）的产生 氧化还原酶1（NQ 01），特别是超氧化物和过氧化氢。由于氧化剂 应激和随之而来的DNA损伤，Cip/Kip家族的表达增加， 有丝分裂抑制剂这些Cip/Kip蛋白的表达增加导致G1期阻滞， 抑制细胞周期进程和上皮细胞增殖。细胞周期中的这种停顿 用于随后的DNA修复，随后恢复上皮增殖。 具体目的是：1）检测NE是否诱导DNA损伤和G1期细胞周期 2）确定NE诱导的细胞凋亡是否与细胞内的活性氧有关， 有丝分裂抑制剂Cip/Kip家族p21、p27和p57的调节和表达由以下介导： 3）测定NE是否抑制DNA合成和细胞增殖 周期阻滞是由有丝分裂抑制剂Cip/Kip家族-p21、p27和p57介导的。 从这些实验中获得的信息将为上皮细胞的研究提供基本的见解。 对损伤的反应，并提供研究正常上皮再生与病理性 重塑