Resolution of Epithelial Cell Hyperplasia

描述（由申请方提供）：炎症反应引起的上皮细胞增生伴有粘液细胞化生（MCM），即气道中出现粘液细胞，而这些细胞通常不存在。MCM是慢性支气管炎粘液高分泌增加的主要因素之一。我们已经表明，Bcl-2，细胞凋亡抑制剂，维持MCM在大鼠和小鼠，并可能具有临床意义，因为它是在囊性纤维化或慢性支气管炎患者的粘液细胞表达。p53基因第72位密码子的多态性（Arg/Pro）对Bcl-2的表达有不同的影响; p53 Pro比p53 Arg更有效地增加，p53 Arg是促凋亡蛋白Noxa，可降低Bcl-2 mRNA的半衰期。删除或突变小鼠p53相应区域内的脯氨酸残基在小鼠胚胎成纤维细胞中具有相同的效果。与这些研究结果一致，p53Pro/Pro基因型吸烟者患慢性支气管炎的风险低于p53Arg/Arg基因型吸烟者。来自5个Arg/Arg和5个Pro/Pro个体的正常人支气管上皮细胞（NHBEC）的初步结果显示，在未处理状态下，NHBECArg/Arg培养物与NHBECPro/Pro培养物相比显示出降低的Noxa mRNA水平，并且当用环境香烟烟雾（ETS）处理培养物时，显示出降低的Puma mRNA水平。因此，我们想测试的中心假设，即支气管细胞与p53 Pro/Pro变异解决上皮细胞增生和MCM比支气管细胞与p53 Arg/Arg更有效地诱导Noxa表达介导的Bcl-2水平的降低。具体目标1将确定分化的NHBECArg/Arg、NHBECArg/Pro和NHBECPro/Pro培养物在暴露于ETS提取物之前和之后是否显示分泌粘液、MUC5AC、Noxa和Puma mRNA表达以及MCM发展的差异。类似地，将测试来自具有野生型和突变型p53的小鼠的小鼠气管上皮细胞的分泌粘液，以及Muc5ac、Noxa和Puma mRNA和MCM在未处理状态下和用ETS提取物处理后的表达。此外，将分别用表达Ad-p53Pro或Ad-p53Arg的腺病毒载体感染五种代表性NHBECArg/Arg和NHBECPro/Pro培养物，以确定这些NHBEC的表型是否将被调节。特异性目标2将增加和减少NHBECPro/Pro和NHBECPrg/Arg培养物中的Noxa和/或Puma表达，以确定它们在MCM发展中的作用。此外，将通过将noxa-/-、puma-/-和野生型小鼠暴露于ETS以及用Noxa-和/或puma衍生肽处理NHBECArg/Arg培养物来检查这些蛋白质的作用。这些研究将提供新的治疗策略，可以促进异常修复过程的恢复，减少慢性支气管炎上皮细胞增生和MCM。公共卫生相关性：了解为什么某些人容易患上慢性支气管炎将有助于早期发现疾病并提供更好的治疗。我们发现上皮细胞数量的增加与粘液细胞数量的增加有关，粘液细胞是慢性支气管炎粘液分泌过多增加的来源。拟议的研究将阐明遗传变异的基础上持续增加的上皮粘液细胞的数量在气道。此外，这些研究将开发更有效的治疗方法，这些方法基于通常减少气道上皮中粘液细胞数量的机制。减少上皮细胞增生也可能有助于降低患肺癌的风险。

DESCRIPTION (provided by applicant): Epithelial cell hyperplasia in response to an inflammatory response is accompanied by mucous cell metaplasia (MCM), the appearance of mucous cells in airways that are normally devoid of these cells. MCM is one of the major factors for increased mucous hypersecretion in chronic bronchitis. We have shown that Bcl-2, an inhibitor of apoptosis, sustains MCM in rats and mice and may have clinical significance because it is expressed in mucous cells of patients with cystic fibrosis or chronic bronchitis. A polymorphism in the p53 gene at codon 72 (Arg/Pro) differentially affects Bcl-2 expression; p53Pro increases more efficiently than p53Arg, the pro-apoptotic protein Noxa that reduces the bcl-2 mRNA half-life. Deleting or mutating the proline residue within the corresponding region of murine p53 had the same effect in mouse embryo fibroblasts. Consistent with these findings, smokers with the p53Pro/Pro genotype were at lower risk of developing chronic bronchitis than smokers with the p53Arg/Arg genotype. Preliminary results with normal human bronchial epithelial cells (NHBECs) from five Arg/Arg and five Pro/Pro individuals showed that in the untreated state NHBECArg/Arg cultures showed reduced Noxa mRNA levels compared to NHBECPro/Pro cultures and reduced Puma mRNA levels when cultures were treated with environmental cigarette smoke (ETS). Therefore, we want to test the central hypothesis that bronchial cells with the p53Pro/Pro variant resolve epithelial cell hyperplasia and MCM more efficiently than bronchial cells with p53Arg/Arg by inducing Noxa expression that mediates the reduction of Bcl- 2 levels. Specific Aim 1 will determine whether differentiated NHBECArg/Arg, NHBECArg/Pro, and NHBECPro/Pro cultures will show differences in secreted mucus, MUC5AC, Noxa, and Puma mRNA expression, and the development of MCM before and after exposure to ETS extract. Similarly, mouse tracheal epithelial cells from mice with wild-type and mutated p53 will be tested for secreted mucus, and expression of Muc5ac, Noxa and Puma mRNAs, and MCM in the untreated state and after treatment with ETS extract. In addition, five representative NHBECArg/Arg and NHBECPro/Pro cultures will be infected with adenoviral vectors expressing Ad-p53Pro or Ad-p53Arg, respectively, to determine whether the phenotypes for these NHBECs will be modulated. Specific Aim 2 will increase and reduce Noxa and/or Puma expression in NHBECPro/Pro and NHBECArg/Arg cultures to determine their roles in the development of MCM. In addition, the role of these proteins will be examined by exposing noxa-/-, puma-/- and wild-type mice to ETS and by treating NHBECArg/Arg cultures with Noxa- and/or Puma-derived peptides. These studies will provide strategies for novel therapies that may facilitate the restoration of the aberrant repair process and reduce epithelial cell hyperplasia and MCM in chronic bronchitis. PUBLIC HEALTH RELEVANCE: Understanding why certain people are susceptible to developing chronic bronchitis will help identify the disease early and provide better treatment. We found that increased number of epithelial cells is associated with an increased number of mucous cells that are a source for increased mucous hypersecretion in chronic bronchitis. The proposed studies will elucidate the genetic variation that underlies sustained increase in the number of epithelial mucous cells in the airways. In addition, these studies will develop more effective treatments that are based on the mechanisms that normally reduce the number of mucous cells in the airway epithelium. Reducing epithelial cell hyperplasia may also help reduce the risk of developing lung cancer.