Resolution of Epithelial Cell Hyperplasia

描述（由申请人提供）：炎症反应引起的上皮细胞增生伴随着粘膜细胞化生（MCM），气道中通常缺乏这些细胞的粘膜细胞的外观。MCM是慢性支气管炎中粘液分泌增多的主要因素之一。我们发现凋亡抑制剂Bcl-2在大鼠和小鼠中维持MCM，可能具有临床意义，因为它在囊性纤维化或慢性支气管炎患者的黏液细胞中表达。p53基因密码子72 （Arg/Pro）多态性差异影响Bcl-2的表达；p53Pro比p53Arg更有效地增加，p53Arg是减少bcl-2 mRNA半衰期的促凋亡蛋白Noxa。删除或突变小鼠p53相应区域的脯氨酸残基在小鼠胚胎成纤维细胞中具有相同的效果。与这些发现一致，携带p53Pro/Pro基因型的吸烟者患慢性支气管炎的风险低于携带p53Arg/Arg基因型的吸烟者。来自5个Arg/Arg和5个Pro/Pro个体的正常人支气管上皮细胞（NHBECs）的初步结果表明，在未处理状态下，NHBECArg/Arg培养物与NHBECPro/Pro培养物相比，Noxa mRNA水平降低，而当培养物被环境香烟烟雾（ETS）处理时，Puma mRNA水平降低。因此，我们希望通过诱导Noxa表达介导Bcl- 2水平的降低，来验证p53Pro/Pro变异体支气管细胞比p53Arg/Arg支气管细胞更有效地解决上皮细胞增生和MCM的中心假设。特异性目的1将确定分化后的NHBECArg/Arg、NHBECArg/Pro和NHBECPro/Pro培养是否在分泌粘液、MUC5AC、Noxa和Puma mRNA表达以及暴露于ETS提取物前后MCM的发展方面存在差异。同样，我们将检测野生型和突变型p53小鼠的气管上皮细胞在未处理状态和ETS提取物处理后的分泌粘液、Muc5ac、Noxa和Puma mrna以及MCM的表达。此外，将五个具有代表性的NHBECArg/Arg和NHBECPro/Pro培养物分别用表达Ad-p53Pro或Ad-p53Arg的腺病毒载体感染，以确定这些NHBECs的表型是否会被调节。特异性Aim 2将增加和减少NHBECPro/Pro和NHBECArg/Arg培养中Noxa和/或Puma的表达，以确定它们在MCM发展中的作用。此外，这些蛋白的作用将通过将noxa-/-、puma-/-和野生型小鼠暴露于ETS和用noxa-和/或puma衍生肽处理NHBECArg/Arg培养物来检验。这些研究将为新疗法提供策略，这些新疗法可能促进慢性支气管炎异常修复过程的恢复，减少上皮细胞增生和MCM。公共卫生相关性：了解为什么某些人易患慢性支气管炎将有助于及早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黏液细胞数量的增加有关，黏液细胞数量的增加是慢性支气管炎黏液分泌增多的一个来源。提出的研究将阐明气道上皮黏液细胞数量持续增加的遗传变异。此外，这些研究将基于通常减少气道上皮粘液细胞数量的机制，开发更有效的治疗方法。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.