Regulation of Mucous Cell Metaplasia in Asthma

描述（由申请人提供）：哮喘患者在气道中出现粘液细胞，而气道通常缺乏这些细胞。这种表型被称为粘液细胞化生（MCM）。MCM具有严重的风险，因为急性分泌的粘液会减少气流，有时会完全阻塞气道，导致哮喘患者死亡。此外，流行病学研究表明，慢性MCM患者患肺癌的风险更高。在之前的资助期内，我们发现在长时间暴露于过敏原IFN？通过STAT1激活需要Bik，一种锚定在内质网（ER）中的蛋白质。Bak对IFN如此重要？来自bak-/-或甚至bak+/-原代细胞的气道上皮细胞具有抗性。我们发现Bik与160 kDa死亡相关蛋白激酶1 （DAPk）相互作用，拴住内质网和线粒体，协调ERK1/2和Bak的组装，激活Bak，并促进内质网钙的释放。我们生成了一种bik衍生的肽，类似于IFN？Bik激活Bak。在两项队列研究中，我们还在BIK基因的含子区发现了一个单核苷酸多态性（a G），该多态性降低了分化的正常人支气管上皮细胞（NHBEs）中BIK的表达水平，并与肺功能的显著下降有关。因此，这项更新应用的重点是描述Bik和DAPk的分子相互作用，以帮助优化生成改进的Bik衍生肽，这些肽可以恢复Bik变异个体的Bik功能，导致Bik水平降低并解决MCM。我们提出以下假设：IFN？-诱导的细胞死亡是通过Bik与DAPk结合介导的，从而促进Bak的激活，将ER Ca2+释放到线粒体，并引发细胞凋亡。此外，在具有导致Bik水平降低的Bik基因变体的受试者中恢复Bik水平可能是减少MCM的有效方法。我们将通过以下方法来验证这些假设：(1)阐明Bik和DAPk的分子相互作用，促进ERK1/2和Bak的组装，从而引起Bak的激活，并测试DAPk是否连接内质网和线粒体以协调内质网钙向线粒体的转移并破坏线粒体外膜的完整性。(2)与AA基因型相比，确定Bik衍生肽是否能恢复GG和AG NHBEs中MCM的溶解，以及Bik衍生肽是否能降低MCM并逆转暴露于过敏原的Bik +/+、Bik +/-和Bik- /-小鼠的肺功能下降。了解MCM分解过程的分子机制将使我们能够开发出更有效的治疗支气管炎哮喘和BIKGG基因型患者的肽。这种策略可能有助于最大限度地减少易感个体过多粘液的效果，并有助于靶向可能在增生性细胞中存活的肿瘤前细胞，以防止高危个体肺癌的发展。

DESCRIPTION (provided by applicant): In patients with asthma mucous cells appear in airways that are normally devoid of these cells. This phenotype is called mucous cell metaplasia (MCM). MCM poses a serious risk because acutely secreted mucus can reduce airflow or can sometimes completely obstruct the airways and lead to death of asthmatics. In addition, epidemiological studies suggest that patients with chronic MCM are at a higher risk of developing lung cancer. During the previous funding period, we discovered that that for the resolution of allergen-induced MCM during prolonged exposure to an allergen, IFN? through STAT1 activation requires Bik, a protein that is anchored in the endoplasmic reticulum (ER). Bak is so crucial for IFN?- or Bik-induced cell death that airway epithelial cells from bak-/- or even bak+/- primary cells are resistant. We found that Bik interacts with the 160 kDa death-associated protein kinase 1 (DAPk), to tether ER and mitochondria, orchestrate the assembly of ERK1/2 and Bak to activate Bak, and facilitate the release of ER calcium. We generated a Bik-derived peptide that similar to IFN? and Bik activates Bak. We also identified a single nucleotide polymorphism (A G) in the intronic region of the BIK gene that reduces Bik expression levels in differentiated normal human bronchial epithelial cells (NHBEs) and is associated with a significant decline in lung function in two cohort studies. Therefore, this renewal application is focused on delineating the molecular interaction of Bik and DAPk to help optimize the generation of improved Bik-derived peptides that restore Bik function in individuals with the BIK variant that causes reduced Bik levels and resolve MCM. We propose the following hypotheses: IFN?- induced cell death is mediated by Bik binding to DAPk to facilitate the activation of Bak, release ER Ca2+ to mitochondria, and initiation of apoptosis. Furthermore, restoring Bik levels in subjects with a BIK gene variant that causes reduced Bik levels can be an effective way to reduce MCM. We will test these hypotheses by: (1) Elucidating the molecular interaction of Bik and DAPk that facilitates assembly of ERK1/2 and Bak to cause activation of Bak, and by testing whether DAPk tethers ER and mitochondria to coordinate the transfer of ER calcium to mitochondria and disrupt the integrity of the mitochondrial outer membrane. (2) Determining whether a peptide derived from Bik restores resolution of MCM in NHBEs with GG and AG compared to AA genotype and whether a Bik-derived peptide reduces MCM and reverses decline in lung function in bik+/+, bik+/-, and bik-/- mice exposed to allergen. Understanding the molecular mechanisms of the resolution process of MCM will allow us to develop more effective peptides for patients with bronchitic asthma and the BIKGG genotype. This strategy may help maximize the effect of reducing excessive mucus in susceptible individuals and help target pre-neoplastic cells that may survive among hyperplastic cells to prevent the development of lung cancer in high-risk individuals.