Regulation of Mucous Cell Metaplasia in Asthma

描述（由申请人提供）：在哮喘患者中，粘液细胞出现在通常没有这些细胞的气道中。这种表型称为粘液细胞化生（MCM）。MCM造成严重的风险，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。在之前的资助期间，我们发现，为了在长期暴露于过敏原期间解决过敏原诱导的MCM，IFNγ通过STAT 1激活需要Bik，一种锚定在内质网（ER）中的蛋白质。巴克对IFNγ或Bik诱导的细胞死亡至关重要，因此来自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的死亡相关蛋白激酶1（DAPk）相互作用，将ER和线粒体拴在一起，协调ERK 1/2和巴克的组装以激活巴克，并促进ER钙的释放。我们产生了一种类似于IFNγ和Bik的Bik衍生肽，其激活巴克。我们还确定了BIK基因内含子区的单核苷酸多态性（A→G），该多态性降低了分化的正常人支气管上皮细胞（NHBE）中的Bik表达水平，并与两项队列研究中肺功能的显著下降相关。因此，该更新申请的重点是描述Bik和DAPk的分子相互作用，以帮助优化改进的Bik衍生肽的生成，这些肽在具有导致Bik水平降低的BIK变体的个体中恢复Bik功能并解决MCM。我们提出以下假设：IFNγ诱导的细胞死亡是由Bik与DAPk结合介导的，以促进巴克的激活，释放ER Ca 2+到线粒体，并启动凋亡。此外，恢复具有导致Bik水平降低的BIK基因变体的受试者的Bik水平可以是减少MCM的有效方式。我们将通过以下方式检验这些假设：（1）阐明Bik和DAPk的分子相互作用，其促进ERK 1/2和巴克的组装以引起巴克的活化，并通过测试DAPk是否束缚ER和线粒体以协调ER钙向线粒体的转移并破坏线粒体外膜的完整性。(2)确定与AA基因型相比，衍生自Bik的肽是否恢复具有GG和AG的NHBE中MCM的消退，以及衍生自Bik的肽是否减少暴露于过敏原的bik+/+、bik+/-和bik-/-小鼠中的MCM并逆转肺功能的下降。了解MCM的分解过程的分子机制将使我们能够为支气管哮喘和BIKGG基因型患者开发更有效的肽。这种策略可能有助于最大限度地减少易感个体中过量粘液的效果，并有助于靶向可能在增生细胞中存活的肿瘤前细胞，以防止高危个体发生肺癌。

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, IFNγ through STAT1 activation requires Bik, a protein that is anchored in the endoplasmic reticulum (ER). Bak is so crucial for IFNγ- 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 IFNγ 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: IFNγ-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.