Bik and Bcl-2 regulate Nuclear Factor-kappaB-mediated inflammation

Bik 和 Bcl-2 调节核因子 kappaB 介导的炎症

基本信息

批准号：
9403355
负责人：
Jane Tully Jones
金额：
$ 0.14万
依托单位：
LOVELACE BIOMEDICAL & ENVIRONMENTAL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-30 至 2018-06-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9403355
关键词：
Address Affect Alanine Alleles Allergens Allergic Reaction Allergic inflammation Amino Acids Anti-Inflammatory Agents Anti-inflammatory Apoptosis Apoptotic Attenuated BCL-2 Protein BCL2 gene BH3 Domain BH3 peptide Bacterial Infections Binding Cell Culture Techniques Cell Death Cell Line Cells Chronic Chronic Bronchitis Data Disease Eosinophilia Epithelial Epithelial Cells Exposure to Extrinsic asthma Family Gene Expression Microarray Analysis Genetic Transcription Health Human Immunofluorescence Immunologic In Vitro Infiltration Inflammation Inflammatory Inflammatory Response Laboratories Lasers Lead Leucine Lung Lung diseases Lymphocyte Mediating Metaplasia Metaplastic Microdissection Modeling Molecular Molecular Conformation Mucous body substance Mus Nuclear Nuclear Translocation Pathologic Pathway interactions Patients Peptides Pneumonia Production Protein Family Proteins Pyroglyphidae Regulation Role Scanning Signal Transduction Structure of parenchyma of lung TNF gene Testing Therapeutic Transgenic Organisms Translational Research Wild Type Mouse airway epithelium airway inflammation base cigarette smoke-induced cigarette smoking cytokine experimental study expression vector in vivo inflammatory lung disease macrophage mimetics mouse model mutant novel overexpression public health relevance response small molecule transcription factor translational impact

项目摘要

DESCRIPTION (provided by applicant): Chronic inflammation and mucous cell metaplasia are common pathologic features of the majority of pulmonary diseases, including allergic asthma, pneumonia, and chronic bronchitis. The number of metaplastic mucous cells is regulated by Bcl-2 family-mediated cell death, specifically Bcl-2 and Bcl-2 interacting killer (Bik), in airway epithelia. However, it is not known whether Bcl-2 and Bik regulate inflammation, or the mechanism by which this regulation occurs. Preliminary results demonstrated that cigarette smoke -induced inflammation is significantly increased in bik-/- compared to bik+/+ mice, shown by increased numbers of macrophages and lymphocytes in the lung. Additionally, transgenic expression of Bik in airway epithelia significantly attenuated allergen-induced eosinophilia in comparison to controls, demonstrating an anti- inflammatory role for Bik. In order to determine of Bik modulates inflammation through inhibition of the pro- inflammatory transcription factor, Nuclear Factor-kappaB (NF-κB), its activation was assessed after adenoviral expression of GFP, wild type Bik, mutant Bik (BH3mutBik, Leucine 61to alanine substitution renders Bik unable to induce cell death), or a peptide containing the mutant BH3 domain of Bik . Interestingly, expression of both Bik and BH3mutBik equally in an airway cell line suppressed NF-κB activation in response to TNF-α stimulation. Inhibition of NF-κB in the presence of BH3mutBik occurred in conjunction with NF-κB's association with Bcl-2, a known binding partner of Bik. Because Bik and Bcl-2 associate via their BH3 domains, the effect on NF-κB activation was examined after blocking of the BH3 domain in Bcl-2 using the BH3 mimetic, ABT-263. ABT-263 reduced activation of NF-κB and enhanced its association to Bcl-2 in comparison to controls. Collectively, these data suggest that binding of the BH3 domain of Bcl-2, either by Bik or a BH3 mimetic, blocks nuclear translocation of NF-κB and inflammation. Specific aims of this proposal will (1) demonstrate the molecular mechanism by which Bik and Bcl-2 regulate NF-κB activation and inflammation and (2) test the role of Bik and Bcl-2 following House Dust Mite (HDM)-induced inflammation in vivo. Expression of Bik and Bcl-2 will be modulated in in vitro airway epithelial models, and Bik/Bcl-2 peptides and truncation mutants will be generated in order to test their impact on NF-κB activation. Additionally, adenoviral constructs of Bik and BH3mutBik, and ABT-263, will be administered in the mouse lung during HDM exposure to evaluate their impact on HDM-induced inflammation. According to the hypothesis, it is anticipated that both overexpression of BH3mutBik and inhibition of Bcl-2 will dampen HDM-induced inflammation in the lung. Based on preliminary data demonstrating that the BH3mutBik dampens NF-κB signaling independent of cell death, a novel role for Bcl-2 proteins will be established. Completion of these aims will not only delineate a novel mechanism for Bcl- 2 proteins and NF-κB signaling, but will also result in highly relevant translational research for inflammatory diseases of the lung.

描述（申请人提供）：慢性炎症和粘液细胞化生是大多数肺部疾病的常见病理特征，包括过敏性哮喘、肺炎和慢性支气管炎。化生粘液细胞的数量受Bcl-2家族介导的细胞调节。气道上皮细胞中的死亡，特别是 Bcl-2 和 Bcl-2 杀伤剂 (Bik)。然而，尚不清楚 Bcl-2 和 Bik 是否调节炎症或其机制。初步结果表明，与 bik+/+ 小鼠相比，bik-/- 小鼠中香烟烟雾引起的炎症显着增加，这可以通过肺部巨噬细胞和淋巴细胞数量的增加来证明。此外，气道上皮细胞中 Bik 的转基因表达也有所增加。与对照相比，显着减弱了过敏原诱导的嗜酸性粒细胞增多，证明了 Bik 的抗炎作用。为了确定 Bik 通过抑制促炎转录因子来调节炎症，核因子-kappaB (NF-κB)，在腺病毒表达 GFP、野生型 Bik、突变型 Bik（BH3mutBik，亮氨酸 61 至丙氨酸取代使 Bik 无法诱导细胞死亡）或包含突变型 BH3 结构域的肽后评估其激活Bik 表明，Bik 和 BH3mutBik 在气道细胞系中的表达同样抑制了 TNF-α 的 NF-κB 激活。 BH3mutBik 存在时对 NF-κB 的抑制作用与 NF-κB 与 Bcl-2（Bik 的已知结合伙伴）的结合有关，因为 Bik 和 Bcl-2 通过其 BH3 结构域结合，从而对 NF-κB 产生影响。使用 BH3 模拟物 ABT-263 阻断 Bcl-2 中的 BH3 结构域后，检查了激活情况，ABT-263 减少了 NF-κB 和 NF-κB 的激活。总体而言，这些数据表明，Bik 或 BH3 模拟物与 Bcl-2 的 BH3 结构域的结合可阻断 NF-κB 的核易位和炎症。该提案将 (1) 证明 Bik 和 Bcl-2 调节 NF-κB 激活和炎症的分子机制，以及 (2) 测试 Bik 和 Bcl-2 在屋尘螨 (HDM) 诱导的炎症后的作用Bik 和 Bcl-2 的表达将在体外气道上皮模型中进行调节，并且将生成 Bik/Bcl-2 肽和截短突变体，以测试它们对 NF-κB 激活的影响。 BH3mutBik 和 ABT-263 将在 HDM 暴露期间注射到小鼠肺部，以评估它们对 HDM 诱导的炎症的影响。预计 BH3mutBik 的过度表达和 Bcl-2 的抑制都会抑制 HDM 诱导的肺部炎症。初步数据表明，BH3mutBik 抑制 NF-κB 信号传导与细胞死亡无关，因此 Bcl-2 蛋白的新作用将是。这些目标的完成不仅将描绘出 Bcl-2 蛋白和 NF-κB 信号传导的新机制，还将导致肺部炎症疾病的高度相关的转化研究。