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

 描述（由申请人提供）：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。在气道上皮中，化生粘液细胞的数量受Bcl-2家族介导的细胞死亡，特别是Bcl-2和Bcl-2相互作用杀伤细胞（Bik）的调节。然而，尚不清楚Bcl-2和Bik是否调节炎症，或者这种调节发生的机制。初步结果表明，与bik+/+小鼠相比，香烟烟雾诱导的炎症在bik-/-小鼠中显著增加，表现为肺中巨噬细胞和淋巴细胞数量增加。此外，与对照相比，气道上皮中Bik的转基因表达显著减弱了变应原诱导的嗜酸性粒细胞增多症，证明了Bik的抗炎作用。为了确定Bik通过抑制促炎性转录因子核因子-κ B（NF-κB）来调节炎症，在GFP、野生型Bik、突变体Bik（BH 3 mutBik，亮氨酸61取代为丙氨酸使Bik不能诱导细胞死亡）或含有Bik的突变体BH 3结构域的肽的腺病毒表达后评估其活化。有趣的是，Bik和BH 3 mutBik在气道细胞系中的表达同等地抑制了响应于TNF-α刺激的NF-κB活化。在BH 3 mutBik存在下，NF-κ B的抑制与NF-κB与Bcl-2（Bik的已知结合伴侣）的结合一起发生。由于Bik和Bcl-2通过其BH 3结构域结合，因此在使用BH 3模拟物ABT-263阻断Bcl-2中的BH 3结构域后检查对NF-κB活化的影响。与对照组相比，ABT-263降低了NF-κB的活化，并增强了其与Bcl-2的结合。总的来说，这些数据表明Bcl-2的BH 3结构域的结合，无论是通过Bik还是BH 3模拟物，都阻断了NF-κB的核转位和炎症。本提案的具体目的是（1）证明Bik和Bcl-2调节NF-κB活化和炎症的分子机制，以及（2）测试Bik和Bcl-2在室内尘螨（HDM）诱导的体内炎症中的作用。将在体外气道上皮模型中调节Bik和Bcl-2的表达，并将产生Bik/Bcl-2肽和截短突变体以测试它们对NF-κB活化的影响。此外，Bik和BH 3 mutBik以及ABT-263的腺病毒构建体将在HDM暴露期间在小鼠肺中施用，以评价它们对HDM诱导的炎症的影响。根据该假设，预期BH 3 mutBik的过表达和Bcl-2的抑制将抑制肺中的HDM诱导的炎症。基于初步数据表明BH 3 mutBik抑制NF-κB信号传导而不依赖于细胞死亡，将确立Bcl-2蛋白的新作用。这些目标的实现不仅将描绘Bcl- 2蛋白和NF-κB信号传导的新机制，而且还将导致与肺部炎症性疾病高度相关的转化研究。

 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.