TRUSS as a novel regulator of inflammatory genes in asthma

TRUSS 作为哮喘炎症基因的新型调节剂

• 批准号: 8702540
• 负责人: OMAR TLIBA
• 金额: $ 19.38万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702540
• 关键词: Acetylation; Address; Affect; Allergic Reaction; Animals; Anti-Tumor Necrosis Factor Therapy; Antibodies; Asthma; Biochemical; Biochemistry; Biological Assay; Biology; Cells; Chronic; Clinical Trials; DNA; Development; Disease; Disease model; Eotaxin; Event; Foundations; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Goals; Healthcare; Human; IL8 gene; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Interferons; Interleukin-13; Interleukin-5; Knowledge; Laboratories; Link; Lung; Mediating; Mission; Molecular; Morbidity - disease rate; Muscle function; Mutate; NF-kappa B; Nature; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; RANTES; Regulation; Research; Research Personnel; Research Proposals; Role; Safety; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Small Interfering RNA; Smooth Muscle Myocytes; Stimulus; TNF gene; TNFRSF1A gene; TRADD gene; TRAF2 gene; Therapeutic; Time; Transactivation; Transcriptional Regulation; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-Beta; United States National Institutes of Health; airway inflammation; autocrine; base; burden of illness; cell transformation; cell type; chemokine; cytokine; design; expression vector; human RIPK1 protein; in vivo; insight; interest; mortality; novel; public health relevance; respiratory smooth muscle; scaffold; therapeutic target; trafficking

DESCRIPTION (provided by applicant): TNF-¿ is an inflammatory cytokine that affects multiple cells types in numerous inflammatory diseases. In asthma, TNF-¿ mediates inflammation through its action on cells that traffic into the lung and on resident lung cells, including airway smooth muscle (ASM) cells. Unfortunately, therapeutic targeting of TNF-¿ using anti-TNF-¿ antibodies has been disappointing due to safety concerns. Herein we propose to elucidate novel signaling pathways transduced by TNF-¿ via the scaffold protein TNFR1-Ubiquitous Scaffolding and Signaling Protein (TRUSS). We will extend previous limited analysis of TRUSS restricted to transformed cells to identify new signaling interactions and pathways in physiologically relevant cells, primary ASM cells. Importantly, we will also link these signaling events to regulation of expression of genes important in airway inflammation in the lung, thus identifying new mechanisms and targets through which ASM functions as an immunomodulatory cell. Our goal is to explore novel components of the TNF-¿ signaling pathways that may offer safer alternative therapeutic options for anti-TNF-¿ therapy. To this end, two aims in ASM cells will be explored: determine the unique contribution of endogenous TRUSS to the regulation of NF-kB activation (Aim 1) and ii) evaluate the selective contribution of endogenous TRUSS to the regulation of NF-kB-associated genes (Aim 2). In both aims, siRNA, mutated constructs and expression vectors will be used to modulate the expression of TRUSS, after which NF-kB-mediated transactivation activities, NF-kB phosphorylation and acetylation, in vitro kinase assays to assess IKK activity, phosphorylation of IKK¿/¿, NF-kB-DNA interactions, and the transcriptional regulation of NF-kB-dependent genes will be analyzed using state-of-the-art molecular and biochemical approaches already established in our laboratories. This comprehensive molecular approach is expected to expand our understanding of how TNF-¿ mechanistically regulates NF-kB-associated genes in physiologically relevant cells. Elucidating TRUSS-dependent signaling events opens up the opportunity for more selective targeting of TNF-driven events. Findings from these studies will provide new insight into fundamental TNF biochemistry, and provide an empirical basis to explore the role of TRUSS in more integrative disease model, with the ultimate goal of developing (safer) therapeutics that target specific TNF-¿ signals critical to inflammatory disease pathogenesis.

描述（由申请人提供）：TNF-¿ TNF-¿ 是一种炎症细胞因子，影响多种炎症疾病中的多种细胞类型。不幸的是，TNF-¿ 的治疗目标是通过其对进入肺部的细胞和驻留肺细胞（包括气道平滑肌 (ASM) 细胞）的作用来介导炎症。使用抗TNF-¿由于安全性问题，抗体一直令人失望。在此，我们建议阐明 TNF-¿ 转导的新信号通路。通过支架蛋白 TNFR1-无处不在的支架和信号蛋白 (TRUSS)，我们将扩展之前对转化细胞的 TRUSS 的有限分析，以识别生理相关细胞（原代 ASM 细胞）中的新信号相互作用和途径。我们的目标是探索 ASM 作为免疫调节细胞发挥作用的新机制和靶点。肿瘤坏死因子-¿信号通路可能为抗 TNF-¿ 提供更安全的替代治疗选择为此，将探索 ASM 细胞的两个目标：确定内源性 TRUSS 对 NF-kB 激活调节的独特贡献（目标 1）和 ii) 评估内源性 TRUSS 对 NF-kB 调节的选择性贡献。 kB 相关基因（目标 2），siRNA、突变构建体和表达载体将用于调节 TRUSS 的表达，然后使用 NF-kB 介导的反式激活。活性、NF-kB 磷酸化和乙酰化、评估 IKK 活性的体外激酶测定、IKK 磷酸化¿ /¿ 、NF-kB-DNA 相互作用以及 NF-kB 依赖性基因的转录调控将使用我们实验室已经建立的最先进的分子和生化方法进行分析，这种全面的分子方法有望扩大我们的理解。 TNF-¿阐明 TRUSS 依赖性信号传导事件的机制为更选择性地靶向 TNF 驱动的事件提供了机会，这些研究结果将为 TNF 的基本生物化学提供新的见解，并提供经验。为探索 TRUSS 在更综合的疾病模型中的作用奠定基础，最终目标是开发针对特定 TNF-¿ 的（更安全）疗法对炎症性疾病发病机制至关重要的信号。