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(申请人提供)：肿瘤坏死因子是一种炎性细胞因子，在多种炎症性疾病中影响多种细胞类型。在哮喘中，肿瘤坏死因子通过作用于进入肺部的细胞和驻留在肺内的细胞，包括呼吸道平滑肌(ASM)细胞来调节炎症。不幸的是，由于安全方面的考虑，使用抗肿瘤坏死因子抗体进行肿瘤坏死因子的靶向治疗一直令人失望。在这里，我们建议阐明新的信号转导途径的肿瘤坏死因子-β通过支架蛋白TNFR1-无所不在的支架和信号蛋白(Truss)。我们将把先前对Truss的有限分析扩展到转化细胞，以确定生理相关细胞、原代ASM细胞中新的信号相互作用和通路。重要的是，我们还将把这些信号事件与调节肺部呼吸道炎症中重要基因的表达联系起来，从而确定ASM作为免疫调节细胞发挥作用的新机制和新靶点。我们的目标是探索肿瘤坏死因子信号通路的新成分，为抗肿瘤坏死因子治疗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对调节NF-kB激活的独特贡献(目标1)和评估内源性Truss对调节NF-kB相关基因的选择性贡献(目标2)。在这两个目标中，将使用siRNA、突变的构建体和表达载体来调节Truss的表达，之后将使用我们实验室已建立的最先进的分子和生化方法来分析NF-kB介导的反式激活活性、NF-kB的磷酸化和乙酰化、体外激酶试验以评估IKK活性、IKK/β的磷酸化、NF-kB-DNA的相互作用以及NF-kB依赖的基因的转录调控。这一全面的分子方法有望扩大我们对肿瘤坏死因子-1是如何机械地调节生理相关细胞中的核因子-kB相关基因的理解。阐明Truss依赖的信号事件为更有选择性地靶向肿瘤坏死因子驱动的事件打开了机会。这些研究的结果将为基础的肿瘤坏死因子生物化学提供新的见解，并为探索Truss在更综合的疾病模型中的作用提供经验基础，最终目标是开发针对炎症性疾病发病机制关键的特定肿瘤坏死因子-β信号的(更安全的)疗法。