TRUSS as a novel regulator of inflammatory genes in asthma

描述（由申请人提供）：TNF-是一种炎症细胞因子，在许多炎症性疾病中影响多种细胞类型。在哮喘中，TNF-¿通过作用于进入肺部的细胞和驻留的肺细胞，包括气道平滑肌（ASM）细胞，介导炎症。不幸的是，由于安全问题，使用抗TNF-抗体靶向治疗TNF-¿一直令人失望。在此，我们提出通过支架蛋白tnfr1 -无处不在的支架和信号蛋白（TRUSS）阐明由TNF-¿转导的新信号通路。我们将扩展之前局限于转化细胞的TRUSS的有限分析，以识别生理相关细胞（原代ASM细胞）中的新的信号相互作用和途径。重要的是，我们还将这些信号事件与肺部气道炎症中重要基因的表达调节联系起来，从而确定ASM作为免疫调节细胞功能的新机制和靶点。我们的目标是探索TNF-信号通路的新成分，这可能为抗TNF-治疗提供更安全的替代治疗选择。为此，我们将探索ASM细胞中的两个目标：确定内源性TRUSS对NF-kB活化调节的独特贡献（目的1）和ii)评估内源性TRUSS对NF-kB相关基因调节的选择性贡献（目的2）。在这两个目标中，siRNA、突变构建体和表达载体将用于调节TRUSS的表达，之后，NF-kB介导的转激活活性、NF-kB磷酸化和乙酰化、体外激酶分析以评估IKK活性、IKK磷酸化、NF-kB- dna相互作用以及NF-kB依赖基因的转录调节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对TNF-¿如何在生理相关细胞中机制调节nf - kb相关基因的理解。阐明依赖于truss的信号事件为更有选择性地靶向tnf驱动事件提供了机会。这些研究的发现将为TNF生化基础提供新的见解，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（更安全的）靶向炎性疾病发病机制中关键的特定TNF-信号的治疗方法。

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.