IN VIVO 2-COLOR BIOLUMINESCENT IMAGING OF CLASSICAL AND ALTERNATIVE NF-KB

描述（由申请人提供）：我们建议开发一种体内报告系统，该系统将专门监测疾病状态下体内 NF-κB 通路的激活。一般来说，NF-κB 转录因子代表了许多疾病（包括炎症性关节炎和癌症）的一组重要但复杂的治疗靶点。 NF-κB 使用两种不同的激活途径：经典途径和替代途径。在经典途径中，配体诱导的 IKK2 激活导致 IκB1 降解。旁路途径中的受控步骤是 IκB 蛋白 p100 的部分降解，由激酶 NIK 启动。经典的 NF-κB 信号传导在正常生理学中普遍存在，这使其在疾病状态下的靶向变得复杂。替代性 NF-:B 激活似乎受到更多限制，为药物靶向提供了潜在的好处。我们已经证明，缺乏 NIK 的小鼠对淋巴细胞介导的关节炎起始和与炎症性关节炎相关的骨吸收具有抵抗力，因此强调了疾病反应期间替代 NF-κB 激活途径的重要性。我们假设，由于 I?B1 和 p100 的降解分别代表经典和替代 NF-?B 途径激活的主要调节步骤，监测这些事件的报告基因将成为研究关节炎背景下 NF-?B 信号传导以及评估靶向 NF-?B 药物的有力工具。为此，我们将 VP16-Gal4BD (VG)（一种有效的转录激活剂）融合到 p65 或 p100 的 N 末端。在静息细胞中，报告构建体将通过 p65 和 p100 的正常控制机制保留在细胞质中。在细胞因子刺激等途径激活后，报告分子将前往细胞核，在那里 VG 成分将反式激活 Gal4 响应元件驱动的 Fluc 基因。在该系统中，Fluc 信号与通路激活成正比，具有广泛的动态范围。目标 1：体外 p100 加工和 I?B1 降解依赖性报告基因构建体的生成和验证。目标 2：生成 VG-p100 和 VG-p65 报告小鼠，并分析体内炎症性关节炎期间的替代和经典 NF-κB 信号传导。公共健康相关性：这些研究提供了一种实时 2 色生物发光成像的新用途，用于无创监测 NF-κB 通路，已知该通路在关节炎等炎症性疾病中很重要。可以对个体进行多次成像，生成疾病背景下 NF-κB 激活的新动态图像，并了解各种干预措施如何在分子层面、体内实时改变疾病进展。这将代表着理解疾病病理生理学和药物作用机制的重大进展。

DESCRIPTION (provided by applicant): We propose to develop an in vivo reporter system that will specifically monitor activation of NF-?B pathways in vivo in disease states. In general, NF-?B transcription factors represent an important, but complex, set of therapeutic targets for many diseases, including inflammatory arthritis and cancer. NF-?B uses two distinct routes of activation, the classical and alternative pathways. In the classical pathway, ligand-induced activation of IKK2 leads to degradation of I?B1. The controlled step in the alternative pathway is the partial degradation of the I?B protein p100, initiated by the kinase NIK. Classical NF-?B signaling is ubiquitous in normal physiology, complicating its targeting in disease states. Alternative NF-:B activation appears to be much more restricted, providing potential benefits for drug targeting. We have shown that mice lacking NIK are resistant to both lymphocyte-mediated initiation of arthritis, and to bone resorption associated with inflammatory arthritis, thus emphasizing the importance of the alternative NF-?B activation pathway during the disease response. We hypothesize that, since the degradation of I?B1 and p100 represent the principal regulated steps in activation of the classical and alternative NF-?B pathways, respectively, reporters monitoring these events will be powerful tools for the study of NF-?B signaling in the context of arthritis, and for evaluating drugs targeting NF-?B. To this end, we will fuse VP16-Gal4BD (VG), a potent transcriptional activator, to the N-terminus of either p65 or p100. In resting cells the reporter constructs will be retained in the cytoplasm by the normal control mechanisms for p65 and p100. Upon pathway activation such as with cytokine stimulation, the reporters will travel to the nucleus where the VG component will transactivate a Gal4-response element driven Fluc gene. In this system, the Fluc signal is proportional to pathway activation, with a broad dynamic range. Aim 1: Generation and validation of p100-processing and I?B1-degradation dependent reporter constructs in vitro. Aim 2: Generation of VG-p100 and VG-p65 reporter mice and analysis of alternative and classical NF-?B signaling during inflammatory arthritis in vivo. PUBLIC HEALTH RELEVANCE: These studies provide a novel use of real-time 2-color bioluminescence imaging to non-invasively monitor NF-?B pathways known to be important in inflammatory diseases such as arthritis. Individuals can be imaged multiple times, generating a new dynamic picture of NF-?B activation in the context of disease, and to understand how various interventions modify disease progression in molecular terms, in vivo and in real time. This will represent a major advance in understanding the mechanisms of both the pathophysiology of disease and drug actions.