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，该系统将在疾病状态下特异性地监测体内核因子-βB通路的激活。总体而言，核因子？B转录因子代表着一组重要但复杂的治疗靶点，可用于许多疾病，包括炎症性关节炎和癌症。核因子-βB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I？B1的降解。另一种途径中的控制步骤是I？B蛋白P100的部分降解，由激酶Nik启动。经典的核因子-β信号在正常生理中普遍存在，使其在疾病状态下的靶向变得复杂。替代的核因子：B激活似乎受到更多的限制，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的启动以及与炎症性关节炎相关的骨吸收都具有抵抗力，因此强调了替代的NF-B激活途径在疾病反应中的重要性。我们假设，由于I？B1和P100的降解分别代表了经典和替代的NF-B通路激活的主要调控步骤，因此监测这些事件的记者将成为研究关节炎背景下的NF-B信号转导和评估针对NF-B的药物的有力工具。为此，我们将VP16-Gal4BD(VG)，一种有效的转录激活因子，融合到P65或P100的N端。在静息细胞中，报告结构将通过p65和p100的正常控制机制保留在细胞质中。一旦通路激活，如细胞因子刺激，报告者将旅行到细胞核，在那里VG成分将反式激活Gal4反应元件驱动的Fluc基因。在这个系统中，Fluc信号与通路的激活成正比，具有很大的动态范围。目的1：体外构建P100加工和I？B1降解依赖的报告载体并进行验证。目的：建立VG-P100和VG-P65报告小鼠，分析炎性关节炎过程中替代的和经典的核因子-β信号转导途径。公共卫生相关性：这些研究提供了一种实时双色生物发光成像的新用途，可以非侵入性地监测已知在关节炎等炎症性疾病中重要的核因子-B通路。个体可以多次成像，在疾病的背景下生成新的核因子-β激活的动态图像，并了解各种干预措施如何在分子方面、在体内和实时地改变疾病的进展。这将代表着在理解疾病的病理生理学和药物作用的机制方面的重大进展。

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.