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，将专门监测NF-？疾病状态下的体内B途径。一般来说，NF-？B转录因子代表了许多疾病（包括炎性关节炎和癌症）的一组重要但复杂的治疗靶点。NF-？B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IKK 2激活导致I？B1.在替代途径的控制步骤是部分降解的I？B蛋白p100，由激酶NIK启动。经典NF-？B信号传导在正常生理学中普遍存在，使其在疾病状态中的靶向复杂化。替代NF-：B活化似乎受到更多限制，为药物靶向提供潜在益处。我们已经表明，小鼠缺乏NIK是抵抗淋巴细胞介导的关节炎的启动，并与炎症性关节炎骨吸收，从而强调了替代NF-？B激活途径在疾病反应。我们假设，既然I？B1和p100代表的经典和替代NF-？B途径，分别，记者监测这些事件将是强大的工具，NF-？B信号的背景下，关节炎，并评估药物靶向NF-？B。为此，我们将VP 16-Gal 4 BD（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。在途径激活后，例如在细胞因子刺激下，报告基因将行进至细胞核，在那里VG组分将反式激活Gal 4应答元件驱动的Fluc基因。在该系统中，Fluc信号与通路激活成比例，具有宽的动态范围。目的1：生成和验证p100处理和I？体外B1降解依赖性报告基因构建体。目的2：建立VG-p100和VG-p65报告基因小鼠，并分析替代和经典NF-？体内炎症性关节炎期间的B信号传导。公共卫生相关性：这些研究提供了一种新的使用实时2色生物发光成像非侵入性监测NF-？已知B途径在炎性疾病如关节炎中很重要。个人可以多次成像，产生一个新的动态图片NF-？疾病背景下的B激活，并了解各种干预措施如何在分子水平、体内和真实的时间内改变疾病进展。这将是理解疾病和药物作用的病理生理机制的一个重大进展。

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.