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，专门监测NF-？B途径在疾病状态下的体内。一般来说，NF-？B转录因子是许多疾病的重要但复杂的治疗靶点，包括炎症性关节炎和癌症。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IKK2激活导致ib1的降解。替代途径中的受控步骤是I？B蛋白p100，由激酶NIK启动。古典NF - ?B信号在正常生理中普遍存在，使其在疾病状态中的靶向变得复杂。替代的NF-:B激活似乎更受限制，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和与炎症性关节炎相关的骨吸收都有抵抗力，从而强调了NF-？B激活途径在疾病反应中的作用。我们假设，自从I？B1和p100代表经典和替代NF-?激活的主要调控步骤。监测这些事件的记者将成为研究NF-？B信号在关节炎中的作用，以及对NF- B靶向药物的评价。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端。在静息细胞中，报告基因通过p65和p100的正常调控机制保留在细胞质中。当通路被激活（如细胞因子刺激）时，报告子将到达细胞核，在那里VG成分将反激活gal4反应元件驱动的Fluc基因。在该系统中，Fluc信号与通路激活成正比，具有较宽的动态范围。目标1：生成和验证p100-processing和I？b1降解依赖性报告基因的体外构建。目的2:VG-p100和VG-p65报告小鼠的生成及替代性和经典NF-？体内炎性关节炎中的B信号传导。公共卫生相关性：这些研究为实时双色生物发光成像无创监测NF-？已知B通路在关节炎等炎症性疾病中很重要。个体可以多次成像，生成新的NF-？疾病背景下的B活化，以及了解各种干预措施如何从分子角度、体内和实时地改变疾病进展。这将在理解疾病的病理生理和药物作用的机制方面取得重大进展。

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.