Gene Specific Regulation of NFkB by Nuclear IkBa in Inflammation and Cancer

描述(由申请者提供)：我们的长期目标是了解依赖于核因子？B的抗凋亡和促炎基因的转录调控机制。由于在许多炎症性疾病中，以及在许多类型的人类癌症和白血病中，核因子B的活性和受核因子B调节的促炎和抗凋亡基因的表达增加，因此抑制核因子B依赖的转录成为一个重要的治疗靶点。然而，关于核因子？B抑制剂的主要问题之一是它们的特异性，因为导致核因子？B激活的许多步骤对其他细胞功能也是重要的。我们以前的研究表明，诱导核转位和核转录因子B抑制剂I？B？能抑制体外培养的人白细胞和癌细胞的核转录因子B DNA结合活性，并诱导其凋亡。重要的是，我们最近的数据表明，在体内抑制核转录因子B依赖的转录。是基因特异性的：虽然人白细胞中促炎细胞因子TNF？、IL-12和IL-6的转录受到核I？B？的抑制，但IL-8的转录不受抑制。然而，目前调控I、B、B细胞体内核相互作用的机制尚不清楚。对于核因子？B蛋白和依赖于核因子？B的启动子是未知的。这一建议的中心假设是，核转录因子I、B、B对促炎症基因和抗细胞凋亡基因转录的调节作用。是基因特异性的，因此可以为针对核I、B、B核特异性抑制核因子B活性的新型抗炎和抗癌治疗提供基础。具体目的在于分析I、B、B、C、C三种化合物的体内结合机制。与核因子？B二聚体和核因子？B启动子结合，从而调节核因子？B依赖的转录。在目标1中，我们将通过染色质免疫沉淀法测定NF？B和I？B？人U937巨噬细胞和白血病Hut-78细胞中核因子B调节的促炎和抗凋亡基因启动子的蛋白质。我们将确定核子I、B、B的能力。抑制核因子B依赖的转录依赖于核因子B二聚体的亚基组成。在目标2中，我们将确定在U937巨噬细胞和HUT-78白血病细胞中调控核I？B-NF？B相互作用的机制，并验证可能的机制涉及p65 NF？B的翻译后修饰(S)和/或蛋白酶体的基因特异性募集。核转录调控核转录因子B依赖基因特异性转录机制的研究可能会提供一种使用核弹的新战略？作为依赖于核因子？B的转录的特异性调节因子。这种方法可以同时适用于抗炎和抗癌治疗。此外，该项目将通过为本科生提供大量学习生物医学研究基础知识的机会来改善圣约翰大学的研究环境。 与公共卫生的相关性：这项建议侧重于核抑制蛋白I、B、B的能力调节机制。结合核因子？B调节的启动子，抑制核因子？B依赖的炎症和促生存基因的转录。由于包括炎症性疾病、癌症和白血病在内的许多人类疾病中的核因子？B活性升高，因此对核转录因子I？B？B的作用机制进行了研究。抑制核因子B依赖的转录将导致更特异的抗炎和抗癌治疗的发展。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms regulating transcription of NF?B-dependent anti-apoptotic and pro-inflammatory genes. Since NF?B activity and expression of NF?B-regulated pro-inflammatory and anti-apoptotic genes are increased in many inflammatory diseases, as well as in many types of human cancer and leukemia, inhibition of NF?B-dependent transcription thus represents an important therapeutic target. However, one of the main concerns regarding the NF?B inhibitors is their specificity, since many steps leading to NF?B activation are important for other cellular functions as well. Our previous studies have shown that an induction of nuclear translocation and accumulation of the NF?B inhibitor, I?B?, inhibits the in vitro NF?B DNA binding activity and induces apoptosis in human leukocytes and cancer cells. Importantly, our recent data indicate that the in vivo inhibition of NF?B-dependent transcription by nuclear I?B? is gene specific: while the transcription of pro-inflammatory cytokines TNF?, IL-12 and IL-6 in human leukocytes is inhibited by nuclear I?B?, transcription of IL-8 is not. However, at present, the mechanisms that regulate the in vivo nuclear interaction of I?B? with NF?B proteins and NF?B- dependent promoters are unknown. The central hypothesis of this proposal is that the regulation of NF?B-dependent transcription of pro-inflammatory and anti-apoptotic genes by nuclear I?B? is gene specific, and could thus provide a basis for novel anti-inflammatory and anti-cancer therapies aimed at the specific inhibition of NF?B activity by nuclear I?B?. The specific aims focus on analyzing the mechanisms that regulate the in vivo binding of I?B? to NF?B dimers and NF?B promoters, thus regulating NF?B-dependent transcription. In Aim 1, we will measure by chromatin immunoprecipitation the recruitment of NF?B and I?B? proteins to NF?B-regulated promoters of pro-inflammatory and anti-apoptotic genes in human U937 macrophages and leukemia Hut-78 cells. We will determine whether the ability of nuclear I?B? to inhibit NF?B-dependent transcription depends on the subunit composition of NF?B dimers. In Aim 2, we will identify the mechanisms that regulate the nuclear I?B?-NF?B interaction in U937 macrophages and Hut-78 leukemia cells, and we will test the hypothesis that the responsible mechanisms involve post-translational modification(s) of p65 NF?B, and/or gene specific recruitment of the proteasome. Identification of the mechanisms that regulate the gene specific transcription of NF?B-dependent genes by nuclear I?B? might provide a new strategy that would use the nuclear I?B? as a specific regulator of NF?B-dependent transcription. This approach could be applicable in both anti-inflammatory and anti-cancer therapies. In addition, this project will enhance the research environment at St. John's University by providing undergraduate students with numerous opportunities to learn the fundamentals of biomedical research. PUBLIC HEALTH RELEVANCE: This proposal focuses on the mechanisms that regulate ability of the nuclear inhibitory protein I?B? to associate with NF?B-regulated promoters and inhibit transcription of NF?B-dependent inflammatory and pro-survival genes. Since NF?B activity is increased in many human diseases including inflammatory disorders, cancer, and leukemia, identification of the mechanisms by which the nuclear I?B? inhibits NF?B-dependent transcription will lead to the development of more specific anti-inflammatory and anti-cancer therapies.