Nuclear Translocation of I-kappaB-alpha as a Therapeutic Target

描述(申请人提供)：NFkB是一种关键的转录因子，调节促炎和抗凋亡基因的表达。本实验室以前的研究表明，与其他类型的细胞不同，静息状态的人中性粒细胞在细胞核中含有大量的NFkB抑制剂IkBA，这种增加的核积聚导致NFkB活性的抑制和中性粒细胞凋亡率的增加。我们最近的数据表明，蛋白酶体抑制诱导白血病HL-60和U-937细胞以及癌症HeLa细胞中的IkBA转位到细胞核；然而，其机制目前尚不清楚。这一假设的中心假设是，诱导IkBA的核积聚抑制了NFkB的活性和NFkB调节的抗凋亡和促炎基因的表达，从而为以抑制核IkBA的NFkB活性为目标的新型抗癌和抗炎治疗提供了基础。具体目的是分析核IkBA抑制白血病HL-60和U-937细胞中NFkB活性和NFkB调控基因表达的机制。在目标1中，我们将通过将抑制的Ikba RNA导入HL-60和U-937细胞来验证这样的假设，即蛋白酶体抑制导致IkBA的核转位导致NFkB活性的抑制和NFkB调节的抗凋亡基因的表达减少。在目标2中，我们将使用染色质免疫沉淀来验证核IkBA通过与NFkB调节基因的启动子结合来抑制NFkB活性的假设。在目标3中，我们将研究蛋白酶体抑制诱导的IkBA核转位的调控机制。通过核IkBA识别控制NFkB活性的关键分子事件将拓宽我们对NFkB活性调控机制的理解，并可能为调控NFkB介导的促炎和抗凋亡基因的表达提供一类新的药物靶点。这一建议的重点是确定IkBA的核转位调节转录因子NFkB活性的机制。由于NFkB活性在包括炎症性疾病、癌症和白血病在内的许多人类疾病中都升高，因此确定核IkBA抑制NFkB活性的机制将有助于开发新的抗癌和抗炎疗法。

DESCRIPTION (provided by applicant): NFkB is a critical transcription factor regulating expression of pro-inflammatory and anti- apoptotic genes. Previous studies from our laboratory demonstrated that as opposed to other cell types, resting human neutrophils contain predominant amount of NFkB inhibitor, IkBa, in the nucleus, and this increased nuclear accumulation of IkBa results in the inhibition of NFkB activity and increased rate of neutrophil apoptosis. Our recent data have shown that proteasome inhibition induces translocation of IkBa to the nucleus in leukemia HL-60 and U- 937 cells, and in cancer HeLa cells; however, the mechanisms are currently unknown. The central hypothesis of this proposal is that induction of nuclear accumulation of IkBa inhibits NFkB activity and expression of NFkB-regulated anti-apoptotic and pro-inflammatory genes, and could thus provide a basis for novel anti-cancer and anti-inflammatory therapies aimed at the inhibition of NFkB activity by the nuclear IkBa. The specific aims focus on analyzing the mechanisms by which the nuclear IkBa inhibits NFkB activity and expression of NFkB-regulated genes in leukemia HL-60 and U-937 cells. In Aim 1, we will test the hypothesis that the proteasome inhibition-induced nuclear translocation of IkBa results in the inhibition of NFkB activity and decreased expression of NFkB-regulated anti-apoptotic genes, by using transfection of HL-60 and U-937 cells with inhibitory IkBa RNA. In Aim 2, we will use chromatin immunoprecipitation to test the hypothesis that the nuclear IkBa inhibits NFkB activity by associating with the promoters of NFkB-regulated genes. In Aim 3, we will investigate the mechanisms that regulate the proteasome inhibition-induced nuclear translocation of IkBa. Identification of the key molecular events that control NFkB activity by the nuclear IkBa will broaden our understanding of the mechanisms regulating NFkB activity, and might provide a new class of drug targets to regulate the NFkB driven pro-inflammatory and anti-apoptotic gene expression. The focus of this proposal is to identify the mechanisms by which the nuclear translocation of IkBa regulates activity of the transcription factor NFkB. Since NFkB activity is increased in many human diseases including inflammatory disorders, cancer, and leukemia, identification of the mechanisms by which the nuclear IkBa inhibits NFkB activity will contribute to the development of novel anti-cancer and anti-inflammatory therapies.