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

描述（由申请人提供）：NF κ B是调节促炎和抗凋亡基因表达的关键转录因子。我们实验室的先前研究表明，与其他细胞类型相反，静息的人中性粒细胞在细胞核中含有主要量的NFkB抑制剂IkBa，并且这种增加的IkBa的核积累导致NFkB活性的抑制和中性粒细胞凋亡速率的增加。我们最近的数据表明，蛋白酶体抑制诱导IkBa易位到白血病HL-60和U- 937细胞的细胞核，在癌症HeLa细胞，然而，目前的机制是未知的。该提议的中心假设是诱导IkBa的核积累抑制NFkB活性和NFkB调节的抗凋亡和促炎基因的表达，并且因此可以为旨在通过核IkBa抑制NFkB活性的新型抗癌和抗炎疗法提供基础。本研究的具体目的是分析核内IkBa抑制白血病HL-60和U-937细胞NF-κ B活性和NF-κ B调控基因表达的机制。在目的1中，我们将通过用抑制性IkBa RNA转染HL-60和U-937细胞来检验蛋白酶体抑制诱导的IkBa核转位导致NF κ B活性抑制和NF κ B调节的抗凋亡基因表达降低的假设。在目标2中，我们将使用染色质免疫沉淀来检验核IkBa通过与NF κ B调控基因的启动子相关联来抑制NF κ B活性的假设。在目的3中，我们将研究调节蛋白酶体抑制诱导的IkBa核转位的机制。通过核IkBa调控NF κ B活性的关键分子事件的鉴定将拓宽我们对NF κ B活性调控机制的理解，并可能提供一类新的药物靶点来调控NF κ B驱动的促炎和抗凋亡基因表达。该建议的重点是确定IkBa核转位调节转录因子NF κ B活性的机制。由于NFkB活性在许多人类疾病（包括炎性病症、癌症和白血病）中增加，因此核IkBa抑制NFkB活性的机制的鉴定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.