Antiapoptotic Role of NF-kB in Calcium Signaling

NF-kB 在钙信号传导中的抗凋亡作用

• 批准号: 7005670
• 负责人: MARTY W MAYO
• 金额: $ 28.67万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7005670
• 关键词: Antiapoptotic; Role; NF; kB; Calcium; Antiapoptotic; Role; NF; kB; Calcium

Late stage prostate cancer (CAP) tumors are resistant to anticancer therapies that cause cell death through the induction of apoptosis. To understand how CaP cells become resistant to apoptotic stimuli, it is important to characterize the molecular pathways that control apoptosis. This proposal focuses on the requirement of the transcription factor NF-kappaB for cell survival in CaP cells in response to interleukin-1 (IL-1)beta-dependent Ca 2+ signaling and apoptosis. Preliminary data presented in this proposal demonstrates that IL-1beta-induced NF-kappaB activity will overcome growth factor induced apoptosis in CaP cells. However, IL-1beta will also induce apoptosis in CaP cells through a Ca2+ -dependent pathway, following the inactivation of NF-kappaB. These results suggest that NF-kappaB activity is required to maintain Ca 2+ homeostasis in CaP cells following IL-1beta stimulation. This is supported by the fact that IL-1beta activates NF-kappaB, in part, through Ca2+/calmodulin-dependent kinases (CaMKs), namely CaMKK and CaMKIV. IL-1beta-induced NF-kappaB activity is required to upregulate transcripts encoding Ca2+-binding proteins (CaBP), including calreticulin, calbindin-3, and DSCR1. Expression of calreticulin blocked Ca 2+ -induced apoptosis in our model system. The overall hypothesis of this proposal is that IL-1beta-induced NF-kappaB activity is required to upregulate gene products that feedback to regulate Ca 2+ levels and modulate apoptosis in CaP cells. To address this hypothesis, the following aims will be explored. Aim 1 will establish whether CaP patients display, elevated IL-1beta. Experiments' proposed in Aim 1 will also identify whether intracellular release of Ca2+ is responsible for inducing apoptosis, and will determine if this effect impacts cytochrome c release, caspase and/or calpain activation, and subsequent cleavage of CaMK and IkappaB/NF-kappaB substrates. Aim 2 will identify the CaMK signaling pathways responsible for upregulating NF-kappaB transcriptional activity and cell survival. Experiments outlined in Aim 2 will use dominant negative and constitutively active kinases to identify CaMK pathways required to activate NF-kappaB in response to IL-1beta, and will determine the impact of these signaling pathways on the transactivation potential of NF-kappaB. Aim 3 will elucidate whether the calreticulin promoter is a direct target of NF-kappaB and determine if the expression of calreticulin alone or in combination with other NF-kappaB-regulated CaBPs rescues apoptosis. Experiments described in Aim 3 will determine whether NF-kappaB directly upregulates the calreticulin promoter, and will potentially identify regulated Ca2+ -binding proteins. Additional experiments proposed in Aim 3 will determine whether the expression of these proteins alone or in combination with calreticulin will rescue Ca2+ -induced apoptosis. Collectively, the experiments outlined in this proposal will offer insight into the anti-apoptotic role of NF-kappaB in Ca 2+ signaling in CaP cells, and will potentially provide a better understanding of how NF-kappaB overcomes Ca2+ -induced apoptotic cues initiatcd by growth factors and cytokines.

晚期前列腺癌（CAP）肿瘤对通过诱导细胞凋亡导致细胞死亡的抗癌疗法具有抵抗力。为了了解帽细胞如何抗凋亡刺激，重要的是要表征控制凋亡的分子途径。该提案的重点是转录因子NF-kappab对Cap细胞中细胞生存的需求，响应白介素-1（IL-1）β依赖性Ca 2+信号传导和凋亡。该提案中提供的初步数据表明，IL-1Beta诱导的NF-kappab活性将克服生长因子诱导的CAP细胞凋亡。但是，在NF-kappab失活之后，IL-1Beta还将通过Ca2+依赖性途径诱导CAP细胞中的凋亡。这些结果表明，在IL-1BETA刺激后，需要NF-kappab活性来维持CAP细胞中的Ca 2+稳态。 IL-1Beta部分通过Ca2+/钙调蛋白依赖性激酶（CAMK），即CAMKK和CAMKIV激活NF-kappab的事实支持了这一点。需要IL-1BETA诱导的NF-kappab活性来上调编码CA2+结合蛋白（CABP）的转录本，包括钙网蛋白，Calbindin-3和DSCR1。钙网蛋白阻断Ca 2+诱导的凋亡的表达。该提议的总体假设是，需要IL-1Beta诱导的NF-kappab活性来上调反馈以调节Ca 2+水平并调节CAP细胞中凋亡的基因产物。为了解决这一假设，将探讨以下目标。 AIM 1将确定CAP患者是否显示出升高IL-1Beta。 AIM 1中提出的实验还将确定Ca2+的细胞内释放是否负责诱导凋亡，并确定这种影响是否影响细胞色素C释放，caspase和/或Calpain激活，以及随后的CAMK和IKAPPAB/IKAPPAB/NF-KAPPAB/NF-KAPPABBAB BABRATES。 AIM 2将确定负责上调NF-kappab转录活性和细胞存活的CAMK信号通路。 AIM 2中概述的实验将使用主要的负阴性和组成性活性激酶来识别激活NF-kappab对IL-1Beta响应IL-1Beta所需的CAMK途径，并将确定这些信号通路对NF-KAPPAB的反式激活潜力的影响。 AIM 3将阐明钙网蛋白启动子是NF-kappab的直接靶标，并确定钙网蛋白的表达是单独的还是与其他NF-kappab调节的CABPS结合的表达。 AIM 3中描述的实验将确定NF -kappab是否直接上调钙网蛋白启动子，并有可能识别受调节的Ca2+结合蛋白。 AIM 3中提出的其他实验将确定这些蛋白质的表达是单独的还是与钙网蛋白结合的表达会挽救Ca2+诱导的凋亡。 总的来说，该提案中概述的实验将洞悉NF-kappab在CAP细胞中Ca 2+信号传导中的抗凋亡作用，并有可能更好地理解NF-kappab对NF-kappab的克服CA2+诱导的凋亡提示如何由生长因子和细胞因素来启动。