Yan Sen Rong, Joseph Robbie Randle, Rosen Kirill, Reginato Mauricio J, Jackson Amanda, Allaire Norman, Brugge Joan S, Jobin Christian, Stadnyk Andrew W
Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3H4R2.
Oncogene. 2005 Sep 29;24(43):6482-91. doi: 10.1038/sj.onc.1208810.
We reported earlier that IL-1beta, an NF-kappaB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappaB-regulated products and therefore aimed to determine whether NF-kappaB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IkappaBalpha becomes phosphorylated immediately following detachment and that levels of phospho-IkappaBalpha peaked within 20 min. Phosphorylation of IkappaBalpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappaB activity following detachment was confirmed using the detection of NF-kappaB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IkappaBalpha protein and pharmacological inhibitors of NF-kappaB resulted in the failure to phosphorylate IkappaBalpha, a more rapid activation of caspases and earlier apoptosis. We also detected that IkappaB kinase alpha (IKKalpha) and not IKKbeta became phosphorylated following detachment. Since IKKalpha is activated by NF-kappaB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IkappaBalpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappaB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.
我们之前报道过，白细胞介素-1β（IL-1β）是一种受核因子-κB（NF-κB）调控的细胞因子，在肠道上皮细胞脱离诱导的凋亡（失巢凋亡）过程中产生，并且IL-1对脱离的细胞具有抗凋亡作用。由于在癌症进展和转移过程中，细胞存活于失巢凋亡状态是一个先决条件，因此我们正在进一步探索失巢凋亡与细胞因子之间的联系。在此，我们确定了细胞脱离后多个基因会表达，其中包括一些受NF-κB调控的产物，因此旨在确定NF-κB信号通路在调节细胞凋亡中是否发挥作用。通过蛋白质印迹法，我们检测到IκBα在细胞脱离后立即发生磷酸化，且磷酸化IκBα的水平在20分钟内达到峰值。IκBα磷酸化之后，Rel A（p65）发生核转位。通过检测腺病毒感染传递的NF-κB促进的荧光素酶基因表达，证实了细胞脱离后NF-κB活性增强。用表达超抑制性IκBα蛋白的腺病毒感染细胞以及使用NF-κB的药理学抑制剂，导致IκBα无法磷酸化，半胱天冬酶激活更快且凋亡更早。我们还检测到细胞脱离后IκB激酶α（IKKα）而非IKKβ发生磷酸化。由于IKKα由NF-κB诱导激酶（NIK）激活，我们使用腺病毒载体过表达天然NIK，与对照脱离细胞相比，这导致脱离细胞中磷酸化IκBα和核p65增强，但并未使存活至24小时的细胞数量显著增加。我们得出结论，细胞脱离直接激活NF-κB，这除了引发炎症细胞因子浪潮外，还导致肠道上皮细胞凋亡延迟。
