Wang Hua, Cebotaru Liudmila, Lee Ha Won, Yang QingFeng, Pollard Bette S, Pollard Harvey B, Guggino William B
Department of Physiology, Medicine, School of Medicine, The Johns Hopkins University, Baltimore, USA.
Cell Physiol Biochem. 2016;40(5):1063-1078. doi: 10.1159/000453162. Epub 2016 Dec 14.
BACKGROUND/AIMS: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function.
We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB.
We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation.
We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.
背景/目的：囊性纤维化中的慢性肺部感染会引发炎症反应，由于细菌的长期存在，这种炎症反应会持续存在，并最终导致肺功能的灾难性衰竭。
我们结合生物化学、细胞和分子生物学方法，研究肿瘤坏死因子α（TNFα）信号传导中的关键衔接分子TRADD与囊性纤维化跨膜传导调节因子（CFTR）在核因子κB（NFκB）调控中的相互作用。
我们发现野生型CFTR与TRADD结合并共定位。TRADD是连接TNFα与NFκB激活的关键信号中间体。相比之下，ΔF508 CFTR不与TRADD结合。在表达ΔF508 CFTR的CFBE细胞中，NF-κB的激活水平高于表达野生型CFTR的细胞。然而，当TRADD水平被敲低时，这种差异效应就会消失。将野生型CFTR转染到CFBE细胞中会降低NF-κB活性。然而，CFTR氯离子转运抑制剂-172会消除这种降低作用。同样，转染正确转运的CFTR传导突变体G551D或S341A也无法降低NFκB活性。因此，CFTR若要调节NFκB活性，必须具备功能。我们还发现，TNFα在CFBE细胞中比在野生型CFTR校正后的同一细胞中能使NF-κB活性有更大的增加。同样，当通过短发夹RNA（shRNA）敲低TRADD时，这种效应也会消失。因此，野生型CFTR对TRADD的调控调节了TNFα对NFκB的生理性激活。基于蛋白酶体和溶酶体抑制剂的研究，野生型CFTR而非ΔF508 CFTR抑制TRADD的机制是通过溶酶体降解。
我们发现了一种新机制，即野生型CFTR通过增强TRADD降解来调节TNFα信号传导。因此，通过降低TRADD水平，野生型CFTR抑制下游促炎NFκB信号传导。相比之下，在表达ΔF508 CFTR的CF细胞中，NF-κB激活的抑制作用失败。
