PDF(Pubmed)
Abstract：
Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11\'s role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.
摘要：
目的:研究香烟烟雾(CS)对丝氨酸/苏氨酸激酶11(STK11)的影响,探讨STK11在CS诱导的气道上皮细胞毒性中的作用。方法：采用免疫印迹法和RT-PCR法检测有或没有COPD的吸烟者和暴露于CS或室内空气（RA）的小鼠肺组织中STK11的表达水平。BEAS-2Bs-人支气管气道上皮细胞暴露于CS提取物（CSE），通过免疫印迹和RT-PCR检测STK11表达水平的变化。用STK11特异性siRNA或STK11表达质粒转染BEAS-2B细胞，并测定CSE对气道上皮细胞细胞毒性的影响。为了确定特定的STK11降解-蛋白水解途径，用环己酰亚胺单独或与MG132或leupeptin组合处理BEAS-2B。最后,为了鉴定介导STK11降解的F-box蛋白，使用一组FBXLE3连接酶亚基转染进行筛选测定.结果：与未患COPD的吸烟者相比，患有COPD的吸烟者的肺组织中STK11蛋白水平显着降低。与RA相比,暴露于CS的小鼠肺组织中的STK11蛋白水平也显著降低。暴露于CSE将BEAS-2B细胞中的STK11mRNA和蛋白质半衰期缩短至4小时。STK11蛋白过表达减弱了CSE诱导的细胞毒性；相反，其敲低增强了CSE诱导的细胞毒性。FBXL19在培养的BEAS-2B细胞中通过泛素-蛋白酶体途径介导CSE诱导的STK11蛋白降解。FBXL19过表达以剂量依赖性方式导致加速的STK11泛素化和降解。结论：我们的结果表明，CSE通过FBXL19介导的泛素-蛋白酶体途径增强了气道上皮细胞中STK11蛋白的降解，导致细胞死亡增加。COPD吸烟者的HIGHLIGHTSLung组织显示STK11RNA和蛋白质表达降低。STK11过表达减弱CS诱导的气道上皮细胞细胞毒性。STK11耗竭增加CS诱导的气道上皮细胞细胞毒性。CS通过FBXL19介导的泛素-蛋白酶体降解减少STK11。
