GADD45a in ventilator-induced lung injury: role of Akt signaling

We explored the mechanistic involvement of the growth arrest and DNA damageinducible gene, GADD45a, in LPS- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated GADD45a-/- mice to be modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume ventilation-induced lung injury (VILI) with increases in microvascular permeability and levels of inflammatory cytokines in bronchoalveolar lavage. Expression profiling of lung tissues from GADD45a-/- mice revealed strong dysregulation in the B cell receptor signaling pathway suggesting involvement of PI3 kinase/Akt signaling components while the wild type controls depicted no observable changes. Western blot analyses of lung homogenates confirmed ~50% reduction in Akt protein levels in GADD45a-/- mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling. Experiment Overall Design: 4 experimental groups, each with 3 mouses. Wild type control group, VILI group, GADD-/- group, GADD-/- and VILI group.本研究探讨了生长阻滞与DNA损伤诱导基因45α（GADD45a）在脂多糖（Lipopolysaccharide, LPS）及呼吸机诱导性肺损伤（Ventilator-induced lung injury, VILI）中的机制性作用。通过炎症性肺损伤的多项生化及基因组学参数评估，结果显示GADD45a基因敲除（GADD45a-/-）小鼠对气管内滴注LPS诱导的肺损伤呈轻度易感性，而对高潮气量通气诱导的肺损伤（VILI）则呈显著易感性，表现为支气管肺泡灌洗液（bronchoalveolar lavage）中微血管通透性升高及炎症细胞因子水平上调。对GADD45a-/-小鼠肺组织的表达谱分析显示，B细胞受体（B cell receptor）信号通路存在显著失调，提示磷脂酰肌醇3-激酶/蛋白激酶B（PI3 kinase/Akt）信号通路组分参与该病理过程；而野生型对照组未观察到任何明显变化。肺组织匀浆的蛋白质印迹（Western blot）分析证实，GADD45a-/-小鼠的Akt蛋白水平下调约50%，同时Akt泛素化水平显著升高。对GADD45a或Akt表达经小干扰RNA（small interfering RNA, siRNA）下调的人肺内皮细胞单层进行跨膜电阻检测，结果显示LPS诱导的人肺内皮屏障功能障碍显著增强，而过表达组成型活性Akt1转基因（constitutively active Akt1 transgene）可缓解该效应。本研究证实GADD45a是炎症性肺损伤的新型候选基因，并通过调控Akt介导的内皮信号通路，在血管屏障调控中发挥重要作用。实验总体设计：设置4组实验，每组包含3只小鼠，分别为野生型对照组、VILI组、GADD45a基因敲除组以及GADD45a基因敲除联合VILI组。