Buckley S, Driscoll B, Barsky L, Weinberg K, Anderson K, Warburton D
Developmental Biology, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027, USA.
Am J Physiol. 1999 Jul;277(1):L159-66. doi: 10.1152/ajplung.1999.277.1.L159.
The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-D-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1beta-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-D-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 microM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.
高氧损伤后肺中2型肺泡上皮细胞（AEC2）的存活受细胞环境信号的调节。角质形成细胞生长因子和基质胶可改善在塑料上培养24小时后的高氧AEC2中出现的凋亡特征[S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L714-L720, 1998]。我们使用相同的体内短期高氧模型来表征底物附着的保护作用。与在塑料上生长相比，在各种生物粘附底物上培养高氧AEC2显示，在所有生物底物上生长的细胞中DNA末端标记减少。相反，合成底物聚-D-赖氨酸没有提供保护作用。与在塑料上培养相比，在层粘连蛋白上培养的高氧AEC2显示Bcl-2与白细胞介素-1β转换酶的表达比值增加。层粘连蛋白还部分恢复了高氧耗尽的谷胱甘肽水平，并通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）测定法显示出改善的最佳线粒体活力。相反，附着于非生理性底物聚-D-赖氨酸则没有这种保护作用，这表明针对高氧诱导损伤的保护作用可能与整合素信号传导有关。在高氧AEC2中，一旦细胞开始附着于层粘连蛋白，就可检测到细胞外信号调节激酶（ERK）酪氨酸磷酸化增加所检测到的ERK激活增加，并且与对照AEC2相比，在培养24小时后持续存在。为了证实ERK激活赋予了对DNA链断裂和凋亡的保护作用，细胞也在50μM PD-98059（一种ERK激活丝裂原激活激酶的抑制剂）存在下接种。用PD-98059培养24小时消除了层粘连蛋白的保护作用。我们推测，在高氧肺损伤后，通过基底膜的信号通过ERK激活依赖性途径赋予针对氧诱导的DNA链断裂和凋亡的特异性保护作用。
