Boardman Kendrick C, Aryal Ashish M, Miller William M, Waters Christopher M
Department of Biomedical Engineering, Northwestern University, Evanston, Illinois, USA.
J Cell Physiol. 2004 Apr;199(1):57-66. doi: 10.1002/jcp.10451.
Reactive oxygen species (ROS) disrupt the barrier function of airway epithelial cells through a mechanism that appears to involve remodeling of the actin cytoskeleton. Similarly, keratinocyte growth factor (KGF) has been shown to protect against ROS-induced loss of barrier function through a mechanism that may also involve the actin cytoskeleton. To further determine the role of the actin cytoskeleton in ROS-induced barrier injury, we quantified the relative amount of total actin associated with the cytoskeleton following exposure to hydrogen peroxide (H(2)O(2)) and pretreatment with KGF. We also determined the role of the actin-myosin contractile mechanism in the process by quantifying the relative amount of myosin heavy chain (MHC) associated with the cytoskeleton. While the transepithelial resistance (TER) of a monolayer of airway epithelial cells (Calu-3) decreased after 2 h of continuous exposure to 0.5 mM H(2)O(2), actin and MHC, both dissociated from the cytoskeleton within 15 min of H(2)O(2) exposure. The TER of the monolayers remained depressed although both actin and myosin returned to the cytoskeleton by 4 h after the initiation of H(2)O(2) exposure. Filamentous actin (f-actin) staining suggested that the re-associating actin took the form of short fibers associated with cortical actin rather than long stress fibers. Furthermore, pretreatment with KGF prevented the loss of actin and MHC from the actin cytoskeleton but did not prevent the decrease in TER. These studies suggest that actin disassembly from the cytoskeleton is important in the loss of barrier function, but that it is not the overall amount of actin that is associated with the cytoskeleton that is important, rather it is the contribution this actin makes to the architectural cohesiveness of the cell that contributes to the barrier function.
活性氧（ROS）通过一种似乎涉及肌动蛋白细胞骨架重塑的机制破坏气道上皮细胞的屏障功能。同样，角质形成细胞生长因子（KGF）已被证明可通过一种可能也涉及肌动蛋白细胞骨架的机制来保护细胞免受ROS诱导的屏障功能丧失。为了进一步确定肌动蛋白细胞骨架在ROS诱导的屏障损伤中的作用，我们在暴露于过氧化氢（H₂O₂）并经KGF预处理后，对与细胞骨架相关的总肌动蛋白的相对量进行了量化。我们还通过量化与细胞骨架相关的肌球蛋白重链（MHC）的相对量来确定肌动蛋白 - 肌球蛋白收缩机制在此过程中的作用。虽然连续暴露于0.5 mM H₂O₂ 2小时后，气道上皮细胞单层（Calu - 3）的跨上皮电阻（TER）降低，但在暴露于H₂O₂的15分钟内，肌动蛋白和MHC均从细胞骨架上解离。尽管在开始暴露于H₂O₂后4小时，肌动蛋白和肌球蛋白都恢复到细胞骨架，但单层的TER仍保持降低。丝状肌动蛋白（f - 肌动蛋白）染色表明，重新结合的肌动蛋白采取与皮质肌动蛋白相关的短纤维形式，而不是长的应力纤维。此外，用KGF预处理可防止肌动蛋白和MHC从肌动蛋白细胞骨架上丢失，但不能防止TER的降低。这些研究表明，肌动蛋白从细胞骨架上的拆卸在屏障功能丧失中很重要，但与细胞骨架相关的肌动蛋白的总量并非重要因素，重要的是这种肌动蛋白对细胞结构凝聚力的贡献，而这种凝聚力有助于屏障功能。
