Banan A, Zhang L J, Shaikh M, Fields J Z, Farhadi A, Keshavarzian A
Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA.
Am J Physiol Cell Physiol. 2003 Oct;285(4):C977-93. doi: 10.1152/ajpcell.00121.2003. Epub 2003 Jun 4.
Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-gamma appears to be essential for monolayer integrity. We thus hypothesized that PLC-gamma activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC-gamma or to inhibit its activation and were then pretreated with EGF +/- oxidant (H2O2). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC-gamma activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC-gamma (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC-gamma prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC-gamma and downregulation of iNOS pathway; 2) activation of PLC-gamma is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.
诱导型一氧化氮合酶（iNOS）的上调是氧化剂诱导肠道（Caco-2）单层屏障破坏的关键，而表皮生长因子（EGF）通过稳定细胞骨架来防止这种破坏。磷脂酶C-γ（PLC-γ）似乎对单层完整性至关重要。因此，我们推测PLC-γ的激活对于EGF保护免受iNOS上调以及随之而来的细胞骨架氧化、紊乱和单层破坏至关重要。将肠道细胞转染以稳定过表达PLC-γ或抑制其激活，然后用EGF±氧化剂（过氧化氢）进行预处理。野生型（WT）肠道细胞也进行类似处理。相对于暴露于氧化剂的WT单层，用EGF预处理可通过以下方式保护单层：增加天然PLC-γ活性；降低六个与iNOS相关的变量（iNOS活性/蛋白、一氧化氮水平、氧化应激、肌动蛋白氧化/硝化）；增加稳定的F-肌动蛋白；维持肌动蛋白稳定性；并增强屏障完整性。相对于暴露于氧化剂的WT细胞，过表达PLC-γ的转染单层（增加2.3倍）受到保护，这表现为iNOS驱动途径的所有指标降低以及肌动蛋白和屏障完整性增强。低剂量的EGF可增强过表达诱导的iNOS抑制作用。稳定抑制PLC-γ可阻止EGF对iNOS上调的所有保护作用。我们得出结论：1）EGF通过稳定F-肌动蛋白来防止肠道屏障的氧化应激破坏，主要是通过激活PLC-γ和下调iNOS途径；2）PLC-γ的激活本身对于细胞抵抗iNOS氧化应激至关重要；3）抑制iNOS驱动反应以及细胞骨架氧化和分解的能力是一种以前未归因于PLC同工型家族的新机制。
