Banan A, Zhang L J, Shaikh M, Fields J Z, Farhadi A, Keshavarzian A
Division of Digestive Diseases, Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University of Chicago School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA.
J Pharmacol Exp Ther. 2004 Apr;309(1):356-68. doi: 10.1124/jpet.103.062232. Epub 2004 Jan 14.
Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 microg) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H(2)O(2)). Stably overexpressing PLC-gamma (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma (-99%) substantially prevented all measures of EGF protection against NF-kappaB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappaB; 2) Activation of PLC-gamma by itself is protective against NF-kappaB activation; 3) the ability to modulate the dynamics of NF-kappaB/I-kappa Balpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.
我们之前利用单层肠上皮（Caco - 2）细胞作为研究炎症性肠病（IBD）的模型，发现氧化剂诱导的细胞骨架和屏障完整性破坏需要核因子 - κB（NF - κB）激活。表皮生长因子（EGF）可稳定F - 肌动蛋白细胞骨架并防止氧化剂损伤，但其机制尚不清楚。我们推测该机制涉及磷脂酶C - γ（PLC - γ）的激活，它可阻止NF - κB激活及其激活所带来的后果，即细胞骨架和屏障破坏。我们研究了野生型细胞和转染细胞。后者用不同水平（1 - 5微克）的cDNA转染，以稳定过表达PLC - γ或抑制其激活。细胞在暴露于氧化剂（H₂O₂）之前先用EGF预处理。稳定过表达PLC - γ（增加2.0倍）或与EGF预孵育可保护细胞免受氧化剂损伤，表现为：1）几个与NF - κB相关的变量降低[NF - κB（p50/p65亚基）核转位、NF - κB亚基活性、抑制性κBα（I - κBα）磷酸化和降解]；2）F - 肌动蛋白增加，G - 肌动蛋白减少；3）肌动蛋白细胞骨架结构稳定；4）屏障功能增强。EGF增强了过表达诱导的NF - κB失活。发现PLC - γ主要存在于膜和细胞骨架部分（胞质部分中<9%），表明其被激活。对内源性PLC - γ的显性负性抑制（-99%）基本上阻止了EGF对NF - κB激活的所有保护作用。我们得出结论：1）EGF通过激活PLC - γ保护细胞免受氧化剂诱导的屏障破坏，而PLC - γ可使NF - κB失活；2）PLC - γ自身的激活对NF - κB激活具有保护作用；3）调节NF - κB/I - κBα动态变化的能力是一种以前未归因于细胞中PLC同工型家族的新机制；4）开发PLC - γ模拟物可能代表一种针对IBD的新治疗策略。
