Watari Akihiro, Hasegawa Maki, Yagi Kiyohito, Kondoh Masuo
Laboratories of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.
PLoS One. 2016 Jan 4;11(1):e0145631. doi: 10.1371/journal.pone.0145631. eCollection 2016.
Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.
已知有几种应激源会影响上皮紧密连接（TJ）的完整性，但DNA损伤与TJ完整性之间的关联仍不清楚。在此，我们研究了两种诱导DNA损伤的抗肿瘤化学物质柔红霉素和瑞贝克霉素对人肠上皮细胞TJ完整性的影响。柔红霉素和瑞贝克霉素在剂量依赖性地增强了Caco-2细胞单层的跨上皮电阻（TER），并降低了4 kDa FITC-葡聚糖的通量。柔红霉素或瑞贝克霉素诱导的TJ屏障功能增强部分挽救了炎性细胞因子TNF-α和IFN-γ对屏障功能的减弱。柔红霉素和瑞贝克霉素增加了claudin-5的表达，且产物分布在富含TJ蛋白的肌动蛋白细胞骨架部分。咖啡因是共济失调毛细血管扩张突变蛋白（ATM）和共济失调毛细血管扩张突变与Rad3相关蛋白（ATR）的抑制剂，Chk1抑制剂抑制了柔红霉素和瑞贝克霉素诱导的TER增加，而Chk2抑制剂则没有。用Chk1 siRNA处理也显著抑制了TER的增加。Chk1抑制剂和siRNA处理均抑制了claudin-5表达的诱导。我们的结果表明，柔红霉素和瑞贝克霉素激活Chk1诱导了Caco-2细胞单层中claudin-5的表达并增强了TJ屏障功能，这表明人肠道中DNA损伤与TJ完整性之间存在联系。
