Naugler Kaitlin M, Baer Kathy A, Ropeleski Mark J
Department of Medicine, Gastrointestinal Diseases Research Unit, Queen's University, Kingston, Ontario, Canada.
Am J Physiol Gastrointest Liver Physiol. 2008 Mar;294(3):G728-37. doi: 10.1152/ajpgi.00002.2007. Epub 2008 Jan 17.
Interleukin-11 (IL-11) displays epithelial cytoprotective effects during intestinal injury. Antiapoptotic effects of IL-11 have been described, yet mechanisms remain unclear. Fas/CD95 death receptor signaling is upregulated in ulcerative colitis, leading to mucosal breakdown. We hypothesized that IL-11 inhibits Fas ligand (FasL)-mediated apoptosis in intestinal epithelia. Cell death was monitored in IEC-18 cells by microscopy, caspase and poly(ADP-ribose) polymerase cleavage, mitochondrial release of cytochrome c, and abundance of cytoplasmic oligonucleosomal DNA. RT-PCR was used to monitor Fas, cIAP1, cIAP2, XIAP, cFLIP, survivin, and Bcl-2 family members. Fas membrane expression was detected by immunoblot. Inhibitors of JAK2, phosphatidylinositol 3-kinase (PI3-kinase), Akt 1, MEK1 and MEK2, and p38 MAPK were used to delineate IL-11's antiapoptotic mechanisms. IL-11 did not alter Fas expression. Pretreatment with IL-11 for 24 h before FasL reduced cytoplasmic oligonucleosomal DNA by 63.2%. IL-11 also attenuated caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage without affecting expression of activated caspase-8 p20 or cytochrome c release. IL-11 did not affect mRNA expression of the candidate antiapoptotic genes. The MEK1 and MEK2 inhibitors U-0126 and PD-98059 significantly attenuated the protection of IL-11 against caspase-3 and caspase-9 cleavage and cytoplasmic oligonucleosomal DNA accumulation. Although Akt inhibition reversed IL-11-mediated effects on caspase cleavage, it did not reverse the protective effects of IL-11 by DNA ELISA. We conclude that IL-11-dependent MEK1 and MEK2 signaling inhibits FasL-induced apoptosis. The lack of reversal of the IL-11 effect on DNA cleavage by Akt inhibition, despite antagonism of caspase cleavage, suggests that IL-11 inhibits caspase-independent cell death signaling by FasL in a MEK-dependent manner.
白细胞介素-11（IL-11）在肠道损伤期间表现出上皮细胞保护作用。IL-11的抗凋亡作用已有报道，但机制仍不清楚。在溃疡性结肠炎中，Fas/CD95死亡受体信号上调，导致黏膜破坏。我们推测IL-11抑制Fas配体（FasL）介导的肠道上皮细胞凋亡。通过显微镜、半胱天冬酶和聚（ADP-核糖）聚合酶裂解、细胞色素c的线粒体释放以及细胞质寡核小体DNA的丰度来监测IEC-18细胞中的细胞死亡。采用逆转录聚合酶链反应（RT-PCR）监测Fas、细胞凋亡抑制蛋白1（cIAP1）、细胞凋亡抑制蛋白2（cIAP2）、X连锁凋亡抑制蛋白（XIAP）、细胞凋亡相关蛋白（cFLIP）、生存素和Bcl-2家族成员。通过免疫印迹检测Fas膜表达。使用Janus激酶2（JAK2）、磷脂酰肌醇3激酶（PI3-激酶）、蛋白激酶B（Akt）1、丝裂原活化蛋白激酶激酶1（MEK1）和MEK2以及p38丝裂原活化蛋白激酶（p38 MAPK）的抑制剂来阐明IL-11的抗凋亡机制。IL-11不改变Fas表达。在FasL作用前用IL-11预处理24小时可使细胞质寡核小体DNA减少63.2%。IL-11还可减弱半胱天冬酶-3、半胱天冬酶-9和聚（ADP-核糖）聚合酶的裂解，而不影响活化的半胱天冬酶-8 p20的表达或细胞色素c的释放。IL-11不影响候选抗凋亡基因的mRNA表达。MEK1和MEK2抑制剂U-0126和PD-98059显著减弱了IL-11对半胱天冬酶-3和半胱天冬酶-9裂解以及细胞质寡核小体DNA积累的保护作用。尽管抑制Akt可逆转IL-11对半胱天冬酶裂解的作用，但通过DNA酶联免疫吸附测定（ELISA）并未逆转IL-11的保护作用。我们得出结论，IL-11依赖的MEK1和MEK2信号传导抑制FasL诱导的细胞凋亡。尽管抑制半胱天冬酶裂解，但抑制Akt并未逆转IL-11对DNA裂解的作用，这表明IL-11以MEK依赖的方式抑制FasL诱导的非半胱天冬酶依赖性细胞死亡信号传导。
