CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes
CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes
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DOI：
10.3748/wjg.v20.i45.17049
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发表时间：
2014-12-07
影响因子：
4.3
通讯作者：
Schulze-Bergkamen, Henning
中科院分区：
文献类型：
--
作者：
Urbanik, Toni;Koehler, Bruno Christian;Schulze-Bergkamen, Henning
目的：方法：应用CD 95-Jo 2）和肿瘤坏死因子（TNF）-α-[D-GalN/LPS]诱导的肝损伤模型，分析CYLD基因敲除小鼠（CYLD-/-）肝细胞死亡情况。通过血清转氨酶测定和组织学分析评估肝损伤。通过裂解的PARP染色和活化的半胱天冬酶的蛋白质印迹定量细胞凋亡诱导。评估核因子（NF）-κ B、ERK、Akt和jun氨基末端激酶信号传导。通过两步胶原酶灌注分离原代肝细胞，并用重组TNF-α和CD 95配体Jo 2处理。结果：CYLD-/-小鼠肝脏中NF-κ B信号通路增强，抗凋亡NF-κ B信号通路增强。在两种应用的肝损伤模型中，CYLD-/-小鼠显示出显著降低的凋亡敏感性。在D-GalN/LPS处理后，CYLD-/-小鼠表现出显著较低水平的丙氨酸氨基转移酶（ALT）（295 U/L对859 U/L，p < 0.05）和天冬氨酸氨基转移酶（AST）（560 U/L对1025 U/L，p < 0.01）。注射Jo后，与WT小鼠相比，CYLD-/-小鼠显示出2倍低的ALT（50 U/L vs 110 U/L，p < 0.01）和低的AST（250 U/L vs 435 U/L，p < 0.01）血清水平。此外，分离的CYLD-/-原代鼠肝细胞（PMH）对死亡受体介导的细胞凋亡不太敏感，并且在TNF和CD 95受体触发后分别显示出Bcl-2、XIAP、cIAP 1/2、生存素和c-FLIP表达水平升高。NF-κ B磷酸化抑制剂BAY 11-7085抑制NF-κ B活化，抑制抗凋亡蛋白的表达，使CYLD-/- MH对TNF-和CD 95-受体介导的细胞死亡重新敏感。结论：CYLD通过控制NF-κ B依赖的抗凋亡信号，是小鼠肝细胞凋亡的中枢调节因子。(C)14百世登出版集团股份有限公司All rights reserved.
AIM: To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models.METHODS: Hepatocyte cell death in CYLD knockout mice (CYLD-/-) was analyzed by application of liver in-jury models for CD95- Jo2) and tumor necrosis factor (TNF)-alpha-[D-GalN/lipopolysaccharide (LPS)] induced apoptosis. Liver injury was assessed by measurement of serum transaminases and histological analysis. Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases. nuclear factor (NF)-kappa B, ERK, Akt and jun amino-terminal kinases signaling were assessed. Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-alpha and with the CD95-ligand Jo2. Cell viability was analyzed by MTT-assay.RESULTS: Livers of CYLD-/- mice showed increased anti-apoptotic NF-kappa B signaling. In both applied liver injury models CYLD-/- mice showed a significantly reduced apoptosis sensitivity. After D-GalN/LPS treatment CYLD-/- mice exhibited significantly lower levels of alanine aminotransferase (ALT) (295 U/L vs 859 U/L, p < 0.05) and aspartate aminotransferase (AST) (560 U/L vs 1025 U/L, p < 0.01). After Jo injection CYLD-/-mice showed 2-fold lower ALT (50 U/L vs 110 U/L, p < 0.01) and lower AST (250 U/L vs 435 U/L, p < 0.01) serumlevels compared to WT mice. In addition, isolated CYLD-/- primary murine hepatocytes (PMH) were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2, XIAP, cIAP1/2, survivin and c-FLIP expression upon TNF-and CD95receptor triggering, respectively. Inhibition of NF-kappa B activation by the inhibitor of NF-kappa B phosphorylation inhibitor BAY 11-7085 inhibited the expression of antiapoptotic proteins and re-sensitized CYLD-/- MH towards TNF- and CD95-receptor mediated cell death.CONCLUSION: CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-kappa B dependent anti-apoptotic signaling. (C)14 Baishideng Publishing Group Inc. All rights reserved.