CYLD基因缺失会触发核因子-κB信号通路，并增强小鼠肝细胞的抗细胞死亡能力。
CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes.
作者信息
Urbanik Toni, Koehler Bruno Christian, Wolpert Laura, Elßner Christin, Scherr Anna-Lena, Longerich Thomas, Kautz Nicole, Welte Stefan, Hövelmeyer Nadine, Jäger Dirk, Waisman Ari, Schulze-Bergkamen Henning
机构信息
Toni Urbanik, Bruno Christian Koehler, Laura Wolpert, Christin Elßner, Anna-Lena Scherr, Nicole Kautz, Stefan Welte, Dirk Jäger, Henning Schulze-Bergkamen, National Center for Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, 69120 Heidelberg, Baden-Wuerrtemberg, Germany.
出版信息
World J Gastroenterol. 2014 Dec 7;20(45):17049-64. doi: 10.3748/wjg.v20.i45.17049.
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 injury models for CD95- (Jo2) and tumor necrosis factor (TNF)-α- [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)-κB, ERK, Akt and jun amino-terminal kinases signaling were assessed. Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-α and with the CD95-ligand Jo2. Cell viability was analyzed by MTT-assay.
RESULTS
Livers of CYLD(-/-) mice showed increased anti-apoptotic NF-κB 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) serum-levels 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 CD95-receptor triggering, respectively. Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of anti-apoptotic proteins and re-sensitized CYLD(-/-) PMH towards TNF- and CD95-receptor mediated cell death.
CONCLUSION
CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.
目的
分析CYLD在急性肝损伤模型中对小鼠肝细胞受体介导的细胞死亡的作用。
方法
通过应用针对CD95（Jo2）和肿瘤坏死因子（TNF）-α[D-半乳糖胺/脂多糖（LPS）]诱导的凋亡的肝损伤模型，分析CYLD基因敲除小鼠（CYLD(-/-)）中的肝细胞死亡情况。通过测量血清转氨酶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法定量凋亡诱导。评估核因子（NF）-κB、细胞外信号调节激酶（ERK）、蛋白激酶B（Akt）和c-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CD95配体Jo2处理。通过MTT法分析细胞活力。
结果
CYLD(-/-)小鼠的肝脏显示抗凋亡NF-κB信号增强。在两种应用的肝损伤模型中，CYLD(-/-)小鼠的凋亡敏感性均显著降低。在D-半乳糖胺/LPS处理后，CYLD(-/-)小鼠的丙氨酸转氨酶（ALT）水平（295 U/L对859 U/L，P < 0.05）和天冬氨酸转氨酶（AST）水平（560 U/L对1025 U/L，P < 0.01）显著更低。在注射Jo后，与野生型小鼠相比，CYLD(-/-)小鼠的ALT血清水平降低了2倍（50 U/L对110 U/L，P < 0.01），AST血清水平更低（250 U/L对435 U/L，P < 0.01）。此外，分离的CYLD(-/-)原代小鼠肝细胞（PMH）对死亡受体介导的凋亡敏感性较低，并且在TNF和CD95受体触发后，分别显示Bcl-2、X连锁凋亡抑制蛋白（XIAP）、细胞凋亡抑制蛋白1/2（cIAP1/2）、生存素和细胞凋亡抑制蛋白（c-FLIP）表达水平升高。通过NF-κB磷酸化抑制剂BAY 11-7085抑制NF-κB激活，抑制了抗凋亡蛋白的表达，并使CYLD(-/-) PMH对TNF和CD95受体介导的细胞死亡重新敏感。
结论
CYLD通过控制NF-κB依赖性抗凋亡信号传导，是小鼠肝细胞凋亡性细胞死亡的中心调节因子。
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