Growth Control of Normal and Cirrhotic Hepatocytes

描述（由申请人提供）： 在美国，肝硬化的发病率和患病率越来越高，并且与肝细胞癌的发展密切相关，肝细胞癌具有令人沮丧的长期预后。导致肝细胞生长失调的机制尚不清楚。在我们的K 08和R 03奖项中，我们假设肝细胞变性对促凋亡剂不太敏感。我们证明，小鼠肝细胞凋亡的转化生长因子β（TGF β），肿瘤坏死因子α（TNF α）和紫外线照射后表现出显着减少。基因表达谱显示抗氧化基因表达降低。这是一个奇怪的发现，面对已知的增加活性氧（ROS）在四氯化碳处理的肝细胞。在正常肝细胞中，我们发现TGF β诱导的细胞凋亡依赖于增加的ROS，caspase-8激活和线粒体通透性转换（MPT）。肝硬化肝细胞没有表现出ROS爆发、半胱天冬酶激活或MPT。然而，用抗氧化剂trolox预处理允许TGF β诱导的肝细胞凋亡，表明肝细胞凋亡中ROS的显著增加可以防止细胞凋亡。此外，在SmadS野生型和敲除小鼠中，我们表明SmadS是ROS产生和凋亡所必需的。此外，肝硬化肝细胞对细胞凋亡敏感性的变化可能是由于肝硬化肝脏中细胞外基质和整合素表达的改变。在初步实验中，我们已经表明，四氯化碳处理的小鼠肝细胞有新的表达的整合素α 6 β 1相比，正常肝细胞。这些变化可能通过黏着斑激酶和相关通路介导生长相关信号。本研究拟从以下几个方面进行研究：（1）探讨TGF β诱导的凋亡信号通路与Smad信号通路在肝细胞凋亡抵抗中的差异。(2)评估正常肝细胞和肝硬化肝细胞中整合素表达和细胞外基质相关生存信号通路的改变。(3)确定肝硬化人肝细胞是否通过在小鼠模型中起作用的机制对TGF β诱导的细胞凋亡具有抵抗力。

DESCRIPTION (provided by applicant): Liver cirrhosis has an increasing incidence and prevalence in the United States and is closely associated with the development of hepatocellular carcinoma, which has a dismal long-term prognosis. The mechanisms that lead to dysregulated cirrhotic hepatocyte growth are unknown. In our K08 and R03 awards we hypothesized that cirrhotic hepatocytes would be less sensitive to pro-apoptotic agents. We demonstrated that murine cirrhotic hepatocytes exhibited markedly decreased apoptosis after treatment with transforming growth factor beta (TGFbeta), tumor necrosis factor alpha (TNFalpha), and ultraviolet radiation. Gene expression profiles revealed decreased anti-oxidant gene expression. This is a curious finding in face of known increased reactive oxygen species (ROS) in CCI4-treated hepatocytes. In normal hepatocytes, we found that TGFbeta-induced apoptosis was dependent on increased ROS, caspase-8 activation, and the mitochondrial permeability transition (MPT). Cirrhotic hepatocytes exhibited no ROS burst, caspase activation, or MPT. Pre-treatment with the anti-oxidant, trolox, however, permitted TGFbeta-induced apoptosis in cirrhotic hepatocytes suggesting that markedly increased ROS in cirrhotic hepatocytes may protect against apoptosis. Furthermore in SmadS wild-type and knockout mice, we showed that SmadS was necessary for ROS generation and apoptosis. Also, changes in cirrhotic hepatocyte sensitivity to apoptosis may be due to altered extracellular matrix and integrin expression in the cirrhotic liver. In preliminary experiments, we have shown that CCI4-treated mouse hepatocytes have novel expression of the integrin alpha6beta1 compared to normal hepatocytes. These changes may mediate growth related signaling through focal adhesion kinase and related pathways. In this proposal we plan to investigate the following: (1) To determine differences between the TGFbeta-induced apoptotic and Smad signaling pathways that account for cirrhotic hepatocyte resistance to apoptosis. (2) To assess alterations in integrin expression and extracellular matrix related survival signaling pathways in normal and cirrhotic hepatocytes. (3) To determine if cirrhotic human hepatocytes are resistant to TGFbeta-induced apoptosis via mechanisms that are operative in a murine model.