Growth Control of Normal and Cirrhotic Hepatocytes

描述(由申请人提供)： 在美国，肝硬变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，肝细胞癌的长期预后很差。导致肝硬变肝细胞生长失调的机制尚不清楚。在我们的K08和R03奖项中，我们假设肝硬变的肝细胞对促凋亡药物不那么敏感。我们发现，经转化生长因子β(TGFbeta)、肿瘤坏死因子α(TNFpha)和紫外线照射治疗后，小鼠肝硬变肝细胞的凋亡率明显减少。基因表达谱显示抗氧化基因表达降低。面对已知的CCI4处理的肝细胞中活性氧物种(ROS)的增加，这是一个奇怪的发现。在正常肝细胞中，我们发现TGFbeta诱导的细胞凋亡依赖于ROS的增加、caspase-8的激活和线粒体通透性转变(MPT)。肝硬变肝细胞未见ROS爆发、半胱氨酸天冬氨酸酶激活或MPT。然而，抗氧化剂曲洛昔单抗可使TGFbeta诱导的肝硬变肝细胞发生凋亡，提示肝硬变肝细胞内ROS的显著增加可能具有抗凋亡作用。此外，在Smads野生型和基因敲除小鼠中，我们发现Smads对于ROS的产生和细胞凋亡是必需的。此外，肝硬变肝细胞对凋亡敏感性的改变可能是由于肝硬变中细胞外基质和整合素表达的改变。在初步实验中，我们已经证明，与正常肝细胞相比，经CCI4处理的小鼠肝细胞具有新的整合素α6β1的表达。这些变化可能通过粘着斑激酶及其相关通路介导生长相关信号。在这项提议中，我们计划研究以下内容：(1)确定TGFbeta诱导的凋亡信号通路和Smad信号通路之间的差异，以解释肝硬变肝细胞对凋亡的抵抗。(2)探讨正常肝细胞和肝硬变肝细胞整合素表达和细胞外基质相关生存信号通路的变化。(3)通过在小鼠模型中可操作的机制，确定肝硬变的人肝细胞是否对转化生长因子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.