PROHIBITIN 1 IN LIVER INJURY AND CANCER

描述（由申请人提供）：禁止蛋白1 （PHB1）是一种高度保守的、普遍表达的蛋白，参与多种过程，包括线粒体伴侣、生长和凋亡。PHB1在体内的作用尚不清楚，因为胚胎缺失是致命的。我们报道，缺乏蛋氨酸腺苷转移酶1A （MAT1A）的小鼠在蛋白质水平上降低了PHB1的表达，线粒体功能受损，并自发发展为脂肪性肝炎和肝细胞癌（HCC）。为了观察PHB1表达降低是否会导致Mat1a基因敲除（KO）表型，我们产生了肝脏特异性PHB1 KO小鼠。3周时，肝脏特异性Phb1 KO小鼠表现出生化和组织学上的肝损伤。免疫组织化学显示氧化应激，纤维化，肝细胞发育不良，肿瘤前标记物染色增加。线粒体功能受损。Phb1 KO小鼠对多种形式的肝损伤敏感，但其关键机制似乎独立于其作为线粒体伴侣的作用。我们的初步数据显示，PHB1缺乏导致总和核组蛋白去乙酰化酶4 （HDAC4）的表达和活性升高。有趣的是，抑制HDAC活性可防止多种毒物诱导的Phb1 KO肝细胞死亡增加。我们还发现，在小鼠和人类的胆汁淤积性肝损伤中，PHB1蛋白水平下降，这表明这可能进一步使肝损伤永久化。许多Phb1 KO小鼠在35周时发生多灶性HCC。我们的初步数据也支持PHB1可以直接影响与肝癌发生有关的基因。在4周龄雄性KO小鼠肝脏中高度上调的两个基因是H19和胰岛素样生长因子2 （IGF2）。小鼠非转化AML12细胞中PHB1的急性敲低提高了cyclin D1、H19和IGF2的表达，增加了E2F与cyclin D1启动子的结合，增加了增殖。PHB1过表达则相反。这些结果支持PHB1在肝细胞中作为肿瘤抑制因子。目前的应用是检查这些高度新颖的领域，并确定PHB1在肝损伤和HCC中的作用。提出了三个具体目标：1)研究PHB1在肝损伤中的作用；2)研究PHB1如何影响HCC的发展；3)确定PHB1下调是否有助于Mat1a KO小鼠的线粒体功能障碍、肝损伤和HCC的形成。我们的主要假设是：1)在胆汁淤淤性肝损伤期间，PHB1蛋白稳定性下降，这是损伤机制的一部分；2)PHB1通过hdac介导的表观遗传改变调节肝细胞死亡；3)PHB1直接调节HDAC4、H19和IGF2的表达，并诱导其在肝脏特异性PHB1 KO小鼠中形成HCC； 4) PHB1表达降低导致Mat1a KO小鼠线粒体功能受损和HCC形成。该应用是假设驱动的和机制的，代表了一个新的研究领域，因为人们对PHB在肝脏中的生物学功能知之甚少。最终目标是将实验室结果转化为床边优化PHB1功能，这对于预防肝损伤和HCC至关重要，这与公共卫生高度相关。

DESCRIPTION (provided by applicant): Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear as embryonic deletion is lethal. We reported that mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression at the protein level, impaired mitochondrial function, and spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC). To see if reduced PHB1 expression can contribute to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. At 3 weeks, liver-specific Phb1 KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed oxidative stress, fibrosis, hepatocyte dysplasia, and increased staining of preneoplastic markers. Mitochondrial function is impaired. Phb1 KO mice are sensitized to multiple forms of liver injury but the key mechanism appears to be independent of its role as a mitochondrial chaperone. Our preliminary data show PHB1 deficiency leads to higher total and nuclear histone deacetylase 4 (HDAC4) expression and activity. Interestingly, inhibiting HDAC activity prevented increased cell death in Phb1 KO hepatocytes induced by a variety of toxicants. We also found PHB1 protein level falls in cholestatic liver injury in mouse and humans, suggesting this could further perpetuate liver damage. Many Phb1 KO mice develop multifocal HCC by 35 weeks. Our preliminary data also support PHB1 can directly influence genes implicated in hepatocarcinogenesis. Two genes highly up-regulated in 4-week-old male KO mice livers are H19 and insulin-like growth factor 2 (IGF2). Acute knockdown of PHB1 in murine non-transformed AML12 cells raised cyclin D1, H19 and IGF2 expression, increased E2F binding to the cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. These results support PHB1 as a tumor suppressor in hepatocytes. The current application is to examine these highly novel areas and define the role of PHB1 in liver injury and HCC. Three specific aims are proposed: 1) examine the role of PHB1 in liver injury, 2) examine how PHB1 influences HCC development, and 3) determine if PHB1 down-regulation contributes to mitochondrial dysfunction, liver injury and HCC formation in the Mat1a KO mice. Our main hypotheses are 1) PHB1 protein stability falls during cholestatic liver injury and this is part of the mechanism of injury, 2) PHB1 regulates hepatocyte death via HDAC-mediated epigenetic changes, 3) PHB1 regulates HDAC4, H19 and IGF2 expression directly and their induction contribute to HCC formation in the liver-specific Phb1 KO mice, and 4) decreased PHB1 expression contributes to impaired mitochondrial function and HCC formation in the Mat1a KO mice. The application is hypothesis-driven and mechanistic and represents a new area of investigation as little is known of the biological functions of PHB in the liver. The ultimate goal is to translate results from the laboratory to bedside to optimize PHB1 function, which is essential to prevent liver injury and HCC, topics that are highly relevant to public health.