PROHIBITIN 1 IN LIVER INJURY AND CANCER

摘要 抑制素1（Prohibitin 1，PHB1）是一种高度保守、广泛表达的蛋白质，参与多种生物学过程 包括线粒体伴侣、生长和凋亡。PHB1在体内的作用尚不清楚， 删除是致命的。我们报道了缺乏甲硫氨酸腺苷转移酶1A（MAT1A）的小鼠， PHB1在蛋白质水平表达，线粒体功能受损，并自发发展 脂肪性肝炎和肝细胞癌（HCC）。为了观察PHB1表达的减少是否有助于 Mat1a敲除（KO）表型，我们产生了肝脏特异性Phb1 KO小鼠。第3周，肝脏特异性Phb 1 KO小鼠表现出生化和组织学肝损伤。免疫组化显示氧化应激， 纤维化、肝细胞发育不良和肿瘤前标志物染色增加。线粒体功能是 受损Phb1 KO小鼠对多种形式的肝损伤敏感，但关键机制似乎是 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 更高的总和核组蛋白脱乙酰酶4（HDAC 4）表达和活性。有趣的是，抑制HDAC 活性阻止了由多种毒物诱导的Phb1 KO肝细胞中细胞死亡的增加。我们也 在小鼠和人类胆汁淤积性肝损伤中发现PHB1蛋白水平福尔斯下降，这表明这可能进一步 使肝脏永久受损许多Phb1 KO小鼠在35周内发生多灶性HCC。我们的初步数据还 支持PHB1可以直接影响肝癌发生相关基因。两个基因高度上调 在4周龄雄性KO小鼠中，肝脏是H19和胰岛素样生长因子2（IGF 2）。PHB1的急性敲除 在小鼠非转化AML 12细胞中，细胞周期蛋白D1、H19和IGF 2表达升高，E2F结合增加， 细胞周期蛋白D1启动子和增殖。PHB1过表达则相反。这些结果 支持PHB1作为肝细胞中肿瘤抑制因子。目前的应用是检查这些高度新颖的 区域，并确定PHB1在肝损伤和HCC中的作用。提出了三个具体目标：1）审查 PHB1在肝损伤中的作用，2）检查PHB1如何影响HCC的发展，3）确定 PHB1下调导致线粒体功能障碍、肝损伤和HCC形成 Mat1a KO小鼠。我们的主要假设是：1）在胆汁淤积性肝损伤期间，PHB1蛋白稳定性福尔斯下降， 2）PHB1通过HDAC介导的表观遗传途径调节肝细胞死亡 3）PHB1直接调控HDAC4、H19和IGF 2的表达，它们的诱导参与了HCC的发生 在肝脏特异性Phb 1 KO小鼠中形成，和4）PHB1表达减少有助于受损的 线粒体功能和肝细胞癌的形成。该应用程序是假设驱动的， 机制，并代表了一个新的研究领域，因为很少有人知道的生物功能的聚羟基丁酸酯在 肝脏最终目标是将结果从实验室转化到床边，以优化PHB1功能， 这对于预防肝损伤和HCC是必不可少的，这是与公共卫生高度相关的主题。

ABSTRACT 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.