DYSREGULATION OF GSH SYNTHESIS DURING LIVER INJURY AND FIBROSIS

描述（由申请人提供）：谷胱甘肽（GSH）在异生物质代谢中起关键作用，防御氧化应激并调节病理过程，如炎症反应和纤维化。谷氨酸-半胱氨酸连接酶（GCL）的活性是决定GSH合成的关键因素。GCL由催化亚基和修饰亚基（GCLC和GCLM）组成，前者具有全酶的所有催化活性，而后者使酶的功能更有效。GSH合成酶（GSH synthase，GS）是GSH合成的第二个酶，它与GCL亚基共同调控GSH的合成，并能进一步增强GSH的合成能力。在慢性肝损伤中，肝脏GSH水平经常下降，并且被认为主要是由于营养缺乏和氧化应激。然而，我们最近发现，在慢性胆汁淤积性肝损伤，肝GSH合成酶的表达显着减少，由于置换转录因子Nrf 2的MafG和c-Maf从结合到抗氧化反应元件（ARE），这是重要的GSH合成酶的正调控。MafG和c-Maf的表达在胆汁淤积性肝损伤期间增加，并且这发生在转录水平。阻断MafG和c-Maf表达的增加可防止GSH合成酶表达和GSH水平的下降，并显着改善胆汁淤积性肝损伤。相反，降低GCLC表达显著恶化胆汁淤积性肝损伤和纤维化。我们还发现GSH合成酶的表达被脂多糖（LPS）抑制。在这个提议中，我们的目标是了解GSH合成在各种肝损伤中是如何失调的，并阐明Maf蛋白诱导的机制。肝保护剂，如熊去氧胆酸（UDCA）和S-腺苷甲硫氨酸（SAMe）防止胆汁淤积期间肝细胞（UDCA和SAMe）和LPS治疗期间巨噬细胞（SAMe）中GCL亚基mRNA水平的下降。我们已经克隆或获得小鼠、大鼠和人GCLC、GCLM和GS启动子。我们还克隆了小鼠和人MafG和c-Maf启动子。我们准备使用这些工具来实现我们的目标，目的如下：1）阐明胆汁淤积性肝损伤期间Nrf 2向MafG/c-Maf转换与ARE核结合的分子机制; 2）阐明UDCA和SAMe阻止GSH合成酶表达下降的机制以及胆汁淤积期间保护GSH的意义; 3）确定LPS诱导GSH合成酶抑制的分子机制。这些研究将使用小鼠和人的肝细胞和巨噬细胞。它们将补充用胆管结扎或LPS治疗的小鼠的体内研究。这是为了确保啮齿动物中的发现也发生在人类细胞中，并且体外发现适用于相关的体内模型。最终目标是将结果从实验室转化到床边，设计更有效的治疗方法来对抗各种肝损伤，其中GSH合成的改变起着主要的致病作用，这是一个与公共卫生高度相关的主题。

DESCRIPTION (provided by applicant): Glutathione (GSH) plays a key role in xenobiotic metabolism, defends against oxidative stress and modulates pathological processes such as inflammatory response and fibrogenesis. The activity of glutamate-cysteine ligase (GCL) is a key factor that determines GSH synthesis. GCL is made up of a catalytic and a modifier subunit (GCLC and GCLM), the former exhibits all of the catalytic activity of the holoenzyme but the latter makes the enzyme function more efficiently. GSH synthase (GS), the second enzyme in GSH synthesis, is often regulated in a coordinated manner as GCL subunits and can further enhance the GSH synthetic capacity. Hepatic GSH level is often decreased in chronic liver injury and was thought largely to be due to nutritional deficiency and oxidative stress. However, we showed recently that in chronic cholestatic liver injury, the expression of hepatic GSH synthetic enzymes is reduced markedly due to displacement of transcription factor Nrf2 by MafG and c-Maf from binding to the anti-oxidant response element (ARE) that is important in positive regulation of the GSH synthetic enzymes. The expression of MafG and c-Maf is increased during cholestatic liver injury and this occurs at the transcriptional level. Blocking the increasein expression of MafG and c-Maf prevented the fall in expression of GSH synthetic enzymes and GSH levels and significantly ameliorated cholestatic liver injury. In contrast, lowering GCLC expression markedly worsened cholestatic liver injury and fibrosis. We have also found that expression of GSH synthetic enzymes is inhibited by lipopolysaccharide (LPS). In this proposal, our goals are to understand how GSH synthesis is dysregulated in various liver injuries and elucidate mechanisms for induction of Maf proteins. Hepatoprotective agents such as ursodeoxycholic acid (UDCA) and S-adenosylmethionine (SAMe) prevented the fall in GCL subunits mRNA levels in hepatocytes (UDCA and SAMe) during cholestasis, and in macrophages (SAMe) during LPS treatment. We have cloned or obtained mouse, rat and human GCLC, GCLM and GS promoters. We have also cloned the mouse and human MafG and c-Maf promoters. We are poised with these tools to achieve our goal with the following aims: 1) elucidate the molecular mechanism(s) of Nrf2 to MafG/c-Maf switch in nuclear binding to ARE during cholestatic liver injury; 2) elucidate the mechanism(s) by which UDCA and SAMe prevent the fall in expression of GSH synthetic enzymes and the significance of preserving GSH during cholestasis; 3) identify molecular mechanisms of LPS-induced inhibition of GSH synthetic enzymes. These studies will use hepatocytes and macrophages from mouse and human. They will complement in vivo studies in mice treated with bile duct ligation or LPS. This is to make sure that findings in rodents also occur in human cells and that in vitro findings are applicable t relevant in vivo models. The ultimate goal is to translate results from the laboratory to bedside i designing more effective therapy against various liver injuries where altered GSH synthesis plays a major pathogenetic role, a topic that is highly relevant to public health.