DYSREGULATION OF GSH SYNTHESIS DURING LIVER INJURY AND FIBROSIS

描述(申请人提供)：谷胱甘肽(GSH)在异种生物代谢中起关键作用，抵御氧化应激，调节炎症反应和纤维化等病理过程。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由一个催化亚基和一个修饰亚基(GCLC和GCLM)组成，前者具有全酶的所有催化活性，但后者使酶的功能更有效。谷胱甘肽合成酶(GSH Synthase，GS)是谷胱甘肽合成的第二种酶，常以GCL亚基的形式协同调节，可进一步提高GSH的合成能力。在慢性肝损伤中，肝脏GSH水平经常下降，被认为很大程度上是由于营养缺乏和氧化应激。然而，我们最近发现，在慢性淤胆性肝损伤中，肝脏GSH合成酶的表达显著减少，这是由于转录因子Nrf2被MafG和c-Maf取代而与抗氧化反应元件(ARE)结合，后者在GSH合成酶的正向调节中起重要作用。在胆汁淤积性肝损伤过程中，MafG和c-Maf的表达增加，这发生在转录水平。阻断MafG和c-Maf的表达可阻止GSH合成酶的表达和GSH水平的下降，从而显着减轻淤胆性肝损伤。相反，降低GCLC的表达明显加重了淤胆性肝损伤和纤维化。我们还发现，GSH合成酶的表达受到脂多糖(LPS)的抑制。在这项建议中，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蛋白的诱导机制。熊去氧胆酸和S-腺苷蛋氨酸等保肝药可阻止胆汁淤积时肝细胞(熊去氧胆酸和熊去氧胆酸)和巨噬细胞(熊去氧胆酸和S腺苷蛋氨酸)GCL亚单位基因表达的下降。我们已经克隆或获得了小鼠、大鼠和人的GCLC、GCLM和GS启动子。我们还克隆了鼠和人的MafG和c-Maf启动子。我们准备利用这些工具来实现我们的目标，目的如下：1)阐明NRF2到MafG/c-Maf开关在淤胆性肝损伤中与ARE核结合的分子机制(S)；2)阐明UDCA及其类似物阻止GSH合成酶表达下降的机制(S)以及在胆汁淤积期保留GSH的意义；3)确定内毒素抑制GSH合成酶的分子机制。这些研究将使用小鼠和人类的肝细胞和巨噬细胞。它们将补充对胆管结扎或脂多糖治疗的小鼠的体内研究。这是为了确保在啮齿动物身上的发现也出现在人类细胞中，并且体外发现适用于体内相关模型。最终目标是将实验室的结果转化为床边I设计更有效的治疗各种肝脏损伤的方法，在这些损伤中，GSH合成变化起着主要的致病作用，这是一个与公众健康高度相关的话题。

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.