Glutathione (GSH) plays a vital defensive role 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. Regulation of GCL expression lies predominantly at the transcriptional level. Over the past 15 years we characterized how hormones, oxidants, and rapid liver growth regulate GCL subunits. We also showed that the second enzyme in GSH synthesis, GSH synthase (GS), is regulated by many of the same treatments in a coordinated manner and can further enhance the GSH synthetic capacity. Over the past reporting period we identified signaling pathways involved in their regulation and uncovered novel cross-talks among different families of transcription factors. In this proposal, our goal is to understand why GSH synthesis is dysregulated in various liver injuries and how hepatoprotective agents, alone or together, work at the molecular level. Our preliminary data showed that 1) expression of GSH synthetic enzymes is inhibited during later stages of cholestasis in hepatocytes, 2) expression of GSH synthetic enzymes in whole liver and macrophages is acutely down-regulated by lipopolysaccharide (LPS) treatment in vivo and in vitro, respectively, and 3) GCLC, GCLM and GS mRNA levels fall during activation of hepatic stellate cells (HSCs) in vivo and in vitro. Consistently, GSH levels in HSCs fell during in vitro activation. Importantly, we have uncovered hepatoprotective agents such as ursodeoxycholic acid (UDCA), S-adenosylmethionine (SAMe), methylthioadenosine (MTA), and (-)-epigallocatechin-3-gallate (EGCG), can prevent the fall in GCL subunits mRNA levels in hepatocytes (UDCA and SAMe), macrophages (SAMe and MTA), and HSC (SAMe and ECGC). Our laboratory has cloned or obtained mouse, rat and human GCLC, GCLM and GS promoters. We are poised with these tools and inducible GCL transgenic mice to achieve our goal with the following aims: 1) identify molecular mechanisms of cholestasis-induced down-regulation of GSH synthetic enzymes and the optimal therapeutic approach; 2) identify molecular mechanisms of LPS-induced down-regulation of GSH synthetic enzymes and how SAMe and MTA prevent this; 3) identify molecular mechanisms of GSH synthetic enzymes down-regulation during fibrogenesis and the optimal therapeutic strategy. These studies will use hepatocytes and macrophages from mouse and human, and HSCs from rat and human. They will complement in vivo studies in mice and rat treated with bile duct ligation or LPS. The use of both rodent and human cells is to make sure that findings in rodents also occur in human. The ultimate goal is to translate results from the laboratory to bedside in 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.
Glutathione (GSH) is a key intracellular antioxidant that also modulates diverse critical cellular processes such as inflammatory and fibrogenic responses. Patients with chronic liver diseases have low hepatic GSH levels that up to now have mostly been attributed to nutritional deficiency and oxidative stress. Our preliminary data suggest that a key reason for the low GSH level is lower expression of the genes involved in GSH synthesis. This can further contribute to liver injury by lowering the anti-oxidant defense, allowing inflammatory cytokines to be released and fibrosis to progress. The goal of this project is to understand why the expression of these genes fall during these injuries and to examine a number of hepatoprotective agents, many already widely used as supplements, in preventing this fall. The hope is that we will gain novel insights to help develop strategies to treat many of these liver diseases where effective treatment is still largely unavailable.
