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.

Public Health Relevance

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 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 how two hepatoprotective agents, ursodeoxycholic acid and S- adenosylmethionine, prevent 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK092407-01A1
Application #
8289973
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Doo, Edward
Project Start
2012-04-01
Project End
2017-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$320,541
Indirect Cost
$124,791
Name
University of Southern California
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Tomasi, Maria Lauda; Ryoo, Minjung; Yang, Heping et al. (2014) Molecular mechanisms of lipopolysaccharide-mediated inhibition of glutathione synthesis in mice. Free Radic Biol Med 68:148-58
Lu, Shelly C (2013) Glutathione synthesis. Biochim Biophys Acta 1830:3143-53