The overall objective of this work is to define the molecular mechanisms of hormone-mediated regulation of hepatic GSH synthesis and its physiologic and toxicologic implications.
The specific aims are to: 1) Determine the molecular mechanisms of signal transduction-mediated hormone-induced inhibition of gamma-glutamylcysteine synthetase (GCS). We will determine if GCS is phosphorylated by activation of the various signal transduction pathways using immunoprecipitation, gel electrophoresis, autoradiography and densitometry. Phosphoamino acid analysis and tryptic mapping by HPLC of phosphopeptides will reveal the site(s) of phosphorylation and if different peptides are phosphorylated by activating different signal transduction pathways. If enzyme phosphorylation is demonstrated, then how phosphorylation affects the enzyme activity (Vmax or Km or both) will be determined. Finally, we will correlate changes in the activity of GCS with phosphorylation and dephosphorylation, and by using selective phosphatase inhibitors, we hope to define the phosphoprotein phosphatases(s) that may be involved in regulating GCS. 2) Assess the toxicologic significance of hepatic GCS inhibition. GSH is vitally important in detoxification, impairment in GSH synthesis during hepatotoxicity may further lower the cell's defensive capacity and contribute to cell death. We will examine if a relationship exists between [Ca2+]i and GCS activity/phosphorylation. If so, we will study GCS inhibition in the course of acetaminophen, CC14 and t-butyl hydroperoxide-mediated hepatotoxicity. Finally, we will use selective calmodulin antagonist to examine if phosphorylation of GCS and inhibition of its activity are mediated by activation of Ca-calmodulin pathway. 3) Assess the role of cytokines on hepatic GSH synthesis. Increased sinusoidal GSH efflux and inhibition of hepatic GSH synthesis may be part of the hepatic """"""""stress"""""""" response. Interleukin-1, interleukin-6 and tumor necrosis factor-alpha are regarded as the major mediators of the acute phase response. We will examine the effects of these agents on hepatic GSH synthesis, cysteine availability, GCS activity both in cultured hepatocytes and in vivo. 4) Determine the molecular mechanisms of insulin and glucocorticoids-mediated induction of GCS. We will make cDNA probe from known sequence of the rat kidney GCS and correlate hormone treatments with GCS mRNA level by Northern blot analysis. Nuclear run-on studies will examine the rate of GCS gene transcription. Tyrosine kinase inhibitor and glucocorticoid receptor antagonist will be used to confirm that the induction of GCS requires binding to insulin and glucocorticoid receptors, respectively. 5) Assess the hepatic acinar heterogeneity of GCS activity and response to hormones. Hepatic zonal heterogeneity exists for various enzyme systems which may profoundly affect susceptibility to toxicity. We will compare the rate of GSH synthesis, availability of cysteine, activity of GCS, levels of GCS and GCS mRNA, and response to different hormones in freshly isolated periportal and perivenular-enriched hepatocytes after in vitro and in vivo treatments. The ultimate goal of this work is to utilize this information to impact on the treatment and prevention of various complications that may result from altered hepatic GSH synthesis.

Project Start
1992-09-30
Project End
1997-09-29
Budget Start
1994-09-30
Budget End
1995-09-29
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Southern California
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Ko, Kwang Suk; Peng, Jian; Yang, Heping (2013) Animal models of cholangiocarcinoma. Curr Opin Gastroenterol 29:312-8
Yang, Heping; Li, Tony W H; Peng, Jian et al. (2011) A mouse model of cholestasis-associated cholangiocarcinoma and transcription factors involved in progression. Gastroenterology 141:378-88, 388.e1-4
Yang, Heping; Ko, Kwangsuk; Xia, Meng et al. (2010) Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice. Hepatology 51:1291-301
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Yang, Heping; Ramani, Komal; Xia, Meng et al. (2009) Dysregulation of glutathione synthesis during cholestasis in mice: molecular mechanisms and therapeutic implications. Hepatology 49:1982-91
Yang, Heping; Li, Tony W H; Ko, Kwang Suk et al. (2009) Switch from Mnt-Max to Myc-Max induces p53 and cyclin D1 expression and apoptosis during cholestasis in mouse and human hepatocytes. Hepatology 49:860-70