Cell signaling and bile acid metabolism

1. Bernard Bouscarel, Ph.D., D.Sc.Associate Professor of Biochemistry and Molecular Biology and of Medicine Director, Digestive Diseases CenterDirector, Molecular Medicine Programbbouscarel@mfa.gwu.edu202-994-2114

2. 1988 Bile acids, hormone AA responses and cirrhosis/diabetes Hepatic fibrosis prevention Bile acid and chemoprevention 2006 Relationship between fibroblasts and HSC Bile acids & Taurine transport Bile acids and hepatic cell proliferation Bile acids and fibroblast proliferation Elucidation of hepatic hormonal and AA response UDCA as a chemotherapeutic agent 2000 UDCA and cholestatic liver diseases Bile acids and skin fibroblast signaling Chemotherapy and TopoI inhibitor 1994 Bile acids and hepatic signaling 1992 Cell signaling and bile acid metabolism

3. cAMP Catalytic domain Ca2+ PS PKC AC ATP DAG Pseudosubstrate Bile Acid    GS Ca2+ ? ? P ? ? P ? P P CDCA (25M) CTL P ? GR glucagon DAG PIP2 PKC translocation IP3 Total PKC PLC (1) (2.13) P-PKC 0’ (1) (4.45) -actin C M C M PKC phosphorylation GR phosphorylation CTL CDCA 6’ (1) (3.0) Co-localization of GR & PKC PL WB 10’ 0’ 20’ Green: GR, Red: PKC, Yellow; merge Cholestasis: Bile acids/ hormonal response Background: • Cholestatic and cirrhotic patients (50-80%) display: • glucose intolerance • decreased gluconeogenic response to glucagon • hepatic resistance to glucagon • attenuation of glucagon-induced cAMP production • Bile acids attenuate glucagon-induced cAMP production through a mechanism involving PKC Hypothesis: Bile acids are responsible for the decreased hepatic glucagon responsiveness in cholestasis through a mechanism involving PKC and GR phosphorylation. Methods: Genomics, proteomics, and pharmacologic approaches to identify the relevant amino acids phosphorylated by bile acids leading to activation of PKC and inhibition of GR cellular response. GR: glucagon receptor AC: adenylyl cyclase PLC: phospholipase C Le et al. Am J Physiol. 2006 Ikegami et al.Endocrinology. 2006 Krilov et al. (in preparation)

4. Fibrosis: Bile acids/ fibroproliferation Bile acids PI3K FXR PKC ILK P38 Normal Cox2 AKT Proliferation Cirrhosis HSC Cox-2 ILK CTL CDCA CTL Chol Proliferation survival Growth arrest /death Growth arrest /death Proliferation survival Cox2 ILK ILK Cox2 Normal HSC Fibrotic HSC Background: Liver • Chronic liver disease and cirrhosis in particular, is the 12th leading cause of death in US. • Fibrosis is a hallmark of cirrhosis • Hepatic stellate cells (HSC) are key in the development of liver fibrosis. Hypothesis: FXR= farnesoid X receptor PKC= protein kinase C P38= p38 MAPK ILK= integlin-liked kinase BA stimulate COX-2-mediated cell cycle arrest/death. In fibrosis, this signal is blunted through increased ILK expression/ activation fibroproliferation. Methods: • Isolation of HSC and Human skin fibroblasts (HSF) from control and patients with acute/chronic cholestasis. • Pharmacologic, genomic, and proteomic methods to identify key determinants of fibroproliferation. Co-Inv. and Collaborators: Drs. Rojkind, Ceryak and Kashanchi Drs. Lin, Latham and Piper Zhang et al. Hepatology. 2006 Meng et al. Am J Physiol. 2006 Meng & Bouscarel (submitted)

5. UDCA drinking CPT-11 I.P. injection Fluorescence intensity Spleen Lung Heart Liver Kidney Spleen Liver Cancer: Bile acids/ topoisomerase I inhibitors MC-26 cells transfected with GFP Background: BALB/c mice BALB/c mice • Colorectal cancer (CRC) is a leading cause of cancer-related death. • Bile acids play an important role in the etiology of CRC. • Certain bile acids (UDCA) may have chemotherapeutic properties against CRC. • CPT-11 (Irinotecan, Camptostar) is one of the leading anti-cancer drugs for CRC therapy. Liver Liver Spleen Spleen Hypothesis: 2 weeks later UDCA enhances the antitumor chemotherapeutic action of CPT-11. Purpose/ Methods: • Compare the effect of various agents, BA, H+-pump inhibitor, PLC, CPT-11 analogs, CPT-11…. on: • Tumor cell metastasis • Survival rate • Cell proliferation, apoptosis, cell cycle factors • Tumor gene expression Ikegami et al.Cancer Res. 2002 Ikegami et al.Mol Cancer Ther. 2006 Ikegami et al. (submitted)

6. www.gwu.edu/~ddc/ Gastroenterology Research Laboratory Bernard Bouscarel, PI Jiamping Meng,Senior Scientist Teruo Miyazaki,Post Doc Maryam Alrashid,5th year IBS student Lada Krilov,4th year IBS student Amy Nguyen,2nd year IBS student Helen Johnston,1st year IBS student (Rotation) Nara Lee,Undergraduate student Ivy Akid,Undergraduate student