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Bernard Bouscarel, Ph.D., D.Sc. Associate Professor of Biochemistry and Molecular Biology and of Medicine Director, Digestive Diseases Center Director, Molecular Medicine Program [email protected] 202-994-2114 2006 Bile acids, hormone Hepatic fibrosis AA responses and cirrhosis/diabetes prevention Relationship between fibroblasts and HSC Bile acids & Bile acids and Taurine transport hepatic cell Bile acids and fibroblast proliferation proliferation 2000 Elucidation of hepatic hormonal and AA response UDCA and cholestatic liver diseases Bile acids and skin fibroblast signaling 1994 1992 1988 Bile acid and chemoprevention Bile acids and hepatic signaling UDCA as a chemotherapeutic agent Chemotherapy and TopoI inhibitor Cell signaling and bile acid metabolism Cholestasis: Bile acids/ hormonal response Catalytic domain Background: Ca2+ PS DAG Cholestatic and cirrhotic patients (50-80%) display: glucose intolerance decreased gluconeogenic response to glucagon hepatic resistance to glucagon attenuation of glucagon-induced cAMP production Pseudosubstrate Bile Acid Ca2+ Bile acids attenuate glucagon-induced cAMP production through a mechanism involving PKC 0’ PKC phosphorylation Co-localization of GR & PKC Total PKC 6’ (1) (2.13) (1) 10’ Green: GR, Red: PKC, Yellow; merge ? DAG Le et al. Am J Physiol. 2006 Ikegami et al. Endocrinology. 2006 Krilov et al. (in preparation) 20’ ? ? ? 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. 0’ ? ? Hypothesis: Bile acids are responsible for the decreased hepatic glucagon responsiveness in cholestasis through a mechanism involving PKC and GR phosphorylation. PKC translocation PKC C M CTL (4.45) C M CDCA PIP2 IP3 PLC GR: glucagon receptor AC: adenylyl cyclase PLC: phospholipase C GR phosphorylation (1) (3.0) P-PKC PL -actin WB CTL CDCA (25M) Fibrosis: Bile acids/ fibroproliferation Background: Liver Bile acids Chronic liver disease and cirrhosis in particular, is the 12th leading cause of death in US. PI3K FXR Fibrosis is a hallmark of cirrhosis PKC P38 Cox2 Hepatic stellate cells (HSC) are key in the development of liver fibrosis. ILK Normal AKT Proliferation 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. Cirrhosis HSC Cox-2 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. CTL Growth arrest /death CDCA Proliferation survival ILK CTL Chol Growth arrest /death Proliferation survival Cox2 ILK Cox2 Co-Inv. and Collaborators: Drs. Rojkind, Ceryak and Kashanchi Drs. Lin, Latham and Piper ILK Zhang et al. Hepatology. 2006 Meng et al. Am J Physiol. 2006 Meng & Bouscarel (submitted) Normal HSC Fibrotic HSC Cancer: Bile acids/ topoisomerase I inhibitors MC-26 cells Background: Colorectal cancer (CRC) is a leading cause of cancerrelated death. Bile acids play an important role in the etiology of CRC. Certain bile acids (UDCA) may have chemotherapeutic properties against CRC. UDCA BALB/c mice transfected with GFP drinking Liver CPT-11 Spleen CPT-11 (Irinotecan, Camptostar) is one of the leading anti-cancer drugs for CRC therapy. I.P. injection Hypothesis: 2 weeks later UDCA enhances the antitumor chemotherapeutic action of CPT-11. Fluorescence intensity 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) Spleen Lung Heart Liver Kidney Spleen Liver www.gwu.edu/~ddc/ Bernard Bouscarel, PI Jiamping Meng, Senior Scientist Teruo Miyazaki, Post Doc Maryam Alrashid, Lada Krilov, 5th year IBS student 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