Molecular regulation of hepatocellular transporters

The overall objective of the proposed research project is to understand the regulation of hepatocellular transport systems at a molecular level and their alterations under cholestatic conditions. Disturbances of transporter expression play an important role in the pathogenesis of cholestasis, but the molecular mechanisms leading to these changes are poorly understood. The experimental design should enable the identification of mediators (bile acids and their interactions with cytokines), the level of regulation (transcriptional vs. post-transcriptional) and the involved molecular mechanisms (role of specific nuclear transcription factors). This project aims specifically to investigate the role of proinflammatory cytokines and cytokine-activated pathways in mediating the molecular effects of bile acids on expression of 4 major hepatocellular transporter genes (Ntcp, Bsep, Mrp2, Mrp3). These questions will be adressed in vivo using cytokine-resistant mice, mice treated with a pharmacological inhibitor of cytokine synthesis (rosiglitazone), and TNF-a receptor and nuclear bile acid receptor knockout mice, which will be subjected to (exogenous) bile acid feeding or bile duct ligation (with retention of endogenous bile acids). Expression and activity of hepatocellular transport systems, cytokines, and transcription factors will be determined by RT-PCR, Western blotting, nuclear run-off assays and gelshift mobility assays. Transfection studies with Ntcp (major bile acid uptake system) and Mrp2 (bilirubin conjugate export pump) promoter constructs and nuclear transcription factors (e.g., c-Jun, SHP-1) will enable the identification of the transcriptional mechanisms in vitro. The proposed project has potential pathophysiological and therapeutic relevance. Identification of the transcriptional mechanisms by which cytokines mediate or modulate the molecular effects of bile acids on hepatocellular transporter expression may provide an attractive target for the design of novel therapeutics for cholestatic liver diseases. Suppression of cytokine action could prevent potential detrimental effects of bile acids on transporter expression and, thereby, counteract cholestasis. Given the importance of bile secretion for cholesterol elimination (directly or indirectly after conversion into bile acids), the proposed studies may not only be relevant for the field of cholestasis, but could also advance our understanding of hypercholesterinemia and atherosclerosis. Therefore, the proposed project should contribute to new knowledge in areas of potential importance to clinical therapeutics.

The overall aim of the project was to understand the molecular regulation of hepatocellular transporters by bile acids and proinflammatory cytokines under cholestatic conditions with impaired bile secretion and jaundice. We could demonstrate, that the liver responds to bile acid challenge (e.g. bile acid feeding or accumulating endogenous bile acids in cholestasis) with repression of hepatocellular bile acid uptake systems and induction of alternative overflow pumps facilitating hepatic and subsequently renal elimination of bile acids and other potentially hepatotoxic biliary compounds. This response helps hepatocytes to survive under cholestatic conditions. These transporter changes are mediated mainly by bile acids, while induction of pro-inflammatory cytokines does not play a critical role in cholestasis (e.g. biliary obstruction). Repression of bile acid uptake systems is regulated by the nuclear bile acid receptor FXR, while the induction of alternative overflow/escape pumps and bile acid- detoxifying pathways is independent from FXR. Interestingly, therapeutic approaches can stimulate bile secretion and alternative overflow pathways. As such, therapeutically used ursodeoxycholic acid (UDCA) induces the gene expression of both normal hepatobiliary transport systems and alternative overflow pumps. The search for appropriate animal models for chronic cholestatic disorders / human cholangiopathies has led to the discovery of Mdr2 knockout mice as reliable and highly reproducible animal model of sclerosing cholangitis. Unfortunately, UDCA (the current clinical standard in the treatment of cholestasis) does not improve cholestatic liver injury in these animals despite some minor, isolated beneficial effects on biliary fibrosis. This model will however be useful to develop and test novel and more effective therapeutic approaches targeting adaptive bile acid detoxification and elimination pathways.