We utilize live cell fluorescent, biochemical, genetic and molecular techniques to study mechanisms responsible for trafficking of ATP binding cassette transporters selectively to the apical domain of hepatocytes and other polarized cells. The goal is to identify components and regulation of these processes, their role in creating and maintaining hepatocyte polarity, and molecular defects responsible for inheritable and acquired bile secretory failure (cholestasis). To date, numerous specific proteins and lipids have been identified that are critical for trafficking, membrane fusion and recycling of the apical transporters. Genetic and pharmacologic manipulation of these factors results in defective biliary secretion (cholestasis). Our studies indicate a dependent relationship between cellular polarity and the apical trafficking systems. It is likely that acquired cholestasis, due to drugs, viruses, hyperalimentation and other factors, results from an intracellular traffic jam. Specific accomplishments during the past year are: (a) Development of a culture system (up to 80 days) for rat and human hepatocytes that retain gene expression and functional activity. Using these cells, we characterized development of polarity and its dependence on AMP kinase, the metabolic sensor of the cell. Formation of tight junctions, canalicular morphology and trafficking of apical ABC transporters paralelled AMPK activity as influenced by activators or inhibitors. These observations directly link cellular metabolism with polarity. Taurocholate was shown to induce canalicular formation and act through a cAMP-dependent pathway via Epac-MEK to activate AMPK.(b) ABC transporters traffic as cargo in endosomes which move along MTs and transfer to an actin-based system before fusion with the apical membrane. We established that the MT plus end of is at the apical region and that, using a specific inhibitor, dynamic microtubules are required for the trafficking process. Of interest is that several drugs and chemicals which produce cholestasis in man and animals have a structure similar to the dynamic MT specific inhibitor.(c) Hepatic endothelial cells are heavily fenestrated which, in asence of a basement membrane, provides direct contact between hepatocytes and plasma. Fenestrae measure approx. 140 nm diameter and are dynamic structures. Caveolin-1 has been proposed to participate in their formation;however, we observed that they are unrelated.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$569,280
Indirect Cost
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State
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Fu, Dong; Wakabayashi, Yoshiyuki; Lippincott-Schwartz, Jennifer et al. (2011) Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc Natl Acad Sci U S A 108:1403-8
Baranova, Irina N; Bocharov, Alexander V; Vishnyakova, Tatyana G et al. (2010) CD36 is a novel serum amyloid A (SAA) receptor mediating SAA binding and SAA-induced signaling in human and rodent cells. J Biol Chem 285:8492-506
Warren, Alessandra; Cogger, Victoria C; Arias, Irwin M et al. (2010) Liver sinusoidal endothelial fenestrations in caveolin-1 knockout mice. Microcirculation 17:32-8
Cullinane, Andrew R; Straatman-Iwanowska, Anna; Zaucker, Andreas et al. (2010) Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization. Nat Genet 42:303-12
Fu, Dong; Wakabayashi, Yoshiyuki; Ido, Yasuo et al. (2010) Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1. J Cell Sci 123:3294-302