In collaboration with Jennifer Lippincott-Schwartz and members of her laboratory, we use live-cell fluorescent, biochemical, genetic, and molecular techniques to study mechanisms responsible for the selective trafficking of proteins to the apical domain of hepatocytes and other polarized cells. Our goal is to identify components and regulation of these processes, their role in creating and maintaining cellular polarity, and the molecular defects responsible for heritable and acquired bile secretory failure (cholestasis).
Intracellular pathways for trafficking ATP binding cassette (ABC) transporters to the bile canalicular domain
Wakabayashi, Ortiz4
Previously, we discovered two pathways by which apical membrane proteins traffic from the Golgi to the bile canaliculus in mammalian hepatocytes and in polarized WIFB9 cells, which are a hybrid of a rat hepatoma and human fibroblasts. Canalicular ATP binding cassette (ABC) proteins, such as BSEP (bile acid transporter), MRP2 (nonbile acid organic anion transporter), and MDR 1 (organic cation transporter), enter a large intracellular rab 11a–enriched endosomal pool from which they cycle to the apical plasma membrane. In contrast, single transmembrane proteins, such as cCAM105 and 5´nucleotidase, traffic from the Golgi to the basolateral plasma membrane domain from which they undergo transcytosis to the apical membrane. We have identified critical roles for tubulin, actin, GGA, hax 2, myosin vb, PI 3-kinase, and rab 11a in the direct trafficking pathway. Live-cell imaging of BSEP-YFP constructs reveals downstream docking sites in the canalicular membrane, sites that we seek to identify.
Chan W, Calderon G, Arias IM, Ortiz D. Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby Canine Kidney cells. J Biol Chem 2005;280:23741-23747.
Mochizuki K, Kagawa T, Arias IM. Role of N-linked glycosylation in folding, trafficking and function of bile salt export pump. Am J Physiol (in press).
Ortiz DF, Mosely J, Calderon G, Swift AL, Arias IM. Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of MDCK cells. J Biol Chem 2004;279:32761-32770.
Wakabayashi Y, Lippincott-Schwartz J, Arias IM. Intracellular trafficking of bile salt expert pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab 11-positive endosomes. Mol Biol Cell 2004;15:3485-3496.
The role in canalicular polarity of rab 11a, myosin Vb, and other proteins
Wakabayashi, Larkin, Elkind
While studying mechanisms of apical targeting in WIFB9 cells, we observed that rab 11a and myosin Vb are required for canalicular formation. Expression of dominant negative constructs or RNAi prevented polarization and resulted in trafficking patterns found in nonpolarized cells. These observations prompted revision of current polarity concepts and suggest that polarization is initiated upon delivery of rab 11a– myosin Vb–containing vesicles to the surface, causing plasma membrane at the site of delivery to differentiate into the apical domain (bile canaliculus).
Wakabayashi Y, Dutt P, Lippincott-Schwartz J, Arias IM. Rab11a and myosinVb are required for polarization of WIF-B9 cells. Proc Natl Acad Sci USA 2005;102:115087-15092.
Physiologic effect of in vivo expression of adenoviral rab 11a–YFP and myosin Vb–CFP dominant negative constructs in rat liver
Veulleux,5 Ortiz,4 Wakabayashi
Using adenoviral YFP and CFP constructs of rab 11a and BSEP, we expanded our in vivo cell-biologic studies in rats. The viral constructs are abundantly expressed in most hepatocytes, but not in other cells. Changes in ABC transporter distribution and function were similar to that observed in cell cultures. The in vivo studies provide an exciting opportunity to explore molecular mechanisms of bile-secretory failure (cholestasis) and the effect of various cholestatic drugs, viruses, diets, and development and to investigate possible new therapies.
Role of rab 3D in transcytosis
Larkin; in collaboration with Remaly
Transcytosis of membrane proteins in polarized cells is functionally important; however, the underlying molecules and cellular mechanisms and their regulation are poorly understood. Live-cell imaging and biochemical studies suggest that rab 3D may be critical in transcytosis. We are studying transcytosis with molecular knockdown methodology and expression of dominant negative constructs and in mice in which rab3D has been deleted.
Regulation of intracellular localization and trafficking of ABC transporters of the G subfamily
Elkind; in collaboration with Kruth
Using transfected cell lines, we propose to expand previous observations made with MDR-GFP and BSEP-YFP to other ABC transporters, particularly ABCG1 and ABCG2, whose functions and regulation are less well understood. To this end, we produced adenoviral GFP-ABCG2 constructs and dominant negatives and the lentiviral-expressed ABC transporters ABCG1 and ABCG2. We will simultaneously express ABC transporters and RNAi for specific knockdown of gene products involved in trafficking of the ABC transporter in question.
To understand the role of the ABCG1 transporter in foam cell production and cholesterol homeostasis, we will also express ABCG1 from a lentiviral vector in human monocyte–derived macrophages and foam cells. ABCG1 is highly induced upon macrophage uptake of LDL cholesterol and causes cholesterol efflux from macrophages directly to HDL, which represents a different mechanism for cholesterol efflux than that used by ABCA1. The working hypothesis is that ABCGl regulates cholesterol efflux and that ABCG1 overexpression in response to cholesterol/lipid loading causes macrophage death in the atherosclerotic plaque, resulting in thrombosis.
Biology and pathobiology of fenestrae in hepatic endothelial cells
Coggin; in collaboration with Leapman
Hepatic endothelial cells are heavily fenestrated. Our previous studies indicated that the fenestrae are formed on an actin-myosin–based cytoskeleton and that their contraction can be regulated physiologically. Given the absence of basement membrane in hepatic sinusoids, fenestrae constitute the only barrier between the circulation and the plasma membrane of hepatocytes. Using a newly described hepatic endothelial cell line with regulatable fenestrae, we are exploring the cell biology and physiology of fenestrae.
Function of MRP6 in pseudoxanthomatosis elasticum
Arias; in collaboration with Rojkind
MRP6, an ABC transporter restricted to the basolateral plasma membrane of hepatocytes, is mutated in patients with pseudoxanthomatosis elasticum, a disease of impaired elastic tissue in blood vessels, eye, and skin. We are exploring the possibility that the hepatocyte normally secretes an elastase inhibitor into the serum. A sensitive, specific assay for elastase activity functions as a screen for a battery of candidate small peptides that may be biologic substrates for MRP6 and serve as elastase inhibitors in vivo.
Role of specific decapeptide in regulating PI 3 kinase activity and its role in intracellular trafficking of ABC transporters
Arias; in collaboration with Cantley, Jamney, Leveille-Webster
We are exploring the role of a decapeptide that enhances PI 3-kinase activity in cells and in vivo by rendering the substrate, PI 45 P2, more susceptible to the enzyme. Our studies reveal that 3´ phospho-inositides are required for trafficking of ABC transporters and for their activity in the plasma membrane. Furthermore, rhodamine-conjugated decapeptide was shown to be a potent choleretic agent in vivo that may be useful therapeutically.
Gene expression in cholestasis associated with hyperalimentation
Arias; in collaboration with Gottesman
We are exploring gene expression patterns in clinical and experimental cholestasis associated with hyperalimentation. The Gottesman laboratory has developed an array procedure that permits examination of all known hepatocyte transporters, drug-metabolizing enzymes, and other critical genes.
Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1
Arias; in collaboration with Harris, Schneider
We are studying the cellular and molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1 (PFIC 1). FIC1 encodes a P-type ATPase, which, as we previously showed, functions as an aminophospho-lipid flippase in the basolateral plasma membrane of hepatocytes and small intestinal cells. FIC1 regulates FXR, a nuclear transcription factor, which in turn regulates the activity of BSEP and other apical ABC transporters. Using molecular and imaging techniques, we seek to elucidate how the P-ATPase and its lipid traffic regulate bile acid secretion.
Harris MJ, Arias IM. FIC1, a P-type ATPase linked to cholestatic liver disease, has homologues (ATP8B2 and ATP8B3) expressed throughout the body. Biochim Biophys Acta 2003;1633:127-131.
Effect of hepatitis B replicon expression on BSEP trafficking in WIF-B9 cells
Arias; in collaboration with Rice
Infection of hepatocytes with hepatitis B or C viruses frequently produces prolonged cholestasis without cellular necrosis. We propose that viral replication is associated with altered intracellular trafficking of canalicular ABC transporters, particularly Bsep. In collaboration with Charles Rice and colleagues, we are investigating the effect of several HBV replicons on trafficking of ABC transporters in WIFB-9 cells.
Sexual dimorphic expression of ABC transporters in mouse liver
Arias; in collaboration with Simon
We are examining the mechanism responsible for sexual dimorphic expression of mrp2 and other ABC transporters in mice. Our studies reveal that the process is mediated by growth hormone and modified by thyroid hormone.
1Assistant to the Director, Intramural Program, NIHG, Bethesda, MD; Professor of Physiology and Medicine, Tufts School of Medicine, Boston, MA
2Also ANZAC Research Institute, University of Sydney, Australia
3Also Tufts School of Medicine, Boston, MA
4Daniel Ortiz, PhD, Research Assistant Professor, Tufts School of Medicine, Boston, MA
5Michael Veilleux, MA, Research Technician, Tufts School of Medicine, Boston, MA
collaborators
Lewis Cantley, PhD, Harvard Medical School, Boston, MA
Michael Gottesman, MD, Laboratory of Cell Biology, NCI, Bethesda, MD
Matt Harris, PhD, Avastra, Inc., Sydney, Australia
Paul Jamney, PhD, University of Pennsylvania School of Medicine, Philadelphia, PA
Howard Kruth, PhD, Cardiovascular Branch, NHLBI, Bethesda, MD
Richard Leapman, PhD, Division of Bioengineering and Physical Science, NIH, Bethesda, MD
Cynthia Leveille-Webster, DVM, Tufts School of Veterinary Medicine, Grafton, MA
Alan Remaly, MD, PhD, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, Bethesda, MD
Charles Rice, PhD, The Rockefeller University, New York, NY
Marcos Rojkind, MD, PhD, George Washington University, Washington, DC
Benjamin Schneider, MD, Mount Sinai Medical School, New York, NY
Franz Simon, MD, University of Colorado School of Medicine, Denver, CO
For further information, contact ariasi@mail.nih.gov.