MOLECULAR AND CELLULAR MECHANISMS OF HEPATOCELLULAR POLARITY AND BILIARY SECRETION IN HEALTH AND CHOLESTASIS
, Head, Unit on Cellular Polarity
Yoshiyuki Wakabayashi, PhD, Senior Research Fellow
Dong Fu, PhD, Postdoctoral Fellow
Andrew Cullinane, MS, Graduate Student1
Rebecca Shein, Summer High School Student
Intracellular pathways for trafficking ATP-binding cassette transporters to the bile canalicular domain
Previously, we discovered two pathways by which apical membrane proteins traffic from Golgi to the bile canaliculus in mammalian hepatocytes and polarized WIFB9 cells; the latter cells 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 Golgi to the basolateral plasma membrane domain, from which they undergo transcytosis to the apical membrane. We have identified critical roles for dynamic and stable microtubules, actin, HAX1, N-linked glycans, myosin Vb, PI 3-kinase, and rab 11a in the direct trafficking pathway. Live-cell imaging of BSEP-YFP (yellow fluorescent protein) constructs reveals downstream docking sites in the canalicular membrane, which we are now seeking to identify.
Kagawa T, Watanabe N, Mochizuki K, Numari A, Ikeno Y, Itoh J, Tanaka H, Arias IM, Mine T. Phenotypic differences in PFIC22 and BRIC2 correlate with protein stability of mutant BSEP and impaired taurocholate secretion in MDCK II cells. Amer J Physiol Gastrointest Liver Physiol 2007 [E-pub ahead of print].
Mochizuki K, Kagawa T, Watanabe N, Arias IM. Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells. Amer J Physiol 2007;292:G818-28.
Wakabayashi Y, Chua J, Larkin J, Lippincott-Schwartz J, Arias IM. Four-dimensional imaging of filter grown polarized epithelial cells. Histochem Cell Biol 2007;127:463-72.
Wakabayashi Y, Kipp H, Arias IM. Transporters on demand: intracellular reservoirs and cycling of bile canalicular ABC transporters. J Biol Chem 2006;281:27699-72.
The role of rab 11a, myosin Vb, and other proteins in canalicular polarity
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. Our observations prompted a revision of current polarity concepts and suggest that polarization is initiated upon delivery of rab 11a-myosin Vb–containing vesicles to the surface, causing the plasma membrane at the site of delivery to differentiate into the apical domain (bile canaliculus).
Wakabayashi Y, Dutt P, Lippincott-Schwartz J, Arias IM. Rab 11a and myosin Vb are required for polarization in WIFB Cells. Proc Natl Acad Sci USA 2005;102:15087-92.
Physiologic effect of in vivo expression of adenoviral rab 11a-YFP and myosin Vb-CFP dominant negative constructs in rat liver
Using adenoviral YFP and CFP constructs of rab 11a, BSEP, and dominant negative constructs of rab 11a and myosin Vb, we expanded our cell-biologic studies in vivo in rats. As previously observed in cell culture, the viral constructs are abundantly expressed in most hepatocytes and change ABC transporter distribution and function. The in vivo studies provide an exciting opportunity to explore the molecular mechanisms of bile secretory failure (cholestasis) and the effect of various cholestatic drugs, viruses, and diets and to develop possible new therapies.
Structural and functional characterization of rat and human hepatocytes maintained in unique long-term cultures
Despite years of effort by many laboratories, a major difficulty in studies of mammalian hepatocytes remains the long-standing inability to maintain polarized primary cultures of the hepatocytes. Colleagues at University of North Carolina and the Massachusetts Institute of Technology developed novel culture systems that hold considerable promise for overcoming earlier difficulties. One system maintains hepatocytes in a collagen sandwich and retains the cells’ structure and function for over 14 days. Another system permits preparation of hepatocytes by using a robotic high-throughput platform involving a feeder layer, specific matrix, and cell number; the cells maintain structure and function for over 70 days. We characterized gene expression of important hepatocyte ABC transporters and their transcriptional regulation and performed live-cell imaging of canalicular ABC-GFP trafficking in cells cultured in both systems. Our goal is to identify and quantify normal intracellular trafficking mechanisms for canalicular ABC transporters and for formation and maintenance of apical polarity.
Role of dynamic microtubules in constitutive canalicular trafficking of BSEP and their interaction with actin
In WIFB9 cells, endosomes containing BSEP traffic along microtubules (MTs) throughout the cytosol but target only the canalicular membrane, from which they cycle to rab11a endosomes. We investigated the role of dynamic MTs (t ½ about 10 minutes) and stable MTs (t ½ several hours) in this trafficking process by using three-dimensional reconstruction of MT distribution and quantitative live-cell imaging of BSEP-YFP trafficking combined with fluorescence recovery after photobleaching. Whereas nocodazole inhibited all MT-dependent trafficking, 202F, a marine sponge product, specifically inhibited dynamic MTs; as a consequence, endosomes containing BSEP remained on stable MTs but were not transferred to the apical membrane. Additional studies revealed that MT+ end interacts with actin through several proteins that localize around the bile canaliculus. Our studies may provide the long-sought link between MTs and actin-based endosome trafficking systems that surround the bile canaliculus.
Role of rab 3D in transcytosis
Mechanisms responsible for transcytosis of membrane proteins in polarized cells are poorly understood. Live-cell imaging and biochemical studies of WIB9 and MDCK cells suggest that rab 3D may be critical in transcytosis. We are studying this process by using molecular knockdown methodology, expression of dominant negative constructs, and mice in which rab3D has been deleted. The mutant mice have markedly reduced transcytosis of horseradish peroxidase, confirming a role for rab 3D in transcytosis.
Biology and pathobiology of fenestrae in hepatic endothelial cells
Our previous studies revealed that the fenestrae of hepatic endothelial cells require an actin-myosin–based cytoskeleton and that contraction of the fenestrae may be regulated physiologically. Given the absence of a basement membrane in hepatic sinusoids, fenestrae constitute the only barrier between the circulation and plasma membrane of hepatocytes and regulate transfer of many substances between the plasma and hepatocytes. Using scanning electron microscopy of hepatic endothelial cells from mice in which caveolin I was deleted, we observed reduced numbers and abnormal shapes of cell fenestrae. In a collaborative study, we are exploring the relation between caveolin 1 and fenestra biology.
Function of MRP6 in pseudoxanthomatosis elasticum
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 provides a screen for candidate small peptides that may serve as biologic substrates for mrp6 and elastase inhibitors in vivo. To date, we have identified no such molecules
Gene expression in cholestasis associated with hyperalimentation
We are exploring gene expression patterns in clinical and experimental cholestasis associated with hyperalimentation. In particular, we are quantifying expression of canalicular ABC transporters and transcriptional regulators. Our working hypothesis posits that hyperalimentation alters intracellular trafficking of canalicular ABC transporters, particularly BSEP, resulting in cholestasis.
Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1
Previously, we showed that FIC1 encodes a P-type ATPase and 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. In a collaborative study, we are seeking to elucidate how the P-ATPase and its lipid traffic regulate bile acid secretion.
How vsp33 mutations are responsible for inheritable cholestasis and proximal tubular and neurologic abnormalities in the ARC syndrome
We are performing live-cell imaging of GFP-vps33b to identify its subcellular localization and specific role in canalicular formation, maintenance, and ABC transporter activity.
Publication Related to Other Work
Listowsky I, Arias IM. Ligandin and glutathione S-transferases: historical milestones. In: Awasthi Y, ed. Toxicology of Glutathione Transferases. CRC Press, 2006;1-10.
1 Birmingham, UK2 Janet Larkin, PhD, former Postdoctoral Fellow
3 Victoria Cogger, PhD, former Visiting Scientist, University of Sydney, Sydney, Australia
COLLABORATORS
Marcelo Amar, MD, Molecular Disease Branch, NHBLI, Bethesda, MD
Sangeeta Bhatia, MD, PhD, Massachusetts Institute of Technology, Cambridge, MA
Kim Brauer, PhD, University of North Carolina School of Medicine, Chapel Hill, NC
Anna Calgano, PhD, Laboratory of Cell Biology, NCI, Bethesda, MD
Lewis Cantley, PhD, Harvard Medical School, Boston, MA
Thomas Fishbein, MD, Georgetown University Hospital, Washington, DC
Paul Gissen, MD, PhD, University of Birmingham School of Medicine, Birmingham, UK
Michael Gottesman, MD, Laboratory of Cell Biology, NCI, Bethesda, MD
Tatehiro Kagawa, MD, Tokai University School of Medicine, Kanagawa, Japan
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Daniel Ortiz, PhD, Tufts University, Boston, MA
Alan Remaley, PhD, Department of Laboratory Medicine, NIH Clinical Center, Bethesda, MD
Marcos Rojkind, MD, PhD, The George Washington University, Washington, DC
Benjamin Schneider, MD, Mount Sinai Medical School, New York, NY
Daniel Teitelbaum, MD, University of Michigan Health System, Ann Arbor, MI
Allan Wolkoff, MD, Albert Einstein College of Medicine, New York, NY
For further information, contact ariasi@mail.nih.gov.
