Molecular and Cellular Mechanisms of Hepatocellular Polarity and Biliary Secretion In Health and Cholestasis

Irwin M. Arias, MD, Head, Unit on Cellular Polarity
Yoshiyuki Wakabayashi, PhD, Senior Research Fellow
Dong Fu, PhD, Postdoctoral Fellow
Malte Renz, MD, Postdoctoral Fellow
Divny Narendra, Summer Program College Student

In collaboration with Jennifer Lippincott-Schwartz’s laboratory, we use biochemical, genetic, molecular, and live-cell imaging techniques to study mechanisms responsible for selective trafficking of proteins to the apical domain of hepatocytes and other polarized cells. Our goal is to identify the components and regulation of these trafficking processes, their role in creating and maintaining cellular polarity, and the molecular defects responsible for inheritable and acquired bile secretory failure (cholestasis).

Intracellular pathways for trafficking ATP-binding cassette (ABC) transporters to the bile canalicular domain

Wakabayashi, Fu, Narendra; in collaboration with Bhatia, Brauer, Calgano, Gottesman, Kagawa, Wolkoff

The lack of a stable primary culture system previously limited studies on intracellular trafficking in hepatocytes. However, a recent advance in the development of two systems (a “collagen sandwich” and a platform bioengineered by the massachusetts institute of technology) now permits long-term (10–60 day) cultures of primary rat and human hepatocytes. In collaboration with the pioneers of these systems (Brauer and Bhatia), we used live-cell imaging and immunocytochemistry to characterize such cultured hepatocytes maintained with respect to the expression profiles of aBc transporters and intracellular trafficking. We established the time-dependent pattern of formation of tight junctions, canalicular structure, and trafficking of aBc transporters to the canalicular membrane. sequential live-cell FRap (fluorescence recovery after photobleaching) and Flip (fluorescence loss in photobleaching) studies confirmed previous findings obtained in WiFB9 cells and revealed the direct golgi-to-canalicular trafficking pathway for atp cassette transporters and rab11a-myosin Vb–dependent cycling to the apical membrane.

Previously, we discovered two pathways by which apical membrane proteins traffic from the golgi to the bile canaliculus in mammalian hepatocytes and polarized WiFB9 cells (the latter cells are a hybrid of rat hepatoma and human fibroblasts). Canalicular aBc proteins, such as aBcB11 (bile acid transporter), aBc1 (non–bile acid organic anion transporter), and aBcB1 (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 ccam 105 and 5′ nucleotidase, traffic from golgi to the basolateral plasma membrane domain, from which they undergo transcytosis to the apical membrane. We 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 aBcB11-YFp constructs revealed downstream docking sites in the canalicular membrane; we are now in the process of identifying those sites.

Kagawa T, Watanabe N, Mochizuki K, Numari A, Ikeno Y, Itoh J, Tanaka H, Arias IM, Mine T. Phenotypic differences in pFic2 and BRic2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in mdcK ii cells. Amer J Physiol Gastrointest Liver Physiol 2008;294:g58-g67.
Mochizuki K, Kagawa T, Watanabe N, Arias IM. Two n-linked glycans are required to maintain the transport activity of the bile export pump (aBcB11) in mdcK ii cells. Amer J Physiol 2007;292:g818-g828.
Wakabayashi Y, Chua J, Larkin J, Lippincott-Schwartz J, Arias IM. Four-dimensional imaging of filter grown polarized epithelial cells. Histochem Cell Biol 2007;127:463-472.
Wakabayashi Y, Kipp H, Arias IM. Transporters on demand: intracellular reservoirs and cycling of bile canalicular aBc transporters. J Biol Chem 2006;281:27669-27672.

The metabolic sensor AMPK and its upstream activator LKB1 participate in bile canalicular and tight junction formation and maintenance and apical content of ABCB11

Fu, Wakabayashi, Renz, Arias; in collaboration with Cantley, Ruderman, Brauer, Cal

An increased amp:atp ratio activates ampK, which increases atp synthesis (glucose uptake, mitochondria and fatty acid oxidation) and downregulates atp consumption (protein, lipid synthesis). ampK is also activated by lKB1-dependent phosphorylation, which responds to stress and growth factor–dependent signals. Studies in Drosophila and mammalian cell lines demonstrate a role for lKB1 and ampK in polarization.

In sandwich cultures of rat hepatocytes, agonists of ampK phosphorylation enhanced apical polarization, whereas inhibitors delayed maturation and prevented development of apical polarization. Removal of calcium from the medium prompted depolarization; however, co-incubation with ampK agonists prevented depolarization. Our discovery links metabolism (amp:atp ratio) to polarization and aBc transporter trafficking. as such, it provides a potential link with bile secretory failure (cholestasis) in hyperalimentation, starvation, diabetes, pregnancy, and liver regeneration, a link that we are currently exploring. Our goal is to determine the pathway, components, and mechanism whereby the ampK-lKB pathway is linked to tight junction structure and function, apical polarization, and the direct pathway for trafficking of aBc transporters to the canalicular membrane.

Role of dynamic microtubules in apical trafficking of the bile acid transporter ABCB11 in WIFB9 cells

Wakabayashi, Arias; in collaboration with Lippincott-Schwartz

Microtubules are required for aBcB11 trafficking from rab11a-positive endosomes to the bile canaliculus. However, the specific contributions of microtubule subsets remain unknown. Using polarized WiF-B9 cells, we investigated the role of dynamic microtubules in canalicular targeting of aBcB11. At the steady state, aBcB11 traffics to the canalicular membrane. After specifically disassembling dynamic microtubules by using a marine sponge–derived quinone, we observed that aBcB11-containing endosomal movement continued along stable microtubules; however, canalicular targeting was abolished. Immunostaining of alpha-tubulin and eB1, a dynamic microtubular plus-end tracking protein, revealed a pericanalicular web that includes the plus end of dynamic microtubules. To explore how dynamic microtubules regulate canalicular targeting of aBcB11, we performed immunostaining of iQgap, Rac, apc, and eB1, which link the dynamic microtubular plus end and actin. iQgap, Rac, and apc surrounded the bile canaliculus in association with actin and eB1. Our results demonstrate that canalicular targeting of aBcB11 depends on dynamic microtubules whose plus ends may establish the long-sought bridge between microtubules and actin, which is required for endosome trafficking to the bile canaliculus. Our goal is to determine whether depolymerization of dynamic microtubules is the mechanism whereby various quinone based drugs and chemicals cause cholestasis in humans and/or animals. We are also exploring the mechanism and sites for microtubular assembly after depolymerization.

The role of rab 1a, myosin Vb, and other proteins in canalicular polarity Wakabayashi, Fu, Narendra; in collaboration with Cantley, Wolkoff, Bhatia, Brauer

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 non-polarization 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).

Our goal is to confirm preliminary results in long-term non-dividing rat hepatocyte cultures by using similar constructs and quantitative live-cell imaging of aBc transporter movement. We are exploring molecular mechanisms of polarization and the specific role of the direct atB transporter pathway in the polarization process.

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 USA2005;102:15087-15092.

Biology and pathobiology of fenestrae in hepatic endothelial cells

Arias; in collaboration with Cogger

Our previous studies revealed that the fenestrae of hepatic endothelial cells require an actinmyosin–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, regulating 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. Additional studies in sK hep1 cells revealed fenestrae that are structurally and functionally similar to those in hepatic endothelial cells associated with caveolin-1. Our immediate goal is to measure the functional effects of caveolin-1 deletion on throughput kinetics in rat liver by using a multiple isotope technique.

Cogger V, Lecouter D, Arias IM. Response of fenestrations, actin and caveolin-1 to vascular endothelial growth factor in sK hep1 cells. Amer J Physiol 2008, in press.

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 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.

Thus far, we have tested a large number of candidate peptides, none of which is an elastase inhibitor. A more recent proposal is that mRp6 transports vitamin K components, which are secreted as gla-modified peptides that function as regulators of peripheral elastases. Our goal is to test this hypothesis in MRP6^−/− mice by determining gla peptides in serum, urine, and bile.

Gene expression in cholestasis associated with hyperalimentation

Arias; in collaboration with Calgano, Gottesman, Fishbein, Teitelbaum

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 the bile salt export pump (Bsep), resulting in cholestasis.

Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1

Arias; in collaboration with Shneider, Paulusma, Perez-Victoria

Previously, we showed that FIC1 encodes a p-type atpase, which functions as an aminophospholipid 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 the 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.

In collaboration with Shneider, Paulusma, and Perez-Victoria, we seek to identify cytosolic factors for vesicle formation. Thus far, we have demonstrated only that several yeast p4-atpases are involved in membrane trafficking. Of interest is that one P-type ATPase, drs2p, regulates retrograde trafficking from the plasma membrane and endosomes to the trans-golgi network. We propose that the retrograde route, which is controlled by several recently identified proteins and is critical for membrane homeostasis, may be linked to Fic1 function. Our goal is to determine the possible role of Fic1 in retrograde trafficking in hepatocytes and, more specifically, whether Fic1 regulates membrane protein sorting at the trans-golgi network, regulates recycling from the plasma membrane, and/or interacts with members of the retrograde machinery.

Frankenbert T, Miloh T, Chen F, Ananthanarayanan M, Sun AQ, Balasubramaniyan N, Arias I, Setchell KdR, Suchy FJ, Shneider Bl. The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor. Hepatology 2008; [E-pub ahead of print].

Publications Related To Other Work

Arias IM. Demystifying medicine: selected topics from 2005-2006. National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development.
Arias IM. perspective: five decades of cholestasis research and the brave new world. Hepatology 2008; 47:777-785.
Listowsky I, Arias IM. Ligandin and glutathione s-transferases: historical milestones. in: Awasthi Y, ed. Toxicology of Glutathione Transferases. CRC press, 2006;1-10.

Collaborators

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
Victoria Cogger, PhD, University of Sydney, Sydney, Australia
Thomas Fishbein, MD, Georgetown University Hospital, Washington, DC
Michael Gottesman, MD, Laboratory of Cell Biology, NCI, Bethesda, MD
Tatehiro Kagawa, MD, Tokai University School of Medicine, Kanagwa, Japan
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Coen Paulusma, PhD, Academic Medical Center, Amsterdam, The Netherlands
Javier Perez-Victoria, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Alan Remaley, PhD, Department of Laboratory Medicine, NIH Clinical Center, Bethesda, MD
Marcos Rojkind, MD, PhD, George Washington University, Washington, DC
Neil Ruderman, MD, DPhil, Boston University School of Medicine, Boston, MA
Benjamin Shneider, 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 or visit http://cbmp.nichd.nih.gov/ucp/index.html