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

Irwin M. Arias, MD
  • Irwin M. Arias, MD, Head, Unit on Cellular Polarity
  • Dong Fu, PhD, Postdoctoral Fellow
  • Malte Renz, MD, Postdoctoral Fellow

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 heritable and acquired bile secretory failure (cholestasis).

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

Previously, the lack of a stable primary culture system limited studies on intracellular trafficking in hepatocytes. However, a recent advance in the development of two systems (a "collagen sandwich" and a platform bioengineered at 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 (Kim Brauer and Sangeeta 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 documented 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 the Golgi to the basolateral plasma membrane domain, from which they undergo trans-cytosis to the apical membrane. We identified critical roles for dynamic and stable microtubules, actin, HAX1, N-linked glycans, myosin Vb, PI 3-kinase, and rab11a in the direct trafficking pathway.

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

An increased AMP:ATP ratio activates AMPK, which increases ATP synthesis (required for glucose uptake, mitochondria and fatty acid oxidation) and downregulates ATP consumption (required for 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. Recently, we discovered that collagen-sandwich cultures of rat hepatocytes undergo sequential development of bile canaliculi, as evidenced by tight junction, microtubular, and actin distribution, and cycling of ABC transporters; the process parallels activation of AMPK and LKBK and is accelerated by agonists of AMPK but blocked by expression of a dominant negative viral construct. It is of great interest that taurocholate is the endogenous activator of the process. We demonstrated that the taurocholate effect is mediated by activation of adenyl cyclase and cAMP production and acts through the Epac pathway and MEK. We are now seeking to establish the link between this pathway and AMPK activation. We established that this pathway is linked to activation of MAeK which, in turn, activates AMPK. In addition, we discovered three downstream targets of AMPK activation that are involved in apical polarization: Par 1, rab11a Flp1, and CLIP170.

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

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 with 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 the actin that 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. Discovery that CLIp 170 is a phosphorylation target of AMPK and live-cell imaging studies in LKB1 Cre-albumin knockout mouse liver reveal that dynamic microtubules participate in polarization. We plan to study possible mechanisms.

The role of rab11a, myosin Vb, and other proteins in canalicular polarity

While studying mechanisms of apical targeting in WIFB9 cells, we observed that rab11a 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 rab11a/myosin Vb–containing vesicles to the cell surface, causing the plasma membrane at the site of delivery to differentiate into the apical domain (bile canaliculus). Similar results were obtained in comparable studies of rat and mouse hepatocytes in collagen sandwich cultures. Furthermore, knockdown of rab 11a flp adapter protein impaired apical trafficking in cell cultures.

Publications

  • Cogger VC, Arias IM, Warren A, McMahon AC, Kiss DL, Avery VM, Le Couteur DG. he response of fenestrations, actin and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells. Amer J Physiol Gastrointest Liver. Physiol 2008 295:G137-G145.
  • Fu D, Wakabayashi Y, Ido Y, Lippincott-schwartz J, Arias IM. Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1. J Cell Sci. 2010 123:3294-3302.
  • Cullinane AR, Straatman-Iwanowska A, Zaucker A, Wakabayashi Y, Bruce CK, Luo G, Rahman F, Gürakan F, Utine E, Ozkan TB, Denecke J, Vukovic J, Di Rocco M, Mandel H, Cangul H, Matthews RP, Thomas SG, Rappoport JZ, Arias IM, Wolburg H, Knisely AS, Kelly DA, Müller F, Maher ER, Gissen P. Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization. Nat Genet. 2010 42:303-312.

Collaborators

  • Sangeeta Bhatia, MD, PhD, Massachusetts Institute of Technology, Cambridge, MA
  • Kim Brauer, PhD, University of North Carolina School of Medicine, Chapel Hill, NC
  • Lewis Cantley, PhD, Harvard Medical School, Boston, MA
  • 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
  • Marcos Rojkind, MD, George Washington University, Washington, DC
  • Niel Ruderman, MD, Boston University School of Medicine, Boston, MA
  • Allan Wolkoff, MD, Albert Einstein College of Medicine, New York, NY

Contact

For further information, contact ariasi@mail.nih.gov or visit cbmp.nichd.nih.gov/ucp.

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