title: Vps33b is crucial for structural and functional hepatocyte polarity.
abstract: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion.
  
  
  
  To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments.
  
  
  
  Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects.
  
  
  
  Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy.
  
  
  
  Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Joanna
authors.initials: J
authors.lastname: Hanley
authors.affiliation: Organ Transplantation Centre and Comparative Medicine Department, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia; MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Dipok Kumar
authors.initials: DK
authors.lastname: Dhar
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Francesca
authors.initials: F
authors.lastname: Mazzacuva
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Fiadeiro
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Jemima J
authors.initials: JJ
authors.lastname: Burden
authors.affiliation: UCL Department of Statistical Science, University College London, London WC1E 6BT, UK.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lyne
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Holly
authors.initials: H
authors.lastname: Smith
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Straatman-Iwanowska
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.
authors.forename: Blerida
authors.initials: B
authors.lastname: Banushi
authors.affiliation: Histopathology Department, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Virasami
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Mills
authors.affiliation: Université Catholique de Louvain, de Duve Institute, 1200 Brussels, Belgium.
authors.forename: Frédéric P
authors.initials: FP
authors.lastname: Lemaigre
authors.affiliation: Institut für Pathologie, Medizinische Universität Graz, 8036 Graz, Austria.
authors.forename: A S
authors.initials: AS
authors.lastname: Knisely
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Howe
authors.affiliation: Histopathology Department, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Sebire
authors.affiliation: UCL Institute for Women's Health, University College London, London WC1E 6AU, UK; Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witswatersrand, Johannesburg 2193, South Africa.
authors.forename: Simon N
authors.initials: SN
authors.lastname: Waddington
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, 1105 BK Amsterdam, Netherlands.
authors.forename: Coen C
authors.initials: CC
authors.lastname: Paulusma
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Clayton
authors.affiliation: UCL Institute of Child Health, University College London, London WC1N 1EH, UK; MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK; Inherited Metabolic Disease Unit, Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, UK. Electronic address: p.gissen@ucl.ac.uk.
authors.forename: Paul
authors.initials: P
authors.lastname: Gissen
mesh_terms: D000818:Animals; D001176:Arthrogryposis / Q000473:pathology / Q000628:therapy; D001647:Bile Acids and Salts / Q000097:blood; D016764:Cell Polarity; D002779:Cholestasis / Q000473:pathology / Q000628:therapy; D002784:Cholesterol / Q000097:blood; D015316:Genetic Therapy; D022781:Hepatocytes / Q000502:physiology*; D008099:Liver / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D051437:Renal Insufficiency / Q000473:pathology / Q000628:therapy; D019108:Tight Junctions / Q000502:physiology; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D001647:Bile Acids and Salts; C000601834:VPS33B protein, mouse; D033921:Vesicular Transport Proteins; D002784:Cholesterol
keywords: Bile; Bile canaliculi; Canalicular membrane; Cholestasis; Gene transfer; Hepatocyte polarity; Liver diseases; Protein trafficking; Rab11a; Vps33b