A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type 1, is required for apical protein expression and microvillus formation in polarized epithelial cells

Authors

  • Patricia M. Verhulst,

    1. Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, the Netherlands
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    • These authors contributed equally to this work.

  • Lieke M. van der Velden,

    1. Department of Metabolic and Endocrine Diseases, UMC Utrecht, and Netherlands Metabolomics Centre, the Netherlands
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    • These authors contributed equally to this work.

  • Viola Oorschot,

    1. Cell Microscopy Centre, Department of Cell Biology, Institute of Biomembranes, University Medical Centre Utrecht, The Netherlands
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  • Ernst E. van Faassen,

    1. Faculty of Science, Department of Interface Physics, Utrecht University, The Netherlands
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  • Judith Klumperman,

    1. Cell Microscopy Centre, Department of Cell Biology, Institute of Biomembranes, University Medical Centre Utrecht, The Netherlands
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  • Roderick H. J. Houwen,

    1. Department of Pediatrics, UMC Utrecht, The Netherlands
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  • Thomas G. Pomorski,

    1. Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark
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  • Joost C. M. Holthuis,

    1. Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, the Netherlands
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    • These authors share senior authorship.

  • Leo W. J. Klomp

    Corresponding author
    1. Department of Metabolic and Endocrine Diseases, UMC Utrecht, and Netherlands Metabolomics Centre, the Netherlands
    • University Medical Center, Department of Metabolic and Endocrine Diseases, Room KC.02.069.1, P.O. Box 85090, 3508AB Utrecht, The Netherlands
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    • These authors share senior authorship.

    • fax: (31)-88-7554295


  • Potential conflict of interest: Nothing to report.

Abstract

Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P4 P-type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cell-fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency. (HEPATOLOGY 2010)

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