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.
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 P(4) 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.


CONCLUSIONS
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.
Verhulst
Patricia M
PM
Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, the Netherlands.
van der Velden
Lieke M
LM
Oorschot
Viola
V
van Faassen
Ernst E
EE
Klumperman
Judith
J
Houwen
Roderick H J
RH
Pomorski
Thomas G
TG
Holthuis
Joost C M
JC
Klomp
Leo W J
LW
D000251:Adenosine Triphosphatases / Q000172:deficiency* / Q000235:genetics; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D016764:Cell Polarity; D002780:Cholestasis, Intrahepatic / Q000235:genetics; D004847:Epithelial Cells / Q000201:enzymology / Q000648:ultrastructure*; D006801:Humans; D008871:Microvilli / Q000502:physiology; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional
D049294:Phospholipid Transfer Proteins; D000251:Adenosine Triphosphatases; C111067:ATP8B1 protein, human