TY - JOUR
T1 - A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type1, is required for apical protein expression and microvillus formation in polarized epithelial cells
AU - Verhulst, P.M.
AU - van der Velden, L.M.
AU - Oorschot, V.M.J.
AU - van Faassen, E.E.H.
AU - Klumperman, J.
AU - Houwen, R.H.J.
AU - Pomorski, T.
AU - Holthuis, J.C.M.
AU - Klomp, L.W.J.
PY - 2010/6
Y1 - 2010/6
N2 - 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, cellfractionation,
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
AB - 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, cellfractionation,
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
U2 - 10.1002/hep.23586
DO - 10.1002/hep.23586
M3 - Article
SN - 0270-9139
VL - 51
SP - 2049
EP - 2060
JO - Hepatology
JF - Hepatology
IS - 6
ER -