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

Patricia M. Verhulst; Lieke M. van der Velden; Viola Oorschot; Ernst E. van Faassen; Judith Klumperman; Roderick H.J. Houwen; Thomas Günther-Pomorski; Joost C.M. Holthuis; Leo W.J. Klomp

doi:10.1002/hep.23586

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

Patricia M. Verhulst, Lieke M. van der Velden, Viola Oorschot, Ernst E. van Faassen, Judith Klumperman, Roderick H.J. Houwen, Thomas Günther-Pomorski, Joost C.M. Holthuis, Leo W.J. Klomp

55 Citations (Scopus)

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, 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.

Original language	English
Journal	Hepatology
Volume	51
Issue number	6
Pages (from-to)	2049-2060
Number of pages	12
ISSN	0270-9139
DOIs	https://doi.org/10.1002/hep.23586
Publication status	Published - Jun 2010

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Verhulst, P. M., Velden, L. M. V. D., Oorschot, V., Faassen, E. E. V., Klumperman, J., Houwen, R. H. J., Günther-Pomorski, T., Holthuis, J. C. M., & Klomp, L. W. J. (2010). 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. Hepatology, 51(6), 2049-2060. https://doi.org/10.1002/hep.23586

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. / Verhulst, Patricia M.; Velden, Lieke M. van der; Oorschot, Viola et al.
In: Hepatology, Vol. 51, No. 6, 06.2010, p. 2049-2060.

Research output: Contribution to journal › Journal article › Research › peer-review

Verhulst, PM, Velden, LMVD, Oorschot, V, Faassen, EEV, Klumperman, J, Houwen, RHJ, Günther-Pomorski, T, Holthuis, JCM & Klomp, LWJ 2010, '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', Hepatology, vol. 51, no. 6, pp. 2049-2060. https://doi.org/10.1002/hep.23586

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title = "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",

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, 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.",

author = "Verhulst, {Patricia M.} and Velden, {Lieke M. van der} and Viola Oorschot and Faassen, {Ernst E. van} and Judith Klumperman and Houwen, {Roderick H.J.} and Thomas G{\"u}nther-Pomorski and Holthuis, {Joost C.M.} and Klomp, {Leo W.J.}",

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T1 - 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

AU - Verhulst, Patricia M.

AU - Velden, Lieke M. van der

AU - Oorschot, Viola

AU - Faassen, Ernst E. van

AU - Klumperman, Judith

AU - Houwen, Roderick H.J.

AU - Günther-Pomorski, Thomas

AU - Holthuis, Joost C.M.

AU - Klomp, Leo 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.

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DO - 10.1002/hep.23586

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C2 - 20512993

SN - 0270-9139

VL - 51

SP - 2049

EP - 2060

JO - Hepatology

JF - Hepatology

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ER -