Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Paulusma, Coen C.; Groen, Annemiek; Kunne, Cindy; Ho-Mok, Kam S.; Spijkerboer, Astrid L.; Waart, Dirk R. de; Hoek, Frans J.; Vreeling, Heleen; Hoeben, Kees A.; Marle, J. van; Pawlikowska, Ludmila; Bull, Laura N.; Hofmann, Alan F.; Knisely, Alexander S.; Elferink, Ronald P.J. Oude

doi:10.1002/hep.21212

Cited by 215 publications

(217 citation statements)

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“…14,15 Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary recovery of PS and cholesterol. 16 We hypothesized that ATP8B1 is a flippase for PS in the canalicular membrane, but direct evidence for this hypothesis remained elusive in our hands. Here we have shown, using the CHO-K1 mutant cell line UPS-1, that ATP8B1 mediates the translocation of PS from the exoplasmic leaflet to the cytoplasmic leaflet of the plasma membrane.…”

Section: Discussionmentioning

confidence: 97%

“…Total RNA was extracted from mouse livers with the Trizol reagent (Invitrogen). cDNA was synthesized from total RNA with an oligo-dT [12][13][14][15][16][17][18] primer and Superscript II reverse transcriptase (Invitrogen). Realtime PCR measurements were performed at 60°C in a Lightcycler apparatus (Roche) with Lightcycler Faststart DNA Master Plus SYBR Green I (Roche).…”

Section: Methodsmentioning

confidence: 99%

“…14,15 We have previously shown in mice that Atp8b1 is essential for maintaining the detergent-resistant properties of the canalicular membrane. 16 When challenged with bile salts, Atp8b1-deficient mice displayed enhanced extraction of cholesterol and ectoenzymes from the canalicular membrane. In addition, Atp8b1-deficient mice had significant amounts of phosphatidylserine (PS) in their bile, whereas PS is essentially absent from wild-type bile.…”

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ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

et al. 2007

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Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%-500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%-25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF-B9 cells resulted in colocalization of both proteins in the canalicular membrane. Conclusion: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1-related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential ␤-subunit or chaperone for ATP8B1 in hepatocytes. (HEPATOLOGY 2008;47: 268-278.)

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Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

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ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

et al. 2007

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“…As a result, the canalicular membrane becomes more sensitive to extraction of cholesterol by hydrophobic bile salts that impairs the activity of the bile salt export pump (ABCB11) and, as a consequence, causes cholestasis. 6 PFIC3 patients suffer from a chronic and progressive cholestasis with high serum g-glutamyltransferase levels; liver histology reveals fibrosis that progresses into cirrhosis with portal inflammation and strong bile duct proliferation. In addition, milder mutations in ABCB4 have been shown to predispose to intrahepatic cholestasis of pregnancy, cholelithiasis, adult biliary cirrhosis, primary sclerosing cholangitis, and drug-and cytokine-induced cholestatic injury.…”

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confidence: 99%

Hepatic cytochrome P450 deficiency in mouse models for intrahepatic cholestasis predispose to bile salt-induced cholestasis

Kunne

Graaff

Duijst

et al. 2014

Laboratory Investigation

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Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1 G308V/G308V and Abcb4 À / À mice with Hrn mice that have a liverspecific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1 G308V/G308V /Hrn and Abcb4 À / À /Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1 G308V/G308V /Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4 À / À /Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1 G308V/G308V /Hrn but was more severe in Abcb4 À / À /Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

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“…At this point, instead of simply determining the expression levels of the remaining thirteen putative human alpha subunits, we adopted a more straightforward approach involving the simultaneous transfection of CDC50A-V5 and murine Atp8b1 (a well-studied P4-ATPase, 95% homologous to its human counterpart [34]) as a Cterminal myc-tagged protein in HeLa and HEK-293T cells (Fig. 8A).…”

Section: Cdc50a and Atp8b1 Associate To Exit The Er And Traffic To Thmentioning

confidence: 99%

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells

Muñoz-Martínez

Torres

Castanys

et al. 2010

Biochemical Pharmacology

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. CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells. Biochemical Pharmacology, Elsevier, 2010, 80 (6) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 ABSTRACT:Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkylphospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosinesensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient)cells, but was only widely distributed in the early and late endosomes in KB cells.Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi.However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.

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Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Cited by 215 publications

References 51 publications

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

Hepatic cytochrome P450 deficiency in mouse models for intrahepatic cholestasis predispose to bile salt-induced cholestasis

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells

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