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.)

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/ index.html). (HEPATOLOGY 2006;44:195-204.)

The enterohepatic circulation of bile salts is an important physiological route to recycle bile salts and ensure intestinal absorption of dietary lipids. The Na 1 -taurocholate cotransporting polypeptide SLC10A1 (NTCP) plays a key role in this process as the major transporter of conjugated bile salts from the plasma compartment into the hepatocyte. Here we present the first patient with NTCP deficiency, who was clinically characterized by mild hypotonia, growth retardation, and delayed motor milestones. Total bile salts in plasma were extremely elevated (up to 1,500 lM, ref. <16.3) but there were no clinical signs of cholestatic jaundice, pruritis, or liver dysfunction. Bile salt synthesis and intestinal bile salt signaling were not affected, as evidenced by normal plasma 7a-hydroxy-4-cholesten-3-one (C4) and FGF19 levels. Importantly, the presence of secondary bile salts in the circulation suggested residual enterohepatic cycling of bile salts. Sequencing of the SLC10A1 gene revealed a single homozygous nonsynonymous point mutation in the coding sequence of the gene, resulting in an arginine to histidine substitution at position 252. Functional studies showed that this mutation resulted in a markedly reduced uptake activity of taurocholic acid. Immunofluorescence studies and surface biotinylation experiments demonstrated that the mutant protein is virtually absent from the plasma membrane. Conclusion: We describe the identification of NTCP deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype. The identification of NTCP deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence. (HEPATOLOGY 2015;61:260-267)

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1), forming a spectrum of cholestatic disease. Whereas PFIC1 is a progressive, endstage liver disease, BRIC1 patients suffer from episodic periods of cholestasis that resolve spontaneously. At present it is not clear how the type and location of the mutations relate to the clinical manifestations of PFIC1 and BRIC1. ATP8B1 localizes to the canalicular membrane of hepatocytes where it mediates the inward translocation of phosphatidylserine. ATP8B1 interacts with CDC50A, which is required for endoplasmic reticulum exit and plasma membrane localization. In this study we analyzed a panel of missense mutations causing PFIC1 (G308V, D554N, G1040R) or BRIC1 (D70N, I661T). In addition, we included two mutations that have been associated with intrahepatic cholestasis of pregnancy (ICP) (D70N, R867C). We examined the effect of these mutations on protein stability and interaction with CDC50A in Chinese hamster ovary cells, and studied the subcellular localization in WIF-B9 cells. Protein stability was reduced for three out of six mutations studied. Two out of three PFIC1 mutant proteins did not interact with CDC50A, whereas BRIC1/ICP mutants displayed reduced interaction. Importantly, none of the PFIC1 mutants were detectable in the canalicular membrane of WIF-B9 cells, whereas all BRIC1/ICP mutants displayed the same cellular staining pattern as wild-type ATP8B1. Our data indicate that PFIC1 mutations lead to the complete absence of canalicular expression, whereas in BRIC1/ICP residual protein is expressed in the canalicular membrane. Conclusion: These data provide an explanation for the difference in severity between the phenotypes of PFIC1 and BRIC1. (HEPATOLOGY 2009;50: 1597-1605

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-016-2360-5) contains supplementary material, which is available to authorized users.

ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ~65% was conjugated and ~35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na+-taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels. Conclusions ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome.

Deficiency of the phospholipid flippase ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1) causes progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1). Apart from cholestasis, many patients also suffer from diarrhea of yet unknown etiology. Here we have studied the hypothesis that intestinal ATP8B1 deficiency results in bile salt malabsorption as a possible cause of PFIC1/BRIC1 diarrhea. Bile salt transport was studied in ATP8B1-depleted intestinal Caco-2 cells. Apical membrane localization was studied by a biotinylation approach. Fecal bile salt and electrolyte contents were analyzed in stool samples of PFIC1 patients, of whom some had undergone biliary diversion or liver transplantation. Bile salt uptake by the apical sodium-dependent bile salt transporter solute carrier family 10 (sodium/bile acid cotransporter), member 2 (SLC10A2) was strongly impaired in ATP8B1-depleted Caco-2 cells. The reduced SLC10A2 activity coincided with strongly reduced apical membrane localization, which was caused by impaired apical membrane insertion of SLC10A2. Moreover, we show that endogenous ATP8B1 exists in a functional heterodimer with transmembrane protein 30A (CDC50A) in Caco-2 cells. Analyses of stool samples of post-transplant PFIC1 patients demonstrated that bile salt content was not changed, whereas sodium and chloride concentrations were elevated and potassium levels were decreased. The ATP8B1-CDC50A heterodimer is essential for the apical localization of SLC10A2 in Caco-2 cells. Diarrhea in PFIC1/BRIC1 patients has a secretory origin to which SLC10A2 deficiency may contribute. This results in elevated luminal bile salt concentrations and consequent enhanced electrolyte secretion and/or reduced electrolyte resorption.

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.