RESUMEN
AIMS: Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. METHODS: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS: ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION: ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.
Asunto(s)
Colitis Ulcerosa , Funcion de la Barrera Intestinal , Animales , Femenino , Humanos , Masculino , Ratones , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Células CACO-2 , Colestasis Intrahepática/metabolismo , Colestasis Intrahepática/genética , Claudina-4/metabolismo , Claudina-4/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colitis Ulcerosa/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Funcion de la Barrera Intestinal/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Uniones Estrechas/metabolismoRESUMEN
ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Fosfatidiletanolaminas , Carcinoma Hepatocelular/genética , Fosforilación Oxidativa , Fosfolípidos/metabolismo , Neoplasias Hepáticas/genética , Colesterol , Fosfatidilcolinas , Lipoproteínas LDL/metabolismoRESUMEN
ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases (P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.
Asunto(s)
Adenosina Trifosfatasas , Fosfolípidos , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de la Membrana/metabolismo , LisosomasRESUMEN
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 ß-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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Adenosina Trifosfatasas/inmunología , Endocitosis , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Receptor Toll-Like 4/inmunología , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Macrófagos/citología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , FN-kappa B/inmunología , Transducción de SeñalRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Activación del Canal Iónico , Pulmón/citología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de la Membrana/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Ácidos y Sales Biliares/metabolismo , Transporte Biológico/genética , Western Blotting , Células CACO-2 , Membrana Celular/metabolismo , Colestasis Intrahepática/genética , Colestasis Intrahepática/metabolismo , Heces/química , Humanos , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación , Multimerización de Proteína , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacocinéticaRESUMEN
UNLABELLED: 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.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Hepatocitos/metabolismo , Mutación Missense/genética , Animales , Línea Celular , Colestasis Intrahepática/metabolismo , Colestasis Intrahepática/patología , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Femenino , Hepatocitos/patología , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , RatasRESUMEN
UNLABELLED: 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 beta-subunit or chaperone for ATP8B1 in hepatocytes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/metabolismo , Células CHO , Cricetinae , Cricetulus , ADN Complementario/aislamiento & purificación , Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hemaglutininas/inmunología , Humanos , Hígado/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Fosfatidilserinas/metabolismo , ARN Mensajero/metabolismoRESUMEN
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.