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She HY, Qiu YL, Feng JY, Cheng Y, Chi H, van IJzendoorn SCD, Xing QH, Wang JS. A liver-specific mouse model for MYO5B-associated cholestasis reveals a toxic gain-of-function as underlying disease mechanism. Biochem Biophys Res Commun 2025; 758:151669. [PMID: 40127562 DOI: 10.1016/j.bbrc.2025.151669] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2025] [Revised: 03/18/2025] [Accepted: 03/19/2025] [Indexed: 03/26/2025]
Abstract
Myosin Vb (MYO5B) deficiency, referring to the loss of protein expression or function, causes microvillus inclusion disease (MVID) and/or progressive familial intrahepatic cholestasis-type 10 (PFIC10) in humans. MYO5B plays a role in intracellular trafficking, but the mechanisms by which it contributes to cholestasis are not understood. The aim of this study was to generate a liver-specific mouse model and investigate the mechanism of MYO5B-associated cholestasis. In this study, we generated a liver-specific Myo5b cKO mice via CRISPR/Cas9 genome editing in conjunction with albumin-cre recombinase. Cholestatic stress was induced by dietary-administration of cholic acid (CA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). To investigate the frequently recurring MYO5B variant (c.2470C > T/p.(Arg824Cys)), adenoviral vectors encoding either the missense variant or blank control sequence were delivered to wild-type and Myo5b cKO mice through tail-vein injection. Serum and liver tissues were harvested from all mice for biochemical and histological analysis. Our findings indicated that loss of Myo5b expression did not cause cholestatic liver disease and did not augment CA or DDC feeding-induced cholestatic stress. By contrast, expression of the MYO5B c.2470C > T/p. (Arg824Cys) variant induced cholestasis, evidenced by elevated levels of serum alanine aminotransferase, alkaline phosphatase and bilirubin, mild hepatocellular injury, and altered bile salt export pump (Bsep) localization, resembling that observed in human PFIC10. In summary, we have developed a mouse model of MYO5B-associated cholestasis. The expression of the MYO5B-p. (Arg824Cys) variant but not the loss of Myo5b expression caused cholestasis, indicating a toxic gain-of-function as underlying disease mechanism.
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Affiliation(s)
- Hui-Yu She
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai, 201102, China
| | - Yi-Ling Qiu
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai, 201102, China
| | - Jia-Yan Feng
- Department of Pathology, Children's Hospital of Fudan University, Shanghai, 201102, China
| | - Ye Cheng
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai, 201102, China; Institutes of Biomedical Sciences of Fudan University and Children's Hospital of Fudan University, Shanghai, 201102, China
| | - Hao Chi
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai, 201102, China
| | - Sven C D van IJzendoorn
- Department of Biomedical Sciences, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Qing-He Xing
- Institutes of Biomedical Sciences of Fudan University and Children's Hospital of Fudan University, Shanghai, 201102, China.
| | - Jian-She Wang
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai, 201102, China.
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Collier GE, Lavado R. An in-depth examination of Per- and Polyfluoroalkyl (PFAS) effects on transporters, with emphasis on the ABC superfamily: A critical review. Toxicology 2024; 508:153901. [PMID: 39094918 DOI: 10.1016/j.tox.2024.153901] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 07/27/2024] [Accepted: 07/29/2024] [Indexed: 08/04/2024]
Abstract
Per- and polyfluoroalkyl (PFAS) substances are a type of chemical compound unique for their multiple carbon-fluorine bonds, imbuing them with strength and environmental permanence. While legacy substances have been phased out due to human health risks, short-chain and alternative PFAS remain omnipresent. However, a detailed explanation for the pathways through which PFAS interact on a cellular and molecular level is still largely unknown, and the human health effects remain mechanistically unexplained. Of particular interest when focusing on this topic are the interactions between these exogenous chemicals and plasma and membrane proteins. Such proteins include serum albumin which can transport PFAS throughout the body, solute carrier proteins (SLC) and ATP binding cassette (ABC) transporters which are able to move PFAS into and out of cells, and proteins and nuclear receptors which interact with PFAS intracellularly. ABC transporters as a family have little available human data despite being responsible for the export of endogenous substances and drugs throughout the body. The multifactorial regulation of these crucial transporters is affected directly and indirectly by PFAS. Changes, which can include alterations to membrane transport activity and differences in protein expression, vary greatly depending on the specific PFAS and protein of interest. Together, the myriad of changes caused by understudied PFAS exposure to a class of understudied proteins crucial to cellular function and drug treatments has not been fully explored regarding human health and presents room for further exploration. This critical work aims to provide a novel framework of existing human data on PFAS and ABC transporters, allowing for future advancement and investigation into human transporter activity, mechanisms of regulation, and interactions with emerging contaminants.
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Affiliation(s)
- Gracen E Collier
- Department of Environmental Science, Baylor University, Waco, TX 76798, United States
| | - Ramon Lavado
- Department of Environmental Science, Baylor University, Waco, TX 76798, United States.
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Ghanem CI, Manautou JE. Role and Regulation of Hepatobiliary ATP-Binding Cassette Transporters during Chemical-Induced Liver Injury. Drug Metab Dispos 2022; 50:1376-1388. [PMID: 35914951 PMCID: PMC9513844 DOI: 10.1124/dmd.121.000450] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Accepted: 07/20/2022] [Indexed: 11/22/2022] Open
Abstract
Severity of drug-induced liver injury (DILI) ranges from mild, asymptomatic, and transient elevations in liver function tests to irreversible liver damage, often needing transplantation. Traditionally, DILI is classified mechanistically as high-frequency intrinsic DILI, commonly dose dependent or DILI that rarely occurs and is idiosyncratic in nature. This latter form is not dose dependent and has a pattern of histopathological manifestation that is not always uniform. Currently, a third type of DILI called indirect hepatotoxicity has been described that is associated with the pharmacological action of the drug. Historically, DILI was primarily linked to drug metabolism events; however, the impact of transporter-mediated rates of drug uptake and excretion has gained greater prominence in DILI research. This review provides a comprehensive view of the major findings from studies examining the contribution of hepatic ATP-binding cassette transporters as key contributors to DILI and how changes in their expression and function influence the development, severity, and overall toxicity outcome. SIGNIFICANCE STATEMENT: Drug-induced liver injury (DILI) continues to be a focal point in drug development research. ATP-binding cassette (ABC) transporters have emerged as important determinants of drug detoxification, disposition, and safety. This review article provides a comprehensive analysis of the literature addressing: (a) the role of hepatic ABC transporters in DILI, (b) the influence of genetic mutations in ABC transporters on DILI, and (c) new areas of research emphasis, such as the influence of the gut microbiota and epigenetic regulation, on ABC transporters.
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Affiliation(s)
- Carolina I Ghanem
- Instituto de Investigaciones Farmacológicas (ININFA-UBA-CONICET) (C.I.G.) and Cátedra de Fisiopatología (C.I.G.), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina; and Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (J.E.M.)
| | - Jose E Manautou
- Instituto de Investigaciones Farmacológicas (ININFA-UBA-CONICET) (C.I.G.) and Cátedra de Fisiopatología (C.I.G.), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina; and Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (J.E.M.)
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Wang L, Qiu YL, Xu HM, Zhu J, Li SJ, OuYang WX, Yang YF, Lu Y, Xie XB, Xing QH, Wang JS. MYO5B-associated diseases: Novel liver-related variants and genotype-phenotype correlation. Liver Int 2022; 42:402-411. [PMID: 34811877 DOI: 10.1111/liv.15104] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Revised: 10/23/2021] [Accepted: 11/15/2021] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS Biallelic pathogenic variants in MYO5B cause microvillus inclusion disease (MVID), or familial intrahepatic cholestasis (FIC). The reported FIC patients are scarce and so the genotype-phenotype correlation has not been fully characterised. This study aimed to report more MYO5B-associated FIC patients and correlate genotypes to phenotypes in more detail. METHODS The phenotype and genetic data of 12 newly diagnosed MYO5B-associated (including 11 FIC) patients, as well as 118 previously reported patients with available genotypes, were summarised. Only patients with biallelic MYO5B variants were enrolled. Nonsense, frameshift, canonical splice sites, initiation codon loss, and single exon or multiexon deletion were defined as null MYO5B variants. RESULTS Phenotypically, 50 were isolated MVID, 47 involved both liver and intestine (combined), and 33 were isolated FIC (9 persistent, 15 recurrent, 3 transient, and 6 un-sub-classified) patients. The severity of intestinal manifestation was positively correlated to an increased number of null variants (ρ = 0.299, P = .001). All FIC patients carried at least one non-null variant, and the severity of cholestasis was correlated to the presence of a null variant (ρ = 0.420, P = .029). The proportion of FIC patients (16/29, 55%) harbouring missense/in-frame variants affecting the non-motor regions of MYO5B was significantly higher than that of MVID (3/25, 12%, P = .001) and combined patients (3/31, 10%, P = .000). 10 of the 29 FIC patients harboured missense/in-frame variants at the IQ motifs comparing to none in the 56 MVID and combined patients (P = .000). CONCLUSIONS The phenotype of MYO5B deficiency was associated with MYO5B genotypes, the nullity or the domain affected.
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Affiliation(s)
- Li Wang
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, China
| | - Yi-Ling Qiu
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, China
| | - Hong-Mei Xu
- Department of Infectious Diseases, Children's Hospital of Chongqing Medical University, Chongqing, China
| | - Jing Zhu
- Department of Infectious Diseases, Children's Hospital of Chongqing Medical University, Chongqing, China
| | - Shuang-Jie Li
- Department of Hepatopathy, Hunan Children's Hospital, Changsha, China
| | - Wen-Xian OuYang
- Department of Hepatopathy, Hunan Children's Hospital, Changsha, China
| | - Yong-Feng Yang
- Department of Hepatology, The Second Hospital of Nanjing, Nanjing, China
| | - Yi Lu
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, China
| | - Xin-Bao Xie
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, China
| | - Qing-He Xing
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China
| | - Jian-She Wang
- The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, National Children's Medical Center, Shanghai, China
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Stalke A, Sgodda M, Cantz T, Skawran B, Lainka E, Hartleben B, Baumann U, Pfister ED. KIF12 Variants and Disturbed Hepatocyte Polarity in Children with a Phenotypic Spectrum of Cholestatic Liver Disease. J Pediatr 2022; 240:284-291.e9. [PMID: 34555379 DOI: 10.1016/j.jpeds.2021.09.019] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 09/13/2021] [Accepted: 09/14/2021] [Indexed: 01/03/2023]
Abstract
KIF12 has been identified as a cholestasis-associated candidate gene. We describe 6 cases from 4 unrelated families with diverse cholestatic phenotypes carrying 2 different homozygous KIF12 truncating variants. Immunofluorescence investigations of paraffin-embedded liver sections suggest that KIF12-associated impaired functional cell polarity may be the underlying cause.
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Affiliation(s)
- Amelie Stalke
- Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany; Department of Human Genetics, Hannover Medical School, Hannover, Germany.
| | - Malte Sgodda
- Translational Hepatology and Stem Cell Biology, Department of Gastroenterology, Hepatology and Endocrinology, REBIRTH-Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Tobias Cantz
- Translational Hepatology and Stem Cell Biology, Department of Gastroenterology, Hepatology and Endocrinology, REBIRTH-Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany
| | - Britta Skawran
- Department of Human Genetics, Hannover Medical School, Hannover, Germany
| | - Elke Lainka
- Department for Pediatric Nephrology, Gastroenterology, Endocrinology and Transplant Medicine, University Children's Hospital Essen, Essen, Germany
| | - Björn Hartleben
- Institute of Pathology, Hannover Medical School, Hannover, Germany
| | - Ulrich Baumann
- Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany
| | - Eva-Doreen Pfister
- Pediatric Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany
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6
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RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic. Int J Mol Sci 2021; 22:ijms22137087. [PMID: 34209301 PMCID: PMC8268348 DOI: 10.3390/ijms22137087] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 06/28/2021] [Accepted: 06/29/2021] [Indexed: 12/11/2022] Open
Abstract
ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.
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Hepatic bile acid transport increases in the postprandial state: A functional 11C-CSar PET/CT study in healthy humans. JHEP Rep 2021; 3:100288. [PMID: 34095797 PMCID: PMC8165435 DOI: 10.1016/j.jhepr.2021.100288] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 03/21/2021] [Accepted: 03/24/2021] [Indexed: 12/22/2022] Open
Abstract
Background & Aims It is not known how hepatic bile acids transport kinetics changes postprandially in the intact liver. We used positron emission tomography (PET)/computed tomography (CT) with the tracer [N-methyl-11C]cholylsarcosine (11C-CSar), a synthetic sarcosine conjugate of cholic acid, to quantify fasting and postprandial hepatic bile acid transport kinetics in healthy human participants. Methods Six healthy human participants underwent dynamic liver 11C-CSar PET/CT (60 min) during fasting and from 15 min after ingestion of a standard liquid meal. Hepatobiliary secretion kinetics of 11C-CSar was calculated from PET data, blood samples (arterial and hepatic venous) and hepatic blood flow measured using indocyanine green infusion. Results In the postprandial state, hepatic blood perfusion increased on average by 30% (p <0.01), and the flow-independent hepatic intrinsic clearance of 11C-CSar from blood into bile increased by 17% from 1.82 (range, 1.59–2.05) to 2.13 (range, 1.75–2.50) ml blood/min/ml liver tissue (p = 0.042). The increased intrinsic clearance of 11C-CSar was not caused by changes in the basolateral clearance efficacy of 11C-CSar but rather by an upregulated apical transport, as shown by an increase in the rate constant for apical secretion of 11C-CSar from hepatocyte to bile from 0.40 (0.25–0.54) min−1 to 0.67 (0.36–0.98) min−1 (p = 0.03). This resulted in a 33% increase in the intrahepatic bile flow (p = 0.03). Conclusions The rate constant for the transport of bile acids from hepatocytes into biliary canaliculi and the bile flow increased significantly in the postprandial state. This reduced the mean 11C-CSar residence time in the hepatocytes. Lay summary Bile acids are important for digestion of dietary lipids including vitamins. We examined how the secretion of bile acids by the liver into the intestines changes after a standard liquid meal. The transport of bile acids from liver cells into bile and bile flow was increased after the meal.
Following a meal, the active transport of bile acids from hepatocytes into bile is increased significantly. A meal also increases bile flow out of the liver. The postprandial changes are induced shortly after intake of a meal.
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Wu SH, Chang MH, Chen YH, Wu HL, Chua HH, Chien CS, Ni YH, Chen HL, Chen HL. The ESCRT-III molecules regulate the apical targeting of bile salt export pump. J Biomed Sci 2021; 28:19. [PMID: 33750401 PMCID: PMC7941988 DOI: 10.1186/s12929-020-00706-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 12/30/2020] [Indexed: 11/17/2022] Open
Abstract
Background The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. Methods The human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. Results We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. Conclusions Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.
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Affiliation(s)
- Shang-Hsin Wu
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100, Taiwan
| | - Mei-Hwei Chang
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100, Taiwan.,Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan.,Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan
| | - Ya-Hui Chen
- Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan
| | - Hui-Lin Wu
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100, Taiwan.,Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan
| | - Huey-Huey Chua
- Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan
| | - Chin-Sung Chien
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100, Taiwan
| | - Yen-Hsuan Ni
- Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan.,Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan.,Medical Microbiota Center of the First Core Laboratory, National Taiwan University College of Medicine, Taipei, 100, Taiwan
| | - Hui-Ling Chen
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan.
| | - Huey-Ling Chen
- Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan. .,Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan. .,Department and Graduate Institute of Medical Education and Bioethics, National Taiwan University College of Medicine, Taipei, 100, Taiwan.
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Molecular Regulation of Canalicular ABC Transporters. Int J Mol Sci 2021; 22:ijms22042113. [PMID: 33672718 PMCID: PMC7924332 DOI: 10.3390/ijms22042113] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 02/15/2021] [Accepted: 02/18/2021] [Indexed: 12/17/2022] Open
Abstract
The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.
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10
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A Link between Intrahepatic Cholestasis and Genetic Variations in Intracellular Trafficking Regulators. BIOLOGY 2021; 10:biology10020119. [PMID: 33557414 PMCID: PMC7914782 DOI: 10.3390/biology10020119] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Revised: 01/27/2021] [Accepted: 02/01/2021] [Indexed: 12/20/2022]
Abstract
Simple Summary Cholestasis refers to a medical condition in which the liver is not capable of secreting bile. The consequent accumulation of toxic bile components in the liver leads to liver failure. Cholestasis can be caused by mutations in genes that code for proteins involved in bile secretion. Recently mutations in other genes have been discovered in patients with cholestasis of unknown origin. Interestingly, many of these newly discovered genes code for proteins that regulate the intracellular distribution of other proteins, including those involved in bile secretion. This group of genes thus suggests the deregulated intracellular distribution of bile-secreting proteins as an important but still poorly understood mechanism that underlies cholestasis. To expedite a better understanding of this mechanism, we have reviewed these genes and their mutations and we discuss these in the context of cholestasis. Abstract Intrahepatic cholestasis is characterized by the accumulation of compounds in the serum that are normally secreted by hepatocytes into the bile. Genes associated with familial intrahepatic cholestasis (FIC) include ATP8B1 (FIC1), ABCB11 (FIC2), ABCB4 (FIC3), TJP2 (FIC4), NR1H4 (FIC5) and MYO5B (FIC6). With advanced genome sequencing methodologies, additional mutated genes are rapidly identified in patients presenting with idiopathic FIC. Notably, several of these genes, VPS33B, VIPAS39, SCYL1, and AP1S1, together with MYO5B, are functionally associated with recycling endosomes and/or the Golgi apparatus. These are components of a complex process that controls the sorting and trafficking of proteins, including those involved in bile secretion. These gene variants therefore suggest that defects in intracellular trafficking take a prominent place in FIC. Here we review these FIC-associated trafficking genes and their variants, their contribution to biliary transporter and canalicular protein trafficking, and, when perturbed, to cholestatic liver disease. Published variants for each of these genes have been summarized in table format, providing a convenient reference for those who work in the intrahepatic cholestasis field.
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Sohail MI, Dönmez-Cakil Y, Szöllősi D, Stockner T, Chiba P. The Bile Salt Export Pump: Molecular Structure, Study Models and Small-Molecule Drugs for the Treatment of Inherited BSEP Deficiencies. Int J Mol Sci 2021; 22:E784. [PMID: 33466755 PMCID: PMC7830293 DOI: 10.3390/ijms22020784] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2020] [Revised: 01/11/2021] [Accepted: 01/12/2021] [Indexed: 02/07/2023] Open
Abstract
The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.
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Affiliation(s)
| | - Yaprak Dönmez-Cakil
- Department of Histology and Embryology, Faculty of Medicine, Maltepe University, Maltepe, 34857 Istanbul, Turkey;
| | - Dániel Szöllősi
- Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse, 13A, 1090 Vienna, Austria;
| | - Thomas Stockner
- Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse, 13A, 1090 Vienna, Austria;
| | - Peter Chiba
- Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Waehringerstrasse, 10, 1090 Vienna, Austria
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12
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Sarkadi B, Homolya L, Hegedűs T. The ABCG2/BCRP transporter and its variants - from structure to pathology. FEBS Lett 2020; 594:4012-4034. [PMID: 33015850 DOI: 10.1002/1873-3468.13947] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Revised: 08/27/2020] [Accepted: 09/21/2020] [Indexed: 12/13/2022]
Abstract
The ABCG2 protein has a key role in the transport of a wide range of structurally dissimilar endo- and xenobiotics in the human body, especially in the tissue barriers and the metabolizing or secreting organs. The human ABCG2 gene harbors a high number of polymorphisms and mutations, which may significantly modulate its expression and function. Recent high-resolution structural data, complemented with molecular dynamic simulations, may significantly help to understand intramolecular movements and substrate handling, as well as the effects of mutations on the membrane transporter function of ABCG2. As reviewed here, structural alterations may result not only in direct alterations in drug binding and transporter activity, but also in improper folding or problems in the carefully regulated process of trafficking, including vesicular transport, endocytosis, recycling, and degradation. Here, we also review the clinical importance of altered ABCG2 expression and function in general drug metabolism, cancer multidrug resistance, and impaired uric acid excretion, leading to gout.
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Affiliation(s)
- Balázs Sarkadi
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary.,Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - László Homolya
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
| | - Tamás Hegedűs
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
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Mózner O, Bartos Z, Zámbó B, Homolya L, Hegedűs T, Sarkadi B. Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism. Cells 2019; 8:E1215. [PMID: 31597297 PMCID: PMC6830335 DOI: 10.3390/cells8101215] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Revised: 10/04/2019] [Accepted: 10/05/2019] [Indexed: 02/07/2023] Open
Abstract
The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.
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Affiliation(s)
- Orsolya Mózner
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.
| | - Zsuzsa Bartos
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.
- MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Tűzoltó u. 37-47, 1094 Budapest, Hungary.
| | - Boglárka Zámbó
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.
| | - László Homolya
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.
| | - Tamás Hegedűs
- MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Tűzoltó u. 37-47, 1094 Budapest, Hungary.
- Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary.
| | - Balázs Sarkadi
- Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.
- Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary.
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14
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Jiang LQ, Wang TY, Wang Y, Wang ZY, Bai YT. Co-disposition of chitosan nanoparticles by multi types of hepatic cells and their subsequent biological elimination: the mechanism and kinetic studies at the cellular and animal levels. Int J Nanomedicine 2019; 14:6035-6060. [PMID: 31534335 PMCID: PMC6681437 DOI: 10.2147/ijn.s208496] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Accepted: 07/03/2019] [Indexed: 12/12/2022] Open
Abstract
Background: The clearance of nanomaterials (NMs) from the liver is essential for clinical safety, and their hepatic clearance is primarily determined by the co-disposition process of various types of hepatic cells. Studies of this process and the subsequent clearance routes are urgently needed for organic NMs, which are used as drug carriers more commonly than the inorganic ones. Materials and methods: In this study, the co-disposition of chitosan-based nanoparticles (CsNps) by macrophages and hepatocytes at both the cellular and animal levels as well as their subsequent biological elimination were investigated. RAW264.7 and Hepa1-6 cells were used as models of Kupffer cells and hepatocytes, respectively. Results: The cellular studies showed that CsNps released from RAW264.7 cells could enter Hepa1-6 cells through both clathrin- and caveolin-mediated endocytosis. The transport from Kupffer cells to hepatocytes was also studied in mice, and it was observed that most CsNps localized to the hepatocytes after intravenous injection. Following the distribution in hepatocytes, the hepatobiliary-fecal excretion route was shown to be the primary elimination route for CsNps, besides the kidney-urinary excretion route. The elimination of CsNps in mice was a lengthy process, with a half time of about 2 months. Conclusion: The demonstration in this study of the transport of CsNps from macrophages to hepatocytes and the subsequent hepatobiliary-fecal excretion provides basic information for the future development and clinical application of NMs.
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Affiliation(s)
- Li-Qun Jiang
- Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
| | - Ting-Yu Wang
- Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
| | - Yun Wang
- Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
| | - Zi-Yao Wang
- Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
| | - Yu-Ting Bai
- Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
- School of Stomatology, Xuzhou Medical University, Xuzhou, China
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15
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Fu D, Cardona P, Ho H, Watkins PB, Brouwer KLR. Novel Mechanisms of Valproate Hepatotoxicity: Impaired Mrp2 Trafficking and Hepatocyte Depolarization. Toxicol Sci 2019; 171:431-442. [PMID: 31368504 PMCID: PMC6760262 DOI: 10.1093/toxsci/kfz154] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2019] [Revised: 07/02/2019] [Accepted: 07/03/2019] [Indexed: 12/14/2022] Open
Abstract
Drug-induced liver injury (DILI) remains a major challenge in drug development. Although numerous mechanisms for DILI have been identified, few studies have focused on loss of hepatocyte polarization as a DILI mechanism. The current study investigated the effects of valproate, an antiepileptic drug with DILI risk, on the cellular mechanisms responsible for loss of hepatocyte polarization. Fully polarized collagen sandwich-cultured rat hepatocytes were treated with valproate (1-20mM) for specified times (3-24hr). Hepatocyte viability was significantly decreased by 10mM and 20mM valproate. Valproate depolarized hepatocytes, even at non-cytotoxic concentrations (=5mM). Depolarization was associated with significantly decreased canalicular levels of multidrug resistance-associated protein 2 (Mrp2) resulting in reduced canalicular excretion of the Mrp2 substrate carboxydichlorofluorescein. The decreased canalicular Mrp2 was associated with intracellular accumulation of Mrp2 in Rab11-positive recycling endosomes and early endosomes. Mechanistic studies suggested that valproate inhibited canalicular trafficking of Mrp2. This effect of valproate on Mrp2 appeared to be selective in that valproate had less impact on canalicular levels of the bile salt export pump (Bsep) and no detectable effect on P-glycoprotein (P-gp) canalicular levels. Treatment with valproate for 24hr also significantly downregulated levels of tight junction-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of tight junction proteins claudin 1, claudin 2, occludin, ZO1 and ZO3. These findings reveal that two novel mechanisms may contribute to valproate hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.
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Affiliation(s)
- Dong Fu
- UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Panli Cardona
- UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Henry Ho
- UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Paul B Watkins
- UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Kim L R Brouwer
- UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
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16
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Roma MG, Barosso IR, Miszczuk GS, Crocenzi FA, Pozzi EJS. Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis. Curr Med Chem 2019; 26:1113-1154. [DOI: 10.2174/0929867325666171205153204] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 09/06/2017] [Accepted: 09/07/2017] [Indexed: 12/25/2022]
Abstract
Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of “cholestatic” signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.
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Affiliation(s)
- Marcelo G. Roma
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Ismael R. Barosso
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Gisel S. Miszczuk
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Fernando A. Crocenzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Enrique J. Sánchez Pozzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
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Abstract
Genetic cholestasis has been dissected through genetic investigation. The major PFIC genes are now described. ATP8B1 encodes FIC1, ABCB11 encodes BSEP, ABCB4 encodes MDR3, TJP2 encodes TJP2, NR1H4 encodes FXR, and MYO5B encodes MYO5B. The full spectra of phenotypes associated with mutations in each gene are discussed, along with our understanding of the disease mechanisms. Differences in treatment response and targets for future treatment are emerging.
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Affiliation(s)
- Laura N Bull
- Department of Medicine and Institute for Human Genetics, University of California San Francisco, UCSF Liver Center Laboratory, Zuckerberg San Francisco General, 1001 Potrero Avenue, Building 40, Room 4102, San Francisco, CA 94110, USA.
| | - Richard J Thompson
- Institute of Liver Studies, King's College London, King's College Hospital, Denmark Hill, London SE5 9RS, UK
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18
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Identification of novel loci for pediatric cholestatic liver disease defined by KIF12, PPM1F, USP53, LSR, and WDR83OS pathogenic variants. Genet Med 2018; 21:1164-1172. [PMID: 30250217 DOI: 10.1038/s41436-018-0288-x] [Citation(s) in RCA: 76] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Accepted: 08/17/2018] [Indexed: 12/18/2022] Open
Abstract
PURPOSE Genetic testing in pediatric cholestasis can be very informative but genetic causes have not been fully characterized. METHODS Exome sequencing and positional mapping in seven families with cholestatic liver disease and negative clinical testing for known disease genes. RESULTS KIF12, which encodes a microtubule motor protein with a tentative role in cell polarity, was found to harbor three homozygous likely deleterious variants in three families with sclerosing cholangitis. KIF12 expression is dependent on HNF-1β, deficiency which is known to cause bile duct dysmorphogenesis associated with loss of KIF12 expression. In another extended family, we mapped an apparently novel syndrome of sclerosing cholangitis, short stature, hypothyroidism, and abnormal tongue pigmentation in two cousins to a homozygous variant in PPM1F (POPX2), a regulator of kinesin-mediated ciliary transport. In the fifth family, a syndrome of normal gamma glutamyltransferase (GGT) cholestasis and hearing loss was found to segregate with a homozygous truncating variant in USP53, which encodes an interactor with TJP2. In the sixth family, we mapped a novel syndrome of transient neonatal cholestasis, intellectual disability, and short stature to a homozygous variant in LSR, an important regulator of liver development. In the last family of three affected siblings, a novel syndrome of intractable itching, hypercholanemia, short stature, and intellectual disability was mapped to a single locus that contains a homozygous truncating variant in WDR83OS (C19orf56), known to interact with ATP13A2 and BSEP. CONCLUSION Our results expand the genetic heterogeneity of pediatric cholestatic liver disease and highlight the vulnerability of bile homeostasis to a wide range of molecular perturbations.
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Schlegel C, Weis VG, Knowles BC, Lapierre LA, Martin MG, Dickman P, Goldenring JR, Shub MD. Apical Membrane Alterations in Non-intestinal Organs in Microvillus Inclusion Disease. Dig Dis Sci 2018; 63:356-365. [PMID: 29218485 PMCID: PMC5797493 DOI: 10.1007/s10620-017-4867-5] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2017] [Accepted: 11/22/2017] [Indexed: 12/09/2022]
Abstract
OBJECTIVES Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation. METHODS We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking. RESULTS Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia. CONCLUSIONS These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.
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Affiliation(s)
- Cameron Schlegel
- Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA
- Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
| | - Victoria G Weis
- Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA
- Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
| | - Byron C Knowles
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA
- Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
| | - Lynne A Lapierre
- Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA
- Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
| | - Martin G Martin
- Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - Paul Dickman
- Division of Pathology and Laboratory Medicine, Phoenix Children's Hospital, Phoenix, AZ, USA
- Department of Child Health, University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA
| | - James R Goldenring
- Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA.
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
- Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
| | - Mitchell D Shub
- Division of Gastroenterology, Phoenix Children's Hospital, Phoenix, AZ, USA
- Department of Child Health, University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA
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20
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Yang G, Ge S, Singh R, Basu S, Shatzer K, Zen M, Liu J, Tu Y, Zhang C, Wei J, Shi J, Zhu L, Liu Z, Wang Y, Gao S, Hu M. Glucuronidation: driving factors and their impact on glucuronide disposition. Drug Metab Rev 2017; 49:105-138. [PMID: 28266877 DOI: 10.1080/03602532.2017.1293682] [Citation(s) in RCA: 95] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Glucuronidation is a well-recognized phase II metabolic pathway for a variety of chemicals including drugs and endogenous substances. Although it is usually the secondary metabolic pathway for a compound preceded by phase I hydroxylation, glucuronidation alone could serve as the dominant metabolic pathway for many compounds, including some with high aqueous solubility. Glucuronidation involves the metabolism of parent compound by UDP-glucuronosyltransferases (UGTs) into hydrophilic and negatively charged glucuronides that cannot exit the cell without the aid of efflux transporters. Therefore, elimination of parent compound via glucuronidation in a metabolic active cell is controlled by two driving forces: the formation of glucuronides by UGT enzymes and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surfaces in various drug disposition organs. Contrary to the common assumption that the glucuronides reaching the systemic circulation were destined for urinary excretion, recent evidences suggest that hepatocytes are capable of highly efficient biliary clearance of the gut-generated glucuronides. Furthermore, the biliary- and enteric-eliminated glucuronides participate into recycling schemes involving intestinal microbes, which often prolong their local and systemic exposure, albeit at low systemic concentrations. Taken together, these recent research advances indicate that although UGT determines the rate and extent of glucuronide generation, the efflux and uptake transporters determine the distribution of these glucuronides into blood and then to various organs for elimination. Recycling schemes impact the apparent plasma half-life of parent compounds and their glucuronides that reach intestinal lumen, in addition to prolonging their gut and colon exposure.
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Affiliation(s)
- Guangyi Yang
- a Department of Pharmacy , Institute of Wudang Herbal Medicine Research, Taihe Hospital, Hubei University of Medicine , Shiyan , Hubei , China.,b Hubei Provincial Technology and Research Center for Comprehensive Development of Medicinal Herbs, Hubei University of Medicine , Shiyan , Hubei , China
| | - Shufan Ge
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA
| | - Rashim Singh
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA
| | - Sumit Basu
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA
| | - Katherine Shatzer
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA
| | - Ming Zen
- d Department of Thoracic and Cardiomacrovascular Surgery , Taihe Hospital, Hubei University of Medicine , Shiyan , Hubei , China
| | - Jiong Liu
- e Department of Digestive Diseases Surgery , Taihe Hospital, Hubei University of Medicine , Shiyan , Hubei , China
| | - Yifan Tu
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA
| | - Chenning Zhang
- a Department of Pharmacy , Institute of Wudang Herbal Medicine Research, Taihe Hospital, Hubei University of Medicine , Shiyan , Hubei , China
| | - Jinbao Wei
- a Department of Pharmacy , Institute of Wudang Herbal Medicine Research, Taihe Hospital, Hubei University of Medicine , Shiyan , Hubei , China
| | - Jian Shi
- f Department of Pharmacy , Institute of Translational Chinese Medicine, Guangzhou University of Chinese Medicine , Guangzhou , Guangdong , China
| | - Lijun Zhu
- f Department of Pharmacy , Institute of Translational Chinese Medicine, Guangzhou University of Chinese Medicine , Guangzhou , Guangdong , China
| | - Zhongqiu Liu
- f Department of Pharmacy , Institute of Translational Chinese Medicine, Guangzhou University of Chinese Medicine , Guangzhou , Guangdong , China
| | - Yuan Wang
- g Department of Pharmacy , College of Pharmacy, Hubei University of Medicine , Shiyan , Hubei , China
| | - Song Gao
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA.,g Department of Pharmacy , College of Pharmacy, Hubei University of Medicine , Shiyan , Hubei , China
| | - Ming Hu
- c Department of Pharmacological and Pharmaceutical Sciences , College of Pharmacy, University of Houston , Houston , TX , USA.,g Department of Pharmacy , College of Pharmacy, Hubei University of Medicine , Shiyan , Hubei , China
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Enrich C, Rentero C, Grewal T. Annexin A6 in the liver: From the endocytic compartment to cellular physiology. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2016; 1864:933-946. [PMID: 27984093 DOI: 10.1016/j.bbamcr.2016.10.017] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Revised: 10/25/2016] [Accepted: 10/26/2016] [Indexed: 12/15/2022]
Abstract
Annexin A6 (AnxA6) belongs to the conserved annexin family - a group of Ca2+-dependent membrane binding proteins. AnxA6 is the largest of all annexins and highly expressed in smooth muscle, hepatocytes, endothelial cells and cardiomyocytes. Upon activation, AnxA6 binds to negatively charged phospholipids in a wide range of intracellular localizations, in particular the plasma membrane, late endosomes/pre-lysosomes, but also synaptic vesicles and sarcolemma. In these cellular sites, AnxA6 is believed to contribute to the organization of membrane microdomains, such as cholesterol-rich lipid rafts and confer multiple regulatory functions, ranging from vesicle fusion, endocytosis and exocytosis to programmed cell death and muscle contraction. Growing evidence supports that Ca2+ and Ca2+-binding proteins control endocytosis and autophagy. Their regulatory role seems to operate at the level of the signalling pathways that initiate autophagy or at later stages, when autophagosomes fuse with endolysosomal compartments. The convergence of the autophagic and endocytic vesicles to lysosomes shares several features that depend on Ca2+ originating from lysosomes/late endosomes and seems to depend on proteins that are subsequently activated by this cation. However, the involvement of Ca2+ and its effector proteins in these autophagic and endocytic stages still remains poorly understood. Although AnxA6 makes up almost 0.25% of total protein in the liver, little is known about its function in hepatocytes. Within the endocytic route, we identified AnxA6 in endosomes and autophagosomes of hepatocytes. Hence, AnxA6 and possibly other annexins might represent new Ca2+ effectors that regulate converging steps of autophagy and endocytic trafficking in hepatocytes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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Affiliation(s)
- Carlos Enrich
- Departament de Biomedicina, Unitat de Biologia Cellular, Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.
| | - Carles Rentero
- Departament de Biomedicina, Unitat de Biologia Cellular, Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain
| | - Thomas Grewal
- Faculty of Pharmacy A15, University of Sydney, Sydney, NSW 2006, Australia
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Hegyi Z, Homolya L. Functional Cooperativity between ABCG4 and ABCG1 Isoforms. PLoS One 2016; 11:e0156516. [PMID: 27228027 PMCID: PMC4882005 DOI: 10.1371/journal.pone.0156516] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2016] [Accepted: 05/16/2016] [Indexed: 11/18/2022] Open
Abstract
ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system.
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Affiliation(s)
- Zoltán Hegyi
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
| | - László Homolya
- Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
- * E-mail:
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Gupta A, Schell MJ, Bhattacharjee A, Lutsenko S, Hubbard AL. Myosin Vb mediates Cu+ export in polarized hepatocytes. J Cell Sci 2016; 129:1179-89. [PMID: 26823605 DOI: 10.1242/jcs.175307] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2015] [Accepted: 01/20/2016] [Indexed: 02/06/2023] Open
Abstract
The cellular machinery responsible for Cu(+)-stimulated delivery of the Wilson-disease-associated protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the Cu(+)-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B-myosin Vb interaction reduces the apical surface expression of ATP7B. Overexpression of the myosin Vb tail, which competes for binding of subapical cargos to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular Cu(+) export. The myosin-Vb-dependent targeting step occurred in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on γ-tubulin. ATP7B became selectively arrested in this compartment at high [Cu(+)] in the presence of myosin Vb tail, suggesting that these compartments are precursors of donor-acceptor transfer stations for apically targeted cargos of myosin Vb. Our data suggest that reduced hepatic Cu(+) clearance in idiopathic non-Wilsonian types of disease might be associated with the loss of function of myosin Vb.
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Affiliation(s)
- Arnab Gupta
- Department of Cell Biology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Michael J Schell
- Department of Cell Biology, Johns Hopkins University, Baltimore, MD 21205, USA
| | | | - Svetlana Lutsenko
- Department of Physiology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Ann L Hubbard
- Department of Cell Biology, Johns Hopkins University, Baltimore, MD 21205, USA
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Telbisz Á, Homolya L. Recent advances in the exploration of the bile salt export pump (BSEP/ABCB11) function. Expert Opin Ther Targets 2015; 20:501-14. [PMID: 26573700 DOI: 10.1517/14728222.2016.1102889] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
INTRODUCTION The bile salt export pump (BSEP/ABCB11), residing in the apical membrane of hepatocyte, mediates the secretion of bile salts into the bile. A range of human diseases is associated with the malfunction of BSEP, including fatal hereditary liver disorders and mild cholestatic conditions. Manifestation of these diseases primarily depends on the mutation type; however, other factors such as hormonal changes and drug interactions can also trigger or influence the related diseases. AREAS COVERED Here, we summarize the recent knowledge on BSEP by covering its transport properties, cellular localization, regulation and major mutations/polymorphisms, as well as the hereditary and acquired diseases associated with BSEP dysfunction. We discuss the different model expression systems employed to understand the function of the BSEP variants, their drug interactions and the contemporary therapeutic interventions. EXPERT OPINION The limitations of the available model expression systems for BSEP result in controversial conclusions, and obstruct our deeper insight into BSEP deficiencies and BSEP-related drug interactions. The knowledge originating from different methodologies, such as clinical studies, molecular genetics, as well as in vitro and in silico modeling, should be integrated and harmonized. Increasing availability of robust molecular biological tools and our better understanding of the mechanism of BSEP deficiencies should make the personalized, mutation-based therapeutic interventions more attainable.
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Affiliation(s)
- Ágnes Telbisz
- a Institute of Enzymology, Research Centre for Natural Sciences , Hungarian Academy of Sciences , Magyar tudósok körútja 2, Budapest 1117 , Hungary
| | - László Homolya
- a Institute of Enzymology, Research Centre for Natural Sciences , Hungarian Academy of Sciences , Magyar tudósok körútja 2, Budapest 1117 , Hungary
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Gissen P, Arias IM. Structural and functional hepatocyte polarity and liver disease. J Hepatol 2015; 63:1023-37. [PMID: 26116792 PMCID: PMC4582071 DOI: 10.1016/j.jhep.2015.06.015] [Citation(s) in RCA: 203] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2015] [Revised: 06/14/2015] [Accepted: 06/15/2015] [Indexed: 02/08/2023]
Abstract
Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease. A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases.
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Affiliation(s)
- Paul Gissen
- MRC Laboratory for Molecular Cell Biology, University College London, London, UK; UCL Institute of Child Health, London, UK; Great Ormond Street Hospital, London, UK.
| | - Irwin M Arias
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
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Anwer MS. Role of protein kinase C isoforms in bile formation and cholestasis. Hepatology 2014; 60:1090-7. [PMID: 24700589 PMCID: PMC4141907 DOI: 10.1002/hep.27088] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2013] [Accepted: 02/13/2014] [Indexed: 12/11/2022]
Abstract
Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short-term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC-mediated regulation of plasma membrane localization of Na+-taurocholate cotransporting polypeptide (NTCP) and multidrug resistance-associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine-rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections.
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Affiliation(s)
- M Sawkat Anwer
- Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA
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Girard M, Lacaille F, Verkarre V, Mategot R, Feldmann G, Grodet A, Sauvat F, Irtan S, Davit-Spraul A, Jacquemin E, Ruemmele F, Rainteau D, Goulet O, Colomb V, Chardot C, Henrion-Caude A, Debray D. MYO5B and bile salt export pump contribute to cholestatic liver disorder in microvillous inclusion disease. Hepatology 2014; 60:301-10. [PMID: 24375397 DOI: 10.1002/hep.26974] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2013] [Accepted: 12/04/2013] [Indexed: 12/12/2022]
Abstract
UNLABELLED Microvillous inclusion disease (MVID) is a congenital disorder of the enterocyte related to mutations in the MYO5B gene, leading to intractable diarrhea often necessitating intestinal transplantation (ITx). Among our cohort of 28 MVID patients, 8 developed a cholestatic liver disease akin to progressive familial intrahepatic cholestasis (PFIC). Our aim was to investigate the mechanisms by which MYO5B mutations affect hepatic biliary function and lead to cholestasis in MVID patients. Clinical and biological features and outcome were reviewed. Pretransplant liver biopsies were analyzed by immunostaining and electron microscopy. Cholestasis occurred before (n = 5) or after (n = 3) ITx and was characterized by intermittent jaundice, intractable pruritus, increased serum bile acid (BA) levels, and normal gamma-glutamyl transpeptidase activity. Liver histology showed canalicular cholestasis, mild-to-moderate fibrosis, and ultrastructural abnormalities of bile canaliculi. Portal fibrosis progressed in 5 patients. No mutation in ABCB11/BSEP or ATP8B1/FIC1 genes were identified. Immunohistochemical studies demonstrated abnormal cytoplasmic distribution of MYO5B, RAB11A, and BSEP in hepatocytes. Interruption of enterohepatic BA cycling after partial external biliary diversion or graft removal proved the most effective to ensure long-term remission. CONCLUSION MVID patients are at risk of developing a PFIC-like liver disease that may hamper outcome after ITx. Our results suggest that cholestasis in MVID patients results from (1) impairment of the MYO5B/RAB11A apical recycling endosome pathway in hepatocytes, (2) altered targeting of BSEP to the canalicular membrane, and (3) increased ileal BA absorption. Because cholestasis worsens after ITx, indication of a combined liver ITx should be discussed in MVID patients with severe cholestasis. Future studies will need to address more specifically the effect of MYO5B dysfunction in BA homeostasis.
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Affiliation(s)
- Muriel Girard
- Department of Pediatric Gastroenterology and Hepatology, Necker Enfants-Malades Hospital, Assistance Publique-Hôpitaux de Paris, Université Paris Descartes-Sorbonne Cité, Paris, France; INSERM, UMR 781, Université Paris Descartes-Sorbonne Cité, Institut Imagine, Paris, France
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Thompson RJ, Knisely AS. Microvilli as markers of disordered apical-membrane trafficking and assembly: bowel and liver. Hepatology 2014; 60:34-6. [PMID: 24668851 DOI: 10.1002/hep.27148] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 03/19/2014] [Accepted: 03/22/2014] [Indexed: 01/22/2023]
Affiliation(s)
- Richard J Thompson
- Institute of Liver Studies, Division of Transplantation Immunology and Mucosal Biology, King's College London School of Medicine, London, UK; Institute of Liver Studies, King's College Hospital, London, UK; Paediatric Liver, GI and Nutrition Centre, King's College Hospital, London, UK
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Soroka CJ, Boyer JL. Biosynthesis and trafficking of the bile salt export pump, BSEP: therapeutic implications of BSEP mutations. Mol Aspects Med 2014; 37:3-14. [PMID: 23685087 PMCID: PMC3784619 DOI: 10.1016/j.mam.2013.05.001] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2013] [Revised: 04/25/2013] [Accepted: 05/07/2013] [Indexed: 12/17/2022]
Abstract
The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter.
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Affiliation(s)
- Carol J Soroka
- Yale University School of Medicine, Department of Internal Medicine, New Haven, CT 06520, United States.
| | - James L Boyer
- Yale University School of Medicine, Department of Internal Medicine, New Haven, CT 06520, United States.
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Erlinger S, Arias IM, Dhumeaux D. Inherited disorders of bilirubin transport and conjugation: new insights into molecular mechanisms and consequences. Gastroenterology 2014; 146:1625-38. [PMID: 24704527 DOI: 10.1053/j.gastro.2014.03.047] [Citation(s) in RCA: 141] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2013] [Revised: 03/12/2014] [Accepted: 03/23/2014] [Indexed: 12/11/2022]
Abstract
Inherited disorders of bilirubin metabolism might reduce bilirubin uptake by hepatocytes, bilirubin conjugation, or secretion of bilirubin into bile. Reductions in uptake could increase levels of unconjugated or conjugated bilirubin (Rotor syndrome). Defects in bilirubin conjugation could increase levels of unconjugated bilirubin; the effects can be benign and frequent (Gilbert syndrome) or rare but severe, increasing the risk of bilirubin encephalopathy (Crigler-Najjar syndrome). Impairment of bilirubin secretion leads to accumulation of conjugated bilirubin (Dubin-Johnson syndrome). We review the genetic causes and pathophysiology of disorders of bilirubin transport and conjugation as well as clinical and therapeutic aspects. We also discuss the possible mechanisms by which hyperbilirubinemia protects against cardiovascular disease and the metabolic syndrome and the effects of specific genetic variants on drug metabolism and cancer development.
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Affiliation(s)
| | | | - Daniel Dhumeaux
- Henri Mondor Hospital, Créteil, University of Paris-Est, Créteil, France
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31
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Kagawa T, Orii R, Hirose S, Arase Y, Shiraishi K, Mizutani A, Tsukamoto H, Mine T. Ursodeoxycholic acid stabilizes the bile salt export pump in the apical membrane in MDCK II cells. J Gastroenterol 2014; 49:890-899. [PMID: 23722250 DOI: 10.1007/s00535-013-0833-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 05/03/2013] [Indexed: 02/04/2023]
Abstract
BACKGROUND Ursodeoxycholic acid (UDCA) partly exerts choleretic effects by modifying the function of the bile salt export pump (Bsep, ABCB11). UDCA induces insertion of Bsep into the canalicular membrane of hepatocytes; however, underlying mechanisms remain unknown. We aimed to elucidate molecular mechanisms behind UDCA-induced Bsep activation. METHODS We established MDCK II cells stably expressing both Bsep and Na(+)-taurocholate cotransporting polypeptide, and investigated the effect of UDCA on activity and protein expression of Bsep using these cells. We performed inhibitor study to know the molecules involved in UDCA-induced Bsep activation, and also tested the influence of UDCA on Bsep having a disease-associated mutation. RESULTS UDCA activated Bsep in a dose-dependent manner. UDCA did not affect Bsep protein expression in whole cell lysates but increased its apical surface expression by extending the half-life from 2.4 to 5.0 h. This effect was specific to Bsep because UDCA did not affect other apical and basolateral proteins, and was independent of protein kinase A, adenylate cyclase, p38(MAPK), phosphatidylinositide 3-kinase, Ca(2+), and microtubules. NorUDCA activated Bsep similar to UDCA; however, cholic acid, taurocholic acid, and tauroUDCA had no effect. UDCA significantly increased the activity of Bsep with a benign recurrent intrahepatic cholestasis 2 mutation (A570T) but did not affect Bsep with a progressive familial intrahepatic cholestasis 2 mutation (G982R or D482G). CONCLUSIONS We demonstrated that UDCA stabilizes Bsep protein in the apical membrane and increases its activity in MDCK II cells, presumably by retarding the endocytotic process.
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Affiliation(s)
- Tatehiro Kagawa
- Division of Gastroenterology, Department of Internal Medicine, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa, 259-1193, Japan,
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Homolya L, Fu D, Sengupta P, Jarnik M, Gillet JP, Vitale-Cross L, Gutkind JS, Lippincott-Schwartz J, Arias IM. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes. PLoS One 2014; 9:e91921. [PMID: 24643070 PMCID: PMC3958433 DOI: 10.1371/journal.pone.0091921] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2013] [Accepted: 02/16/2014] [Indexed: 11/19/2022] Open
Abstract
Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
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Affiliation(s)
- László Homolya
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
- Laboratory of Molecular Cell Biology, Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
- * E-mail:
| | - Dong Fu
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
- Faculty of Pharmacy, The University of Sydney, Sydney, Australia
| | - Prabuddha Sengupta
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Michal Jarnik
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Jean-Pierre Gillet
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America
- Laboratory of Molecular Cancer Biology, Molecular Physiology Research Unit – URPhyM, Namur Research Institute for Life Sciences (NARILIS), Faculty of Medicine, University of Namur, Belgium University of Namur, Belgium
| | - Lynn Vitale-Cross
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
| | - J. Silvio Gutkind
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Jennifer Lippincott-Schwartz
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Irwin M. Arias
- Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America
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Aida K, Hayashi H, Inamura K, Mizuno T, Sugiyama Y. Differential roles of ubiquitination in the degradation mechanism of cell surface-resident bile salt export pump and multidrug resistance-associated protein 2. Mol Pharmacol 2014; 85:482-91. [PMID: 24378332 DOI: 10.1124/mol.113.091090] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025] Open
Abstract
We previously showed that ubiquitination, a reversible post-translational modification, facilitates degradation of cell surface-resident bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), ABC transporters that are expressed at the canalicular membrane (CM) of hepatocytes. In the current study, its underlying mechanism was investigated by evaluating the role of ubiquitination in the processes of internalization and subsequent degradation of cell surface-resident BSEP and MRP2. Cell surface biotinylation analysis using Flp-In T-REx 293 cells showed that ectopic expression of Ub(Δ)(GG), which is ubiquitin (Ub) lacking the two C-terminal glycines essential for the Ub conjugation reaction, inhibited the internalization of 3× FLAG-BSEP, but not of MRP2, and the degradation of the internalized MRP2, but not of the internalized 3× FLAG-BSEP. Its inhibitory effect on BSEP internalization was also indicated by a time-lapse imaging analysis using the rat hepatoma cell line McA-RH7777 in which Ub(Δ)(GG) delayed the loss of fluorescence from photoactivated Dronpa-BSEP on the CM. The effect of Ub(Δ)(GG) on BSEP internalization in these experiments was abrogated by treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and the introduction of a Y1311A mutation into BSEP. This mutation eliminates the ability of BSEP to interact with the AP2 adaptor complex, an adaptor protein required for cargo selection in clathrin-mediated endocytosis. In conclusion, our data suggest that ubiquitination facilitates clathrin-mediated endocytosis of BSEP and the degradation of internalized MRP2, leading to the degradation of the cell surface-resident form of both transporters.
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Affiliation(s)
- Kensuke Aida
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (K.A., H.H., K.I., T.M.); and Sugiyama Laboratory, RIKEN Innovation Center, Research Cluster for Innovation, RIKEN, Yokohama, Japan (Y.S.)
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Liu BB, Kong J, Wu SD, Wang Y. Bile acid salt export pump: Molecular mechanisms of transcription and intracellular regulation. Shijie Huaren Xiaohua Zazhi 2014; 22:788-794. [DOI: 10.11569/wcjd.v22.i6.788] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Bile salt export pump (BSEP), a member of ATP binding cassette (ABC), is responsible for transporting bile salt and is located on cholangiole lateral membrane. In humans, BSEP defects can lead to different types of cholestatic diseases, including hereditary or acquired liver diseases. In addition, BSEP is the most likely candidate gene for Lith1 stone. The bile salt plays an important role in many physiological and pathophysiological processes, and the scientific community has attached great importance to the research on the regulatory mechanism of the expression of BSEP. This paper summarizes the research related to transcriptional regulation of BSEP, and expression or functional changes of BSEP on cholangiole lateral membrane caused by intracellular transport changes, including intracellular endoplasmic reticulum and cell membrane ubiquitination-protease mediated protein degradation, short-term phosphorylation of BSEP, glycosylation, ubiquitination, and the regulatory effect of cholangiole lateral membrane-associated proteins.
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Abstract
Bile is a unique and vital aqueous secretion of the liver that is formed by the hepatocyte and modified down stream by absorptive and secretory properties of the bile duct epithelium. Approximately 5% of bile consists of organic and inorganic solutes of considerable complexity. The bile-secretory unit consists of a canalicular network which is formed by the apical membrane of adjacent hepatocytes and sealed by tight junctions. The bile canaliculi (∼1 μm in diameter) conduct the flow of bile countercurrent to the direction of portal blood flow and connect with the canal of Hering and bile ducts which progressively increase in diameter and complexity prior to the entry of bile into the gallbladder, common bile duct, and intestine. Canalicular bile secretion is determined by both bile salt-dependent and independent transport systems which are localized at the apical membrane of the hepatocyte and largely consist of a series of adenosine triphosphate-binding cassette transport proteins that function as export pumps for bile salts and other organic solutes. These transporters create osmotic gradients within the bile canalicular lumen that provide the driving force for movement of fluid into the lumen via aquaporins. Species vary with respect to the relative amounts of bile salt-dependent and independent canalicular flow and cholangiocyte secretion which is highly regulated by hormones, second messengers, and signal transduction pathways. Most determinants of bile secretion are now characterized at the molecular level in animal models and in man. Genetic mutations serve to illuminate many of their functions.
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Affiliation(s)
- James L Boyer
- Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut, USA.
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Reshetnyak VI. Physiological and molecular biochemical mechanisms of bile formation. World J Gastroenterol 2013; 19:7341-7360. [PMID: 24259965 PMCID: PMC3831216 DOI: 10.3748/wjg.v19.i42.7341] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Revised: 09/27/2013] [Accepted: 09/29/2013] [Indexed: 02/06/2023] Open
Abstract
This review considers the physiological and molecular biochemical mechanisms of bile formation. The composition of bile and structure of a bile canaliculus, biosynthesis and conjugation of bile acids, bile phospholipids, formation of bile micellar structures, and enterohepatic circulation of bile acids are described. In general, the review focuses on the molecular physiology of the transporting systems of the hepatocyte sinusoidal and apical membranes. Knowledge of physiological and biochemical basis of bile formation has implications for understanding the mechanisms of development of pathological processes, associated with diseases of the liver and biliary tract.
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Zucchetti AE, Barosso IR, Boaglio AC, Luquita MG, Roma MG, Crocenzi FA, Sánchez Pozzi EJ. Hormonal modulation of hepatic cAMP prevents estradiol 17β-D-glucuronide-induced cholestasis in perfused rat liver. Dig Dis Sci 2013; 58:1602-14. [PMID: 23371010 DOI: 10.1007/s10620-013-2558-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/13/2012] [Accepted: 01/01/2013] [Indexed: 12/09/2022]
Abstract
BACKGROUND Estradiol-17β-D-glucuronide (E17G) induces cholestasis in vivo, endocytic internalization of the canalicular transporters multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11) being a key pathomechanism. Cyclic AMP (cAMP) prevents cholestasis by targeting these transporters back to the canalicular membrane. In hepatocyte couplets, glucagon and salbutamol, both of which increase cAMP, prevented E17G action by stimulating the trafficking of these transporters by different mechanisms, namely: glucagon activates a protein kinase A-dependent pathway, whereas salbutamol activates an exchange-protein activated by cAMP (Epac)-mediated, microtubule-dependent pathway. METHODS The present study evaluated whether glucagon and salbutamol prevent E17G-induced cholestasis in a more physiological model, i.e., the perfused rat liver (PRL). Additionally, the preventive effect of in vivo alanine administration, which induces pancreatic glucagon secretion, was evaluated. RESULTS In PRLs, glucagon and salbutamol prevented E17G-induced decrease in both bile flow and the secretory activity of Abcc2 and Abcb11. Salbutamol prevention fully depended on microtubule integrity. On the other hand, glucagon prevention was microtubule-independent only at early time periods after E17G administration, but it was ultimately affected by the microtubule disrupter colchicine. Cholestasis was associated with endocytic internalization of Abcb11 and Abcc2, the intracellular carriers being partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained colocalized with Rab11a after glucagon treatment. In vivo, alanine administration increased hepatic cAMP and accelerated the recovery of bile flow and Abcb11/Abcc2 transport function after E17G administration. The initial recovery afforded by alanine was microtubule-independent, but microtubule integrity was required to sustain this protective effect. CONCLUSION We conclude that modulation of cAMP levels either by direct administration of cAMP modulators or by physiological manipulations leadings to hormone-mediated increase of cAMP levels (alanine administration), prevents estrogen-induced cholestasis in models with preserved liver architecture, through mechanisms similar to those arisen from in vitro studies.
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Affiliation(s)
- Andrés E Zucchetti
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET, U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
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Boaglio AC, Zucchetti AE, Toledo FD, Barosso IR, Sánchez Pozzi EJ, Crocenzi FA, Roma MG. ERK1/2 and p38 MAPKs are complementarily involved in estradiol 17ß-D-glucuronide-induced cholestasis: crosstalk with cPKC and PI3K. PLoS One 2012; 7:e49255. [PMID: 23166621 PMCID: PMC3498151 DOI: 10.1371/journal.pone.0049255] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2012] [Accepted: 10/04/2012] [Indexed: 12/17/2022] Open
Abstract
Objective The endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E217G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect. Design ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E217G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis. Results E217G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E217G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E217G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2. Conclusions cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E217G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.
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Affiliation(s)
| | | | | | | | | | - Fernando A. Crocenzi
- Institute of Experimental Physiology, National Scientific and Technical Research Council/National University of Rosario, Rosario, Argentina
- * E-mail: (FAC); (MGR)
| | - Marcelo G. Roma
- Institute of Experimental Physiology, National Scientific and Technical Research Council/National University of Rosario, Rosario, Argentina
- * E-mail: (FAC); (MGR)
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A hypothetical model of cargo-selective rab recruitment during organelle maturation. Cell Biochem Biophys 2012; 63:59-71. [PMID: 22328341 DOI: 10.1007/s12013-012-9341-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Rabs constitute a group of small GTPases that confer directionality to intracellular vesicle transport by promoting on the membrane of transport vesicles in the formation of specific protein complexes allowing for efficient fusion with a selected set of target organelles. The molecular mechanism controlling recruitment of the correct Rab at the right time is not fully understood. We propose a model according to which the residence time of a given Rab on the membrane of an organelle is determined by its transient trapping into a Rab effector complex (REC) composed of cargo receptor, SNAREs and further effectors. The stability of REC is controlled by the conformational state of the receptor which may change due to binding and release of cargo or changes in the luminal ion milieu. We use a conceptual mathematical model to calculate temporal changes in the Rab decoration of an organelle brought about by exchange with a cytosolic pool of Rabs or alternatively by budding and uptake of Rab-carrying vesicles. Considering the time-dependent drop in pH as one crucial factor for the conformational change of endocytic cargo receptors, our model provides a good quantitative description of the switch from Rab5 to Rab7 during the early-to-late endosome transition and correctly explains the arrest of this transition at insufficient luminal acidification. Model simulations suggest that a switch from one Rab to another may be continuous or abrupt. We discuss mechanisms, e.g. specific signalling pathways, which may restore an arrested organelle maturation.
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Lam P, Xu S, Soroka CJ, Boyer JL. A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis. Hepatology 2012; 55:1901-11. [PMID: 22161577 PMCID: PMC3319652 DOI: 10.1002/hep.25523] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2011] [Accepted: 11/21/2011] [Indexed: 12/11/2022]
Abstract
UNLABELLED The liver-specific bile salt export pump (BSEP) is crucial for bile acid-dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related adenosine triphosphate (ATP)-binding cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and two large cytoplasmic nucleotide-binding domains (NBDs). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface enzyme-linked immunosorbent assay (ELISA) endocytosis reporter system using a chimera of the interleukin-2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif (1310) YYKLV(1314) (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y(1310) Y(1311) amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity. CONCLUSION Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.
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Affiliation(s)
- Ping Lam
- Liver Center, Yale University School of Medicine, New Haven, CT06520-8019, USA
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Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 2012; 485:465-70. [DOI: 10.1038/nature11133] [Citation(s) in RCA: 274] [Impact Index Per Article: 21.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2011] [Accepted: 04/03/2012] [Indexed: 12/17/2022]
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Blazquez AG, Briz O, Romero MR, Rosales R, Monte MJ, Vaquero J, Macias RIR, Cassio D, Marin JJG. Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta. Mol Pharmacol 2012; 81:273-83. [PMID: 22096226 DOI: 10.1124/mol.111.075143] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025] Open
Abstract
ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17β-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for <2-fold increase in fetal cholanemia despite >14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(-/-) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver.
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Affiliation(s)
- Alba G Blazquez
- Laboratory of Experimental Hepatology and Drug Targeting, National Institute for the Study of Liver and Gastrointestinal Diseases, University of Salamanca, Salamanca, Spain
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Kruglov EA, Gautam S, Guerra MT, Nathanson MH. Type 2 inositol 1,4,5-trisphosphate receptor modulates bile salt export pump activity in rat hepatocytes. Hepatology 2011; 54:1790-9. [PMID: 21748767 PMCID: PMC3205211 DOI: 10.1002/hep.24548] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 06/26/2011] [Indexed: 12/16/2022]
Abstract
UNLABELLED Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short-term regulation of Bsep activity. Ca(2+) regulates targeting and insertion of transporters in many cell systems, and Ca(2+) release near the canalicular membrane is mediated by the type II inositol 1,4,5-trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido-fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen- and endotoxin-induced cholestasis. CONCLUSION These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis.
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Affiliation(s)
| | | | | | - Michael H. Nathanson
- Address for correspondence: Michael H. Nathanson, Section of Digestive Diseases, Yale University School of Medicine, 333 Cedar Street, TAC S241D, New Haven, CT. 06520-8019, Phone: (203) 785-7312. Fax: (203) 785-7273
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Li X, DiFiglia M. The recycling endosome and its role in neurological disorders. Prog Neurobiol 2011; 97:127-41. [PMID: 22037413 DOI: 10.1016/j.pneurobio.2011.10.002] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Revised: 10/14/2011] [Accepted: 10/17/2011] [Indexed: 02/08/2023]
Abstract
The recycling endosome (RE) is an organelle in the endocytic pathway where plasma membranes (proteins and lipids) internalized by endocytosis are processed back to the cell surface for reuse. Endocytic recycling is the primary way for the cell to maintain constituents of the plasma membrane (Griffiths et al., 1989), i.e., to maintain the abundance of receptors and transporters on cell surfaces. Membrane traffic through the RE is crucial for several key cellular processes including cytokinesis and cell migration. In polarized cells, including neurons, the RE is vital for the generation and maintenance of the polarity of the plasma membrane. Many RE dependent cargo molecules are known to be important for neuronal function and there is evidence that improper function of key proteins in RE-associated pathways may contribute to the pathogenesis of neurological disorders, including Huntington's disease. The function of the RE in neurons is poorly understood. Therefore, there is need to understand how membrane dynamics in RE-associated pathways are affected or participate in the development or progression of neurological diseases. This review summarizes advances in understanding endocytic recycling associated with the RE, challenges in elucidating molecular mechanisms underlying RE function, and evidence for RE dysfunction in neurological disorders.
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Affiliation(s)
- Xueyi Li
- Laboratory of Cellular Neurobiology and Department of Neurology, Massachusetts General Hospital, 114 16th Street, Charlestown, MA 02129, USA
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45
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Zucchetti AE, Barosso IR, Boaglio A, Pellegrino JM, Ochoa EJ, Roma MG, Crocenzi FA, Sánchez Pozzi EJ. Prevention of estradiol 17beta-D-glucuronide-induced canalicular transporter internalization by hormonal modulation of cAMP in rat hepatocytes. Mol Biol Cell 2011; 22:3902-15. [PMID: 21865596 PMCID: PMC3192868 DOI: 10.1091/mbc.e11-01-0047] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
In estradiol 17β-d-glucuronide (E17G)-induced cholestasis, the canalicular hepatocellular transporters bile salt export pump (Abcb11) and multidrug-resistance associated protein 2 (Abcc2) undergo endocytic internalization. cAMP stimulates the trafficking of transporter-containing vesicles to the apical membrane and is able to prevent internalization of these transporters in estrogen-induced cholestasis. Hepatocyte levels of cAMP are regulated by hormones such as glucagon and adrenaline (via the β2 receptor). We analyzed the effects of glucagon and salbutamol (a β2 adrenergic agonist) on function and localization of Abcb11 and Abcc2 in isolated rat hepatocyte couplets exposed to E17G and compared the mechanistic bases of their effects. Glucagon and salbutamol partially prevented the impairment in Abcb11 and Abcc2 transport capacity. E17G also induced endocytic internalization of Abcb11 and Abcc2, which partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained internalized and mainly colocalizing with Rab11a in the perinuclear region after incubation with glucagon. Glucagon prevention was dependent on cAMP-dependent protein kinase (PKA) and independent of exchange proteins activated directly by cAMP (Epac) and microtubules. In contrast, salbutamol prevention was PKA independent and Epac/MEK and microtubule dependent. Anticholestatic effects of glucagon and salbutamol were additive in nature. Our results show that increases in cAMP could activate different anticholestatic signaling pathways, depending on the hormonal mediator involved.
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Affiliation(s)
- Andrés E Zucchetti
- Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, S2002LRL Rosario, Argentina
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Molnar A, Haybaeck J, Lackner C, Strnad P. The cytoskeleton in nonalcoholic steatohepatitis: 100 years old but still youthful. Expert Rev Gastroenterol Hepatol 2011; 5:167-77. [PMID: 21476912 DOI: 10.1586/egh.11.5] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The hepatocellular cytoskeleton consists of three filamentous systems: microfilaments, microtubules and keratins (Ks). While the alterations in microfilaments and microtubules during nonalcoholic steatohepatitis (NASH) are largely unexplored, K8/K18 reorganization into Mallory-Denk bodies (MDBs) represents a NASH hallmark, and serological K18 fragments constitute an established tool to monitor NASH severity. To commemorate the 100th anniversary of the first description of MDBs, this article summarizes the composition and function of the hepatocellular cytoskeleton, as well as the importance of cytoskeletal alterations in NASH. The significance of MDBs in clinical routine is illustrated, as are the findings from MDB mouse models, which shape our current view of MDB pathogenesis. Even after 100 years, the cytoskeleton represents a fascinating but greatly understudied area of NASH biology.
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Affiliation(s)
- Agnes Molnar
- Department of Internal Medicine I, University Hospital Ulm, Germany
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Goler-Baron V, Assaraf YG. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance. PLoS One 2011; 6:e16007. [PMID: 21283667 PMCID: PMC3025911 DOI: 10.1371/journal.pone.0016007] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2010] [Accepted: 12/02/2010] [Indexed: 02/04/2023] Open
Abstract
Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.
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Affiliation(s)
- Vicky Goler-Baron
- The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel
| | - Yehuda G. Assaraf
- The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel
- * E-mail:
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Stieger B. The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation. Handb Exp Pharmacol 2011:205-59. [PMID: 21103971 DOI: 10.1007/978-3-642-14541-4_5] [Citation(s) in RCA: 207] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Bile formation is an important function of the liver. Bile salts are a major constituent of bile and are secreted by hepatocytes into bile and delivered into the small intestine, where they assist in fat digestion. In the small intestine, bile salts are almost quantitatively reclaimed and transported back via the portal circulation to the liver. In the liver, hepatocytes take up bile salts and secrete them again into bile for ongoing enterohepatic circulation. Uptake of bile salts into hepatocytes occurs largely in a sodium-dependent manner by the sodium taurocholate cotransporting polypeptide NTCP. The transport properties of NTCP have been extensively characterized. It is an electrogenic member of the solute carrier family of transporters (SLC10A1) and transports predominantly bile salts and sulfated compounds, but is also able to mediate transport of additional substrates, such as thyroid hormones, drugs and toxins. It is highly regulated under physiologic and pathophysiologic conditions. Regulation of NTCP copes with changes of bile salt load to hepatocytes and prevents entry of cytotoxic bile salts during liver disease. Canalicular export of bile salts is mediated by the ATP-binding cassette transporter bile salt export pump BSEP (ABCB11). BSEP constitutes the rate limiting step of hepatocellular bile salt transport and drives enterohepatic circulation of bile salts. It is extensively regulated to keep intracellular bile salt levels low under normal and pathophysiologic situations. Mutations in the BSEP gene lead to severe progressive familial intrahepatic cholestasis. The substrates of BSEP are practically restricted to bile salts and their metabolites. It is, however, subject to inhibition by endogenous metabolites or by drugs. A sustained inhibition will lead to acquired cholestasis, which can end in liver injury.
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Affiliation(s)
- Bruno Stieger
- Division of Clinical Pharmacology and Toxicology, University Hospital, 8091, Zurich, Switzerland.
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Lam P, Soroka CJ, Boyer JL. The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease. Semin Liver Dis 2010; 30:125-33. [PMID: 20422495 PMCID: PMC3008346 DOI: 10.1055/s-0030-1253222] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.
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Affiliation(s)
- Ping Lam
- Liver Center, Yale University School of Medicine, New Haven, Connecticut
| | - Carol J. Soroka
- Liver Center, Yale University School of Medicine, New Haven, Connecticut
| | - James L. Boyer
- Liver Center, Yale University School of Medicine, New Haven, Connecticut
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Xue X, Jaulin F, Espenel C, Kreitzer G. PH-domain-dependent selective transport of p75 by kinesin-3 family motors in non-polarized MDCK cells. J Cell Sci 2010; 123:1732-41. [PMID: 20427314 DOI: 10.1242/jcs.056366] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
A key process during epithelial polarization involves establishment of polarized transport routes from the Golgi to distinct apical and basolateral membrane domains. To do this, the machinery involved in selective trafficking must be regulated during differentiation. Our previous studies showed that KIF5B selectively transports vesicles containing p75-neurotrophin receptors to the apical membrane of polarized, but not non-polarized MDCK cells. To identify the kinesin(s) responsible for p75 trafficking in non-polarized MDCK cells we expressed KIF-specific dominant-negative constructs and assayed for changes in post-Golgi transport of p75 by time-lapse fluorescence microscopy. Overexpression of the tail domains of kinesin-3 family members that contain a C-terminal pleckstrin homology (PH) domain, KIF1A or KIF1Bbeta, attenuated the rate of p75 exit from the Golgi in non-polarized MDCK cells but not in polarized cells. Analysis of p75 post-Golgi transport in cells expressing KIF1A or KIF1Bbeta with their PH domains deleted revealed that vesicle transport by these motors depends on the PH domains. Furthermore, purified KIF1A and KIF1Bbeta tails interact with p75 vesicles and these interactions require the PH domain. Knockdown of canine KIF1A also inhibited exit of p75 from the Golgi, and this was rescued by expression of human KIF1A. Together these data demonstrate that post-Golgi transport of p75 in non-polarized epithelial cells is mediated by kinesin-3 family motors in a PH-domain-dependent process.
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Affiliation(s)
- Xiaoxiao Xue
- Department of Cell and Developmental Biology, Weill Medical College, Cornell University, 1300 York Avenue, New York, NY 10021, USA
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