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Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

ABSTRACT: Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

SUBMITTER: Petrareanu C

PROVIDER: S-EPMC3748114 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

Petrareanu Catalina C Macovei Alina A Sokolowska Izabela I Woods Alisa G AG Lazar Catalin C Radu Gabriel L GL Darie Costel C CC Branza-Nichita Norica N

PloS one 20130820 8

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitr ...[more]

PMID: 23977166

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Project description:BackgroundNAFLD is highly prevalent with limited treatment options. Bile acids (BAs) increase in the systemic circulation and liver during NAFLD progression. Changes in plasma membrane localization and zonal distribution of BA transporters can influence transport function and BA homeostasis. However, a thorough characterization of how NAFLD influences these factors is currently lacking. This study aimed to evaluate the impact of NAFLD and the accompanying histologic features on the functional capacity of key hepatocyte BA transporters across zonal regions in human liver biopsies.MethodsA novel machine learning image classification approach was used to quantify relative zonal abundance and plasma membrane localization of BA transporters (bile salt export pump [BSEP], sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [OATP] 1B1 and OATP1B3) in non-diseased (n = 10), NAFL (n = 9), and NASH (n = 11) liver biopsies. Based on these data, membrane-localized zonal abundance (MZA) measures were developed to estimate transporter functional capacity.ResultsNAFLD diagnosis and histologic scoring were associated with changes in transporter membrane localization and zonation. Increased periportal BSEPMZA (mean proportional difference compared to non-diseased liver of 0.090) and decreased pericentral BSEPMZA (-0.065) were observed with NASH and also in biopsies with higher histologic scores. Compared to Non-diseased Liver, periportal OATP1B3MZA was increased in NAFL (0.041) and NASH (0.047). Grade 2 steatosis (mean proportional difference of 0.043 when compared to grade 0) and grade 1 lobular inflammation (0.043) were associated with increased periportal OATP1B3MZA.ConclusionsThese findings provide novel mechanistic insight into specific transporter alterations that impact BA homeostasis in NAFLD. Changes in BSEPMZA likely contribute to altered BA disposition and pericentral microcholestasis previously reported in some patients with NAFLD. BSEPMZA assessment could inform future development and optimization of NASH-related pharmacotherapies.

Project description:HepaRG is a proliferative human hepatoma-derived cell line that can be differentiated into hepatocyte-like and biliary-like cells. Differentiated HepaRG cultures maintain key hepatic functions including drug transporters and xenobiotic-metabolizing enzymes. To gain insight into proliferative and differentiated HepaRG metabolism we profiled various bioenergetic parameters and investigated cell culture levels of adenosine triphosphate (ATP), lactate, and lactate dehydrogenase (LDH) activity. Compared to differentiated-derived HepaRG, cells from proliferative cultures had increased basal and ATP-linked respiration and decreased maximal and spare respiratory capacities. Basal ATP levels but not lactate or LDH activity were increased in samples from proliferative-derived compared to differentiated-derived HepaRG. Further extracellular acidification rate (ECAR) experiments revealed parameters associated with glycolysis and oxidative phosphorylation. Under basal conditions, cells derived from both cultures had similar ECARs; however, under stressed conditions, proliferative-derived HepaRG had increases in ECAR capacity and apparent glycolytic reserve. The biguanide metformin has been reported to protect differentiated HepaRG against acetaminophen (APAP)-induced cell injury, as well as offer protection against bioenergetic deficiencies; therefore, we studied the outcome of exposure to these drugs in both culture conditions. Proliferative- and differentiated-derived cells were found to have distinct mitochondrial bioenergetic alterations when exposed to the hepatotoxic drug APAP. Metformin offered protection against loss of APAP-induced cellular viability and prevented APAP-induced decreases in bioenergetics in differentiated- but not proliferative-derived HepaRG. Distinguishingly, treatment with metformin alone reduced ATP-linked respiration, maximal respiratory capacity, and basal respiration in proliferative-derived HepaRG. Our results support that HepaRG represents an appropriate model to study drug-induced bioenergetic dysfunction.

Dataset Information

Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

Publications

Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

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