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Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells.

ABSTRACT: Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

SUBMITTER: Sokolowska I

PROVIDER: S-EPMC3527237 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells.

Sokolowska Izabela I Dorobantu Cristina C Woods Alisa G AG Macovei Alina A Branza-Nichita Norica N Darie Costel C CC

Proteome science 20120802 1

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more ...[more]

PMID: 22857383

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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

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells.

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Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells.

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