WIF-B cells as a model for alcohol-induced hepatocyte injury

Schaffert, Courtney S.; Todero, Sandra L.; McVicker, Benita L.; Tuma, Pamela L.; Sorrell, Michael F.; Tuma, Dean J.

doi:10.1016/j.bcp.2004.01.022

Cited by 34 publications

(48 citation statements)

References 41 publications

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“…Not only does this cell line exhibit a polarized phenotype in culture, it also maintains numerous liver-specific activities normally lost in isolated hepatocytes or missing from other hepatic cell lines. Importantly, these cells metabolize ethanol 2 and display alcohol-induced defects in cellular processes as described in situ or in isolated hepatocytes. These impairments include decreased microtubule polymerization, 3 defects in ASGP-R trafficking, and albumin secretion.…”

Section: Discussionmentioning

confidence: 99%

“…In general, cells were treated on day 7 with 50 mM ethanol in medium buffered with 10 mM Hepes (pH 7.0) for 72 hours as described. 2 Cells were additionally treated with 4-MP or TSA as described in the figure captions.…”

Section: Methodsmentioning

confidence: 99%

“…1 Importantly, WIF-B cells metabolize ethanol using alcohol dehydrogenase (ADH) and cytochrome P4502E 1 . 2 Like hepatocytes, ethanol-treated WIF-B cells display a reduced redox state and increased triglycerides. 2 More recently, we discovered that microtubule repolymerization was impaired in ethanol-treated WIF-B cells, 3 consistent with reports performed in vitro and in isolated hepatocytes.…”

mentioning

confidence: 99%

“…2 Like hepatocytes, ethanol-treated WIF-B cells display a reduced redox state and increased triglycerides. 2 More recently, we discovered that microtubule repolymerization was impaired in ethanol-treated WIF-B cells, 3 consistent with reports performed in vitro and in isolated hepatocytes. [4][5][6] We also found that steady-state microtubules in alcohol-treated WIF-B cells were more stable and were acetylated 2-3-fold more than were those in control cells.…”

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confidence: 99%

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Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking

et al. 2007

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We have been using polarized hepatic WIF-B cells to examine ethanol-induced liver injury. Previously, we determined microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells. We proposed that the ethanol-induced alterations in microtubule dynamics may explain the ethanol-induced defects in membrane trafficking that have been previously documented. To test this, we compared the trafficking of selected proteins in control cells and cells treated with ethanol or with the histone deacetylase 6 inhibitor trichostatin A (TSA). We determined that exposure to 50 nM TSA for 30 minutes induced microtubule acetylation (ϳ3-fold increase) and stability to the same extent as did ethanol. As shown previously in situ, the endocytic trafficking of the asialoglycoprotein receptor (ASGP-R) was impaired in ethanol-treated WIF-B cells. This impairment required ethanol metabolism and was likely mediated by acetaldehyde. TSA also impaired ASGP-R endocytic trafficking, but to a lesser extent. Similarly, both ethanol and TSA impaired transcytosis of the single-spanning apical resident aminopeptidase N (APN). For both ASGP-R and APN and for both treatments, the block in trafficking was internalization from the basolateral membrane. Interestingly, no changes in transcytosis of the glycophosphatidylinositol-anchored protein, 5 -nucleotidase, were observed, suggesting that increased microtubule acetylation and stability differentially regulate internalization. We further determined that albumin secretion was impaired in both ethanol-treated and TSA-treated cells, indicating that increased microtubule acetylation and stability also disrupted this transport step. Conclusion: These results indicate that altered microtubule dynamics explain in part alcoholinduced defects in membrane trafficking. (HEPATOLOGY 2008;47:1745-1753 A lcoholic liver disease is a major biomedical health concern in the United States. Although the progression of this disease has been well described clinically, little is known about the molecular basis for liver injury. Research aimed at identifying the molecules and pathways that promote alcohol-induced hepatotoxicity has been hampered by the lack of good in vitro models. Our recent studies have been performed in WIF-B cells, an emerging model system for the study of alcohol-induced liver injury. These hepatic cells are highly differentiated and form discrete apical and basolateral membrane domains in culture. Domain-specific membrane proteins are localized in WIF-B cells as they are in hepatocytes in situ, and many liver-specific functions are maintained. 1 Importantly, WIF-B cells metabolize ethanol using alcohol dehydrogenase (ADH) and cytochrome P4502E 1 . 2 Like hepatocytes, ethanol-treated WIF-B cells display a reduced redox state and increased triglycerides. 2 More recently, we discovered that microtubule repolymerization was impaired in ethanol-treated WIF-B cells, 3 consistent with reports performed in vitro and in isolated hepatocytes. [4][5][6] We also found that steady-state micro...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking

et al. 2007

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“…Therefore, several in vitro culture systems have been developed in order to determine the effects of ethanol administration on the liver. These systems include primary hepatocyte cultures [3,4], HepG2 hepatoma cells expressing stable exogenous alcohol dehydrogenase [5,6] or cytochrome P4502E1 [7], and WIF-B cells [8,9]. Although these models have provided valuable information, numerous limitations and weaknesses are evident in these systems.…”

Section: Introductionmentioning

confidence: 99%

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Klassen

Thiele

Duryee

et al. 2008

Biochemical Pharmacology

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Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96 h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

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Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells

et al. 2006

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Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. T he proteasome is a multicatalytic enzyme crucially important for proteolysis of oxidized and misfolded proteins, peptides and short-lived signal transduction factors. In vivo, the proteasome exists in equilibrium as 2 particles: the 20S proteasome degrades substrate proteins with no prior ubiquitylation and the 26S form degrades ubiquitylated proteins in an ATP-dependent manner.Both 20S and 26S proteasomes generate peptides for MHC class I-restricted antigen presentation. 1,2 Processing of antigens is a necessary step for recognition by cytotoxic T lymphocytes (CTLs) of virally infected cells. To be recognized by CTLs, "nonself" antigens are processed to peptides of 8-10 amino acids, which then bind to MHC class I molecules in the endoplasmic reticulum. 3 Initial processing of antigenic proteins in the cytoplasm is catalyzed by the proteasome and/or leucine aminopeptidase (LAP). 4 Both enzymes are activated by the proinflammatory cytokine interferon gamma (IFN␥), 5 which induces formation of the immunoproteasome that cleaves antigenic proteins to uniformly sized peptides for presentation.The foregoing series of events occurs after hepatitis B or C infections during which CTLs eliminate HBV-infected or HCV-infected hepatocytes are based on ability of immune system to clear infected hepatocytes. Epidemiological studies have provided supporting evidence of

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WIF-B cells as a model for alcohol-induced hepatocyte injury

Cited by 34 publications

References 41 publications

Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking

Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells

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